小分子干扰RNA抑制CyclinD 1表达诱导瘢痕疙瘩成纤维细胞凋亡研究

目的 探讨RNA干扰阻断细胞周期蛋白DI(CyclinDl)基因表达后,对瘢痕疙瘩成纤维细胞细胞周期和细胞增殖、凋亡的影响.方法 针对CyclinDl基因设计并合成特定的小分子干扰RNA(small interfering RNA,siRNA)转染入瘢痕疙瘩成纤维细胞,测定CyelinDl mRNA水平,流式细胞术分析细胞周期,FITC-Annexin V/PI细胞平均百分率为凋亡检测试剂盒检测细胞凋亡情况.DNA梯型观察凋亡细胞DNA变化.结果 特异性siRNA能在mRNA水平下调成纤维细胞的CyclinDl基因的表达.转染特异性siRNA 24、48、72 h后,Gl期细胞平均百分率分别为(59.80±3.06)%、(66.01±4.03)%和(67.43±5.35)%,高于对照组(54.50±5.35)%;S期细胞平均百分率为(18.40±1.42)%、(17.21±1.76)%和(11.07±1.00)%,低于对照组(22.33±1.49)%.细胞凋亡率分别为(7.82±0.45)%、(15.71±1.06)%、(18.32±1.08)%.均显著高于对照组(0.68±0.12)%,细胞DNA断裂成片段状.结论 特异性siRNA分子能够抑制细胞CyclinDl基因的表达,使细胞阻滞于Gl期,并诱导细胞凋亡,为应用RNA干扰技术治疗瘢痕疙瘩提供了初步实验依据.

Apoptosis of human keloid flbroblast induced by small interfering RNA-mediated CyclinD1 gene silencing

Objective To study the inhibition effect of CyclinDl specific small interfering RNA(siRNA) on CyclinDl gene expression in human keloid fibreblast, investigating the effect of CyclinDl specific siRNA (siRNA-CyclinDl) on the cell cycle, multiplication and apoptosis. Methods According to the principle of siRNA design, siRNA-CyclinDl was designed and the keloid fthroblast were transfected. RT-PCR was used to examine CyclinDl mRNA expression. Flow cytometry was used to examine cell cycle. The apoptotic rate was analyzed by using Annexin V-FITC/PI kit. The DNA gragmentation wer measured by DNA ladder analysis. Results After transfection, the expression of CyclinDl mRNA decreased remarkably. Twenty-four, forty-eight and seventy-two hours after transfection, the radio of GI stage cell was (59.80±3.06) ] 、(66.01±4.03) ]and (67.43±5.35)] ,all significantly higher than in the control group(54.50±5.35)] ;the radio of S stage cell was (18.404±1.42)] 、(17.21±1.76)] and (11.07±1.00)] ,significantly lower than in the control group (22.33±1.49)] ;the proportion of the cells in Gl stage increased and those in the S stage decreased in the keloid fibrnhlast transfected with siRNA-CyclinDl .The apoptotic rate of the siRNA-CyclinDl group was (7.82± 0.45)] 、(15.71±1.06)] 、(18.32±1.08)],all significantly higher than in the control group(0.68± 0.12)],and the DNA grngmentation can be seen remarkably. Conclusions Chemically synthesized siRNA- CyclinDl effectively inhibits The expression of CyclinDl in keloid fibroblast thus arresting the cell cycle at GI stage and enhancing cell apoptosis. Our study provided a preliminary results in seaching of a RNAi therapy of keloid.