本文選題：HBx蛋白 + 肝前體細胞��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的 通過篩選穩(wěn)定表達HBx(hepatitis B virus,HBV)基因的肝前體細胞株,進一步構建靶向作用于小鼠肝前體細胞的動物模型。體內研究乙型肝炎病毒X蛋白(HBx)對肝前體細胞上皮間質轉化(Epithelial-mesenchymal transition,EMT)的影響。方法 通過慢病毒篩選穩(wěn)轉細胞株。2 mL/L四氯化碳每周2次給予昆明小鼠灌胃,4周后行經門靜脈注射HBx-14-19細胞和NC-14-19細胞同時切除部分肝臟。術后繼續(xù)每周2次進行灌胃,并分別于術后3天、5天、7天、14天、21天、28天處死小鼠取肝臟標本;實時定量PCR檢測小鼠肝組織HBx的mRNA水平、免疫組織化學染色檢測肝組織中外源性細胞的存活。實時熒光定量PCR檢測上皮鈣黏素(E-cadherin)、神經鈣黏素(N-cadherin)、波形蛋白(Vimentin)和細胞角蛋白18(CK18)mRNA水平,Western blot法檢測E-cadherin、N-cadherin、Vimentin、CK18蛋白水平。結果 1、熒光顯微鏡下觀察HBx-14-19細胞、NC-14-19細胞均可見有綠色熒光表達,傳代3次以后其表達量仍為90%以上,證實2種穩(wěn)定感染細胞均能高表達GFP基因;熒光定量PCR檢測HBx基因的mRNA表達結果顯示HBx-14-19細胞HBx基因表達明顯高于14-19細胞及nc-14-19細胞(p0.05)。證實穩(wěn)轉株hbx-14-19細胞、nc-14-19細胞構建成功。2、熒光顯微鏡下觀察小鼠不同天數(shù)冰凍切片綠色熒光表達情況顯示,實驗組和對照組術后不同天數(shù)均有綠色熒光表達,其表達量隨術后天數(shù)增加呈增多趨勢。3、免疫組化結果顯示,術后肝臟組織明顯有外源性細胞存活。隨注射時間延長,肝組織hbx的含量增加,表達區(qū)域增大。4、實時熒光定量pcr檢測發(fā)現(xiàn)實驗組與對照組相比,第3天、5天、7天、14天、21天、28天的肝臟組織中hbx的mrna均有較高表達(p0.05),隨術后時間的進展,hbx的表達含量顯著增加。證實動物模型構建成功。5、與陰性對照組相比,在注射含hbx基因的肝前體細胞的實驗組不同天數(shù),e-cadherinmrna水平均表現(xiàn)為下降趨勢(p0.05),而n-cadherinmrna在不同天數(shù)都表現(xiàn)為增多趨勢,術后3天、5天增加最為明顯(p0.05)。vimentinmrna表達呈相對上升趨勢,術后7天表達量最高(p0.01)。ck18mrna在術后實驗組與對照組相比整體呈下降趨勢,但術后3d表達量顯著升高(p0.05),術后14天、28天下降最為顯著(p0.01),其余均呈下降趨勢(p0.05)。6、westernblot法檢測實驗組與對照組不同天數(shù)emt相關標志物e-cadherin、n-cadherin、vimentin和上皮標志物ck18的蛋白表達水平顯示。與陰性對照組相比,在注射含hbx基因的肝前體細胞的實驗組不同天數(shù),e-cadherin蛋白含量均表現(xiàn)為下降趨勢,14天下降最為明顯(3天、5天、7天、14天為p0.01,21d、28d為p0.05),而n-cadherin蛋白在術后整體為上升趨勢(p0.05)。vimentin蛋白的表達呈相對上升趨勢,術后7天開始表達量明顯升高(7天、14天、21天為P0.01,其余為P0.05)。CK18蛋白在術后整體呈下降趨勢(3天、5天為P0.05,其余P0.01)。結論 NC-14-19細胞和HBx-14-19細胞均能穩(wěn)定表達綠色熒光蛋白,穩(wěn)轉株構建成功;穩(wěn)定表達HBx基因的動物模型構建成功;本實驗通過動物模型證實HBx在肝前體細胞上皮間質轉化過程中起著重要的調控作用。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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