官内膜干细胞的成肝分化及其对肝损伤修复作用的研究

发布时间：2018-01-20 18:10

本文关键词： 宫内膜干细胞成肝分化肝损伤　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：本研究目的为探讨宫内膜干细胞的体外成肝诱导分化及其在肝脏损伤中发挥的修复作用。首先,我们从招募的志愿者经血中分离出单个核细胞进行体外传代培养并对获得的细胞进行间充质干细胞鉴定。宫内膜干细胞不具有致瘤性,可以呈单层贴壁生长,可以24小时倍增一次,为典型的棒状或长梭状的成纤维细胞样细胞。流式细胞术分析这类细胞均表达CD44,CD73,CD90,和CD29,而不表达CD117,CD34, CD45,和HLA-DR。宫内膜干细胞在体外可以被诱导分化为成骨细胞和成脂细胞。其次,我们研究了宫内膜干细胞的体外成肝诱导分化能力。宫内膜干细胞在与HGF、FGF4、OSM以及DEX等共培养后,逐渐向上皮细胞样细胞形态变化。诱导分化后的细胞能够表达ALB和AFP蛋白,以及基因ALB、AFP、CK18、CK19、CYP1A1和CYP3A4等。表达谱芯片分析可见分化后的细胞能表达33个肝脏相关基因,这些基因在原代肝脏细胞中表达,而宫内膜干细胞中则不表达。另外,诱导分化后细胞功能验证显示,其具有糖原合成、ICG排泌和尿素合成功能等。这些数据可以初步证实,所诱导的细胞是具有肝细胞表型且能发挥部分肝脏细胞功能的较成熟的细胞。最后,为了验证移植细胞能否参与肝损伤修复过程,小鼠肝脏部分切除后,分别移植宫内膜干细胞和诱导分化后细胞,移植后10天,移植分化组小鼠肝功能较移植宫内膜干细胞组恢复明显,具有显著性差异。移植8周后,在小鼠肝脏中可以检测到移植细胞的存在,并且移植宫内膜干细胞在受体肝脏内发生分化。Fah-/小鼠作为移植受体,移植细胞后8周,发现诱导分化组小鼠肝脏可见大量Fah阳性细胞,而移植宫内膜干细胞组则仅有少量散在分布的Fah阳性细胞存在。这些数据说明,分化后细胞可以部分重建小鼠肝脏,未分化细胞可以发生分化但整体水平较低。结果显示,移植细胞可以随血流迁移至小鼠肝脏,并在小鼠肝脏内达到一定程度的重建。综上所述,宫内膜干细胞及其来源的细胞在肝脏疾病治疗中可以发挥一定作用。
[Abstract]:The purpose of this study was to investigate the differentiation of endometrial stem cells in vitro and its role in the repair of liver injury. We isolated mononuclear cells from the blood of recruited volunteers and cultured them in vitro and identified the obtained cells by mesenchymal stem cells. The endometrial stem cells were not tumorigenic and could be monolayer adherent. It can be doubled once in 24 hours and is a typical rod-like or long-fusiform fibroblast-like cell. All of these cells express CD44-CD73 CD90 and CD29 by flow cytometry. However, the endometrial stem cells could be induced to differentiate into osteoblasts and adipoblasts without expression of CD117, CD45, and HLA-DR.In vitro, endometrial stem cells could be induced to differentiate into osteoblasts and adipoblasts. Secondly, we studied the ability of endometrial stem cells to induce liver differentiation in vitro. Endometrial stem cells were co-cultured with HGFFGF4OSM and DEX. The cells induced by differentiation could express ALB and AFP proteins, as well as the gene ALBN-AFPCK18CK-19. CYP1A1 and CYP3A4. Microarray analysis showed that the differentiated cells could express 33 liver-associated genes, which were expressed in primary liver cells. In addition, after induction of differentiation, the cell function test showed that it had the function of glycogen synthesis, ICG excretion, urea synthesis and so on. These data can be preliminarily confirmed. The induced cells are mature cells with hepatocyte phenotype and function of some liver cells. Finally, in order to verify whether the transplanted cells can participate in the repair process of liver injury, after partial hepatectomy, endometrial stem cells and differentiated cells were transplanted, respectively, 10 days after transplantation. The liver function of the transplanted differentiation group was significantly different from that of the transplanted endometrial stem cell group. 8 weeks after transplantation, the presence of transplanted cells could be detected in the mouse liver. And transplanted endometrial stem cells in the recipient liver differentiation. Fah-/ mice as the transplantation receptor, 8 weeks after transplantation, we found that a large number of Fah positive cells were found in the liver of the induced differentiation group. However, there were only a few scattered Fah positive cells in the transplanted endometrial stem cells group. These data showed that the differentiated cells could partially reconstruct the liver of mice. The results showed that the transplanted cells could migrate to the mouse liver with the blood flow, and the transplanted cells could be reconstructed in the mouse liver to a certain extent. In conclusion, endometrial stem cells and their derived cells may play a role in the treatment of liver diseases.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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