诱导型一氧化氮合酶基因转染兔牙周膜细胞的瞬时表达检测

目的检测含有人诱导型一氧化氮合酶（iNOS）基因的重组质粒pEGFP-iNOS体外转染兔牙周膜细胞后的瞬时表达情况,为动物牙周局部导入外源性iNOS基因奠定实验基础。方法通过脂质体介导将重组真核质粒pEGFPiNOS转染兔牙周膜细胞,在荧光显微镜下观察瞬时转染情况。并在转染后12 h与48 h,应用实时定量PCR检测iNOS基因的转录,Western免疫印迹技术检测iNOS蛋白表达。设空质粒转染组和空白组为对照。结果重组质粒转染后12 h即可观察到荧光蛋白的表达,pEGFP-iNOS转染组细胞中有iNOS mRNA转录及iNOS蛋白的表达。结论iNOS基因成功导入兔牙周膜细胞,瞬时转染后可在基因和蛋白水平检测到外源性基因的表达。在兔牙周组织导入iNOS基因从而诱导NO生成是具有可行性的。

Test of transient expression of inducible nitric oxide synthase gene transferred to rabbit periodontal ligament cells

Objective This article tests a recombinant plasmid pEGFP -iNOS encoding human inducible nitric oxide synthase （iNOS） transient expression in rabbit periodontal ligament cells, and this may contribute to transfer iNOS gene to animal periodontal tissue in vivo. Methods Rabbit periodontal ligament cells were transfected with pEGFP -iNOS by means of lipofectamine media methods. Transient transfection were evaluated by fluorescent microscope. After transferring 12 h and 48 h, the transcription of iNOS gene was tested by quantitative real-time PCR and the expression product of pEGFP-iNOS was identified by Western blot. Blank plasmid-transfected group and non-transfected group were used as control. Results The expression of green fluorescence protein was detected in 12 h after transferring. The transcription of iNOS mRNA and expression of iNOS protein in cells transfected with pEGFP -iNOS were detected by real -time PCR and Western blot. Conclusion The iNOS gene was transfected successfully, the exogenetic iNOS mRNA can be correctly transcribed and expressed in rabbit periodontal ligament cells. In vivo gene transfer of iNOS to rabbit periodontal tissue is feasible.