摘要
目的构建过表达大鼠阴茎神经源性一氧化氮合酶(PnNOS)基因大鼠脂肪源性干细胞(ADSCs),为基因修饰ADSCs移植治疗勃起功能障碍(ED)大鼠模型提供种子细胞。方法获取大鼠PnNOS基因,并与线性化的腺病毒真核表达载体pDC315-EGFP定向克隆连接。构建正确的pDC315-PnNOS-EGFP穿梭质粒转染293 T细胞,采用Western Blot检测PnNOS基因在293 T细胞中的表达情况。利用AdMax腺病毒包装系统包装产生重组腺病毒,包装后扩增纯化并测定病毒滴度。PnNOS基因重组腺病毒转染大鼠ADSCs,观察GFP表达情况和Western Blot检测PnNOS基因表达情况。结果经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-PnNOS-EGFP重组腺病毒载体构建成功。重组腺病毒包装成功且病毒滴度为5.0×109 PFU/ml。Western Blot于大鼠ADSCs中检测到约161 KDa大小条带,其与PnNOS蛋白分子量大小基本相一致。结论大鼠PnNOS基因修饰的ADSCs构建成功,从而为基因修饰ADSCs移植治疗ED提供了良好的基因工程细胞。
Objective To construct a recombinant adenovirus carrying rat PnNOS gene and transfeet into adipose-derived stem cells （ADSCs）, providing seed cells for the treatment of erectile dysfunction. Methods PnNOS gene was amplified fi＇om gene bank by PCR and connected with adenovirus linearized eukaryotic expression vector pDC315-EGFP to construct the shuttle plasmid pDC315-PnNOS-EGEP. HEK 293 cells were transfected with recombinant shuttle plasmid pDC315-PnNOS-EGFP and the expression of PnNOS gene was detected by Western Blot at 48 hours. The obtained plasmid pDC315-PnNOS-EGFP was packaged by AdMax system, and the recombinant adenovirus was purified and determined for virus titer. The recombinant adenovirus was transfected into ADSCs and the expression of PnNOS was detected by fluorescence microscope and Western Blot. R^sults pDC315-PnNOS-EGFP was confirmed by PCR analysis, restriction enzymes analysis, sequencing identification and Western Blot, and was successfully constructed and packed, with titer of 5.0×109 PFU/ml. A band of 161 KDa was detected by Western Blot, which matched with the rat PnNOS protein. Conclusion The ADSCs over-expressing rat PnNOS gene have been successfully constructed, providing the preeminent genetic engineering cells for erectile dysfunction therapy.
出处
《中华腔镜泌尿外科杂志（电子版）》
2014年第4期52-56,共5页
Chinese Journal of Endourology(Electronic Edition)
基金
国家自然科学基金资助项目(81070487和81370705)
国家自然科学基金项目(81370702)
中国博士后科学基金资助项目(2013M542191)
广东省自然科学基金资助项目(S2012010009140)
广东省科技计划项目基金资助项目(2010B031600038)
广东省医学科研基金资助项目(B2012114)
广东省科技计划项目(2011B0603000037)
