犬Mnx1基因过表达载体的构建及其对脂肪间充质干细胞向胰岛β细胞分化的影响

本研究旨在构建犬Mnx1基因腺病毒过表达载体,并探究Mnx1基因过表达对犬脂肪间充质干细胞向胰岛β细胞方向分化的影响。本试验构建了腺病毒重组表达载体pAD-Mnx1,经293A细胞包装扩增后产生具有感染性的重组腺病毒,将其按感染复数(MOI)100感染犬ADSCs后,分别在第3、6、9、12和15天观察绿色荧光蛋白的表达情况、细胞形态变化和胰岛β细胞发育相关基因的表达情况。结果显示,ADSCs在感染后24 h出现绿色荧光,形态无显著变化;Mnx1基因表达可激发胰十二指肠同源盒1(Pdx1)基因、神经元素3(Ngn3)基因、肌腱膜纤维肉瘤癌基因同源物A(Mafa)基因、NK2同源盒2(Nkx2.2)基因、NK6同源盒1(Nkx6.1)基因、神经分化因子1(Neurod1)基因、成对合基因4(Pax4)和GATA结合蛋白4(Gata4)基因等内源性基因的表达。结果证实,腺病毒介导的过表达载体pAD-Mnx1可在犬ADSCs超表达Mnx1基因,从而诱导犬ADSCs向胰岛前体细胞分化,具有进一步诱导犬ADSCs形成胰岛β细胞的可能。

Construction of canineMnx1 gene overexpression vector and its effect on differentiation of adipose-derived mesenchymal stem cells into islet beta cells

The aim of this study was to construct a canine Mnx1 gene adenovirus overexpression vector and explore the effect of Mnx1 gene overexpression on the differentiation of canine adipose-derived mesenchymal stem cells into insulin-secreting cells.This experiment constructed an adenovirus recombinant expression vector pAD-Mnx1,which was packaged and amplified by 293A cells to produce an infectious recombinant adenovirus.After infection of canine ADSCs by multiplicity of infection(MOI)100,the expression of green fluorescent protein and cell morphology and the expression of islet beta cell development-related geneswere observed on the 3rd,6th,9th,12th and 15th day respectively.The results showed that ADSCs showed green fluorescence at 24 h after infection,and the morphology did not change significantly.Mnx1 gene expression could stimulate expression of endogenous genes such as Pancreatic duodenal homeobox 1(Pdx1),Neuronal element3(Ngn3),Myofascial fibrosarcoma oncogene homolog A(Mafa),NK2 homeobox 2(Nkx2.2),NK6 homeobox 1(Nkx6.1),Neural differentiation 1(Neurod1),Paired gene 4(Pax4)and GATA binding protein 4(Gata4).The results confirmed that the adenovirus-mediated overexpression vector pAD-Mnx1 overexpresses the Mnx1 gene in canine ADSCs,thereby inducing the differentiation of canine ADSCs into islet precursor cells,which may further induce the formation of insulin-secreting cells in canine ADSCs.