shRNA对瘢痕疙瘩成纤维细胞Smad3及Ⅰ型胶原表达的影响

Effect of the inhibition of smad3 by short hairpin RNA on the expression of smad3 and collagen in keloid fibroblast

目的:构建抑制瘢痕成纤维细胞(keloid fibroblast,KFB)Smad3基因表达的shRNA真核表达载体。研究Smad3 shRNA对KFBSmad3及I型胶原(type I collagen,COL1A2)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Smad3shRNA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、COLlA2在不同时间点(0d至9d)表达的变化。结果:①重组质粒经酶切鉴定和测序、RT-PCR和Western blot的结果均证实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)。COLlA2的mRNA与蛋白表达都明显下降(P<0.05)。结论:体外构建的shRNA-Smad3 RNAi真核表达载体能显著抑制KFBSmad3的表达。并使其I型胶原mRNA和蛋白水平表达出现相同的变化,提示Smad3 shRNA可能是改善皮肤创面愈合和抑制瘢痕增生一个新的治疗方向。

Objective：To construct a plasmid geueratiug short hairpin RNA （shRNA）contaiuing the Smad3 gene segment in keloid fibroblasts＇ （KFB） and to investigate it is effect on the expression of Smad3 and type Ⅰ collagen （COLIA2） in KFB. Methods： A couple of the most effective siRNA .selected from former experiment were used to recombine the plasmids of Smad3 shRNA which were transfeeted into KFB by Lyo Vec. TM. The expressions of Shred3 and COL1A2 at difli：rent time points （0～ 9d） were detected by RT- PCR and Western blotting. Results： （ 1 ） The recombinant Smad3 shRNA vectors were identified by sequence analysis, RT - PCR and Westem blotting. （2） The expressions of mRNA and pratein of Smad3 in KFB decreased significantly with the extension of time after Smacl3 shRNA was transfected and reached the lowest point at 72 hours in experimental group compared with control group by optical density analysis （P 〈 0.05 ）. The expressions of mRNA and protein uf COLIA2 were also significantly and uniquely deere＇asecl following the reduction of Smad3 （ P 〈 0.05）. Conclusion： The rceombinant Smad3 shRNA vector can suppress the expression of Srnad3 in KFB, and decrease the expressions of COL1A2 mRNA and protein. It could be a new promisiag therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions.