乙型肝炎病毒X蛋白诱导肝前体细胞上皮—间质转化的体内研究

发布时间：2018-07-04 14:49

本文选题：HBx蛋白 + 肝前体细胞　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的通过筛选稳定表达HBx(hepatitis B virus,HBV)基因的肝前体细胞株,进一步构建靶向作用于小鼠肝前体细胞的动物模型。体内研究乙型肝炎病毒X蛋白(HBx)对肝前体细胞上皮间质转化(Epithelial-mesenchymal transition,EMT)的影响。方法通过慢病毒筛选稳转细胞株。2 mL/L四氯化碳每周2次给予昆明小鼠灌胃,4周后行经门静脉注射HBx-14-19细胞和NC-14-19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天、5天、7天、14天、21天、28天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 1、荧光显微镜下观察HBx-14-19细胞、NC-14-19细胞均可见有绿色荧光表达,传代3次以后其表达量仍为90%以上,证实2种稳定感染细胞均能高表达GFP基因;荧光定量PCR检测HBx基因的mRNA表达结果显示HBx-14-19细胞HBx基因表达明显高于14-19细胞及nc-14-19细胞(p0.05)。证实稳转株hbx-14-19细胞、nc-14-19细胞构建成功。2、荧光显微镜下观察小鼠不同天数冰冻切片绿色荧光表达情况显示,实验组和对照组术后不同天数均有绿色荧光表达,其表达量随术后天数增加呈增多趋势。3、免疫组化结果显示,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。4、实时荧光定量pcr检测发现实验组与对照组相比,第3天、5天、7天、14天、21天、28天的肝脏组织中hbx的mrna均有较高表达(p0.05),随术后时间的进展,hbx的表达含量显著增加。证实动物模型构建成功。5、与阴性对照组相比,在注射含hbx基因的肝前体细胞的实验组不同天数,e-cadherinmrna水平均表现为下降趋势(p0.05),而n-cadherinmrna在不同天数都表现为增多趋势,术后3天、5天增加最为明显(p0.05)。vimentinmrna表达呈相对上升趋势,术后7天表达量最高(p0.01)。ck18mrna在术后实验组与对照组相比整体呈下降趋势,但术后3d表达量显著升高(p0.05),术后14天、28天下降最为显著(p0.01),其余均呈下降趋势(p0.05)。6、westernblot法检测实验组与对照组不同天数emt相关标志物e-cadherin、n-cadherin、vimentin和上皮标志物ck18的蛋白表达水平显示。与阴性对照组相比,在注射含hbx基因的肝前体细胞的实验组不同天数,e-cadherin蛋白含量均表现为下降趋势,14天下降最为明显(3天、5天、7天、14天为p0.01,21d、28d为p0.05),而n-cadherin蛋白在术后整体为上升趋势(p0.05)。vimentin蛋白的表达呈相对上升趋势,术后7天开始表达量明显升高(7天、14天、21天为P0.01,其余为P0.05)。CK18蛋白在术后整体呈下降趋势(3天、5天为P0.05,其余P0.01)。结论 NC-14-19细胞和HBx-14-19细胞均能稳定表达绿色荧光蛋白,稳转株构建成功;稳定表达HBx基因的动物模型构建成功;本实验通过动物模型证实HBx在肝前体细胞上皮间质转化过程中起着重要的调控作用。
[Abstract]:Objective to further construct an animal model of liver precursor cells targeting the HBx (hepatitis B virus, HBV) gene, and to study the effect of hepatitis B virus X protein (HBx) on the epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT) of the hepatic precursor cells (Epithelial-mesenchymal transition, EMT) in vivo. .2 mL/L carbon tetrachloride was given to Kunming mice 2 times a week. After 4 weeks, the liver was excised by injection of HBx-14-19 cells and NC-14-19 cells by portal vein. After the operation, the liver was performed 2 times a week, and the liver specimens were sacrificed at 3 days, 5 days, 7 days, 14 days, 21 days and 28 days respectively. Quantitative PCR detection was carried out in real time. The mRNA level of HBx in liver tissue of mice and the survival of exogenous cells in liver tissue were detected by immunohistochemical staining. Real-time quantitative PCR was used to detect epithelial calcinein (E-cadherin), nerve calcin (N-cadherin), vimentin (Vimentin) and cytokeratin 18 (CK18) mRNA level. Western blot assay was used to detect E-cadherin, N-cadherin, etc. Results 1, the expression of HBx-14-19 cells in NC-14-19 cells was observed under fluorescence microscope, and the expression of NC-14-19 cells was more than 90% after passage. It was confirmed that all 2 kinds of stable infected cells could express the GFP gene high, and the mRNA table of the fluorescence quantitative PCR detection of HBx gene showed that the expression of HBx gene in HBx-14-19 cells was significantly higher. In 14-19 cells and nc-14-19 cells (P0.05), the stable transgenic hbx-14-19 cells were confirmed and the nc-14-19 cells were successfully constructed.2. Under the fluorescence microscope, the green fluorescence expression of the frozen sections of different days in mice showed that the experimental group and the control group had a green fluorescent meter at different days after operation, and the expression increased with the increase of.3 in the number of days after the operation. The results of immunohistochemical staining showed that the liver tissue was obviously alive after the operation. With the prolongation of the injection time, the content of HBx in the liver tissue increased and the expression area increased.4. The real-time fluorescence quantitative PCR detection found that the experimental group had a higher expression of HBx mRNA in the liver tissues of third days, 5 days, 7 days, 14 days, 21 days and 28 days (P0.05). The expression level of HBx was significantly increased after the time of operation. It was confirmed that the animal model was constructed successfully.5. Compared with the negative control group, the level of e-cadherinmrna in the experimental group with HBx containing HBx gene showed a declining trend (P0.05), while n-cadherinmrna showed an increasing trend in different days, 3 days after the operation, 5 days after the operation. The expression of the most obvious (P0.05).Vimentinmrna showed a relatively rising trend, the highest expression of 7 days after operation (P0.01).Ck18mrna in the experimental group after the operation compared with the control group decreased, but the expression of 3D after the operation was significantly increased (P0.05), 14 days after the operation, the most significant (P0.01), the rest were decreased (P0.05).6, Westernblot test. The protein expression levels of the EMT related markers E-cadherin, N-cadherin, vimentin and CK18 of the epithelial markers in the experimental group and the control group showed that the content of E-cadherin protein in the experimental group containing the HBx gene was decreased in different days compared with the negative control group, and the 14 day was the most obvious (3 days,) 5 days, 7 days, 14 days were p0.01,21d, 28d was P0.05), and the expression of N-cadherin protein in the whole after the operation (P0.05).Vimentin protein expression showed a relatively rising trend, and the expression level was obviously increased in 7 days after operation (7 days, 14 days, 21 days P0.01, and the rest P0.05), the overall decline trend (3 days, 5 days for P0.05, and the rest P0.01). On both NC-14-19 and HBx-14-19 cells, the green fluorescent protein could be expressed stably, the stable transgenic plant was successfully constructed, and the animal model for the stable expression of HBx gene was successfully constructed. This experiment proved that HBx plays an important role in the process of epithelial mesenchymal transition in the hepatic precursor cells through animal models.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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