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  

Effects of fibroblast growth factor on proliferation and differentiation of serially passaged bone marrow mesenchymal stem cells

作　　者：宋明宇 杨勇 吴华[2] 王蓉 Song Ming-yu;Yang Yong;Wu Hua;Wang Rong(Department of Obstetrics and GynecologyTongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,Hubei Province, China;Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,Hubei Province, China;Department of Gastroenterology, General Hospital of the Yangtse River Shipping, Wuhan 430015, Hubei Province, China)

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,湖北省武汉市430030 [2]华中科技大学同济医学院附属同济医院骨科,湖北省武汉市430030 [3]长江航运总医院消化内科,湖北省武汉市430015

出　　处：《中国组织工程研究》2017年第25期3937-3942,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年基金项目(81301552;51707075)~~

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(P<0.05);(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(P<0.05);(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(P<0.05);(5)与对照�BACKGROUND:The source of bone marrow mesenchymal stem cells(BMSCs)is limited,and the cellular morphology,proliferation and multi-directional differentiation capacities can vary during serial passages in BMSCs in vitro.OBJECTIVE:To study the effects of fibroblast growth factor(FGF)on cellular morphology,proliferation and differentiation of serially passaged BMSCs.METHODS:(1)BMSCs were isolated from Sprague-Dawley rats and cultured.These cells were passaged six times in vitro,and the cellular morphology was observed and photographed.(2)BMSCs at passage6were seeded into96-well plates and randomly divided into control group and FGF treatment group.The proliferation of cells in both groups was detected with cell counting kit-8kit at days1,2,3,4,5,6,7after culture.(3)BMSCs at passage6were seeded into6-well plates and randomly divided into control group and FGF treatment group.After7days treatment with growth medium or growth medium containing FGF,the cellular morphology was observed and photographed.And then the cells of both groups were treated with osteogenic induction medium,adipogenic induction medium and chondrogenic induction medium for the next7days.

关 键 词：干细胞 骨髓干细胞 骨髓间充质干细胞 增殖 分化 细胞形态 成骨 成脂 成软骨 干细胞特性 成纤维细胞生长因子 

分 类 号：R394.2[医药卫生—医学遗传学]