人骨髓间充质干细胞的体外培养及其向肝细胞诱导分化的实验研究

发布时间：2018-01-25 05:57

本文关键词： 骨髓间充质干细胞体外培养诱导分化肝细胞生长因子表皮细胞生长因子肝细胞　出处：《江西医学院》2005年硕士论文　论文类型：学位论文

【摘要】：目的:建立一种人骨髓间充质干细胞(Mesenchymal stem cells, MSCs)体外培养方法,探讨肝细胞生长因子(HGF)、表皮细胞生长因子(EGF)诱导MSCs 向肝细胞定向分化的可行性。方法:用Ficoll 淋巴细胞分离液分离出骨髓中的MSCs,接种于含10%胎牛血清的L-DMEM 培养液中进行原代培养,待细胞基本融合后用胰蛋白酶和EDTA 混合液消化传代。在倒置相差显微镜下观察细胞形态,台盼蓝拒染法计数细胞并绘制生长曲线,流式细胞仪检测细胞周期。取第三代人MSCs 分别在含HGF、EGF、HGF+EGF 以及不含生长因子的无血清肝细胞培养基中诱导培养。在倒置相差显微镜下观察细胞形态变化,于诱导培养的第1、7、14、21、28 天取出细胞爬片,应用免疫细胞化学法分别检测甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白18(CK18)的表达,PAS 法进行糖原染色试验,并收集培养上清液用全自动生化分析仪检测ALB 和BUN的含量。结果:1、采用密度梯度离心结合贴壁培养法可成功分离出人骨髓中的MSCs。刚接种时细胞形态呈圆形、三角形或棒杆状,48h 后细胞开始贴壁,4-5d 可见有集落形成,第7d 形成多个细胞克隆,第10d 细胞呈长梭形,2w 左右可达到基本融合;传代细胞呈均匀的纺锤形,基本上无悬浮细胞。生长曲线呈现生长缓滞期、对数增殖期和生长平稳期的典型特征。流式细胞仪检测显示约有86.4%的细胞处于G0、G1 期。2、接种于含HGF、EGF 和HGF+EGF 无血清肝细胞培养基中的MSCs 于诱导第5 天均可观察到部分纺锤形细胞逐渐变圆,换液的同时诱导孔中细胞数目略有减少;第10 天左右基本
[Abstract]:Objective: to establish a culture method of mesenchymal stem cells (MSCs) from human bone marrow mesenchymal stem cells (MSCs) in vitro and to investigate the expression of hepatocyte growth factor (HGF). The feasibility of inducing MSCs to differentiate into hepatocytes by epidermal growth factor (EGF). Methods: MSCs was isolated from bone marrow by Ficoll lymphocyte isolate. Primary culture was carried out in L-DMEM medium containing 10% fetal bovine serum. After cell fusion, trypsin and EDTA mixture were used for digestion and passage. Cell morphology was observed under inverted phase contrast microscope. Trypan blue exclusion method was used to count cells and draw growth curve. Flow cytometry was used to detect the cell cycle. The third generation of human MSCs was detected with HGF EGF. HGF EGF and serum-free hepatocyte medium without growth factor were cultured. The morphological changes of the cells were observed under inverted phase contrast microscope. Cell climbing tablets were taken out for 28 days. The expression of AFPU, ALBN and CK18 were detected by immunocytochemical method in Afetoprotein A (AFP), Albumin (ALBN) and cytokeratin (CK-18). The glycogen staining test was carried out by PAS, and the supernatant of culture was collected and the contents of ALB and BUN were detected by automatic biochemical analyzer. Results: 1. MSCs in human bone marrow could be successfully isolated by density gradient centrifugation and adherent culture. The cells were round in shape at first inoculation, and the cells began to adhere to the wall after 48 hours of triangular or rod-shaped cells. Colony formation was observed at 4-5 days, multiple cell clones were formed on the 7th day, and the cells were fusiform for about 2w on the 10th day. The passage cells showed a uniform spindle shape and basically no suspension cells. The growth curve showed a slow growth phase. The typical characteristics of logarithmic proliferative phase and growth stationary phase. Flow cytometry showed that about 86.4% cells were in G _ 0 / G _ 1 phase and inoculated with HGF. MSCs in EGF and HGF EGF serum-free hepatocytes culture medium could be observed on the 5th day after induction. At the same time, the number of cells in the orifices decreased slightly. About the tenth day
【学位授予单位】：江西医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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