STAT-3基因腺病毒介导感染ARIP、AR42J细胞的表达

Expression of STAT-3 gene by adenovirus assisted in ARIP、AR42J cells

目的:探测4种腺病毒液在胰腺源ARIP、AR42J细胞中的表达,为基因改造干预干细胞分化治疗糖尿病的进一步研究提供基础。方法:①扩增、纯化STAT-3 WT、STAT-3 DN、STAT-3 CA、STAT-3 GF 4种质粒;脂质体转染法转染质粒进入人胚肾293腺病毒包装细胞系,Hela细胞作为包装细胞的对照细胞。②生产含4种基因组合的腺病毒,腺病毒检测、鉴定。③常规与盖玻片法ARIP、AR42J等细胞培养;含目的基因的腺病毒液感染目标细胞。④荧光显微镜观察感染后细胞的基因表达。结果:STAT-3腺病毒基因载体生产了较高滴度(病毒滴度为6 X106pfu/ml)的腺病毒,用含目的基因的腺病毒对靶细胞感染3 d后,荧光显微镜观察到ARIP等细胞中绿色荧光蛋白成功、高效的阳性表达报告。结论:腺病毒介导STAT-3 WT、STAT-3 DN、STAT-3 CA、STAT-3 GF进入ARIP细胞的良好表达为STAT-3基因改造病毒感染干预干细胞分化的进一步相关研究提供了重要依据。

Objective：To explore the STAT-3 wild type （STAT-3 WT）, STAT-3 dominant negative （STAT-3 DN）, STAT-3 constant act （STAT-3 CA） and STAT-3 green fluorescence （STAT-3 GF） expression by adenovirus assisted in ARIP. AR42J cells and provide molecular basis for the mechanism of the gene modified to intervene stem cell differentiation to treat diabetes. Methods：STAT-3 WT, STAT-3 DN, STAT-3 CA and STAT-3 GF plasmids were purified; the plasmids were transfeeted into QBI-293A package cell line by lipofeetamine. Hela cell was the control. The four adenovirus included STAT-3 gene were produced, and checked the titer; ARIP, AR42J ete cells were cultured with general and cover glass methods, and target cells were infected by adenovirus assisted. Expression of the report gene was observed by the fluorescence-microscope after infection. Results：There were strong positive reports of the green fluorescence in ARIP cells after infected by adenovirus assisted STAT-3 genes. Enough four adenovirus suspension was harvested. The titers of adenovirus were 6 × 10^6pfu/ml. Conclusion：The positive expression of STAT-3 Green fluorescences in ARIP cell provides a vital data for the further research of the STAT-3 gene modified to intervened the stem cell differentiation to treat diabetes related subject.