兔骨髓间充质干细胞体外诱导分化为胰岛细胞的动态变化  

Dynamic Change of Lepus brachyurus Bone Marrow Mesenchymal Stem Cells(BMSCs) Induced and Differentiated into Islet Cells In vitro

作　　者：陈晓佩[1] 于文浩[1] 王亚丹[1] 刘远哲[1] 张宁[1] 王新庄[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《农业生物技术学报》2015年第8期1058-1066,共9页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2008AA101010);河南省基础与前沿技术研究计划基金(No.092300410081)

摘　　要：本研究旨在探索生长因子三阶段诱导法体外诱导日本大耳白兔(Lepus brachyurus)骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)定向分化为功能性胰岛β细胞的可能性,并观察其动态变化。采用红细胞裂解法分离纯化兔BMSCs,免疫细胞化学法鉴定表面抗原CD90、CD34、CD44的表达;采用生长因子三步法,将兔BMSCs体外诱导为胰岛细胞,倒置显微镜下观察其形态学变化;双硫腙染色鉴定细胞团;RT-PCR检测胰岛细胞相关基因(Pdx-1、Insulin、Nkx6.1)的表达;免疫细胞化学法检测特异性蛋白的表达;酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测细胞分泌胰岛素的情况。结果显示,分离纯化获得的BMSCs呈旋涡状或放射状生长,细胞形态大小均一,CD90、CD44抗原阳性表达,CD34抗原阴性表达。第一阶段诱导末(3 d),细胞形态无明显变化,部分细胞变圆,双硫腙染色不着色,未检测到任何特异性基因的表达;诱导7 d时,细胞逐渐融合形成团簇状,双硫腙染色细胞团呈棕红色,仅检测到Pdx-1基因的表达,证实其为胰岛前体细胞;第二阶段诱导末(11 d),细胞簇数目增多,细胞边缘突起,检测到Pdx-1、Insulin的表达;第三阶段诱导末(19 d),细胞团状物与周围开始分离,形成团簇状胰岛样结构,双硫腙染色细胞呈红棕色,可检测到Pdx-1、Insulin、Nkx6.1均表达;免疫细胞化学法检测诱导后细胞内胰岛素蛋白的表达情况,实验组细胞胞浆内出现大量棕黄色颗粒,结果呈阳性反应,对照组细胞胞浆内未出现棕黄色颗粒,结果呈阴性反应;ELISA检测诱导后培养上清液中胰岛素含量结果显示,3d时诱导组与对照组相比差异不显著(P>0.05),7、11和19 d差异均显著(P<0.05),表明分离纯化的兔骨髓间充质干细胞在体外可诱导分化为成熟的具有功能性的胰岛细胞。本研究为糖尿病患者实现自体骨髓间充质干细胞移植治疗糖尿病提供了基础资料。The aim of this study was to explore the feasibility of rabbit bone marrow mesenchymal stem cells （BMSCs） into islet cells induced by growth factors in three phase induction method in vitro, and observe their dynamic change. BMSCs were obtained by red blood cell lysis, the CD90, CD44 and CD34 antigen expression were detected by immunocytochemistry technique. Growth factors three-step strategy was adopted to induce BMSCs to differentiate toward islet cells. The growth of BMSCs were observed under inverted microscope. The differentiated cells were stained with dithizone （DTZ）. The express of inslet cells relatedgene（Pdx-l, Insulin, Nkx6.1） was detected by RT-PCR, and the expression of specific protein like Insulin in derived cells was investigated by immunocytochemistry technique. Insulin secretion was assayed by enzyme linked immunosorbent assay （ELISA）. The result showed that the isolated BMSCs were shuttle or gathered into a vortex-like uniform growth, and positive for CD90, CD44 antigen, negative for CD34. At the end of the first- step strategy （3 d）, the cell morphology had no obvious change, and part of cells become round, DTZ staining negative, any specific gene mRNA expression was detected. After inducted 7 d, BMSCs gradually merged to form cluster, DTZ staining positive, only Pdx-1 mRNA expression was observed, which was verified as insulin progenitor cells. At the end of second-step strategy （11 d）, the number of differentiated cells cluster was incteased, RT-PCR showed that Pdx-1 and Insulin were expressed. After inducted 19 d, cell clusters looked like islet-like structure in terminal, DTZ staining positive and detected Pdx-1, Insulin and Nkx6.1 expression. Immunocytocheemistry analysis showed expression of Pdx-1 in the islet-like clusters. A lot of dark brown particles appeared in the cytoplasm of cells in the experimental group, the results showed positive reaction. The control group did not appear brown particles in the cytoplasm of cells, the results showed negative reaction.

关 键 词：兔 骨髓间充质干细胞(BMSCs) 分化 生长因子 胰岛细胞 

分 类 号：S829.1[农业科学—畜牧学]