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题名 肝硬化患者骨髓间充质干细胞体外培养及诱导分化为肝细胞的实验研究
姓名 安方梅
院系 临床医学院
专业 内科学(传染病)
学位名称 法律硕士
外文题名 experimental study on culture and differentiation of bone marrow mesenchymal cells from cirrhosis patients into hepatocytes in vitro
第一导师姓名 陈红
关键词 骨髓间充质干细胞;移植;生长特性;肝细胞生长因子;白介素-6;诱导分化;肝细胞
外文关键词 bone marrow mesenchymal stemcells ;transplantation;biological features;hepatocyte growth factor;interleukin-6;indifferentiation ;hepatocyte
学科 医学
摘要 目的 探讨肝硬化患者骨髓间充质干细胞(Bone marrow mesenchymal stem cells MSCs)的体外扩增培养生长特性及其诱导分化为肝功能细胞的能力,为肝组织工程“种子细胞”的选择提供新的理论依据,为进一步研究自体骨髓间充质干细胞移植治疗肝衰竭时移植细胞在体内的调控机制打下基础。 方法 无菌抽取我科4例肝硬化志愿者骨髓3~4ml,年龄30~40岁,利用密度梯度离心法(1.073g/L ficoll)联合贴壁筛选法逐步筛选MSCs,获取MSCs,倒置显微镜下逐日观察细胞生长特性,以Cell Counter做细胞计数,绘制细胞生长曲线,流式细胞仪检测细胞表面标志CD44、CD45。制作细胞爬片,取第三代培养细胞,将细胞分成两组:A组 HGF(20ng/ml) +IL-6 (10ng/ml)+基础培养基诱导培养。B组(空白对照组)基础培养基(胎牛血清+L-DMEM)培养。倒置显微镜下观察诱导分化过程中细胞形态变化,分别于诱导培养后0、7、14、21、28d时取出爬片免疫细胞化学染色测定肝细胞特征表型甲胎蛋白(AFP)、细胞角蛋白18(CK18)的表达情况并于诱导培养后多时间点测定培养细胞产生的AFP 和CK18的阳性表达率(%),并与空白对照作作以比较,得出结论。 结果 原代培养细胞生长潜伏期0~9d,对数增殖期为10~19d,生长平台期为20~24d;传代细胞生长周期较原代细胞快,细胞生长潜伏期为4~24h,对数增殖期为3~12d,13~15d进入生长平台期。肝硬化患者骨髓 MSCs高表达CD44,低表达表达CD45。分别在诱导培养0、7、14、21、28d经免疫细胞化学方法测细胞浆内AFP和CK18表达情况,A组细胞为阳性表达,而B组则均为阴性表达。 结论 肝硬化患者骨髓MSCs体外生长良好,性质稳定,在体外诱导培养中可分化为肝功能细胞,因此可以作为“种子细胞”应用于自体骨髓干细胞移植和生物人工肝支持系统。
外文摘要 Objective To investigate the growth pattern and surface markers of Bone marrow mesenchyaml stem cells(MSCs) of cirrhosis patient ,And explore whether it can be induced into hepatocytes by Hepatocyte growrh factor(HGF)and Interleukin-6(IL-6) in vitro. Give new concept to the choice of “Seed Cells”for liver tissue engineer. Methods Bone marrow cells were collected from volunteers of cirrhosis patient aged from 30 to 40 in our department.MSCs were separated with Lymophocyte Separating Liquid(density 1.073g/L ficoll),biological features of cultures MSCs were observed under inversion microscope and their growrh curves were investigated, CD44 and CD45 was analyzed by flow cytometry. In this study MSCs passaged 3 were divided into 2 groups:group A wirh 20ng/ml HGF and 10ng/ml IL-6 in basical culture medium(L-DMEM with 10% FBS), group B basical culture medium respectively. The morphological changes of MSCs were observed by phase-contrast microscope and the expressions of alphafetoprotein(AFP),cell keratin 18(CK18) of each group were detected by immunohistochem technique on 0,7,14,21,28 days during differentiation. Results The latent phade of the primary culture cells was 0 to 9days,followed by 10 to 19 days of logarithmical proliferation,the plateau began at day 20 to 24. Subcultures showed faster growrh than primary cells, The latent phade was 4 to 24 hours,followed by 3 to 12 days of logarithmical proliferation,the plateau began at day 13 to 15.The MSCs expressed positive CD44 unanimously,but don’t expressed CD45. Immunocytochemical analysis for AFP and CK18 showed positive staining reaction and negative staining resction in group B. Conclusion The biological features of MSCs of cirrhosis patient are as same as healthy human,and can be induced into hupatocyte lineage in the condition of HGF and IL-6 in vitro. It can be“Seed Cells”for liver tissue engineer and autologous MSCs transplantation .
研究领域 干细胞移植治疗重症肝炎
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