骨髓间充质干细胞培养并向骨骼肌诱导分化的实验研究  

Experimental study of the rat mesenchymal stem cells＇ culture and their differentiation into muscle skeletal cells

作　　者：孙占胜[1] 杨志明[2] 陈振强[1] 王伯珉[1] 张云峰 冯震[1] 穆卫东[1] 李连新[1] 王永会[1] 邓力[2] 王珍[2] 智伟[2] 王杨[2] 

机构地区：[1]山东省立医院创伤骨科,济南250021 [2]四川大学附属华西医院干细胞与组织工程研究室 [3]不详

出　　处：《中华创伤杂志》2007年第8期609-613,共5页Chinese Journal of Trauma

基　　金：山东省重点自然科学基金资助项目（Y2003C35）

摘　　要：目的 研究大鼠骨髓间充质干细胞（BMSCs）的定向分化及其在体内的形态发育情况。方法 Wistar大鼠24只，向其左胫前肌处注射0．2ml无水乙醇，致其无菌坏死，右胫前肌注射等渗盐水0．2ml作为对照，制成动物模型后备用；取大鼠第3代BMSCs，5-氮杂胞苷、成肌调节因子（MyoD）、转化生长因子-β1（TGF-β1）、胰岛素样生长因子-1（IGF-1）联合进行诱导分化。诱导后第9天，收集BMSCs，用BrdU标记并诱导后注射到大鼠双下肢胫前肌处，分别在3，6，9和12d取材进行形态观察和免疫组织化学检查。结果 注射诱导后3d，实验组的成活率明显高于注射单纯注射BMSCs的对照组，BrdU标记细胞向骨骼肌分化，结蛋白、骨骼肌特异肌球蛋白均为弱阳性表达，6，9d可见BrdU标记细胞向骨骼肌分化，结蛋白、骨骼肌特异肌球蛋白阳性表达，第9天更为明显，注射后12d后可见新生骨骼肌细胞形成肌小管，细胞核结蛋白、骨骼肌特异肌球蛋白强阳性表达。结论 大鼠损伤肌肉的微环境中，BMSCs可向骨骼肌定向分化，并伴有结蛋白和骨骼肌肌球蛋白阳性表达；定向诱导后BMSCs在体内成活率高，增殖速率快，向骨骼肌分化纯度更高。Objective To study oriental differentiation and morphological development of the rat mesenchymal stem cells （ BMSCs ）. Methods The animal models were made by injecting dehydrated alcohol 0.2 ml into the middle of the anterior tibial muscle of the left leg and sterilitas normal saline 0.2 ml into the same site of right leg in 24 Wistar rats. The BMSCs were induced and differentiated by affiliation of 5-azacytidine, MyoD, TGF-β1, IGF-1. Nine days after induction, were collected and signed by BrdU and injected into the anterior tibial muscle. Four hours after sign, BMSCs were injected into both legs of the models and the materials drawn from the models at days 3, 6, 9 and 12 for morphous observation and immunohistochemistry examination. Results Three days after injection, the survival rate of the induced cells were more than the BMSCs in vivo; the BrdU signed cells differentiated into skeletal muscle cells, desmine, myosin （skeletal） expressed weakly positively. At days 6 and 9, the BrdU signed cells differentiated into skeletal muscle cells, and desmine and myosin （skeletal） expressed positively, with statistical significance （P 〈0.05）. There could be found the muscle skeletal cells making up sarcotubule in vivo 12 days after injection, with strongly positive expressions of desmine and myosin （skeletal）. Conclusions In the microenviroment of the rat muscle injury, BMSCs can be induced and differentiated oreitally into muscle skeletal cells, accompanied by positive expressions of esmine and myosin （skeletal）. The induced cells have high survival rate, fast proliferation and high purity of differentiation.

关 键 词：骨髓间充质干细胞 氮丙啶类 细胞 骨骼肌 细胞分化 

分 类 号：R686[医药卫生—骨科学]