人生长激素释放激素变异受体1型基因绿色荧光蛋白真核表达载体1的构建及体外表达

目的构建人生长激素释放激素变异受体1型（GHRHR-SVT1）基因绿色荧光蛋白真核表达载体1（pEG—FP—N1）并在鼠成纤维细胞株NIH-3T3细胞中的表达。方法GHRHR-SVT1基因是由人胃癌细胞株AGS中扩增出的DNA片段，克隆入真核表达载体pEGFP-N1中，用高效转染试剂将pEGFP-N1／GHRHR—SVT1转染NIH-3T3细胞，NIH-3T3细胞分3组：非转染组（对照组），只加转染试剂不加质粒；转染空质粒组（pEGFP-N1组），加转染试剂与空质粒；重组质粒转染组（pEGFP-N1／GHRHR-SVT1组），加转染试剂与重组质粒。采用四甲基偶氮唑盐（MTT）法和流式细胞仪检测各组转染后各组NIH-3T3细胞的生长情况。结果（1）pGEFP—N1／GHRHR-SVT1经限制性内切酶XhoⅠ和BarnHⅠ双酶切产生约1080bp和4700bp2条带。（2）构建的GHRHR-SVT1cDNA序列与GHRHR—SVT1原始序列（AF-282259）进行对比，第54位碱基突变（A突变为T），为无义突变。（3）重组质粒转染组NIH-3T3细胞增殖率明显高于对照组（P〈0．01）。（4）重组质粒转染组NIH-3T3细胞增殖指数明显高于对照组（P〈0．01）。结论在适当浓度GHRH（10μmol／L）的培养基中，GHRHR—SVT1能够促进NIH-3T3细胞增殖，并为肿瘤的治疗提供新的思路。

Construction of Eukaryotic Green Fluorescent Protein Expression Vector 1 of Human Growth Hormone Releasing Hormone Receptor Gene Splice Variant Type 1 and Its Expression in Vitro

Objective To construct eukaryotic green fluorescent protein expression vector 1 （pEGFP-N1） of human growth hormone releasing hormone receptor sp RHR-SVT1） gene and Its expression in rat fibroblast line NIH-3T3 cel ice variant s. Methods type 1 （GH- The target gene fragment which had been obtained from human gastric cancer cell AGS,was cloned into a eukaryotic expression vector pEGFP-N1. The vector pEGFP-N1/GHRHR-SVT1 was transfected into NIH-3T3 cell by VigoFect. The NIH-3T3 cells were then randomly divided into three groups： non-transfection group（control group） , empty plasmid transfection group（pEGFP-N1 group）, and recombinant plasmid transfection group （pEGFP-N1/GHRHR-SVT1 group）. For the control group,the cells were treated only plus with transfection reagent without plasmid; For pEGFP-N1 group,the cells were treated with plus transfection reagent and empty plasmid; and for pEGFP- N1/GHRHR-SVT1 group,the cells were treated with plus transfection reagent and the recombinant plasmid. The growth conditions of NIH-3T3 cells in each group was observed with methyl thiazolyl tetrazolium（MTT） and flow cytometry（FCM）. Results （1） Two bands of 1 080 bp and 4 700 bp were obtained after double-enzyme digestion of pGEFP-N1/GHRHR-SVT1 with restriction endonuelease Xho Ⅰ and Barn H Ⅰ. （2）Compared with primitive sequence（AF-282259）,the NO. 54 base of eonstuetion sequence of GHRHR-SVT1 eDNA occurred mutation （A mutation to T） ,whith is nonsense mutation. （3）The proliferation rate of NIH-3T3 cells of pEGFP-N1/GH- RHR-SVT1 group,was significantly higher-than the control group（P〈0.01）. （4）The cell proliferation index of NIH-3T3 cells of pEGFP-N1/GHRHR-SVT1 group, was significantly higher than the control group（P〈0.01）. Conclusion The medium with propriate concentration of GH- RH（10 μmol/L）,GHRHR-SVT1 accelerated the proliferation of NIH-3T3 cells,the study which provide to a new way for treatment of cancer.