同种异体大鼠骨髓间充质干细胞体外诱导分化为肝样细胞的研究
发布时间：2018-11-16 21:33
【摘要】： 目的:本实验通过体外分离培养同种异体大鼠间充质干细胞(Mesenchymal stem cells,MSCs),了解其一般生物特性及低免疫原性,探讨同种异体大鼠MSCs在肝细胞生长因子(hepatocyte growth factor HGF)和成纤维生长因子4(basic fibroblast factor FGF-4)诱导下向肝样细胞(hepatocyte-like cells)横向分化的可能性,以期为临床同种异体骨髓间充质干细胞治疗终末期肝病提供理论基础。 方法: 1.无菌条件下取出大鼠的两侧股骨,冲洗骨髓腔获得细胞,采用全骨髓培养法体外培养细胞,贴壁筛选纯化大鼠MSCs并进行体外培养、扩增。 2.取第三代MSCs进行流式细胞仪测试CD34、CD45、CD29、CD106,并诱导其向脂肪细胞分化。 3.取两只大鼠第三代MSCs混合培养,加入一定浓度的HGF和FGF-4对MSCs诱导培养,未加入生长因子组作为对照组,观察诱导细胞的形态学变化,免疫组化检测培养诱导细胞的细胞角蛋白-18(cytokeratin-18 CK-18)、细胞角蛋白-19(cytokeratin-19 CK-19)及波形蛋白Vimentin;ELISA等方法检测甲胎蛋白(alpha-fetoprotein AFP)、白蛋白(albumin ALB)、碱性磷酸酶(alkaline phosphatase ALP)。 结果: 1.刚分离接种的大鼠骨髓MSCs形态大小均一。大鼠MSCs在24h内贴壁,10天左右达到对数生长期。细胞传至第三代时候,细胞融合成单层,梭形突起变长,排列有明显方向性,呈旋涡状、鱼群样。 2.第三代大鼠MSCs流式细胞仪检测CD34、CD45阴性,CD29、CD106阳性,MSC诱导后分化为脂肪细胞。 3.混合培养的大鼠MSCs经HGF和FGF-4共同诱导后7天后,开始观察到少量细胞变成短梭形,诱导14天后可以观察到细胞变成三角形或是椭圆形,随时间的延长椭圆形细胞越多。而未加诱导因子的对照组未观察到三角形及椭圆形细胞的出现,细胞仍呈梭形生长,并相互融合。当诱导的细胞大部分变成椭圆形后,行免疫组化及ELASA检测,结果显示CK18、CK19、Vimentin表达阳性,AFP、ALB、ALP出现明显改变。 结论: 1. HGF和FGF-4的共同作用可以促进同种异体大鼠MSCs横向分化为肝样细胞。 2. 10%胎牛血清的DMEM低糖培养基适合大鼠间充质干细胞培养和分化。 3.骨髓源性间充质干细胞具有低免疫原性。 4.骨髓源性MSCs可成为肝组织工程主要种子细胞来源。
[Abstract]:Objective: to investigate the general biological characteristics and low immunogenicity of allogeneic rat mesenchymal stem cells (Mesenchymal stem cells,MSCs) isolated and cultured in vitro. To investigate the possibility of transversely differentiation of allogeneic MSCs into hepatoid cells (hepatocyte-like cells) induced by hepatocyte growth factor (hepatocyte growth factor HGF) and fibroblast growth factor (4 (basic fibroblast factor FGF-4). To provide a theoretical basis for the treatment of end-stage liver disease with allogeneic bone marrow mesenchymal stem cells. Methods: 1. The femur of both sides of the rat was removed under aseptic condition, and the cells were obtained by washing the medullary cavity. The cells were cultured in vitro by whole bone marrow culture method. The MSCs of rats was screened and purified by adherent method and cultured and amplified in vitro. 2. The third generation MSCs was used to detect CD34,CD45,CD29,CD106, and induce it to differentiate into adipocytes. 3. The third generation MSCs of two rats were mixed cultured, and the MSCs was induced by adding a certain concentration of HGF and FGF-4. The growth factor group was not added to the control group, and the morphological changes of the induced cells were observed. Immunohistochemical detection of cytokeratin-18 (cytokeratin-18 CK-18), cytokeratin 19 (cytokeratin-19 CK-19) and vimentin Vimentin; in cultured cells Detection of alpha-fetoprotein (alpha-fetoprotein AFP), albumin (albumin ALB), alkaline phosphatase (alkaline phosphatase ALP).) by ELISA and other methods Results: 1. The bone marrow MSCs of the newly isolated and inoculated rats was uniform in size. Rat MSCs adheres to the wall within 24 hours and reaches logarithmic growth stage about 10 days. At the third generation, the cells were fused into monolayer, fusiform protuberances became longer, arranged in obvious directionality, appeared as swirl, fish. 2. The third generation of rat MSCs flow cytometry detected CD34,CD45 negative, CD29,CD106 positive, MSC induced differentiation into adipocytes. 3. After co-induction of MSCs by HGF and FGF-4 for 7 days, a small number of cells were observed to be fusiform. After 14 days of induction, triangular or elliptical cells were observed, and the number of oval cells increased with time. In the control group without induction factor, triangular and oval cells were not observed, and the cells were still fusiform and fused with each other. When most of the induced cells became oval, the positive expression of CK18,CK19,Vimentin and AFP,ALB,ALP were detected by immunohistochemistry and ELASA. Conclusion: 1. The interaction of HGF and FGF-4 can promote the transversely differentiation of MSCs into hepatoid cells in allogeneic rats. 2. DMEM medium with 10% fetal bovine serum was suitable for rat mesenchymal stem cell culture and differentiation. 3. Bone marrow derived mesenchymal stem cells have low immunogenicity. 4. Bone marrow derived MSCs may be the main source of seed cells in liver tissue engineering.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2336694
[Abstract]:Objective: to investigate the general biological characteristics and low immunogenicity of allogeneic rat mesenchymal stem cells (Mesenchymal stem cells,MSCs) isolated and cultured in vitro. To investigate the possibility of transversely differentiation of allogeneic MSCs into hepatoid cells (hepatocyte-like cells) induced by hepatocyte growth factor (hepatocyte growth factor HGF) and fibroblast growth factor (4 (basic fibroblast factor FGF-4). To provide a theoretical basis for the treatment of end-stage liver disease with allogeneic bone marrow mesenchymal stem cells. Methods: 1. The femur of both sides of the rat was removed under aseptic condition, and the cells were obtained by washing the medullary cavity. The cells were cultured in vitro by whole bone marrow culture method. The MSCs of rats was screened and purified by adherent method and cultured and amplified in vitro. 2. The third generation MSCs was used to detect CD34,CD45,CD29,CD106, and induce it to differentiate into adipocytes. 3. The third generation MSCs of two rats were mixed cultured, and the MSCs was induced by adding a certain concentration of HGF and FGF-4. The growth factor group was not added to the control group, and the morphological changes of the induced cells were observed. Immunohistochemical detection of cytokeratin-18 (cytokeratin-18 CK-18), cytokeratin 19 (cytokeratin-19 CK-19) and vimentin Vimentin; in cultured cells Detection of alpha-fetoprotein (alpha-fetoprotein AFP), albumin (albumin ALB), alkaline phosphatase (alkaline phosphatase ALP).) by ELISA and other methods Results: 1. The bone marrow MSCs of the newly isolated and inoculated rats was uniform in size. Rat MSCs adheres to the wall within 24 hours and reaches logarithmic growth stage about 10 days. At the third generation, the cells were fused into monolayer, fusiform protuberances became longer, arranged in obvious directionality, appeared as swirl, fish. 2. The third generation of rat MSCs flow cytometry detected CD34,CD45 negative, CD29,CD106 positive, MSC induced differentiation into adipocytes. 3. After co-induction of MSCs by HGF and FGF-4 for 7 days, a small number of cells were observed to be fusiform. After 14 days of induction, triangular or elliptical cells were observed, and the number of oval cells increased with time. In the control group without induction factor, triangular and oval cells were not observed, and the cells were still fusiform and fused with each other. When most of the induced cells became oval, the positive expression of CK18,CK19,Vimentin and AFP,ALB,ALP were detected by immunohistochemistry and ELASA. Conclusion: 1. The interaction of HGF and FGF-4 can promote the transversely differentiation of MSCs into hepatoid cells in allogeneic rats. 2. DMEM medium with 10% fetal bovine serum was suitable for rat mesenchymal stem cell culture and differentiation. 3. Bone marrow derived mesenchymal stem cells have low immunogenicity. 4. Bone marrow derived MSCs may be the main source of seed cells in liver tissue engineering.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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