RNA干扰Cyclin D1基因表达对瘢痕疙瘩成纤维细胞增殖和G1期调控的影响(英文)

Influence of RNA Interference Mediated CyclinD1 Gene Silencing on The Proliferation and G1 Phase Regulators of Fibroblasts Derived From Keloid

为研究siRNA干扰瘢痕疙瘩成纤维细胞cyclin D1基因表达,对瘢痕疙瘩成纤维细胞的增殖、细胞周期和G1期调控的影响,构建了靶向cyclin D1的siRNA表达质粒.利用LipofectamineTM2000转染体外培养的瘢痕疙瘩成纤维细胞,应用荧光定量PCR、RT-PCR检测cyclin D1 mRNA的干扰效果,应用MTT法、流式细胞仪检测细胞增殖和细胞周期的变化,应用免疫组织化学染色检测成纤维细胞中cyclin D1、CDK4、P16、pRb蛋白表达的影响.主要结果如下:a.靶向cyclin D1的特异性siRNA序列可以高效地抑制成纤维细胞cyclin D1基因表达,对照组与实验组在mRNA水平其表达抑制率分别为63.68%和92.83%(P<0.01);b.可以显著抑制瘢痕疙瘩成纤维细胞的增殖,改变细胞周期分布,G0/G1期细胞比例显著高于各对照组(P<0.05),细胞分裂被阻滞;c.免疫组化染色发现,转染72h后,过表达的cyclin D1、CDK4和pRb蛋白,在瘢痕疙瘩成纤维细胞中均出现了不同程度的表达下调,而低表达的P16则呈上调表现.由上述结果可见,构建的靶向cyclin D1的RNAi表达质粒,可有效地抑制瘢痕疙瘩成纤维细胞cyclin D1基因表达,通过改变G1期相关周期蛋白的水平,影响G1/S期的进程,显著地抑制成纤维细胞的增殖.

In order to investigate the effect of sequence-specific small interfering RNA on suppressing cyclin D1 expression and proliferation and cell cycle and expression of G1 phase regulators of fibroblasts derived from keloid, the plasmid expression vector of siRNA targeted against cyclin D1 was constructed and transfected into fibroblasts with Lipofectamine^TM 2000. The changes of cyclin D1 expression were detected by fluorescent quantitative PCR（FQ-PCR）, semi-quantitative RT-PCR. The effect of sequence-specific small interfering RNA in suppressing the proliferation of fibroblasts was detected by MTT assay. Flow cytometry were used for evaluation of cell cycle. The expression of cyclin D1, CDK4, pRb and P 16 was detected by immunohistochemical method. The results showed that： （1） The sequence- specific siRNA effectively suppressed cyclin D1 expression at both mRNA levels with inhibition rate of 63.68% and 92.83% （P 〈 0.01）. （2） Significantly inhibited the proliferation of fibroblasts, and changed cell cycle in percentage of G0/G1 phase cells was increased compared with the controls groups in fibroblasts（P 〈 0.05）. （3） 72 h after transfection, the expression of cyclin D 1, CDK4 and pRb decreased, and the expression of P 16 increased. It can be concluded that the plasmid expression vector for the RNAi against cyclin D1 constructed in the study can effectively and specifically suppress cyclin D1 expression, and progression of G1/S is effected by G1 phase related regulatory protein, and suppresses proliferation of fibroblasts derived from keloid.