shRNA抑制Smad3基因的表达对瘢痕疙瘩成纤维细胞Smad7表达的影响

Effects of the inhibition of Smad3 by short hairpin RNA on the expression of Smad7 in keloid fibroblasts

目的构建抑制瘢痕疙瘩成纤维细胞(keloid fibroblast,KFB)Smad3基因表达的shRNA真核表达载体。研究Smad3 shRNA对KFB Smad7表达的影响。方法用前期实验筛选出的1对最有效siRNA重组构建Smad3 shRNA表达质粒。通过脂质体介导的方法,将Smad3 shRNA转染到KFB,用RT-PCR及Western blot的方法检测Smad3、Smad7在不同时间点(0～9d)表达的变化。结果①RT-PCR和Western blot的结果证实Smad3 shRNA载体构建成功。②转染Smad3 shRNA后,随着时间的延长,KFB中Smad3的mRNA与蛋白表达都显著减低,到72h作用最强。光密度分析与对照组比较差异有统计学意义(P<0.05)。Smad7的mRNA与蛋白表达都明显升高(P<0.05)。结论体外构建的shRNA-Smad3 RNAi真核表达载体能显著抑制KFB Smad3的表达。Smad3可能对Smad7起反向调节的作用。

Objective To construct the eukaryotic expression vector containing short hairpin RNA （shRNA） targeting the inhibition of Smad3 gene segment in keloid fibroblasts （KFBs） and to investigate the effects on the expression of Smad7 in KFBs. Methods A couple of the most effective siRNA screened from former experiments were used to recombine the plasmids of Smad3 shRNA, and then Smad3 shRNA was trans- fected into KFBs by LyoVec TM. The expressions of Smad3 and Smad7 at different time points （0 -9 d） were detected by RT-PCR and Western blotting. Results （~） The recombinant Smad3 shRNA vectors were identi- fied by RT-PCR and Western blotting. （~） The expressions of mRNA and protein of Smad3 in KFBs decreased significantly in a time-dependent manner after transfection of Smad3 shRNA and reached the lowest point at 72 hours in the experimental group. Optical density analysis revealed a significant difference between the experi- mental group and the control group （ P 〈 0. 05 ）. In contrast, the expressions of mRNA and protein of Smad7 increased significantly （P 〈 0. 05）. Conclusion The recombinant Smad3 shRNA vector can suppress the expression of Smad3 in KFBs, suggesting Smad3 may play a role in reverse regulation of SmadT.