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目的构建靶向作用于小鼠肝前体细胞的动物模型,研究乙型肝炎病毒X蛋白(HBx)对肝前体细胞上皮间质转化(EMT)的影响。方法每周2次给予昆明小鼠2 m L/L四氯化碳灌胃,4周后行经门静脉注射稳定表达HBx的肝前体细胞和稳定表达空载体的肝前体细胞同时切除部分肝脏。术后继续每周2次进行灌胃,并分别于术后3、5、7、14、21、28 d,处死小鼠取肝脏标本;实时定量PCR检测小鼠肝组织HBx的mRNA水平、免疫组织化学染色检测肝组织中外源性细胞的存活。实时荧光定量PCR检测上皮钙黏素(E-cadherin)、神经钙黏素(N-cadherin)、波形蛋白(vimentin)和细胞角蛋白18(CK18)mRNA水平,Western blot法检测E-cadherin、N-cadherin、vimentin、CK18蛋白水平。结果免疫组织化学染色结果显示,术后肝脏组织明显有外源性细胞存活.随注射时间延长,肝组织HBx的含量增加,表达区域增大;实时荧光定量PCR和Western blot结果均显示过表达HBx能够使小鼠肝组织中E-cadherin、CK18水平降低,而N-cadherin、vimentin的水平增加。结论动物模型证实HBx在肝前体细胞EMT过程中起着重要的调控作用。
OBJECTIVE: To construct an animal model targeting mouse hepatic precursor cells and study the effect of hepatitis B virus X protein (HBx) on the epithelial-mesenchymal transition (EMT) of hepatic precursor cells. Methods Kunming mice were orally administered with 2 m L / L carbon tetrachloride twice a week. Four weeks later, the hepatic progenitor cells stably expressing HBx and the hepatic progenitor cells stably expressing the empty vector were simultaneously transplanted to remove some of the liver. The mice were sacrificed on the 3rd, 5th, 7th, 14th, 21st and 28th day after operation, respectively. The liver samples were also taken from the mice. The mRNA levels of HBx in the liver of the mice were detected by real- Histochemical staining detects the survival of exogenous cells in liver tissue. The expressions of E-cadherin, N-cadherin, vimentin and cytokeratin 18 mRNA were detected by real-time fluorescence quantitative PCR. The expressions of E-cadherin, N -cadherin, vimentin, CK18 protein levels. Results The results of immunohistochemistry showed that exogenous cells were obviously survived in liver tissue after operation, with the increase of injection time, the content of HBx in liver tissue increased and the expression area increased. Real-time fluorescence quantitative PCR and Western blot showed that HBx was overexpressed Can reduce the levels of E-cadherin and CK18 in mouse liver tissue, and increase the levels of N-cadherin and vimentin. Conclusion Animal models confirmed that HBx plays an important regulatory role in the process of EMT of liver precursor cells.