促进成骨与抑制成脂基因共转染NIH3T3成纤维细胞中的表达

摘要

目的：检测降钙素基因相关肽基因（CGRP）和沉默过氧化物酶体增殖子活化受体-γ（PPARγ）共转染NIH3T3成纤维细胞中的表达。方法培养 NIH3T3成纤维细胞，收集细胞随机分为五组，正常组：正常培养细胞，不做特殊处理，作为正常对照；空载体组：仅用空载体 pGFP-V-RS 质粒转染细胞，作为阴性对照组；CGRP 组：用重组载体 pGFP-V-RS-exCGRP 质粒转染细胞；siPPARγ组：用重组载体 pGFP-V-RS-siPPARγ质粒转染细胞；双基因组：用重组双基因载体 pGFP-V-RS-siPPARγ-exCGRP 质粒转染细胞。电转染成功后，继续培养细胞48 h，采用逆转录聚合酶链反应（RT-PCR）与蛋白质免疫印迹法（Western blot）分别检测各组细胞内 PPARγ、CGRP mRNA 与蛋白的表达。结果双基因组中 CGRP 基因呈高表达，明显高于正常组、空载体组、siPPARγ组，差异均有统计学意义（P ＜0.05）；与 CGRP 组中 CGRP 基因表达量相近，差异未见统计学意义（P ＞0.05）。双基因组细胞中 PPARγ基因呈低表达，明显低于正常组、空载体组、CGRP 组，差异均有统计学意义（P ＜0.05）；与 siPPARγ组细胞中 PPARγ基因表达量相似，差异未见统计学意义（P ＞0.05）。结论促进成骨基因和抑制成脂基因共转染 NIH3T3成纤维细胞，能够上调细胞中 CGRP 基因表达，同时下调 PPARγ基因表达。%Objective To detect the expression of the recombinant vector silencing peroxisome proliferator-activated receptor-γ(PPAR-γ)and calcitonin gene-related peptide(CGRP)gene in NIH3T3 fibroblasts. Methods The NIH3T3 fibroblasts were cultured,collected and randomly divided into 5 groups. Normal group:the cells were not treated with any vector,and served as control. Empty vector group:the cells were transfected with recombinant vector pGFP-V-RS,and served as negative control. CGRP group:the cells were transfected with recombinant vector pGFP-V-RS-exCGRP. siPPARγ group:the cells were transfected with recombinant vector pGFP-V-RS-siPPARγ. Double genes group:the cells were transfected with recombinant vector pGFP-V-RS-siPPARγ-exCGRP. After successful electroporation transfection,the cells were cultured and collected 48 h after transfection. The expression levels of the PPARγ,CGRP mRNA and their proteins were determined by reverse transcription polymerase chain reac-tion(RT-PCR)and western blot. Results In double genes group,the expressions of CGRP mRNA and its protein gene were significantly higher than those in normal group,empty vector group and siPPARγgroup,the differences were significant(P < 0. 05),and were similar to those in CGRP goup,the differ-ences were not significant(P > 0. 05). In double genes group,the expressions of PPARγ mRNA and its protein were significantly lower than those in normal group,empty vector group and CGRP group,the differences were significant(P < 0. 05),and were similar to those in siPPARγ group,the differences were not significant(P > 0. 05). Conclusions Combined double gene,expressing CGRP and silencing PPARγ gene,co-transfected 3T3 NIH fibroblasts,expression of CGRP gene can be up-regulated and same time the expression of PPAR gene is down-regulated.