IM To determine the incidence of hepatitis B virus (HBV) intrauterine infection and to explore the relationship between HBV viremia level of pregnant women and HBV intrauterine infection.METHODS Sixtynine pregnant w...IM To determine the incidence of hepatitis B virus (HBV) intrauterine infection and to explore the relationship between HBV viremia level of pregnant women and HBV intrauterine infection.METHODS Sixtynine pregnant women were divided into three groups. Group A, 41 HBsAg positive patients, 14 of them were HBeAg positive (group A1), and 27 HBeAg negative (group A2); Group B, 12 HBsAg negative patients, but positive for antiHBs and/or antiHBe and/or antiHBc; and Group C, 16 patients negative for all HBV markers. Blood samples of mothers were taken at delivery, samples of their infants were collected within 24 hours after birth (before injection of HBIG and HBV vaccine). All the serum samples were stored at -20℃. HBV serum markers were tested by radioimmunoassay and HBV NDA were detected by nested polymerase chain reaction.RESULTS In group C, all of 16 newborns were negative for HBsAg and HBV DNA. In group A, 7 infants were HBsAg positive (171%), and 17 (415%) were HBV DNA positive (P<005). The incidence of intrauterine HBV infection was much higher in group A1 than that in group A2 (HBsAg 429% vs 37%, HBV DNA 929% vs 148%, P<005). The incidence of HBV intrauterine infection was significantly different between high and low HBV viremia of mothers (933% vs 429%, P<005).CONCLUSION The incidence of HBV intrauterine infection is high when HBV DNA in newborns detected with nested PCR is used as a marker of HBV infection. It is related to HBV viremia level of mothers.展开更多
AIM:To assess expression of matrix metalloproteinases 2(MMP2)and MMP9 in gastric cancer,superficial gastritis and normal mucosa,and to measure metalloproteinase activity.METHODS:MMP2 and MMP9 mRNA expression was deter...AIM:To assess expression of matrix metalloproteinases 2(MMP2)and MMP9 in gastric cancer,superficial gastritis and normal mucosa,and to measure metalloproteinase activity.METHODS:MMP2 and MMP9 mRNA expression was determined by quantitative real-time polymerase chain reaction.Normalization was carried out using three different factors.Proteins were analyzed by quantitative gelatin zymography(qGZ).RESULTS:18S ribosomal RNA(18SRNA)was very highly expressed,while hypoxanthine ribosyltransferase-1(HPRT-1)was moderately expressed.MMP2 was highly expressed,while MMP9 was not detected or lowly expressed in normal tissues,moderately or highly expressed in gastritis and highly expressed in cancer.Relative expression of 18SRNA and HPRT-1 showed no significant differences.Significant differences in MMP2 and MMP9 were found between cancer and normal tissue,but not between gastritis and normal tissue.Absolute quantification of MMP9 echoed this pattern,but differential expression of MMP2 proved conflictive.Analysis by qGZ indicated significant differences between cancer and normal tissue in MMP-2,total MMP-9,250 and 110 kDa bands.CONCLUSION:MMP9 expression is enhanced in gastric cancer compared to normal mucosa;interpretation of differential expression of MMP2 is difficult to establish.展开更多
AIM To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS Seventy patients with duodenalulcer and fifty healthy controls were examinedfor HLA-DQA1 genotypes.HLA-DQA1 typing wa...AIM To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS Seventy patients with duodenalulcer and fifty healthy controls were examinedfor HLA-DQA1 genotypes.HLA-DQA1 typing wascarried out by digesting the locus specificpolymerase chain reaction amplified productswith alleles specific restriction enzymes(PCR-RFLP),i.e.,Apal Ⅰ,Bsaj Ⅰ,Hph Ⅰ,Fok Ⅰ,Mbo Ⅱ and Mnl Ⅰ.RESULTS The allele frequencies of DQA1 * 0301and DQA1 * 0102 in patients with duodenal ulcerwere significantly higher and lower respectivelythan those in healthy controls(0.40 vs 0.20,P = 0.003,mcorret = 0.024)and(0.05 vs 0.14,P = 0.012,but Pcorret】0.05),respectively.CONCLUSION DQA1 * 0301 is a susceptiblegene for duodenal ulcer in Wuhan Hans,andthere are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients andhealthy controls.展开更多
The gram-negative bacterium Helicobacter pylori(H.pylori)causes chronic gastritis,gastric and duodenal ulcers,gastric cancer and mucosa-associated lymphoid tissue lymphoma.Treatment is recommended in all symptomatic p...The gram-negative bacterium Helicobacter pylori(H.pylori)causes chronic gastritis,gastric and duodenal ulcers,gastric cancer and mucosa-associated lymphoid tissue lymphoma.Treatment is recommended in all symptomatic patients.The current treatment options for H.pylori infection are outlined in this review in light of the recent challenges in eradication success,largely due to the rapid emergence of antibiotic resistant strains of H.pylori.Antibiotic resistance is a constantly evolving process and numerous studies have shown that the prevalence of H.pylori antibiotic resistance varies significantly from country to country,and even between regions within the same country.In addition,recent data has shown that previous antibiotic use is associated with harbouring antibiotic resistant H.pylori.Local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy.Antimicrobial resistance is assessed by H.pylori culture and antimicrobial susceptibility testing.Recently developed molecular tests offer an attractive alternative to culture and allow for the rapid molecular genetic identification of H.pylori and resistance-associated mutations directly from biopsy samples or bacterial culture material.Accumulating evidence indicates that surveillance of antimicrobial resistance by susceptibility testing is feasible and necessary to inform clinicians in their choice of therapy for management of H.pylori infection.展开更多
Objective： MicroRNA-21 （miR-21） has been shown to be a key regulator of carcinogenesis. There were few reports about the comparison of serum miR-21 with conventional tumor markers. This study aimed to explore the d...Objective： MicroRNA-21 （miR-21） has been shown to be a key regulator of carcinogenesis. There were few reports about the comparison of serum miR-21 with conventional tumor markers. This study aimed to explore the diagnostic value of circulating miR-21 as a tumor marker in breast cancer （BC） and compare it with CA15 3 and carcinoembryonic antigen （CEA）. Methods： Circulating miR-16 and miR-21 were amplified and quantitatively detected by real-time PCR in 89 BC patients and 55 healthy controls. The levels of CA153 and CEA were measured through assays. Then the sensitivity in diagnosis of BC was compared among miR-21, CA153 and CEA. Results： The level of serum miR-21 was significantly higher in BC patients than controls （P〈0.001）. The sensitivity and specificity of miR-21 were 87.6% and 87.3%, respectively, whereas the sensitivities of CEA and CA153 were only 22.47% and 15.73%. Con^lusions： Compared with CEA and CA153, serum miR-21 has a higher sensitivity in diagnosis of BC. Although not correlated with the status of ER, PR and clinical stages, serum miR-21 may be a potential diagnostic indicator for BC, especially for the early stage.展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co展开更多
INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship
INTRODUCTIONLiver fibrosis is an excessive deposition ofextracellular matrix(ECM)resulted from bothincreased synthesis and decreased degradation.Matrix metalloproteinases(MMPs)represent agroup of neutral proteinases w...INTRODUCTIONLiver fibrosis is an excessive deposition ofextracellular matrix(ECM)resulted from bothincreased synthesis and decreased degradation.Matrix metalloproteinases(MMPs)represent agroup of neutral proteinases with variable展开更多
The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this...The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this phenomenon is named occult hepatitis B infection(OBI). The presence of anti-HBc antibody in serum is an important key for OBI tracking, although about 20% of OBI cases are negative for anti-HBc antibody. The diagnosis of OBI is mainly based on polymerase chain reaction(PCR) and real-time PCR assays. However, real-time PCR is a more reliable method than PCR. OBI is a great issue for the public health problem and a challenge for the clinical entity worldwide. The persistence of OBI may lead to the development of cirrhosis and hepatocellular carcinoma. With regard to OBI complications, the screening of HBV DNA by the highly sensitive molecular means should be implemented for:(1) patients with a previous history of chronic or acute HBV infection;(2) patients co-infected with hepatitis C virus/human immunodeficiency virus;(3) patients undergoing chemotherapy or anti-CD20 therapy;(4) recipients of organ transplant;(5) blood donors;(6) organ transplant donors;(7) thalassemia and hemophilia patients;(8) health care workers;(9) patients with liver related disease(cryptogenic);(10) hemodialysis patients;(11) patients undergoing lamivudine or interferon therapy; and(12) children in time of HBV vaccination especially in highly endemic areas of HBV. Active HBV vaccination should be implemented for the close relatives of patients who are negative for OBI markers. Thus, the goal of this review is to evaluate the rate of OBI with a focus on status of high risk groups in different regions of the world.展开更多
Helicobacter pylori (H. pylori ) is a major causativebacterium of chronic gastritis, peptic ulcer and mucosaassociated lymphoid tissue lymphoma in humans, and associated with an increased risk of gastric cancer[1 -8]....Helicobacter pylori (H. pylori ) is a major causativebacterium of chronic gastritis, peptic ulcer and mucosaassociated lymphoid tissue lymphoma in humans, and associated with an increased risk of gastric cancer[1 -8]. An important virulant factor of H. pylori is the vacuolating cytotoxin ( VacA ) encoded by vacA that induces cytoplasmic vacuolation in target cells both in vitro and in vivo[9-11]. VacA is produced as a 140 kDa precursor which contains an N-terminal signal peptide and an approximately 33 kDa C-terminal outer membrance exporter. The precursor is cleaved at both N-terminal and C-terminal and secreted into the extracellular milieu as a 95 kDa mature protein. The mature protein futher undergoes specific cleavage to yield 37 kDa and 58 kDa subunits[12-14] Although vacA is present in all H. pylori strains, only about 50% to 60% of strains can induce vacuolation of epithelial cells as assessed by the HeLa cell assay. vacA shows considerable genetic variation in H. pylori isolated from all over the world and contains at least two variable regions. The s region exists as sl or s2 allelic types. Among type sl strains, subtypes sla and slb have been identified. The m region occurs as ml or m2 allelic types. Specific vacA genotype of H. pylori strains are associated with the production of the cytotoxin in vitro, epithelial damage in vivo, and clinical consequences[15-27]. The other virulant factor is the cytotoxin-associated protein (CagA) encoded by the cytotoxin-associated gene (cagA). The cagA gene is present in about 60% to 70% of strains and all of these strains express the cagA. The presence of cagA is also associated with the production of the cytotoxin in vitro, and clinical outcome[24-30]. The aim of this study was (i) to identify vacA genotypes and cagA status of H. pylori isolated from Chinese patients; (ii) to evaluation the relatioship beween vacA genotypes, cagA status and related gastroenterological disorders.展开更多
Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages....Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, <sup>13</sup>C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while <sup>13</sup>C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The <sup>13</sup>C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor’s test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori’s DNA in gastric mucosa, gastric juice, saliva, dental plaques展开更多
AIM:To establish the real time fluorescence polymerase chain reaction(RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.METHODS:Four hundred infants(< 1 year of age) with unexpla...AIM:To establish the real time fluorescence polymerase chain reaction(RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.METHODS:Four hundred infants(< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4.Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed.Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes.After a single RT-PCR,results were obtained by analyzing the take-off curves.Twenty-four positive and 14 negative samples were retested by direct sequencing.RESULTS:Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8%(46/800).Twenty-six and 20 mutant alleles were observed respectively,in 474 and 242 alleles from the intermediate and southern areas of China.No mutant allele was detected in 84 alleles from northern China.Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations,and 14 negative samples were retested by direct sequencing,which confirmed that the accuracy of RTPCR was 100%.CONCLUSION:RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.展开更多
AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in se...AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.展开更多
Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymera...Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.展开更多
AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carr...AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.展开更多
AIM:To study whether selected bacterial 16S ribosomal RNA(rRNA)gene phylotypes are capable of disting- uishing irritable bowel syndrome(IBS). METHODS:The faecal microbiota of twenty volunteers with IBS,subdivided into...AIM:To study whether selected bacterial 16S ribosomal RNA(rRNA)gene phylotypes are capable of disting- uishing irritable bowel syndrome(IBS). METHODS:The faecal microbiota of twenty volunteers with IBS,subdivided into eight diarrhoea-predominant (IBS-D),eight constipation-predominant(IBS-C)and four mixed symptom-subtype(IBS-M)IBS patients,and fifteen control subjects,were analysed at three time-points with a set of fourteen quantitative real-timepolymerase chain reaction assays.All assays targeted 16S rRNA gene phylotypes putatively associated with IBS,based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobac-teria,Bacteroidetes and Firmicutes.Eight of the target phylotypes had less than 95%similarity to cultured bacterial species according to their 16S rRNA gene sequence.The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis(PCA)with linear mixed-effects models applied to the principal component scores. RESULTS:Bacterial phylotypes Clostridium cocleatum 88%,Clostridium thermosuccinogenes 85%,Coprobacillus catenaformis 91%,Ruminococcus bromii-like, Ruminococcus torques 91%,and R.torques 93%were detected from all samples analysed.A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups.The PCA on the first principal component(PC1),explaining 30.36%of the observed variation in the IBS-D patient group,was significantly altered from all other sample groups(IBS-D vs control, P=0.01;IBS-D vs IBS-M,P=0.00;IBS-D vs IBS-C, P=0.05).Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria.A phy- lotype with 85%similarity to C.thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects(-4.08±0.90 vs -3.33±1.16,P=0.04)and IBS-D and IBS-M subjects 展开更多
AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's...AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.展开更多
AIM To certify the relationship between AFP mRNA and some pathological parameters of he-patocellular carcinoma (HCC).METHOD We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffering ...AIM To certify the relationship between AFP mRNA and some pathological parameters of he-patocellular carcinoma (HCC).METHOD We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffering from HCC by RT-PCR method.RESULTS The positive rate of AFP mRNA was 76.9% in the HCC tumor tissues, and 69.4% in the paratumor tissues from the HCC patients with severe cirrhosis. However, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFP mRNA expression was found in the related paratumor tissues.CONCLUSION The AFP protein was specially expressed by HCC cells and mutated hepatocytes. The AFP mRNA was positively related with cirrhosis, but no significant relationship was found between AFP mRNA and tumor size, capsule status and tumor metastasis.展开更多
AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was...AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.展开更多
AIM: To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. METHODS: Fecal samples were collected from 14 con...AIM: To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. METHODS: Fecal samples were collected from 14 control individuals (hemorrhoid patients only) and 26 UC patients (severe: n = 12, moderate: n = 6, remission: n = 8), recruited by the gastroenterologist at the Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India. Disease activity in UC patients was determined by clinical colitis activity index. We employed fluorescent in situ hybridization in combination with flow cytometry to enumerate the clostridium cluster population targeted by 16S rRNA gene probe. Major butyrate-producing species within this cluster were quantified to see if any change existed in control vs UC patients with different disease activity. This observed change was further validated by quantitative polymerase chain reaction. In addition to this,we carried out gas chromatography to evaluate the changes in concentration of major short chain fatty acids (SCFAs), namely acetate, n -butyrate, iso -butyrate, in the above samples. Student t test and Graph pad prism-6 were used to compare the data statistically. RESULTS: There was a significant decrease of Clostridium coccoides (control, 25.69% ± 1.62% vs severe, 9.8% ± 2.4%, P = 0.0001) and Clostridium leptum clusters (control, 13.74% ± 1.05% vs severe, 6.2% ± 1.8%, P = 0.0001) in fecal samples of UC patients. Furthermore, we demonstrated that some butyrateproducing members of the clostridial cluster, like Fecalibacterium prausnitzii (control, 11.66% ± 1.55% vs severe, 6.01% ± 1.6%, P = 0.0001) and Roseburia intestinalis (control, 14.48% ± 1.52% vs severe, 9% ± 1.83%, P = 0.02) were differentially present in patients with different disease activity. In addition, we also demonstrated decreased concentrations of fecal SCFAs, especially of n -butyrate (control, 24.32 ± 1.86 mmol/μL vs severe, 12.74 ± 2.75 mmol/μL, P = 0.003), iso -butyrate (cont展开更多
文摘IM To determine the incidence of hepatitis B virus (HBV) intrauterine infection and to explore the relationship between HBV viremia level of pregnant women and HBV intrauterine infection.METHODS Sixtynine pregnant women were divided into three groups. Group A, 41 HBsAg positive patients, 14 of them were HBeAg positive (group A1), and 27 HBeAg negative (group A2); Group B, 12 HBsAg negative patients, but positive for antiHBs and/or antiHBe and/or antiHBc; and Group C, 16 patients negative for all HBV markers. Blood samples of mothers were taken at delivery, samples of their infants were collected within 24 hours after birth (before injection of HBIG and HBV vaccine). All the serum samples were stored at -20℃. HBV serum markers were tested by radioimmunoassay and HBV NDA were detected by nested polymerase chain reaction.RESULTS In group C, all of 16 newborns were negative for HBsAg and HBV DNA. In group A, 7 infants were HBsAg positive (171%), and 17 (415%) were HBV DNA positive (P<005). The incidence of intrauterine HBV infection was much higher in group A1 than that in group A2 (HBsAg 429% vs 37%, HBV DNA 929% vs 148%, P<005). The incidence of HBV intrauterine infection was significantly different between high and low HBV viremia of mothers (933% vs 429%, P<005).CONCLUSION The incidence of HBV intrauterine infection is high when HBV DNA in newborns detected with nested PCR is used as a marker of HBV infection. It is related to HBV viremia level of mothers.
基金Supported by The National Council on Science and Technology (CONACYT:85675 and 79628)Institute of Public Health(POA: 2008-2010)Research Office of Veracruzana University and Public Education Secretariat(SEP-PROMEP-UV:PTC-319)
文摘AIM:To assess expression of matrix metalloproteinases 2(MMP2)and MMP9 in gastric cancer,superficial gastritis and normal mucosa,and to measure metalloproteinase activity.METHODS:MMP2 and MMP9 mRNA expression was determined by quantitative real-time polymerase chain reaction.Normalization was carried out using three different factors.Proteins were analyzed by quantitative gelatin zymography(qGZ).RESULTS:18S ribosomal RNA(18SRNA)was very highly expressed,while hypoxanthine ribosyltransferase-1(HPRT-1)was moderately expressed.MMP2 was highly expressed,while MMP9 was not detected or lowly expressed in normal tissues,moderately or highly expressed in gastritis and highly expressed in cancer.Relative expression of 18SRNA and HPRT-1 showed no significant differences.Significant differences in MMP2 and MMP9 were found between cancer and normal tissue,but not between gastritis and normal tissue.Absolute quantification of MMP9 echoed this pattern,but differential expression of MMP2 proved conflictive.Analysis by qGZ indicated significant differences between cancer and normal tissue in MMP-2,total MMP-9,250 and 110 kDa bands.CONCLUSION:MMP9 expression is enhanced in gastric cancer compared to normal mucosa;interpretation of differential expression of MMP2 is difficult to establish.
文摘AIM To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS Seventy patients with duodenalulcer and fifty healthy controls were examinedfor HLA-DQA1 genotypes.HLA-DQA1 typing wascarried out by digesting the locus specificpolymerase chain reaction amplified productswith alleles specific restriction enzymes(PCR-RFLP),i.e.,Apal Ⅰ,Bsaj Ⅰ,Hph Ⅰ,Fok Ⅰ,Mbo Ⅱ and Mnl Ⅰ.RESULTS The allele frequencies of DQA1 * 0301and DQA1 * 0102 in patients with duodenal ulcerwere significantly higher and lower respectivelythan those in healthy controls(0.40 vs 0.20,P = 0.003,mcorret = 0.024)and(0.05 vs 0.14,P = 0.012,but Pcorret】0.05),respectively.CONCLUSION DQA1 * 0301 is a susceptiblegene for duodenal ulcer in Wuhan Hans,andthere are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients andhealthy controls.
文摘The gram-negative bacterium Helicobacter pylori(H.pylori)causes chronic gastritis,gastric and duodenal ulcers,gastric cancer and mucosa-associated lymphoid tissue lymphoma.Treatment is recommended in all symptomatic patients.The current treatment options for H.pylori infection are outlined in this review in light of the recent challenges in eradication success,largely due to the rapid emergence of antibiotic resistant strains of H.pylori.Antibiotic resistance is a constantly evolving process and numerous studies have shown that the prevalence of H.pylori antibiotic resistance varies significantly from country to country,and even between regions within the same country.In addition,recent data has shown that previous antibiotic use is associated with harbouring antibiotic resistant H.pylori.Local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy.Antimicrobial resistance is assessed by H.pylori culture and antimicrobial susceptibility testing.Recently developed molecular tests offer an attractive alternative to culture and allow for the rapid molecular genetic identification of H.pylori and resistance-associated mutations directly from biopsy samples or bacterial culture material.Accumulating evidence indicates that surveillance of antimicrobial resistance by susceptibility testing is feasible and necessary to inform clinicians in their choice of therapy for management of H.pylori infection.
基金supported by National High-Tech Research and Development(863)Program of China(No.2012AA02A504)
文摘Objective： MicroRNA-21 （miR-21） has been shown to be a key regulator of carcinogenesis. There were few reports about the comparison of serum miR-21 with conventional tumor markers. This study aimed to explore the diagnostic value of circulating miR-21 as a tumor marker in breast cancer （BC） and compare it with CA15 3 and carcinoembryonic antigen （CEA）. Methods： Circulating miR-16 and miR-21 were amplified and quantitatively detected by real-time PCR in 89 BC patients and 55 healthy controls. The levels of CA153 and CEA were measured through assays. Then the sensitivity in diagnosis of BC was compared among miR-21, CA153 and CEA. Results： The level of serum miR-21 was significantly higher in BC patients than controls （P〈0.001）. The sensitivity and specificity of miR-21 were 87.6% and 87.3%, respectively, whereas the sensitivities of CEA and CA153 were only 22.47% and 15.73%. Con^lusions： Compared with CEA and CA153, serum miR-21 has a higher sensitivity in diagnosis of BC. Although not correlated with the status of ER, PR and clinical stages, serum miR-21 may be a potential diagnostic indicator for BC, especially for the early stage.
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with co
基金the Natural Science Foundation of Anhui Province,No.9741006Natural Science Foundation of Anhui Educational Commission,No.JL-97-077.
文摘INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship
基金Natural Science Foundation of Committee of Science and Technology of Shanghai Municipality(952B14003)Science and Technology Development Foundation of Educational Committee of Shanghai Municipality(96B06).
文摘INTRODUCTIONLiver fibrosis is an excessive deposition ofextracellular matrix(ECM)resulted from bothincreased synthesis and decreased degradation.Matrix metalloproteinases(MMPs)represent agroup of neutral proteinases with variable
文摘The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this phenomenon is named occult hepatitis B infection(OBI). The presence of anti-HBc antibody in serum is an important key for OBI tracking, although about 20% of OBI cases are negative for anti-HBc antibody. The diagnosis of OBI is mainly based on polymerase chain reaction(PCR) and real-time PCR assays. However, real-time PCR is a more reliable method than PCR. OBI is a great issue for the public health problem and a challenge for the clinical entity worldwide. The persistence of OBI may lead to the development of cirrhosis and hepatocellular carcinoma. With regard to OBI complications, the screening of HBV DNA by the highly sensitive molecular means should be implemented for:(1) patients with a previous history of chronic or acute HBV infection;(2) patients co-infected with hepatitis C virus/human immunodeficiency virus;(3) patients undergoing chemotherapy or anti-CD20 therapy;(4) recipients of organ transplant;(5) blood donors;(6) organ transplant donors;(7) thalassemia and hemophilia patients;(8) health care workers;(9) patients with liver related disease(cryptogenic);(10) hemodialysis patients;(11) patients undergoing lamivudine or interferon therapy; and(12) children in time of HBV vaccination especially in highly endemic areas of HBV. Active HBV vaccination should be implemented for the close relatives of patients who are negative for OBI markers. Thus, the goal of this review is to evaluate the rate of OBI with a focus on status of high risk groups in different regions of the world.
基金the National Natural Science Foundation of China,№39670648
文摘Helicobacter pylori (H. pylori ) is a major causativebacterium of chronic gastritis, peptic ulcer and mucosaassociated lymphoid tissue lymphoma in humans, and associated with an increased risk of gastric cancer[1 -8]. An important virulant factor of H. pylori is the vacuolating cytotoxin ( VacA ) encoded by vacA that induces cytoplasmic vacuolation in target cells both in vitro and in vivo[9-11]. VacA is produced as a 140 kDa precursor which contains an N-terminal signal peptide and an approximately 33 kDa C-terminal outer membrance exporter. The precursor is cleaved at both N-terminal and C-terminal and secreted into the extracellular milieu as a 95 kDa mature protein. The mature protein futher undergoes specific cleavage to yield 37 kDa and 58 kDa subunits[12-14] Although vacA is present in all H. pylori strains, only about 50% to 60% of strains can induce vacuolation of epithelial cells as assessed by the HeLa cell assay. vacA shows considerable genetic variation in H. pylori isolated from all over the world and contains at least two variable regions. The s region exists as sl or s2 allelic types. Among type sl strains, subtypes sla and slb have been identified. The m region occurs as ml or m2 allelic types. Specific vacA genotype of H. pylori strains are associated with the production of the cytotoxin in vitro, epithelial damage in vivo, and clinical consequences[15-27]. The other virulant factor is the cytotoxin-associated protein (CagA) encoded by the cytotoxin-associated gene (cagA). The cagA gene is present in about 60% to 70% of strains and all of these strains express the cagA. The presence of cagA is also associated with the production of the cytotoxin in vitro, and clinical outcome[24-30]. The aim of this study was (i) to identify vacA genotypes and cagA status of H. pylori isolated from Chinese patients; (ii) to evaluation the relatioship beween vacA genotypes, cagA status and related gastroenterological disorders.
基金Supported by Council of Scientific and Industrial Research,New Delhi,India in the form of Senior Research Fellowship awarded to Patel SK
文摘Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, <sup>13</sup>C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while <sup>13</sup>C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The <sup>13</sup>C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor’s test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori’s DNA in gastric mucosa, gastric juice, saliva, dental plaques
基金Supported by National Natural Science Foundation of China,No 30672257 and No 30973230Shanghai Public Health Key Subject Construction,No 08GWZX0102
文摘AIM:To establish the real time fluorescence polymerase chain reaction(RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.METHODS:Four hundred infants(< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4.Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed.Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes.After a single RT-PCR,results were obtained by analyzing the take-off curves.Twenty-four positive and 14 negative samples were retested by direct sequencing.RESULTS:Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8%(46/800).Twenty-six and 20 mutant alleles were observed respectively,in 474 and 242 alleles from the intermediate and southern areas of China.No mutant allele was detected in 84 alleles from northern China.Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations,and 14 negative samples were retested by direct sequencing,which confirmed that the accuracy of RTPCR was 100%.CONCLUSION:RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.
基金Supported by a grant of DFG (SFB 402 Teilprojekt C1 (Mihm))by a grant of Hoffmann La Roche (Grenzach-Wyhden, Germany)Part of the data has been presented as poster at the 1999 EASL-meeting in Neaples
文摘AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.
基金This work was supported by the National Basic Research Program(973 Program)the National Programs for High Technology Research and Development Program(863 Program)(Grant Nos.2006CB806503,2006AA020502)+1 种基金National Major Project(Grant Nos.2009ZX10004-304,2009ZX09311-001)Tsinghua University Initiative Scientific Research Program(Grant No,2009THZ01).
文摘Enterovirus 71(EV71),one of the major causative agents for hand-foot-and-mouth disease(HFMD),has caused more than 100 deaths among Chinese children since March 2008.The EV71 genome encodes an RNAdependent RNA polymerase(RdRp),denoted 3D^(pol),which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics.Here we report the crystal structures of EV71 RdRp(3D^(pol))and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4Åresolution.The structure of EV71 RdRp(3D^(pol))has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp.And the EV71 RdRp(3D^(pol))complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding.The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus(FMDV)3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase.These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.
基金Supported by Research Fund for the Control of Infectious Diseases and Research Grant Committee of Hong Kong Government
文摘AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.
基金Supported by The Finnish Funding Agency for Technologyand Innovation,Tekes,grants No.945/401/00 and 40160/05the Finnish Graduate School of Applied Biosciences,the Academy of Finland,Grant No.214 157the Centre of Excellence on Microbial Food Safety Research,Academy of Finland
文摘AIM:To study whether selected bacterial 16S ribosomal RNA(rRNA)gene phylotypes are capable of disting- uishing irritable bowel syndrome(IBS). METHODS:The faecal microbiota of twenty volunteers with IBS,subdivided into eight diarrhoea-predominant (IBS-D),eight constipation-predominant(IBS-C)and four mixed symptom-subtype(IBS-M)IBS patients,and fifteen control subjects,were analysed at three time-points with a set of fourteen quantitative real-timepolymerase chain reaction assays.All assays targeted 16S rRNA gene phylotypes putatively associated with IBS,based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobac-teria,Bacteroidetes and Firmicutes.Eight of the target phylotypes had less than 95%similarity to cultured bacterial species according to their 16S rRNA gene sequence.The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis(PCA)with linear mixed-effects models applied to the principal component scores. RESULTS:Bacterial phylotypes Clostridium cocleatum 88%,Clostridium thermosuccinogenes 85%,Coprobacillus catenaformis 91%,Ruminococcus bromii-like, Ruminococcus torques 91%,and R.torques 93%were detected from all samples analysed.A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups.The PCA on the first principal component(PC1),explaining 30.36%of the observed variation in the IBS-D patient group,was significantly altered from all other sample groups(IBS-D vs control, P=0.01;IBS-D vs IBS-M,P=0.00;IBS-D vs IBS-C, P=0.05).Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria.A phy- lotype with 85%similarity to C.thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects(-4.08±0.90 vs -3.33±1.16,P=0.04)and IBS-D and IBS-M subjects
基金Supported by Henan Distinguished Junior Scholar Grant,No.074100510017
文摘AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.
基金Project supported by the National Natural Science Foundation of China,No.3900050.
文摘AIM To certify the relationship between AFP mRNA and some pathological parameters of he-patocellular carcinoma (HCC).METHOD We detected the expression of AFP in mRNA level in tissue samples from 52 patients suffering from HCC by RT-PCR method.RESULTS The positive rate of AFP mRNA was 76.9% in the HCC tumor tissues, and 69.4% in the paratumor tissues from the HCC patients with severe cirrhosis. However, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFP mRNA expression was found in the related paratumor tissues.CONCLUSION The AFP protein was specially expressed by HCC cells and mutated hepatocytes. The AFP mRNA was positively related with cirrhosis, but no significant relationship was found between AFP mRNA and tumor size, capsule status and tumor metastasis.
基金This study is supported by Science Foundation of the Education Department of Zhejiang Province.
文摘AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.
基金Supported by Council of Scientific and Industrial Research,New Delhi Government of India
文摘AIM: To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. METHODS: Fecal samples were collected from 14 control individuals (hemorrhoid patients only) and 26 UC patients (severe: n = 12, moderate: n = 6, remission: n = 8), recruited by the gastroenterologist at the Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India. Disease activity in UC patients was determined by clinical colitis activity index. We employed fluorescent in situ hybridization in combination with flow cytometry to enumerate the clostridium cluster population targeted by 16S rRNA gene probe. Major butyrate-producing species within this cluster were quantified to see if any change existed in control vs UC patients with different disease activity. This observed change was further validated by quantitative polymerase chain reaction. In addition to this,we carried out gas chromatography to evaluate the changes in concentration of major short chain fatty acids (SCFAs), namely acetate, n -butyrate, iso -butyrate, in the above samples. Student t test and Graph pad prism-6 were used to compare the data statistically. RESULTS: There was a significant decrease of Clostridium coccoides (control, 25.69% ± 1.62% vs severe, 9.8% ± 2.4%, P = 0.0001) and Clostridium leptum clusters (control, 13.74% ± 1.05% vs severe, 6.2% ± 1.8%, P = 0.0001) in fecal samples of UC patients. Furthermore, we demonstrated that some butyrateproducing members of the clostridial cluster, like Fecalibacterium prausnitzii (control, 11.66% ± 1.55% vs severe, 6.01% ± 1.6%, P = 0.0001) and Roseburia intestinalis (control, 14.48% ± 1.52% vs severe, 9% ± 1.83%, P = 0.02) were differentially present in patients with different disease activity. In addition, we also demonstrated decreased concentrations of fecal SCFAs, especially of n -butyrate (control, 24.32 ± 1.86 mmol/μL vs severe, 12.74 ± 2.75 mmol/μL, P = 0.003), iso -butyrate (cont
