Promote osteogenic gene and suppress adipogenetic gene-transfected NIH 3T3 fibroblasts and their expressions

Abstract

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 reaction (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 siPPARγ group, the differences were 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). 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. Key words: Gene; Transfection; Calcitonin gene-related peptide; Peroxisome proliferator-activated receptor-γ; NIH 3T3 fibroblasts