Pdx-1、Ngn3联合MafA诱导大鼠骨髓间充质干细胞分化为胰岛样细胞

Differentiation of bone marrow mesenchymal stem cells into insulin-producing cells via transfection of Pdx-1,Ngn3, and MafA

目的探讨胰十二指肠同源盒1（pancreaticandduodenalhomeobox1，Pdx．1）、神经元素3（neurogenin3，Ngn3）联合V型肌腱膜纤维肉瘤癌基因同源基因A（v—marmuscu—loaponeuroticfibrosarcomaoncogenehomologA，MafA）诱导大鼠骨髓间充质干细胞（bonemarrowmesenchymalstemcells，BMSC）分化为胰岛样细胞（insulin．producingcells，IPC）的机制。方法通过贴壁培养法纯化BMSC，分为4组（A：未感染组；B：MafA感染组；C：Pdx-1-Nsn3感染组；D：联合感染组），高糖条件下培养7d，荧光显微镜观察其感染效率。Western印迹检测各组细胞Pdx．1、NgIl3和MafA表达情况，RT-PCR检测各组胰岛素2、葡萄糖激酶、巢蛋白、胰升糖素及Pdx．1表达水平，ELISA法检测各组胰岛素分泌情况。结果分离的细胞表面高表达CD44、CDl05，而低表达CD34。诱导过程中各感染组BMSC逐渐聚集并形成细胞团块，双硫棕染色呈红棕色；各组在感染第0、3、5、7、9天胰岛素分泌水平逐渐提高，且联合感染组升高最为明显（P〈0．05）；与A组相比，B、C和D组Pdx-1、胰岛素2、胰升糖素及葡萄糖激酶基因表达明显上调（P〈O．05）；与B组相比，C组Pdx-1表达上调，D组Pdx-1和胰岛素2表达上调，胰升糖素表达下调（P〈0．05）；与C组相比，D组胰升糖素表达上调（P〈0．05）。结论Pdx-1、Ngn3联合MafA共同感染BMSC可分化为IPC。

Objective To investigate the effect of pancreatic and duodenal homeoboxl （ Pdx-1 ）, neurogenin 3 （ Ngn3 ） , and v-maf muscu-loaponeurotic fibrosarcoma oncogene homolog A （MafA） on the differentiation of bone marrow mesenchymal stem cells（BMSC） into insulin-producing cells（IPCs）. Methods BMSCs were separated and purified by attachment culture method in vitro, and then they were divided into 4 groups （ A ： un-infection group ; B ： MafA infection group ; C ： Pdx-1-Ngn3 infection group ; D ： co-infection group）. BMSCs were cultured for 7 days under high glucose conditions. The infection efficiency was observed through fluorescence microscope. The protein expression of Pdx-1, Ngn3, and MafA were detected by Western blot. mRNA expression of Insulin, Pdx-1, glucokinase （GK） , nestin, and glucagon were detected by RT- PCR. The secretion of insulin were measured by ELISA. Results The induced effect was pronounced after infected for 7 days. The morphology of most ceils appeared as grape-like aggregation and clustered islet-like cells by the end of induction, and they were confirmed by dithizone （DTZ） staining. The insulin secretion levels of each infection group were gradually improved before and after infected 3rd, 5th, 7th, and 9th day, and in D group, the increment was most significant（ P〈0.05 ）. mRNA expression levels of insulin, Pdx-1, GK, and glucagon in the infection groups were significantly higher than A group （P 〈 0.05 ）. Conclusions BMSCs are able to be induced into IPCs by infection of Pdx-1, Ngn3, and MafA.