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该研究旨在探索二甲基亚砜(dimethyl sulphoxide,DMSO)联合高糖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)分化为胰岛样细胞的可行性及其调控机制。采用不含血清的DMSO联合高糖诱导P3代兔BMSCs分化为胰岛样细胞。倒置显微镜下观察细胞的形态变化;双硫腙染色和免疫荧光染色检测细胞分化;RTq PCR检测胰岛细胞相关基因[Foxa2(forkhead box A2)、Nestin(neuroepithelial stem cell protein)、Pax6(paired box gene 6)、Pdx-1(pancreatic duodenal homeobox-1)、胰岛素]的表达,并以诱导培养前(0 d)细胞、兔骨髓细胞、P3代BMSCs和胰腺组织中胰岛细胞相关基因表达情况作为参照。结果表明,Foxa2、Nestin和Pax6基因不能作为兔BMSCs向胰岛样细胞诱导分化成功的标志基因。DMSO能够激活Pdx-1基因的表达,促进兔BMSCs分化为可分泌胰岛素的胰岛前体细胞。高糖能够促进兔BMSCs分化为胰岛样细胞,并可显著促进Pdx-1和Foxa2基因的表达。
This study was designed to investigate the effects of dimethyl sulphoxide (DMSO) combined with high glucose on the differentiation of Leukemia mesenchymal stem cells (BMSCs) of Lepus brachyurus into islet-like cells in vitro Feasibility and regulation mechanism. The P3 generation of rabbit BMSCs were induced to differentiate into islet-like cells by using serum-free DMSO combined with high glucose. Cell morphology was observed under inverted microscope. Dithizone staining and immunofluorescence staining were used to detect cell differentiation. RT-PCR was used to detect the expression of isoforms of Foxa2 (forkhead box A2), nestin (neuroepithelial stem cell protein), Pax6 (paired box gene 6 ), PDX-1 (pancreatic duodenal homeobox-1) and insulin]. The expression of pancreatic islet cell related genes in pre-culture (0 d), rabbit bone marrow, P3, BMSCs and pancreatic tissues was used as a reference. The results showed that Foxa2, Nestin and Pax6 genes could not be used as markers for the successful differentiation of BMSCs into pancreatic islet-like cells. DMSO can activate Pdx-1 gene expression and promote the differentiation of rabbit BMSCs into insulin-secreting islet precursor cells. High glucose can promote rabbit BMSCs differentiate into islet-like cells, and can significantly promote the expression of Pdx-1 and Foxa2 genes.