成纤维细胞生长因子处理多次传代培养骨髓间充质干细胞的增殖与分化

发布时间：2018-03-02 18:06

  本文选题：骨髓　切入点：间质干细胞　出处：《中国组织工程研究》2017年25期 　论文类型：期刊论文

【摘要】：背景:骨髓间充质干细胞来源有限,而且在多次体外传代培养过程中,其细胞形态、增殖及多向分化能力会发生改变。目的:探讨成纤维细胞生长因子对经过多次传代、细胞形态发生改变的骨髓间充质干细胞形态、增殖及多向分化能力的影响。方法:(1)体外分离培养大鼠骨髓间充质干细胞,连续传代培养6次,观察其细胞形态变化;(2)将第6代细胞接种于96孔板中,随机分为对照组和成纤维细胞生长因子处理组,相应培养1,2,3,4,5,6,7 d后,用CCK-8试剂盒检测细胞增殖状况;(3)将第6代细胞接种于6孔板中,随机分为对照组和成纤维细胞生长因子处理组,分别用普通培养基和含成纤维细胞生长因子培养基培养7 d,换成骨、成脂、成软骨诱导培养基继续培养7 d,应用荧光定量PCR法检测成骨(RUNX2、ALP、OCN)、成脂(PPARγ2、AP2、ADIPOQ)、成软骨(SOX9、Collagen Ⅱ、aggrecan)相关基因的表达,应用蛋白印记法检测RUNX2、PPARγ2、SOX9蛋白的表达;(4)将第6代细胞接种到6孔细胞培养板中,随机分为对照组及成纤维细胞生长因子处理组,分别用普通生长培养基及含成纤维细胞生长因子的生长培养基培养7 d,换用成骨、成脂、成软骨诱导培养基继续培养14 d,进行茜素红染色、油红O染色和阿利新蓝染色。结果与结论:(1)经过连续6次传代培养,骨髓间充质干细胞形态发生明显变化,成纤维细胞生长因子处理7 d后其形态逐渐恢复原代特性;(2)与对照组相比,培养第3-7天成纤维细胞生长因子处理组的细胞增殖速度明显加快,差异有显著性意义(P0.05);(3)与对照组相比,成纤维细胞生长因子处理组细胞成骨相关基因(RUNX2,ALP,OCN)、成脂相关基因(PPARγ2,AP2,ADIPOQ)、成软骨相关基因(SOX9,collagen 2,aggrecan)表达明显升高,差异有显著性意义(P0.05);(4)成纤维细胞生长因子处理组RUNX2、PPARγ2、SOX9蛋白表达明显高于对照组(P0.05);(5)与对照组相比,成纤维细胞生长因子预处理组细胞产生胞外钙结节的数量、细胞内脂滴的数量、细胞酸性黏多糖的表达明显增加;(6)结果表明,成纤维细胞生长因子可以维持多次传代骨髓间充质干细胞的干细胞特性。
[Abstract]:Background: bone marrow mesenchymal stem cells (BMSCs) have limited sources, and their cell morphology, proliferation and multidirectional differentiation ability will change in vitro. Objective: to investigate the effects of fibroblast growth factor (FGF) on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). Effects of Morphogenetic changes on the Morphology, Proliferation and Multidirectional differentiation of Bone Marrow Mesenchymal Stem cells methods Rat Bone Marrow Mesenchymal Stem cells were isolated and cultured in vitro for 6 consecutive times. The sixth passage cells were inoculated into 96 well plate and randomly divided into control group and fibroblast growth factor treated group. The sixth passage cells were inoculated into 6-well plate with CCK-8 kit. The cells were randomly divided into two groups: control group and fibroblast growth factor treatment group. Normal culture medium and fibroblast growth factor medium were used for 7 days, then bone and fat were replaced, and cartilage induction medium was cultured for 7 days. Fluorescence quantitative PCR was used to detect the expression of genes related to osteoblasts RUNX2, APPAR- 纬 2, AP2, ADIPOQN and cartilage SOX9Collagen 鈪，

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