小分子干扰RNA抑制Survivin基因表达诱导瘢痕疙瘩成纤维细胞凋亡的研究

Suppression of Survivin expression by small interference RNA inducing apoptosis of keloid fibroblasts

目的观察应用小分子干扰RNA（siRNA）技术抑制瘢痕疙瘩成纤维细胞中Survivin表达对细胞凋亡和细胞增殖的影响。方法设计、合成特异性抑制Survivin表达的siRNA，转染瘢痕疙瘩成纤维细胞，通过半定量逆转录一聚合酶链反应（RT—PCR）及Western blot检测24h和48h后成纤维细胞内Survivin mRNA和蛋白变化，流式细胞仪检测细胞周期和凋亡率，噻唑蓝（MTF）测定成纤维细胞的增殖。结果重组质粒成功转染瘢痕成纤维细胞，转染效率为67．0％；阳性质粒转染24h后，Survivin mRNA和蛋白表达明显降低，抑制率分别为52．2％和54．8％（P〈0．05），48h后抑制率增至75．7％和76．7％。阳性质粒转染24h凋亡率为11．9％，48h增加至21．2％，显著高于阴性对照组和未处理组；细胞周期检测阳性质粒干扰组s期细胞减少，G2／M期所占比例增高，出现了G2／M期阻滞现象；MTF测定阳性质粒干扰组细胞生长曲线平缓。结论特异性siRNA可以有效干扰瘢痕疙瘩成纤维细胞内Survivin mRNA和蛋白的表达，诱导细胞的凋亡和抑制细胞的增殖。

Objective To evaluate the effect of Survivin small interfering RNA （siRNA） silencing Survivin gene on the apoptosis rate and proliferation of fibroblasts in keloid. Methods The plasmid pSI- REN-siRNA/Survivin was constructed and transfected into fibroblasts via LipofectamineTM 2000. The expression 1 evel of Survivin mRNA and proteio was detected by using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting after thtransfection. Apoptosis rate and cell cycle were measured by flow cytometry. Cellular proliferation was measured by methyl thiazol tetrazolium （MTF） assay. Results siRNA was transfected into fibroblasts effectively and its transfection efficiency was 67.0%. Survivin mRNA and protein were inhibited after transfection for 24 h and the inhibitory rate was 52. 2% and 54. 8% respectively （P 〈 0.05 ）. The inhibitory rate was increased to 75.7% and 76.7% after transfection for 48 h. Amount of S phase cells was decreased, while that of G2/M phase increased. The apoptosis rate 24 and 48 h after transfection was 11.9% and 21.2% respectively, significantly higher than in negative and un- treated groups. MTF asasy showed that keloid fibroblasts proliferation was inhibited after transfection of plasmid pSIREN-siRNA/Survivin. Conclusion siRNA silencing Survivin gene can effectively inhibit its mRNA and protein expression and fibroblast proliferation, and induce apoptosis.