SOX9腺病毒过表达载体的构建及其对犬ADSCs向胰岛样细胞分化的影响

Construction of SOX9 Adenovirus Overexpression Vector and its Effect on the Differentiation of Canine ADSCs to Islet-like Cells

脂肪间充质干细胞(ADSCs)具有多向分化潜能,是治疗自身免疫损伤性糖尿病的理想种子细胞。SOX9是胰腺发育调控网络中的多潜能因子,其功能缺失可以导致胰腺发育不全和祖细胞池枯竭。但到目前为止,SOX9在ADSCs向胰岛样细胞分化过程中是否具有重要作用尚不完全清楚。本试验构建了腺病毒过表达重组载体pAdTrack-SOX9-AdEasy,并获得了具有感染性的腺病毒毒液,将其感染犬ADSCs,感染后4、7、10、13 d和16 d观察绿色荧光蛋白表达情况、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,过表达SOX 9基因的腺病毒毒液感染犬ADSCs后,出现绿色荧光蛋白表达;并在第13天细胞开始出现聚集生长现象,在第16天出现多个似胰岛样细胞团;同时检测到SOX9可外源性刺激Pdx1、Ngn3、Mnx1、MafA、Nkx2.2、Nkx6.1、PCSK 1和PCSK 2等基因的内源性表达。表明SOX 9基因过表达腺病毒毒液可成功感染犬ADSCs,促进其向胰岛样细胞分化。

Adipose-derived mesenchymal stem cells(ADSCs)have multi-directional differentiation potential and are the ideal seed cells for the treatment of autoimmune damaging diabetes.SOX9 is a multipotential factor in the regulatory network of pancreatic developmental processes,and it’s dysfunction can lead to pancreatic hypoplasia and depletion of progenitor cells.But so far,it is not yet clear whether SOX9 plays an important role in the differentiation of ADSCs into islet like cells.Therefore,in this study we constructed adenovirus overexpression recombinant vector pAdTrack-SOX9-AdEasy,and then used the obtained infectious adenovirus vector to infect canine ADSCs.The expression of green fluorescent protein,cell morphology changes and expression of isletβcell development related genes were studied after 4,7,10,13 days and 16 days post infection.The results showed that SOX 9 was successfully expressed in canine ADSCs identified by green fluorescent protein expression.Cells began to show aggregate growth on the 13th day,and multiple cell clusters appeared on the 16th day.Our results also showed that SOX9 exogenously stimulated the endogenous expression of genes such as Pdx1,Ngn3,Mnx1,MafA,Nkx2.2,Nkx6.1,PCSK1 and PCSK 2.This indicates that SOX 9 gene overexpression adenovirus vector could successfully infect canine ADSCs and promote their differentiation into islet like cells.