Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fer...Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.展开更多
AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the...AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36%+/-1.95%), proliferative index (PI, 19.11%+/-6.79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P【0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81%+/-1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25%+/-5.63%, P【0.01). In the phase of dysplasia, AI (2.31%+/-1.10%) in H. pylori-positive group was lower (3.05%+/-1.29%) than that in H. pylori-negative group, but PI (33.89%+/-11.65%) was significantly higher (22.09+/-8018%, P【0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P【0.01), while PIs had an evidently gradual increasing trend (P【0.05 or P【0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellu展开更多
INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation an...INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation and progression at the level of cellular and molecular events have been elucidated in the past two decades[1,2].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
AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the...AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.展开更多
AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by i...AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.展开更多
基金Acknowledgment This work was supported by a grant from the National Nature Science Foundation of China （No. 39970374）. The authors wish to thank Professor Yi-Pong Hu, Second Military Medical University of China, for his kindness in providing us the recombinant plasmid （pBR322-HBV）. We wish to thank Mr. Michael Talion of Shantou University Medical College, English Language Training Section for his assistance in proofreading this manuscript. We gratefully acknowledge the support of the leaders of Shantou University Medical College.
文摘Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.
基金Supported by National Ninth Five-Year Study Program for Tacking Key Scientific Problems.No.96-906-01-04
文摘AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36%+/-1.95%), proliferative index (PI, 19.11%+/-6.79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P【0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81%+/-1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25%+/-5.63%, P【0.01). In the phase of dysplasia, AI (2.31%+/-1.10%) in H. pylori-positive group was lower (3.05%+/-1.29%) than that in H. pylori-negative group, but PI (33.89%+/-11.65%) was significantly higher (22.09+/-8018%, P【0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P【0.01), while PIs had an evidently gradual increasing trend (P【0.05 or P【0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellu
文摘INTRODUCTIONLiver fibrosis or cirrhosis is a common progressively pathological lesion of chronic liver diseases in response to various liver-damaging factors. The main mechanisms of fibrotic or cirrhotic initiation and progression at the level of cellular and molecular events have been elucidated in the past two decades[1,2].
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This work was supported by National NaturalScience Fundation of China(No.39760077).
文摘AIM: The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS: The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS: The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722 x 10(-9)M (Bmax=12810 sites per cell) and 8.931 x 10(-8)M (Bmax=119700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION: The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.
基金Supported by the National Postdoctoral Science Foundation of China,No.199711.
文摘AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.
