大鼠骨髓间充质干细胞培养、鉴定与成胰岛样β细胞诱导  

Cultivation,Identification and Islet β Cells Induction of Bone Marrow Mesenchymal Stem Cells in Rats

作　　者：王鹤霏[1] 任宇[1] 呼啸[1] 张飞旭 李晓聪[1] 仓明[1] 刘东军[1] 

机构地区：[1]内蒙古大学国家动物转基因技术研究中心,呼和浩特010070

出　　处：《中国细胞生物学学报》2016年第5期540-549,共10页Chinese Journal of Cell Biology

基　　金：转基因生物新品种培育重大专项(批准号:2014ZX08008-002)资助的课题~~

摘　　要：为治疗糖尿病寻找新的胰岛β细胞替代物,该研究对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)进行了体外分离培养、鉴定及成胰岛样β细胞诱导。应用全骨髓离心贴壁培养法分离大鼠BMSCs,进行培养、传代、表面标志物检测,利用DMSO和高糖环境对BMSCs进行胰岛样β细胞诱导。结果显示,大鼠BMSCs体外培养细胞形态呈成纤维细胞样,可以稳定传代;CD13、CD44和CD106表达呈阳性,CD49d呈阴性。在成胰诱导条件下,BMSCs可形成胰岛样圆形细胞团,双硫腙染色呈棕红色,PDX1(pancreatic duodenal homeodomain 1)、CK19(cytokeratin 19)、巢蛋白、胰岛素免疫组化染色均呈阳性;经RT-PCR检测发现,成胰诱导后的BMSCs中表达胰岛素、PDX1和glucagon基因;经q-PCR检测发现,成胰诱导后的BMSCs中胰岛素m RNA水平是大鼠胰腺成体干细胞(pancreas adult stem cells,PASCs)的1.09倍(P>0.05);同时,有相应量的胰岛素合成。结果表明,分离得到大鼠BMSCs体外生长稳定,能转分化为胰岛样细胞,该研究结果为糖尿病治疗提供了新的实验依据。To find new substitution of islet β cells for treating diabetes, rat bone marrow mesenchymal stem cells （BMSCs） were isolated, cultured, identified and induced into islet β cells in vitro. Isolation of rat BMSCs by the whole bone marrow centrifugation and adherence method, then BMSCs were cultured, subcultured, detected cell surface markers and induced into islet β cells by DMSO and high glucose environment. The results showed that the morphology of BMSCs was fibroblast like, with active passage ability. CD 13, CD44 and CD 106 antigens were detected, but CD49d antigen was negative. Under the condition of islet β cells induction, BMSCs could form islet- like round cell mass, dithizone staining was brownish red, PDX1, CK19, nestin and insulin antigens were positive using immunohistochemistry staining. BMSCs induction into islet β cells expressed insulin, PDX1 and glucagon detected by RT-PCR. The expression quantity of insulin mRNA in differentiated BMSCs was 1.09 times （P〉0.05） than that of in pancreas adult stem cells （PASCs） detected by q-PCR. At the same time, differentiated BMSCs had the corresponding expression of insulin protein. The results demonstrated that rat BMSCs grow stably in vitro, and can be converted into islet-like cells, providing new experimental basis for diabetes treatment.

关 键 词：骨髓间充质干细胞 体外分离培养 细胞鉴定 成胰岛β细胞分化 

分 类 号：R587.1[医药卫生—内分泌]