转化生长因子β3诱导外周血源性间充质干细胞成软骨分化的量效关系  

Chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells induced by transforming growth factor beta 3:a dose-effect relationship

作　　者：杨腾云 李彦林[1] 刘德健 王国梁[1] 郑竹君 Yang Tengyun;Li Yanlin;Liu Dejian;Wang Guoliang;Zheng Zhujun(Department of Sports Medicine,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan Province,China;Department of Rehabilitation,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan Province,China)

机构地区：[1]昆明医科大学第一附属医院运动医学科,云南省昆明市650032 [2]昆明医科大学第一附属医院康复科,云南省昆明市650032

出　　处：《中国组织工程研究》2022年第1期45-51,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81960409,81760403),项目负责人:李彦林;云南省专家工作站项目(2018IC102),项目负责人:李彦林;云南省骨关节疾病临床医学中心(ZX2019-03-04),项目负责人:李彦林。

摘　　要：背景:随着软骨组织工程技术的发展,国内外已有应用外周血源性间充质干细胞修复软骨缺损的体外培养及动物实验,并取得了不错的进展,转化生长因子β3常被用于细胞体外培养以诱导间充质干细胞成软骨分化。但在实验应用中,很少涉及转化生长因子β3量效探讨,涉及量效的细胞培养实验又经常误用单因素方差分析或t检验来分析这类资料。目的:采用重复测量方法分析转化生长因子β3诱导外周血源性间充质干细胞成软骨分化的量效关系,以期获得较佳的转化生长因子β3用量,获得较好的成软骨分化效果。方法:动员、提取、培养滇南小耳猪外周血源性间充质干细胞,分别加入含不同质量浓度转化生长因子β3(25-500μg/L)的成软骨诱导液进行成软骨诱导分化,对照组不作诱导处理,培养第2,4,6,8,10,12,14天,CCK-8法检测各组细胞增殖情况;培养第3,7,14,21天,收集各组细胞培养上清,ELISA法检测Ⅱ型胶原水平;培养第21天,进行甲苯胺蓝染色观察聚集蛋白聚糖表达;培养第21天,进行免疫细胞化学染色观察Ⅱ型胶原表达。结果与结论:在25-500μg/L范围内,转化生长因子β3质量浓度在40μg/L时外周血源性间充质干细胞生长增殖能力最强,转化生长因子β3质量浓度在40,80μg/L时更容易促进外周血源性间充质干细胞成软骨分化。BACKGROUND:With the development of cartilage tissue engineering technology,there have been in vitro culture and animal experiments on repairing cartilage defects with peripheral blood-derived mesenchymal stem cells,and good progress has been made.Transforming growth factor beta 3 is often used in cell culture in vitro to induce chondrogenic differentiation of mesenchymal stem cells.However,in the experimental application,the dose-effect study of transforming growth factor beta 3 is rarely involved,and the cell culture experiments involving dose-effect often misuse one-way analysis of variance or t-test to analyze this kind of data.OBJECTIVE:To analyze the dose-effect relationship of chondrogenic differentiation potency of peripheral blood-derived mesenchymal stem cells induced by transforming growth factor beta 3 by repeated measurement,in order to obtain a better dosage of transforming growth factor beta 3,and obtain a better effect of chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells.METHODS:After mobilization,extraction and culture of peripheral blood-derived mesenchymal stem cells of Diannan small eared pigs,transforming growth factor beta 3 at different concentrations(25-500μg/L)was added for chondrogenic differentiation or not added(control group).Cell proliferation in each group was detected by CCK8 assay at 2,4,6,8,10,12 and 14 days.At 3,7,14 and 21 days of culture,the supernatant of each group was collected,and the level of type II collagen in the supernatant was detected by ELISA.At 21 day of culture,toluidine blue staining was used to observe the expression of the aggregated proteoglycan.At 21 days,immunocytochemical staining was performed to observe the expression of type II collagen.RESULTS AND CONCLUSION:In the range of 25-500μg/L,the proliferation ability of peripheral blood-derived mesenchymal stem cells was the strongest at the concentration of transforming growth factor beta 3 at 40μg/L.The concentration of transforming growth factor beta 3 was more likely to pro

关 键 词：干细胞 外周血源性间充质干细胞 细胞因子 转化生长因子Β3 重复测量 软骨组织工程 Ⅱ型胶原 聚集蛋白聚糖 软骨修复 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]