重构腺相关病毒转导PDX-1对C2C12分化为胰岛素分泌细胞的作用

Role of Recombinant AAV Vectors with PDX-1 in Transdifferentiation Myoblast C2C12 Cells into Insulin-secreting Cells

【目的】重构含有PDX-1的腺相关病毒载体,将PDX-1转导入C2C12细胞,观察胰岛素分泌量的改变。【方法】应用PCR方法将PDX-1从pZL1质粒中克隆出来,安装到Stratagene公司的腺相关病毒(AAVHelper-FreeSystem)表达系统中,转染HEK293细胞,培养72h,提取病毒悬液,转导PDX-1进入C2C12细胞中,检测细胞培养液中的胰岛素含量。【结果】①用X-gal染色系统检测,可见有蓝色的阳性细胞。②病毒的粗略的浓度经计算后大约为1012/mL,转导的病毒数约为1011。③PCR结果:用重构的腺相关病毒转导后C2C12有胰岛素及PDX-1的mRNA的表达。④用重构的腺相关病毒转导PDX-1进入C2C12后的培养液的胰岛素浓度为(5.5±1.1)μIU/mL,较其他方法也有较明显的增加(P<0.05)。【结论】通过应用含有PDX-1基因的重构腺相关病毒载体可以转导PDX-1在C2C12细胞中表达,且可以提高C2C12分化为胰岛素分泌细胞的分泌量。

[Objective] To transduce PDX-1 gene into C2C12 ceiLs with recombinant adeno-associated virus （AAV） vectors and observe the excretion change of insulin. [Methods ] The PDX-1 gene was cloned from pZL1 plasmids by RT-PCR. A recombinant adeno-assoeiated virus containing the eDNA encoding PDX-1 was prepared using AAV Helper-Free System. 72 h after transfeetion, cell lysate was obtained from the HEK293 cell and transformed into the C2C12 cell. Then we detected the insulin content of supernatant. [Results] ①There were some blue positive ceiLs by means of X-gal staining system after transferring into HEK293 cell. ②Viral titers were about 10^12/mL. The transduetion virus number was about 10^11. ③ PCR results： the mRNA of insulin and PDX-1 were expressed in the C2C12 cells after infecting the recombinant adeno-associated virus which containing PDX-1. ④ The insulin content of supernatant was 5.5±1.1 μIU/mL in the C2C12 ceiLs which were transfeeted with recombinant adeno-associated virus containing the PDX-1 cDNA. It was higher than the control （P〈 0.01） or the groups which were differentiated by other means （P〈 0.05）. [Conclusions]Recombinant AAV vector carrying PDX-1 can not only transduce PDX-1 gene into C2C12 ceiLs, but also increase insulin excretion of insulln-secreting cells differentiated from C2Cl2 cells.