外源性碱性成纤维细胞生长因子对大鼠肌源性干细胞增殖的影响  

Effects of exogenous basic fibroblast growth factor on proliferation of rat muscle-derived stem cells

作　　者：吕丹[1] 姜殿君[1] 张庭深[1] 

机构地区：[1]沈阳医学院基础医学教研室,辽宁省沈阳市110034

出　　处：《中国组织工程研究与临床康复》2009年第27期5271-5275,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：辽宁省教委资助课题(2005L437)~~

摘　　要：背景：肌源性干细胞在体外培养过程中存在纯化、扩增、定向分化等难题,碱性成纤维细胞生长因子作为生物活性因子的一员,因其生物活性的多效性而备受关注。目的：探讨碱性成纤维细胞生长因子对大鼠肌源性干细胞体外增殖的影响。设计、时间及地点：细胞学体外观察,于2008-04/07在沈阳医学院完成。材料：健康雄性Wister大鼠5只,由沈阳医学院实验动物中心提供。碱性成纤维细胞生长因子为珠海生物制品有限公司产品。方法：无菌条件下取大鼠左前肢肱三头肌,采用酶消化法分离肌源性干细胞,应用密度梯度离心法和差速贴壁法进行纯化。取第2代肌源性干细胞,分别用终浓度为6.25,12.50,25.00,50.00,100.00μg/L碱性成纤维细胞生长因子进行干预;以单纯加入200μL生长培养基作为空白对照组,以加入200μL生长培养基＋肌源性干细胞作为阴性对照组。分别于培养24,48,72,96h加入MTT溶液。主要观察指标：免疫细胞化学法鉴定大鼠肌源性干细胞,MTT比色法检测细胞增殖情况。结果：肌源性干细胞多呈Sca-1阳性反应,少数呈Desmin阳性反应。①不同浓度碱性成纤维细胞生长因子对肌源性干细胞的促增殖效应：与阴性对照组比较,各浓度碱性成纤维细胞生长因子组对肌源性干细胞均有明显的促增殖效应（P〈0.05）;浓度为6.25~50.00μg/L时促增殖作用随其质量浓度升高而显著增强（P〈0.01）,至50.00μg/L时其促增殖作用接近顶峰。②碱性成纤维细胞生长因子在不同培养时间点对肌源性干细胞的促增殖效应：随培养时间的延长肌源性干细胞明显增殖,与阴性对照组比较,碱性成纤维细胞生长因子组出现显著促增殖效应（P〈0.01）的时间为培养96h。结论：碱性成纤维细胞生长因子可促进体外培养的大鼠肌源性干细胞增殖,且呈浓度-时间依赖性增加,50.00μg/L与96h为较佳的体外培养质�BACKGROUND： Muscle-derived stem cells have some problems in purification, amplification and directional differentiation during in vitro culture. Basic fibroblast growth factor as a membrane of bioactive factor has bean paid great attention, due to its biological activity. OBJECTIVE： To explore effects of basic fibroblast growth factor on in vitro proliferation of rat muscle-derived stem cells. DESIGN, TIME AND SETTING： The cytological in vitro study was performed at the Shenyang Medical College from April to July 2008. MATERIALS： Five healthy male Wistar rats were supplied by Experimental Animal Center, Shenyang Medical College. Basic fibroblast growth factor was produced by Zhuhai Biological Product Co., Ltd. METHODS： Left anterior limb triceps brachii was sterilely obtained from rats. Muscle-derived stem cells were harvested by isolation with enzyme digestion, purified by density gradient centrifugation and differential attachment. At the second passage, muscle-derived stem cells were treated with basic fibroblast growth factor at final concentration of 6.25, 12.50, 25.00, 50.00, 100.00 μg/L. Cells received 200 μL growth medium as blank control group, and those treated with 200 μL growth medium ＋ muscle-derived stem cells as negative controls. MTT was added at 24, 48, 72 and 96 hours of culture. MAIN OUTCOME MEASURES： Immunocytochemical method was used to determine rat muscle-derived stem cells. MTT assay was utilized to detect cell proliferation. RESULTS： Muscle-derived stem cells were mostly positive for Sca-1, but few was positive for Desmin. Compared with the negative control group, various concentrations of basic fibroblast growth factor had significant proliferation effects on muscle-derived cells （P 〈 0.05）. At 6.25-50.00 μg/L, the proliferation effect became strong with the increased concentration （P 〈 0.01）. At 50.00μg/L, the proliferation promotion effect nearly reached a peak. Muscle-derived stem cells significantly proliferated over time. Compared with the negative c

关 键 词：肌源性干细胞 碱性成纤维细胞生长因子 浓度 培养时间 

分 类 号：R394.2[医药卫生—医学遗传学]