本文選題：肝再生增強因子 + HepG2細胞�。� 參考：《中國人民解放軍軍醫(yī)進修學(xué)院》2010年碩士論文

【摘要】： 人肝再生增強因子(human augmenter of liver regeneration, hALR)是目前已知唯一可以特異性刺激肝源性細胞增殖的細胞因子,是早期表達的肝再生增強因子之一。它能促進肝細胞增殖和肝臟再生,還能夠保護肝臟、預(yù)防肝纖維化以及提高肝移植的成功率。 目的：研究人肝再生增強因子(hALR)對HepG2細胞增殖的影響,探討其促進肝細胞增殖的機制,為生物人工肝細胞反應(yīng)器提供合適的細胞材料。 方法：本研究采用轉(zhuǎn)染重組質(zhì)粒pcDNA3.1(-)hALR的HepG2細胞(前期研究構(gòu)建),經(jīng)G418篩選,在體外培養(yǎng)、傳代后,進行Western blot免疫印跡試驗及免疫熒光實驗,檢測細胞中hALR的表達,并與未轉(zhuǎn)染重組質(zhì)粒的HepG2細胞進行比較;采用蛋白免疫印跡和ELISA方法，檢測兩組細胞培養(yǎng)液中hALR的分泌情況;使用流式細胞儀檢測細胞中增殖細胞相關(guān)核抗原Ki-67,了解重組質(zhì)粒轉(zhuǎn)染前后HepG2細胞的增殖狀況;以蛋白免疫印跡試驗檢測兩組肝細胞及培養(yǎng)液中轉(zhuǎn)化生長因子α(TGFα)、表皮生長因子受體(EGFR)的表達情況,研究hALR是否通過影響TGFα、EGFR的表達促進肝細胞的增殖。 結(jié)果：轉(zhuǎn)染重組質(zhì)粒pcDNA3.1(-)hALR的HepG2細胞功能穩(wěn)定,形態(tài)良好,細胞胞漿中有大量hALR表達,較未轉(zhuǎn)染重組質(zhì)粒的HepG2細胞為多,細胞培養(yǎng)液中亦有hALR分泌,且隨著培養(yǎng)時間的延長,細胞培養(yǎng)液中的hALR含量迅速升高,優(yōu)于未轉(zhuǎn)染重組質(zhì)粒的HepG2細胞;轉(zhuǎn)染重組質(zhì)粒的HepG2細胞中Ki-67陽性細胞計數(shù)明顯高于未轉(zhuǎn)染重組質(zhì)粒的HepG2細胞，說明轉(zhuǎn)染重組質(zhì)粒使肝細胞增殖活躍。轉(zhuǎn)染重組質(zhì)粒pcDNA3.1(-)hALR使得肝細胞及培養(yǎng)液中TGFα含量升高,肝細胞中EGFR含量升高。 結(jié)論：本研究結(jié)果表明,前期研究構(gòu)建的轉(zhuǎn)染了hALR基因的肝細胞系能較高表達hALR,且細胞增殖活躍;根據(jù)結(jié)果推測,hALR可能通過上調(diào)TGFα及其受體EGFR的表達來促進肝細胞的再生;生物人工肝細胞反應(yīng)器中應(yīng)用該細胞系,在完成肝細胞的合成、代謝和解毒功能的同時,還可補充hALR、TGFα等可促進肝細胞生長的細胞因子,為研制生物活性更強的生物人工肝細胞反應(yīng)器提供物質(zhì)基礎(chǔ)。
[Abstract]:Human augmenter of liver Regeneration (hALR) is the only known cytokine that can specifically stimulate the proliferation of hepatogenic cells and is one of the early expressed augmenters of liver regeneration. It can promote hepatocyte proliferation and liver regeneration, protect liver, prevent liver fibrosis and improve the success rate of liver transplantation. Aim: to study the effect of human augmenter of liver regeneration (hALR) on the proliferation of HepG2 cells and to explore the mechanism of promoting the proliferation of hepatocytes. Methods: in this study, HepG2 cells transfected with recombinant plasmid pcDNA3.1(-)hALR were used to detect the expression of hALR by Western blot immunoblotting assay and immunofluorescence assay. The secretion of hALR in the culture medium of the two groups was detected by Western blot and ELISA. The proliferative cell associated nuclear antigen Ki-67 was detected by flow cytometry to investigate the proliferation of HepG2 cells before and after transfection of recombinant plasmid. The expression of transforming growth factor- 偽 (TGF- 偽) and epidermal growth factor receptor (EGFR) in hepatocytes and culture media were detected by Western blot assay. The effect of hALR on the expression of TGF 偽 -EGFR was studied. Results: the HepG2 cells transfected with recombinant plasmid pcDNA3.1(-)hALR had stable function and good morphology. There was a large amount of hALR expression in the cytoplasm of the cells, which was more than that in the HepG2 cells without the recombinant plasmid transfection. HALR was secreted in the cell culture medium, and with the prolongation of the culture time. The hALR content in cell culture medium increased rapidly, which was superior to that in HepG2 cells without recombinant plasmid, and the number of Ki-67 positive cells in HepG2 cells transfected with recombinant plasmid was significantly higher than that in HepG2 cells without recombinant plasmid. The results showed that the proliferation of hepatocytes was active after transfection of recombinant plasmids. Transfection of recombinant plasmid pcDNA3.1(-)hALR increased the content of TGF 偽 in hepatocytes and culture medium, and the content of EGFR in hepatocytes. Conclusion: the results of this study indicate that the liver cell lines transfected with hALR gene can express hALRhighly, and the proliferation of HALR cells is active. It is speculated that HALR may promote the regeneration of hepatocytes by up-regulating the expression of TGF 偽 and its receptor EGFR. When the cell line was used in the biological artificial hepatocyte reactor, the synthesis, metabolism and detoxification function of the hepatocytes could be completed, and the cytokines such as hALRGF-a could promote the growth of hepatocytes. It provides a material basis for the development of bioactive bioartificial hepatocyte reactor.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進修學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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