Objective: To investigate the effect of the lentiviral vector pGCL-GFP transferred connective tissue growth factor (CTGF) short hairpin RNA (CTGF-ShRNA) on CTGF expression, cell proliferation and collagen synthesis induced by hypoxia in rat cardiac fibroblasts (CFs).
Methods: CTGF-ShRNA plasmids successfully constructed and screened. CFs of adult Sprague-Dawley (SD) rats isolated with the method of trypsin digestion and differential anchoring velocity which randomly divided into the control group, the hypoxia group, Hypo+pGCL-GFP group and Hypo+CTGF-ShRNA group. The mRNA and protein levels of CTGF were detected by means of semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot 24 h later. Proliferation of CFs was observed by WST-1 coloricmetric assay and synthesis of collagen was observed by the hydroxyproline.
Results: Successfully constructed and screened CTGF short hairpin RNA. Compared with control group, the expression of CTGF mRNA and protein levels induced by hypoxia in CFs were markedly up-regulated in Hypoxia, Hypo + pGCL-GFP and Hypo + CTGF-ShRNA group (P < 0.05). CFs proliferation and collagen synthesis in Hypoxia, Hypo+pGCL-GFP and Hypo+CTGF-ShRNA group were significantly higher than that of the control group (P < 0.05). In comparison to Hypoxia and Hypo+pGCL-GFP group, the CTGF mRNA and protein levels induced by hypoxia in CFs were markedly down-regulated in Hypo + CTGF-ShRNA group (P < 0.01). CFs proliferation and collagen synthesis in Hypo+CTGF-ShRNA group were significantly lower than that of the Hypoxia and Hypo+pGCL-GFP group (P < 0.01).
Conclusion: CTGF mRNA and protein expression, CFs proliferation and collagen synthesis induced by hypoxia in CFs effectively inhibited by CTGF-ShRNA.