TGF-β3、BMP-2及地塞米松诱导兔滑膜MSCs成软骨分化的研究  

EFFECT OF TRANSFORMING GROWTH FACTOR β_3,BONE MORPHOGENETIC PROTEIN 2,AND DEXAMETHASONE ON CHONDROGENIC DIFFERENTIATION OF RABBIT SYNOVIAL MESENCHYMAL STEM CELLS

作　　者：陈松[1] 符培亮[1] 丛锐军[1] 吴海山[1] 许震宇[2] 

机构地区：[1]上海长征医院关节外科上海,200003 [2]第二军医大学组织胚胎教研室

出　　处：《中国修复重建外科杂志》2014年第1期92-99,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金青年科学基金资助项目(81000798)~~

摘　　要：目的探讨TGF-β3、BMP-2及地塞米松(dexamethasone,DEX)诱导兔滑膜MSCs(synovial MSCs,SMSCs)成软骨分化的作用。方法取5只10~16月龄新西兰大白兔(体重1.8~2.5 kg)膝关节滑膜分离培养SMSCs,并行形态学观察、流式细胞仪检测细胞表面抗原及成脂、成骨诱导分化鉴定。采用PELLET培养系统,将聚丙烯管中的SMSCs团块分成8组,分别加入不同组合的细胞因子进行成软骨诱导培养。A组TGF-β3,B组BMP-2,C组DEX,D组TGF-β3+BMP-2,E组TGF-β3+DEX,F组BMP-2+DEX,G组TGF-β3+BMP-2+DEX,H组为对照组。通过比较各组软骨微球的直径、重量,蛋白聚糖(甲苯胺蓝染色)、Ⅱ型胶原(免疫组织化学染色)表达,以及软骨标志基因表达水平[实时定量RT-PCR(real time quantitative PCR,RT-qPCR)]来评价各组细胞因子诱导SMSCs成软骨能力大小,确定最佳成软骨诱导细胞因子组合;并通过检测各组软骨微球DNA含量来评价软骨微球重量增加与细胞增殖的关系。结果经鉴定,成功从新西兰大白兔膝关节处滑膜分离获得SMSCs。A^F组诱导产生的软骨微球直径较小、重量较轻,Ⅱ型胶原及蛋白聚糖合成量亦较低;而G组诱导产生的软骨微球直径最大,重量最重,蛋白聚糖及Ⅱ型胶原合成量亦最多,均显著高于A^F组(P<0.05);RT-qPCR结果显示,G组软骨相关基因(SOX-9、聚集蛋白聚糖、Ⅱ型胶原、Ⅹ型胶原、BMP受体Ⅱ)的相对表达量显著高于其余各组(P<0.01)。各组软骨微球DNA含量随时间延长不断降低(7 d降低约70%,14 d降低约80%,21 d降低约88%)。结论 SMSCs在TGF-β3、BMP-2及DEX三者联合诱导条件下成软骨分化能力最强;软骨微球重量的增加由细胞外基质合成增多导致,而不是由细胞增殖引起。Objective To study the effect of transforming growth factor β3 （TGF-β3）, bone morphogenetic protein 2 （BMP-2）, and dexamethasone （DEX） on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells （SMSCs）. Methods SMSCs were isolated from the knee joints of 5 rabbits （weighing, 1.8-2.5 kg）, and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups： TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3＋BMP-2 in group C, TGF-β3＋DEX in group E, BMP- 2＋ DEX in group F, and TGF-β3＋BMP-2＋DEX in group G; group H served as control group. The diameter, weight, collagen type II （immuohistochemistry staining）, proteoglycan （toluidine blue staining）, and expression of cartilage related genes [real time quantitative PCR （RT-qPCR） technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. Results SMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G （P 〈 0.05）. RT-qPCR detection results showed that the relative expressions of cartilage related genes （SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II） in group G were significantly higher than those in the other groups （P 〈 0.01）. Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 2

关 键 词：TGF-Β3 BMP-2 地塞米松 滑膜MSCs 成软骨分化 兔 

分 类 号：R965[医药卫生—药理学]