【作者】 潘小平；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2013， 博士

【摘要】 研究背景各种原因引起的肝衰竭病死率高达70%～80%。以肝细胞为生物活性成分的生物型人工肝(bioartificial liver, BAL)成为肝衰竭治疗研究的热点和发展方向。基于原代人肝细胞、原代猪肝细胞、C3A肝肿瘤细胞的三种类型肝细胞的BAL临床试验治疗显示,BAL具有很好的疗效和应用前景。但是,肝细胞源的短缺等问题,依然阻碍着BAL的临床应用和发展。肝脏干细胞可诱导分化为功能成熟的肝细胞,被认为是非常有前景的细胞源。目前,肝脏干细胞在体外诱导分化是受到多种因素影响和调控,分化后的肝细胞尚难达到生物人工肝临床治疗的数量。因此,需要进一步探索肝脏干细胞的规模化培养和诱导分化的新方式和新方法。肝细胞永生化是解决肝细胞来源困难的重要可行性途径之一。然而,永生化人肝细胞的基因表达和细胞功能,与原代人肝细胞功能存在一定的差异。因此,必须在解决肝细胞数量的同时,需要进一步提高永生化人肝细胞的分化程度和细胞功能。细胞与细胞间相互作用在保持和促进细胞功能中起着非常重要作用。在各种共培养体系中,肝细胞与肝非实质细胞之间的相互作用为肝细胞生长提供了微环境,有助于肝细胞保持其结构稳定和细胞功能。肝星状细胞是肝脏中非常重要的非实质细胞。然而,原代肝星状细胞存在着分离繁琐,分离成本较高,容易失去生物学特性和功能等问题。综上所述,我们从以下几个方面进行研究,建立高活性和功能的永生化人肝星状细胞系,探索永生化人肝星状细胞与永生化人肝细胞共培养对肝细胞功能的影响,以及探索永生化人肝星状细胞对肝祖细胞向肝细胞分化的影响。为优化和完善永生化人肝细胞培养的微环境和建立肝祖细胞向肝细胞分化的新方法提供实验依据,将为生物型人工肝提供更高质量的肝细胞奠定基础。第一部分永生化人肝星状细胞系的建立及其鉴定目的：建立高活性和功能的永生化人肝星状细胞系,为肝非实质细胞与肝细胞、肝祖细胞共培养研究提供理想的细胞模型。方法：采用SV40LT重组反转录病毒感染原代人肝星状细胞,获得永生化人肝星状细胞系。采用电镜技术、RT-PCR等分析永生化人肝星状细胞的形态学、生物学特性和功能等。结果：建立一株永生化人肝星状细胞系,命名为HSC-Li。该细胞系具有典型的肝星状细胞形态学特征；HSC-Li细胞的Ⅰ、Ⅱ型胶原、HGF等mRNA表达为阳性；HSC-Li细胞的a-SMA、Vimentin、GFAP和SV40LT蛋白表达为阳性。HSC-Li细胞分泌VEGF、HGF、IL-6、TGF-betal等多种细胞因子和炎症因子。结论：建立了一株永生化人肝星状细胞系HSC-Li,该细胞系具有人肝星状细胞的生物学特性和功能；可为肝细胞与肝星状细胞、肝祖细胞与肝星状细胞共培养研究提供理想的细胞模型。第二部分永生化人肝星状细胞与永生化人肝细胞共培养对肝细胞功能的影响目的：探索永生化人肝星状细胞与永生化人肝细胞共培养对永生化人肝细胞功能的影响；为优化和完善永生化人肝细胞培养的微环境提供实验依据。方法：首先采用SV40LT重组反转录病毒感染原代人肝细胞,获得一株永生化人肝细胞系；通过直接接触共培养、间接接触共培养方式进行永生化人肝细胞与永生化人肝星状细胞共培养研究,测定永生化人肝细胞共培养24h、48h、72h后ALB、 CYP3A5、CYP2E1、UGT2b7等基因的表达水平,以及测定CYP1A2活力。结果：建立了一株永生化人肝细胞系HepLi5,HepLi5细胞的ALB、GS、CYP2E1、 CYP3A5等基因的mRNA表达均为阳性,具有分泌白蛋白等原代人肝细胞的生物学特性和功能。HSC-Li细胞与HepLi5细胞直接接触和间接接触共培养研究结果显示,HepLi5细胞在共培养24h、48h、72h后ALB、CYP3A5、CYP2E1、UGT2b7等基因表达显著增强,以及CYP1A2活力显著增强。与单独培养组相比较,直接接触共培养方式和间接接触共培养方式均显著增强肝细胞基因表达和功能。结论：建立了一株永生化人肝细胞系HepLi5,HepLi5细胞具有分泌白蛋白等原代人肝细胞的生物学特性和功能。直接接触共培养和间接接触共培养方式均可显著增强HepLi5细胞的基因表达和细胞功能。为优化和完善永生化人肝细胞培养的微环境提供实验依据,将为生物型人工肝提供更高质量的肝细胞奠定基础。第三部分永生化人肝星状细胞对肝祖细胞向肝细胞分化的影响目的：探索永生化人肝星状细胞对肝祖细胞BMEL-TAT分化为肝细胞的影响；为建立肝祖细胞向肝细胞分化的新方法提供实验依据。方法：通过永生化人肝星状细胞与肝祖细胞间接接触共培养方式诱导肝祖细胞分化为肝细胞,检测肝祖细胞BMEL-TAT共培养前、后细胞表型和功能。结果：HSC-Li星状细胞与肝祖细胞BMEL-TAT间接接触共培养方式可以诱导肝祖细胞分化为功能成熟的肝细胞。以单独培养BMEL-TAT细胞作为对照,与HSC-Li细胞共培养3天后,BMEL-TAT细胞在细胞形态上开始向肝样细胞转变；与HSC-Li细胞共培养7、14、21天后,BMEL-TAT细胞的ALB、CK18、TAT、G6P等基因表达逐渐增强；细胞免疫荧光染色显示：共培养21天后BMEL-TAT细胞的ALB、CK18表达显著增强,CK19、AFP表达下降。共培养21天后BMEL-TAT细胞的X-gal染色、PAS染色和Dil-LDL吸收实验均为阳性,以及CYP1A2活力和尿素合成也显著增强。结论：建立了肝祖细胞向肝细胞分化的新方法；永生化人肝星状细胞与肝祖细胞间接接触共培养方式可以诱导肝祖细胞BMEL-TAT向肝细胞分化；可为生物型人工肝提供新型、高质量的肝细胞奠定基础。更多还原

【Abstract】 BackgroundFulminant hepatic failure (FHF) has very high mortality rates approaching70-80%. Considering the potential of the liver to regenerate, Liver support system has been considered to the alternative therapy for liver failure in clinical trial as a bridge to orthotropic liver transplantation(OLT) or avoiding liver transplantation. To date, several kind of bioartificial liver (BAL) using primary porcine hepatocyte, primary human hepatocytes and C3A liver tumor cells have been submitted for clinical trials. However, no BAL has been approved by government for clinical treatment, because suitable sources of liver cells are not available. So, it is very urgent and important to find new type of hepatocytes for application in BAL as the biological component of BAL.The immortalized human hepatocytes with indefinitive expansion in vitro and allogenic cells are an ideal suitable for application in BAL. However, its functionality and differentiation is lower than that in primary human hepatocytes. Liver progenitor cells can be expanded and differentiated into functional hepatocytes that are urgently required as potentially alternatives to hepatocytes.Cell-cell interactions such as hepatocytes and stellate cells play a very important role in the stability of liver function of primary hepatocytes. However, human hepatic stellate cells (HSC) are terminally differentiated liver cells, which have a limited proliferation and do not passage in vitro for long term time. Moreover, isolation of HSC is extremely time-consuming and laborious works, their yields are commonly even lower.Here, we aimed to establish an immortalized human hepatic stellate cells line, and to evaluate the effects of co-culture with the immortalized human hepatic stellate cells on hepaocyte functions of immortalized human hepatocytes and differentiation of liver progenitor cells into functional hepatocytes.Part I Establishment and characterization of an immortalized human hepatic stellate cells lineObjective:To establish an immortalized human hepatic stellate cells Line for either co-culture of hepatcotyes and human hepatic stellate cells or co-culture of liver progenitor cells with human hepatic stellate cells in vitro as an ideal cells line.Methods:Primary human Hepatic stellate cells were transfected with recombinant retrovirus containing simian virus40large T antigen (SV40LT). Subsequently, the characteristics of immortalized human hepatic stellate cells was characterized by reverse transcription polymerase chain reactions (RT-PCR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescent cytochemistry staining, and so on.Results:An immortalized human hepatic stellate cells Line, HSC-Li, was harvested after infection of primary human hepatic stellate cells with recombinant retrovirus containing SV40LT. Under phase contrast microscope and electronic microscope, the immortalized human hepatic stellate cells showed classical appearance of hepatic stellate cells. The mRNA expression of human Collagen type I, Collagen type Ⅱ、HGF and SV40LT was observed by RT-PCR in HSC-Li cells. The results of immunofluorescent cytochemistry staining showed that HSC-Li cells expressed α-smooth muscle actin (a-SMA), Vimentin, and glial fibrillary acid protein (GFAP). The cytokines such as HGF, VEGF, IL-6, and TGF-betal were determined in the culture supernatant of HSC-Li cells by ELIS A and protein array. Conclusions:The immortalized human hepatic stellate cells, HSC-Li cells, have the biological and functional characteristics of human hepatic stellate cells, which may be an ideal cells line for either co-culture of hepatocytes with hepatic stellate cells or co-culture of liver progenitor cells with hepatic stellate cells in vitro. Part II Effects of co-culture with immortalized human hepatic stellate cells on the hepatocyte function of immortalization human hepatocytesObjective:To evaluate the effects of co-culture with immortalized human hepatic stellate cells on hepaocyte function of immortalized human hepatocytes for application in bioartificial liver.Methods:Immortalized human Hepatocytes were established by transfection of primary human hepatocytes with recombinant retrovirus containing SV40LT. Characteristics of immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro. After co-culture with the HSC-Li cells employing mixed co-culture and separated co-culture, the mRNA expression of metabolizing enzymes and CYP1A2activity of immortalized human hepatocytes was evaluated.Results:An immortalized human hepatocytes cell line, HepLi5, was successfully established. The expression of SV40LT in HepLi5cells could be detected by RT-PCR and Western blot. The mRNA expression of liver-enriched genes and human cytochrome P450was detectable by RT-PCR in HepLi5cells.When HepLi5cells were co-cultured with HSC-Li cells in either separated co-culture or mixed co-culture, the CYP1A2activity was significantly enhanced. The mRNA expression levels of metabolizing enzymes such as CYP3A5, CYP2E1and UGT2b7of HepLi5cells were significantly improved in both separated co-culture and mixed co-culture with HSC-Li cells. Conclusions:we have successfully established an immortalized human hepatocytes cell line, HepLi5. Our results suggested that co-culture of HepLi5cells with HSC-Li cells significantly improved the gene expression and functional characteristics of HepLi5cells. The hepatocyte function of immortalized human hepatocytes could be enhanced in separated co-culture and in mixed co-culture with HSC-Li cells, which may be greatly helpful for use in BAL in future. Part Ⅲ Effects of co-culture with immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytesObjective:To evaluate the effects of co-culture with the immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytes in vitro.Methods:liver progenitor cells, BMEL-TAT, and the immortalized human hepatic stellate cells, HSC-Li cells, were used in this study. The separated co-culture of BMEL-TAT with HSC-Li cells was established for analyzing the effects of co-culture with immortalized human hepatic stellate cells on the differentiation of liver progenitor cells into functional hepatocytes. The cell morphology of BMEL-TAT cells was observed by light microscope and laser confocal microscope. The mRNA expression of ALB, CK18, TAT and G6P in the BMEL-TAT cells was detected by RT-PCR after co-culture with HSC-Li cells. The CYP1A2activity, Dil-LDL uptake, and urea secretion were also determined in both BMEL-TAT control and after21days co-culture of BMEL-TAT, etc. The BMEL-TAT cultured on normal medium served as control.Results:co-culture with HSC-Li cells significantly enhanced the differentiation of liver progenitor cells into functional hepatocytes. The cell morphology of BMEL-TAT cells was changed to hepatocyte-like cells from days3to days21after co-culture with HSC-Li cells. The mRNA expression of ALB, CK18, TAT and G6P in the BMEL-TAT cells was significantly improved by RT-PCR after co-culture with HSC-Li cells. The results of immunofuorescent cytochemistry staining showed the expression of ALB, CK18was significantly enhanced in BMEL-TAT after21days co-culture with HSC-Li cells, whereas the expression of CK19and AFP apparently decreased. The CYP1A2activity, Dil-LDL uptake, urea secretion was also increased significantly after21days co-culture of BMEL-TAT with HSC-Li cells. X-gal staining and PAS staining in BMEL-TAT after21days co-culture with HSC-Li cells were apparently positive, whereas the BMEL-TAT control was negative.Conclusion:Separated co-culture with HSC-Li cells could successfully induce the differentiation of liver progenitor cells into functional hepatocytes, which may be very important for generation of functional hepatocytes from liver progenitor cells for use in BAL in future.更多还原