重组腺病毒-p21抑制猴视网膜微血管内皮细胞增生的作用研究

目的 观察重组腺病毒-p21 (rAd-p21)对体外培养的猴视网膜微血管内皮细胞(RF/6A)增生的作用。方法 体外培养RF/6A细胞系，分为磷酸盐缓冲液(PBS)组、rAd-p21转染组及阴性对照组，并转入相应的质粒表达载体。应用逆转录聚合酶链反应(RT-PCR)法及蛋白免疫印迹(Western blot)检测p21mRNA及蛋白在RF/6A细胞中的表达；应用流式细胞仪检测p21基因对RF/6A细胞周期的影响；行内皮细胞体外成管实验观察p21基因对RF/6A细胞成管的抑制作用。结果 rAd-p21转染组p21 mRNA及蛋白表达显著增高。细胞周期检测结果显示，PBS组、rAd-p21转染组、阴性对照组G0/G1期细胞百分比分别为（40.76±6.66）％、（67.45±11.61）％、(41.55±8.99)％；rAd-p21转染组RF/6A细胞出现G0/G1期阻滞，G0/G1期细胞比例增多。rAd-p21转染组与PBS组和阴性对照组比较，差异有统计学意义(F=21.284，P=0.000)。体外成管实验显示，PBS组、rAd-p21转染组、阴性对照组每一视野下内皮细胞成管数分别为（8.25±3.19）、（3.86±1.21）、(7.62±2.69)个；rAd-p21转染组与PBS组和阴性对照组比较，差异有统计学意义(F=7.138，P=0.004)。结论 rAd-p21可成功转染RF/6A细胞并稳定表达p21 mRNA及蛋白，并可显著抑制其增生。

Recombined adenovirus of p21 inhibit the proliferation of Rhesus retinal vascular endothelial cells in vitro

Objective To observe the inhibitory effect of recombined adenovirus of p21 (rAd-p21) on the proliferation of Rhesus retinal vascular endothelial cell line (RF/6A). MethodsRF/6A cells were cultured in vitro which divided into phosphate buffered solution (PBS) group, rAd-p21 transfection group and negative control (NC) group. Plasmid vectors were transfected into RF/6A cells. The expression of p21mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively. The cell cycle distribution was analyzed by flow cytometry. Endothelial-cell tube formation assay was performed in Matrigel. Results The expression of p21 mRNA and protein in rAd-p21 transfection group were higher than that in PBS and negative control group. The cell cycle distribution showed that the proportion of G0/G1 cells in rAd-p21 transfection group(67.45 ± 11.61) ％]was apparently higher than that in the negative control group(41.55±8.99)％]and PBS group(40. 76±6.66) ％](F=21.284, P =0. 000 ). The number of endothelial-cell tubes in the rAd-p21 transfection group (3.86±1.21) was apparently less than that in the negative control group (7.62±2.69) and PBS group (8.25±3.19) (F=7.138,P=0.004).Conclusions The p21 mRNA and protein can stably express in RF/6A cells after rAd-p21 transfection.RAd-p21 can inhibit the proliferation of RF/6A cells.