AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepa...AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited th展开更多
The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE ...The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state.展开更多
AIM: To investigate the prognostic role of isoform 165 vascular endothelial growth factor messenger RNA (VEGF165 mRNA)in noncancerous liver tissues from patients with primary hepatocellular carcinoma (HCC).METHODS: Us...AIM: To investigate the prognostic role of isoform 165 vascular endothelial growth factor messenger RNA (VEGF165 mRNA)in noncancerous liver tissues from patients with primary hepatocellular carcinoma (HCC).METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in noncancerous liver tissues from 60 consecutive patients with HCC undergoing curative resection. We categorized the patients with VEGF165 mRNA over 0.500 in noncancerous liver tissues as group A, and those below 0.500 as group B.RESULTS: Among the isoforms of VEGF mRNA by multivariate analysis, a higher level of VEGF165 mRNA in noncancerous liver tissue correlated significantly with a higher risk of HCC recurrence (P = 0.039) and recurrence-related mortality (P= 0.048), but VEGF121 did not. The other significant predictors of recurrence consisted of vascular permeation (P = 0.022),daughter nodules (P = 0.033), cellular dedifferentiation (P = 0.033), an absent or incomplete capsule (P = 0.037).A significant variable of recurrence-related mortality was Vascular permeation (P= 0.012). As to the clinical manifestations of 16 patients who developed recurrence,the recurrent tumor number over 2, recurrent extent over two-liver segments, and the median survival after recurrence,all significantly correlated with group A patients (P = 0.043,0.043, and 0.048, respectively). However, the presence of extrahepatic metastasis was not (P＞0.05). The difference in recurrence after treatment between the two groups had no statistical significance (P＞0.05).CONCLUSION: The higher expression of isoform VEGF165mRNA in noncancerous liver remnant of patients with HCC may be a significant biological indicator of the invasiveness of postoperative recurrence.展开更多
AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions...AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The li展开更多
Background Molecular targeted drugs is now widely used in non-small cell lung cancer （NSCLC） clinical treatment. Icotinib hydrochloride is a new type of oral epidermal growth factor receptor （EGFR） tyrosine kinase...Background Molecular targeted drugs is now widely used in non-small cell lung cancer （NSCLC） clinical treatment. Icotinib hydrochloride is a new type of oral epidermal growth factor receptor （EGFR） tyrosine kinase inhibitors （EGFR-TKIs）. In this study, we examined the role of EGFR, K-RAS, B-RAF somatic mutations and EGFR mRNA expression in tumor specimens from advanced NSCLC patients as predicators of the efficacy of icotinib hydrochloride. Methods We analyzed tumor paraffin-embedded specimens, which were obtained from 14 of 40 patients with advanced NSCLC who enrolled in the stage I clinical trial of icotinib hydrochloride. Somatic mutations were evaluated by mutant-enriched liquidchip （MEL） technology, and EGFR mRNA expression was measured by branched DNA liquidchip （MBL） technology. Results In the 14 specimens, seven patients showed EGFR mutations, exon 19 deletion （3/7） and exon 21 point mutation （4/7）; and two patients showed K-RAS mutation. No mutations in EGFR exon 20. or B-RAF were detected. In patients with EGFR mutation, one patient developed progress disease （PD）, three patients had stable disease （SD）, two patients had partial responses （PR） and one patient had a complete response （CR）. In patients with wild-type EGFR, four patients had PD, three patients acquired SD, and none had PR/CR （P=-0.0407）. EGFR mutations were associated with better progress-free survival （PFS） （141 days vs. 61 days） but without a statistically significant difference （P=0.8597）, and median overall survival （OS） （-〉449 days vs. 140 days）. EGFR mRNA expression levels were evaluated （three high, eight moderate, one low, and two that can not be measured due to insufficient tumor tissue） and no statistically significant relationships was observed with response, PFS or OS. Conclusions The EGFR mutation rate was consistent with that reported in the Asian population, so the MEL technology is reliable for measuring EGFR mutation with high throughput and rapidity展开更多
INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (E...INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].展开更多
AIM To detect the expression of CD44v6 mRNA and nm23-H1 mRNA in hepatocellular carcinoma (HCC) by in situ hybridization, and to evaluate the relationship between their expression and also relationship between their ex...AIM To detect the expression of CD44v6 mRNA and nm23-H1 mRNA in hepatocellular carcinoma (HCC) by in situ hybridization, and to evaluate the relationship between their expression and also relationship between their expressions and tumor invasion and metastasis.METHODS CD44v6 cDNA probe was synthesized with PCR technique and the nm23-H1 cRNA probe by in vitro transcription. The expression of CD44v6 mRNA and nm23-H1 mRNA was detected by in situ hybridization.RESULTS In group with high invasion and metastasis potential, the positive rates of CD44v6 mRNA and nm23-H1 mRNA were 80% (8/10) and 40% (4/10), in group with poor invasion and metastasis potential, they were 21.7% (5/23) and 91.3% (21/23). There was a positive correlation between the expression of CD44v6 mRNA and tumor invasion and metastasis potential in HCC (P＜0.01), and a reverse correlation between the expression of nm23-H1 mRNA and tumor invasion and metastasis potential (P＜0.01) and a reverse correlation in the expression between CD44v6 mRNA and nm23-H1 mRNA in HCC (P＜0.01).CONCLUSION Detection of CD44v6 mRNA and nm23-H1 mRNA may be useful for tumor invasion and metastasis in HCC.INTRODUCTIONCD44 is a cell surface transmembrane glycoprotein. As a kind of adhesive molecule, it participates in cell-cell and cell-matrix adhesion and interactions. Many studies revealed a correlation between high-level expression of CD44, especially CD44v and tumor invasion, metastasis and prognosis. The exon 6v containing isoforms may be an independent diagnostic parameter[1,2]. Some other studies, however, had different results[3,4]. Some researches showed a reverse correlation between the expression of nm23-H1 mRNA and tumor metastasis[5,6]. In order to evaluate the relationship between the expression of CD44v6 mRNA and nm23-H1 mRNA and tumor invasive and metastatic potential in HCC and to evaluate the relationship in the expression between CD44v6 mRNA and nm23-H1 mRNA, we detected their expression in HCC by in situ hybridization.展开更多
AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human...AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.展开更多
基金Supported by the Postdoctoral Science Foundation of China(No.1999-10 State Postdoctoral Foundation Commission)
文摘AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited th
文摘The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state.
基金Supported by the Grants from the Department of Medical Research,Mackay Memorial Hospital, Taiwan, China (MMH9237)
文摘AIM: To investigate the prognostic role of isoform 165 vascular endothelial growth factor messenger RNA (VEGF165 mRNA)in noncancerous liver tissues from patients with primary hepatocellular carcinoma (HCC).METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in noncancerous liver tissues from 60 consecutive patients with HCC undergoing curative resection. We categorized the patients with VEGF165 mRNA over 0.500 in noncancerous liver tissues as group A, and those below 0.500 as group B.RESULTS: Among the isoforms of VEGF mRNA by multivariate analysis, a higher level of VEGF165 mRNA in noncancerous liver tissue correlated significantly with a higher risk of HCC recurrence (P = 0.039) and recurrence-related mortality (P= 0.048), but VEGF121 did not. The other significant predictors of recurrence consisted of vascular permeation (P = 0.022),daughter nodules (P = 0.033), cellular dedifferentiation (P = 0.033), an absent or incomplete capsule (P = 0.037).A significant variable of recurrence-related mortality was Vascular permeation (P= 0.012). As to the clinical manifestations of 16 patients who developed recurrence,the recurrent tumor number over 2, recurrent extent over two-liver segments, and the median survival after recurrence,all significantly correlated with group A patients (P = 0.043,0.043, and 0.048, respectively). However, the presence of extrahepatic metastasis was not (P＞0.05). The difference in recurrence after treatment between the two groups had no statistical significance (P＞0.05).CONCLUSION: The higher expression of isoform VEGF165mRNA in noncancerous liver remnant of patients with HCC may be a significant biological indicator of the invasiveness of postoperative recurrence.
基金the key,project of the tenth-five foundation of PLA,No.01Z011
文摘AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The li
文摘Background Molecular targeted drugs is now widely used in non-small cell lung cancer （NSCLC） clinical treatment. Icotinib hydrochloride is a new type of oral epidermal growth factor receptor （EGFR） tyrosine kinase inhibitors （EGFR-TKIs）. In this study, we examined the role of EGFR, K-RAS, B-RAF somatic mutations and EGFR mRNA expression in tumor specimens from advanced NSCLC patients as predicators of the efficacy of icotinib hydrochloride. Methods We analyzed tumor paraffin-embedded specimens, which were obtained from 14 of 40 patients with advanced NSCLC who enrolled in the stage I clinical trial of icotinib hydrochloride. Somatic mutations were evaluated by mutant-enriched liquidchip （MEL） technology, and EGFR mRNA expression was measured by branched DNA liquidchip （MBL） technology. Results In the 14 specimens, seven patients showed EGFR mutations, exon 19 deletion （3/7） and exon 21 point mutation （4/7）; and two patients showed K-RAS mutation. No mutations in EGFR exon 20. or B-RAF were detected. In patients with EGFR mutation, one patient developed progress disease （PD）, three patients had stable disease （SD）, two patients had partial responses （PR） and one patient had a complete response （CR）. In patients with wild-type EGFR, four patients had PD, three patients acquired SD, and none had PR/CR （P=-0.0407）. EGFR mutations were associated with better progress-free survival （PFS） （141 days vs. 61 days） but without a statistically significant difference （P=0.8597）, and median overall survival （OS） （-〉449 days vs. 140 days）. EGFR mRNA expression levels were evaluated （three high, eight moderate, one low, and two that can not be measured due to insufficient tumor tissue） and no statistically significant relationships was observed with response, PFS or OS. Conclusions The EGFR mutation rate was consistent with that reported in the Asian population, so the MEL technology is reliable for measuring EGFR mutation with high throughput and rapidity
基金Project supported by the National Natural Science Foundation of China, No. 39500138
文摘INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].
文摘AIM To detect the expression of CD44v6 mRNA and nm23-H1 mRNA in hepatocellular carcinoma (HCC) by in situ hybridization, and to evaluate the relationship between their expression and also relationship between their expressions and tumor invasion and metastasis.METHODS CD44v6 cDNA probe was synthesized with PCR technique and the nm23-H1 cRNA probe by in vitro transcription. The expression of CD44v6 mRNA and nm23-H1 mRNA was detected by in situ hybridization.RESULTS In group with high invasion and metastasis potential, the positive rates of CD44v6 mRNA and nm23-H1 mRNA were 80% (8/10) and 40% (4/10), in group with poor invasion and metastasis potential, they were 21.7% (5/23) and 91.3% (21/23). There was a positive correlation between the expression of CD44v6 mRNA and tumor invasion and metastasis potential in HCC (P＜0.01), and a reverse correlation between the expression of nm23-H1 mRNA and tumor invasion and metastasis potential (P＜0.01) and a reverse correlation in the expression between CD44v6 mRNA and nm23-H1 mRNA in HCC (P＜0.01).CONCLUSION Detection of CD44v6 mRNA and nm23-H1 mRNA may be useful for tumor invasion and metastasis in HCC.INTRODUCTIONCD44 is a cell surface transmembrane glycoprotein. As a kind of adhesive molecule, it participates in cell-cell and cell-matrix adhesion and interactions. Many studies revealed a correlation between high-level expression of CD44, especially CD44v and tumor invasion, metastasis and prognosis. The exon 6v containing isoforms may be an independent diagnostic parameter[1,2]. Some other studies, however, had different results[3,4]. Some researches showed a reverse correlation between the expression of nm23-H1 mRNA and tumor metastasis[5,6]. In order to evaluate the relationship between the expression of CD44v6 mRNA and nm23-H1 mRNA and tumor invasive and metastatic potential in HCC and to evaluate the relationship in the expression between CD44v6 mRNA and nm23-H1 mRNA, we detected their expression in HCC by in situ hybridization.
文摘AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.
