针对p27Kip1基因的小发夹RNA对牛角膜内皮细胞增殖的影响

目的 探讨针对p27Kip1基因的小发夹RNA(shRNA)表达质粒对牛角膜内皮细胞(bCEC)增殖能力的影响.方法 实验研究.设计有发夹状结构的3条p27Kip1-shRNA对应模板DNA序列,并构建无关序列HK-shRNA作为阴性对照.构建并鉴定重组质粒Pgenesil-P1、Pgenesil-P2、Pgenesil-P3及Pgenesil-HK.以脂质体法将以上4种质粒分别转染bCEC,并设立空白组.稳定转染后采用RT-PCR和Western免疫印迹法榆测bCEC内p27Kip1的mRNA及其蛋白水平,筛选出抑制效果最好的阳性siRNA质粒.用四甲基偶氮唑盐比色法(MTT)检测该质粒组、Pgenesil-HK组及空白组的细胞生长情况.流式细胞术检测各组细胞周期的分布.以上数据结果均采用单因素方差分析.结果酶切及测序证实4个重组质粒均构建成功.与空白组比较,Pgenesil-P1、Pgenesil-P2、Pgenesil-P3各组mRNA的抑制效率分别为32.71%、67.76%及80.28%(F=453.102,P=0.000),蛋白表达水平降低为29.27%、64.73%及76.13%(F=75.385,P=0.000),以Pgenesil-P3抑制效果最明显.空白组与Pgenesil-HK组比较蛋白水平(P=0.356)和mRNA水平(P=0.246)均差异无统计学意义.与空白对照和阴性siRNA相比,Pgenesil-P3组bCEC增殖能力提高,G1期细胞比例下降(F=134.224,P=0.000),s期比例增加(F=334.957,P=0.000).结论针对p27Kip1的shRNA可有效降低p27Kip1基因及其蛋白的表达水平,并可提高bCEC增殖能力.RNA干扰介导的p27Kip1基因沉默可能是促进角膜内皮细胞再生的有效手段.(中华眼科杂志,2009,45:254-259)

Effect on the proliferation of bovine corneal endothelial cells by small hairpin RNA interference targeting p27Kip1

Objective To clarify the proliferation of bovine corneal endothelial cells(bCEC)by interference with the recombinant plasmid of short hairpin RNA(shRNA)against p27Kip1,a kind of cyclin dependent kinase inhibitor(CKI).MethodsIt was an experimental study.Three p27Kip1-shRNA template DNA sequences containing small hairpin structure were designed and synthesized as experimental groups.Plasmid expressing irrelevant shRNA with a random combination was used as negative shRNA.The products were inserted into the Pgensil-1 plasmid and the recombinant plasmid of Pgenesil-P1,Pgenesil-P2,Pgenesil-P3 and Pgenesil-HK were constructed.The recombinant plasmids were transfected into bCEC cells with lissome and a blank group.The expression of mRNA and protein of p27 Kip1 was detected by RT-PCR and Western blot after stable transfection,and the plasmid with the best inhibitory effect was selected.The growth of the experimental group.Pgenesil-HK group and blank group were assessed by MTT.The influence of shRNA-p27Kip1 on bCEC cell cycle was deteceted by flow cytometry(FCM).All statistical analyses were performed using one-way ANOVA.Results Restrictive enzyme digestion and sequence analysis showed that four recombinant plamids were constructed successfully and the aim sequence was obtained.The expression of p27Kip1 mRNA and p27Kip1 protein of Pgenesil-P1 group.Pgenesil-P2 group and Pgenesil-P3 group were all lower than that in the control group,including blank group and negative siRNA group.The inhibitive rate of mRNA reached 32.71%,67.76% and 80.28%(F=453.102,P=0.000 in each group)and the inhibitive rate of protein reached 29.27%,64.73% and 76.13%(F=75.385,P=0.000 in each group)compared with the blank group.As the lowest expression among the three positive shRNA group,Pgenesil P3 was selected for the next steps.There was no significant difference between blank group and negative Pgenesil-HK of the expression of p27Kip1 protein(P=0.356)and the express of p27Kip1 mRNA(P=0.246).Compared with the control group and the blank group,the growth of the bCEC transfected by Pgenesil-P3 was significantly promoted with increased cell percent of S-phrase(F=334.957,P=0.000)and decreased cell percent of G1-phrase(F=134.22,4,P=0.000).Conclusions shRNA-p27Kipl can down-regulate the expression of bCEC effectively and increase the growth of bCEC.shRNA-p27 Kip1 RNA interference may be an effective method to promote the proliferation of CEC.(Chin J Ophthalmol,2009,45:254-259)