本文選題：轉(zhuǎn)基因HBV小鼠 + 肝損傷��； 參考：《新疆農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：研究背景與目的：肝臟疾病是影響人類健康的最為常見疾病之一。各種有害因素導(dǎo)致的肝損傷主要有病毒性肝炎、酒精性肝炎和藥物性肝炎等類型，其臨床表現(xiàn)為肝壞死、脂肪肝、膽汁淤積、肝纖維化、肝硬化以及肝癌等。目前對肝損傷的治療仍是一個全球性的嚴(yán)峻課題，而干細(xì)胞移植用于治療肝損傷是一種新方法并顯示出良好的應(yīng)用前景。骨髓間充質(zhì)干細(xì)胞具有極強的多向分化潛能和自我復(fù)制能力,有望成為治療肝損傷的一種新型種子細(xì)胞。本課題基于上述問題,采用HGF和EGF聯(lián)合誘導(dǎo)BMSCs21天為肝樣細(xì)胞，再將肝樣細(xì)胞移植到肝損傷的HBV小鼠肝臟來治療，并通過鑒定來確定細(xì)胞移植治療肝損傷的效果，以期為臨床研究提供實驗基礎(chǔ)。方法：(1)分別建立HBV轉(zhuǎn)基因小鼠和C57BL/6小鼠骨髓間充質(zhì)干細(xì)胞BMSCs的分離、培養(yǎng)體系，并進行比較研究。(2)將純化后的BMSCs在肝樣細(xì)胞生長因子HGF和表皮生長因子EGF的作用下進行誘導(dǎo)培養(yǎng)后,免疫細(xì)胞化學(xué)檢測肝樣細(xì)胞表面標(biāo)志物角蛋白CK18及糖原PAS的表達(dá),明確是否可分化為肝樣細(xì)胞（3）制作小鼠肝損傷模型，，將標(biāo)記后的BMSCs移植入肝損傷模型小鼠體內(nèi),熒光顯微鏡檢測干細(xì)胞在肝內(nèi)的定植情況,檢測血清肝功能及免疫組化檢測肝組織角蛋白的表達(dá)情況。結(jié)果：(1) C57BL/6小鼠的原代BMSCs生長迅速，按1:3傳代后呈漩渦狀生長，形態(tài)類似成纖維細(xì)胞。HBV轉(zhuǎn)基因小鼠原代BMSCs生長緩慢；以1:2傳代后生長緩慢，并且有明顯的血清依賴性及密度依賴性。(2) BMSCs經(jīng)HGF和EGF誘導(dǎo)培養(yǎng)后,免疫細(xì)胞化學(xué)檢測肝樣細(xì)胞表面標(biāo)志物角蛋白CK18及糖原PAS的表達(dá)均為陽性。(3)血清ALT及AST活性結(jié)果，模型組A2與正常對照A1組存在顯著差異（P0.05）；模型組A2與干預(yù)組B1、B2在第四周有顯著差異（P0.05）；干預(yù)組B1、B2在第四周有顯著差異（P0.05）。結(jié)論：(1)與C57BL/6小鼠BMSCs比較，HBV轉(zhuǎn)基因小鼠BMSCs體外生長存在顯著的血清依賴性及密度依賴性。(2) BMSCs經(jīng)HGF和EGF誘導(dǎo)培養(yǎng)后,可分化為肝樣細(xì)胞。(3)在第四周時兩個干預(yù)組BMSCs組、BMSCs誘導(dǎo)為肝樣細(xì)胞組之間也存在顯著差異，從ALT及AST活性及熒光標(biāo)記結(jié)果來看，BMSCs誘導(dǎo)為肝樣細(xì)胞組對受損肝臟的功能恢復(fù)上有更好的作用。
[Abstract]:Background and objective: liver disease is one of the most common diseases affecting human health. The main types of liver injury caused by various harmful factors are viral hepatitis, alcoholic hepatitis and drug hepatitis. Their clinical manifestations are liver necrosis, fatty liver, cholestasis, liver fibrosis, liver cirrhosis and liver cancer. At present, the treatment of liver injury is still a severe global problem, and stem cell transplantation is a new method for the treatment of liver injury and has shown a good application prospect. Bone marrow mesenchymal stem cells (BMSCs) have strong multidirectional differentiation potential and self-replicating ability and are expected to be a new type of seed cells for the treatment of liver injury. Based on the above problems, we used HGF and EGF to induce BMSCs21 cells to be hepatoid cells, then transplanted hepatoid cells into the liver of HBV mice with liver injury, and confirmed the effect of cell transplantation on liver injury by identification. In order to provide experimental basis for clinical research. Methods the isolation and culture system of BMSCs from bone marrow mesenchymal stem cells of HBV transgenic mice and C57BL/6 mice were established. The purified BMSCs was induced by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). The expression of keratin CK18 and glycogen PAS was detected by immunocytochemistry. To determine whether it is possible to differentiate into hepatocyte-like cells, and to make the liver injury model in mice, the labeled BMSCs was transplanted into the liver injury model mice, and the colonization of stem cells in the liver was detected by fluorescence microscope. Serum liver function and immunohistochemistry were used to detect the expression of keratin in liver tissue. Results the primary BMSCs of C57BL/6 mice grew rapidly, and grew in whirlpool form after subculture at 1:3. The primary BMSCs of HBV-like fibroblasts grew slowly, but at 1:2, it grew slowly. The expression of keratin CK18 and glycogen PAS on the surface of hepatoid cells was detected by immunocytochemistry. The results of serum ALT and AST activity were also observed. There were significant differences between model group A2 and normal control group A1 (P 0.05), model group A 2 and intervention group B 1 B 2 in the fourth week (P 0.05), and intervention group B 1 B 2 had significant difference in the fourth week (P 0.05). Conclusion compared with BMSCs of C57BL/6 mice, the growth of BMSCs in BMSCs transgenic mice is significantly serum-dependent and density-dependent. 2) BMSCs was induced by HGF and EGF. At the fourth week, there was also significant difference between the two intervention groups in inducing BMSCs into hepatoid cells. From the results of ALT and AST activity and fluorescence labeling, it was found that ALT induced by hepatocyte-like cells had a better effect on the recovery of damaged liver function.
【學(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332;R575

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1 饒金才;黃蘭珍;肖純;彭澤華;;小鼠早期肝硬化模型和組織學(xué)觀察[J];江西醫(yī)藥;1988年04期

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