pIRES-GFP-前脑啡肽原载体在体内外的表达及生物学效应

目的检测pIRES-GFP-前脑啡肽原载体在体内外的表达,以探索脑啡肽基因镇痛的生物学效应与临床应用的可行性。方法将大鼠前脑啡肽原基因与绿色荧光蛋白真核质粒连接构建重组质粒,体外转染机体细胞,通过阳性细胞绿色荧光表达的强弱与细胞上清液脑啡肽水平的放射免疫测定,观察重组质粒在细胞内的合成与表达;大鼠蛛网膜下腔注射重组质粒,观察大鼠热痛阈值的变化,确定重组质粒在机体细胞内合成外源性脑啡肽的生物学活性。结果体外试验表明重组质粒转染24 h后COS-7细胞内开始有绿色荧光表达,48 h达高峰,持续27 d开始减弱,动物实验大鼠热痛阈值的变化与体外试验基本相符。结论重组质粒在机体细胞内合成的外源性脑啡肽具有与内源性脑啡肽相同的生物学活性,可以作为一种长效的基因镇痛方法应用于临床。

Biological effect and expression of pIRES-GFP-preproenkephalin vector in vivo and vitro

Objective To detect the expression of pIRES-GFP-preproenkephalin(PENK) vector in vivo and vitro in order to research the biological effect and the feasibility of clinical application of enkephalin gene.Methods Recombinant plasmid was constructed by connecting green fluorescence protein eukaryotic plasmid with the prepro-enkephalin gene of rats,and then the organism cells were transfected in vitro.The synthesis and expression of recombinant plasmid were observed by the expression level of the green fluorescence in positive cells and the radioimmunoassay of the content of enkephalin in the cell supernatants.The change of the rats′ heat and pain threshold was observed by injecting recombinant plasmid of enkephalin in subarachnoid space of the rats to determine its biological activity of synthetic exogenous enkephalin in the body cells.Results The experiment in vitro showed green fluorescence of COS-7 cell expressed 24 hours after recombinant plasmidit transfection,reached peak in 48 hours and weakened in 27 days.The change of heat and pain threshold of the rats in animal experiment matched the effect of the vitro study.Conclusion The exogenous enkephalin,which is synthesized by recombinant plasmid in organism cells,possesses identical biological effect of endogenous enkephalin and can be used as a gene analgesic method of long-term in clinical application.