目的:建立干扰素-(interferon-,IFN-)基因敲除小鼠慢性乙型肝炎病毒(hepatitis B virus,HBV)复制模型.方法:IFN-基因敲除(IFN--/-)小鼠繁育并抽提组织DNA进行聚合酶链反应(PCR)及凝胶电泳鉴定基因型.IFN--/-小鼠纯合子9只与野生型C57BL/...目的:建立干扰素-(interferon-,IFN-)基因敲除小鼠慢性乙型肝炎病毒(hepatitis B virus,HBV)复制模型.方法:IFN-基因敲除(IFN--/-)小鼠繁育并抽提组织DNA进行聚合酶链反应(PCR)及凝胶电泳鉴定基因型.IFN--/-小鼠纯合子9只与野生型C57BL/6小鼠9只同时高压水注射pAAV/HBV1.2质粒,按既定时间点采血检测乙型肝炎表面抗原(hepatitis B virus surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B virus e antigen,HBeAg)和HBV DNA.血清HBsAg和HBeAg表达水平由电化学发光法进行定量检测.经抽提血清总DNA后,血清HBV DNA由定量PCR进行检测.结果:本实验室繁殖的IFN--/-小鼠均为纯合子基因型.IFN--/-小鼠和野生型C57BL/6小鼠血清中HBsAg、HBeAg和HBV DNA持续存在,转染后第40天仍阳性.但是,IFN--/-小鼠血清HBsAg表达水平高于C57BL/6野生小鼠(40天时,P=0.042);IFN--/-小鼠血清HBV DNA持续高水平复制,明显高于C57BL/6野生小鼠(第25天时,P=0.012;第40天时,P=0.039).两组小鼠血清HBeAg表达水平无差异.结论:IFN--/-小鼠慢性HBV复制模型成功建立,并揭示了IFN-在慢性HBV感染中可抑制HBV复制.展开更多
Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad...Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations （0-10 maol/L）. Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFI31, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different con- centrations （1, 2, 3 μtmol/L） or RPMI1640 alone （serving as control）. The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC50 of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 nol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased （P〈0.05）, but the Smad7 mRNA expression had no significant change （P〉0.05）. There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 （P〈0.05）. However, the expression of Smad7 protein was substantially increased when compared with the control group （P〈0.05）. It was concluded that the inhibition of TGFi/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors （TGFI31, Smad3） and promote the expression of anti-fibrosis factor （Smad7）, suggesting that MG132 may become a po- tential therapeutic alternative for liver fibrosis.展开更多
Summary： The type I interferon and IFNAR play an important role in hepatitis B virus （HBV） infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more in- sights into t...Summary： The type I interferon and IFNAR play an important role in hepatitis B virus （HBV） infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more in- sights into the role of type I interferon and type I interferon receptor （IFNAR） in HBV infection, we established an HBV persistent replication IFNAR knockout （IFNAR-/-） mouse model and preliminarily applied this model. At first, the progeny of IFNAR-/- mouse was reproduced. Then hydrodynamic injec- tion with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR-/- mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir （ETV） on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline （NS） treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days af- ter the ETV treatment [P=0.035, P=0.00, P=0.149 and P=-0.084, IFNAR knockout （KO） control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=-0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR/- and C57BL/6 mice. This result suggests that HBV sup- pression during ETV treatments doesn＇t depend on type Ⅰinterferon and IFNAR. Collectively, persis- tent HBV replication IFNAR/ mouse model that we established is a useful and convenient tool to detect the function of the type Ⅰ interferon and IFNAR in HBV infection and anti-HBV treatments.展开更多
目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基...目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。展开更多
文摘目的:建立干扰素-(interferon-,IFN-)基因敲除小鼠慢性乙型肝炎病毒(hepatitis B virus,HBV)复制模型.方法:IFN-基因敲除(IFN--/-)小鼠繁育并抽提组织DNA进行聚合酶链反应(PCR)及凝胶电泳鉴定基因型.IFN--/-小鼠纯合子9只与野生型C57BL/6小鼠9只同时高压水注射pAAV/HBV1.2质粒,按既定时间点采血检测乙型肝炎表面抗原(hepatitis B virus surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B virus e antigen,HBeAg)和HBV DNA.血清HBsAg和HBeAg表达水平由电化学发光法进行定量检测.经抽提血清总DNA后,血清HBV DNA由定量PCR进行检测.结果:本实验室繁殖的IFN--/-小鼠均为纯合子基因型.IFN--/-小鼠和野生型C57BL/6小鼠血清中HBsAg、HBeAg和HBV DNA持续存在,转染后第40天仍阳性.但是,IFN--/-小鼠血清HBsAg表达水平高于C57BL/6野生小鼠(40天时,P=0.042);IFN--/-小鼠血清HBV DNA持续高水平复制,明显高于C57BL/6野生小鼠(第25天时,P=0.012;第40天时,P=0.039).两组小鼠血清HBeAg表达水平无差异.结论:IFN--/-小鼠慢性HBV复制模型成功建立,并揭示了IFN-在慢性HBV感染中可抑制HBV复制.
基金supported by a grant from the Natural Science Foundation of Hubei Province(No.2010CHB00401)
文摘Summary： The activation of hepatic stellate cells （HSCs） and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-J3 （TGFβ）/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations （0-10 maol/L）. Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFI31, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different con- centrations （1, 2, 3 μtmol/L） or RPMI1640 alone （serving as control）. The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC50 of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 nol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased （P〈0.05）, but the Smad7 mRNA expression had no significant change （P〉0.05）. There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 （P〈0.05）. However, the expression of Smad7 protein was substantially increased when compared with the control group （P〈0.05）. It was concluded that the inhibition of TGFi/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors （TGFI31, Smad3） and promote the expression of anti-fibrosis factor （Smad7）, suggesting that MG132 may become a po- tential therapeutic alternative for liver fibrosis.
基金supported by grants from the National Natural Science Foundation of China (No. 81001313)China Postdoctoral Science Foundation (No. 2009046094)+2 种基金National Science and Technology Major Projects (No. 2008ZX10002-011)National Key Basic Research Program of China (Nos. 2007CB512804 and 2009CB522506)International Science and Technology Cooperation Program (No. 2011DFA31030)
文摘Summary： The type I interferon and IFNAR play an important role in hepatitis B virus （HBV） infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more in- sights into the role of type I interferon and type I interferon receptor （IFNAR） in HBV infection, we established an HBV persistent replication IFNAR knockout （IFNAR-/-） mouse model and preliminarily applied this model. At first, the progeny of IFNAR-/- mouse was reproduced. Then hydrodynamic injec- tion with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR-/- mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir （ETV） on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline （NS） treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days af- ter the ETV treatment [P=0.035, P=0.00, P=0.149 and P=-0.084, IFNAR knockout （KO） control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=-0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR/- and C57BL/6 mice. This result suggests that HBV sup- pression during ETV treatments doesn＇t depend on type Ⅰinterferon and IFNAR. Collectively, persis- tent HBV replication IFNAR/ mouse model that we established is a useful and convenient tool to detect the function of the type Ⅰ interferon and IFNAR in HBV infection and anti-HBV treatments.
文摘目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。
