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

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

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p21tuRNA及蛋自在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p21mRNA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）％；tAdp21转染组RF／6A细胞出现GO／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p21mRNA及蛋白，并可显著抑制其增生。

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）. Methods RF/6A cells were cultured in vitro which divided into phosphate buffered solution （PBS） group, tAd-p21 transfection group and negative control （NC） group. Plasmid vectors were transfected into RF/6A cells. The expression of p21 mRNA 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 tAd-p21 transfection group were higher than that in PBS and negative control group. The cell eyele distribution showed that the proportion of GO/ 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 tAd-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.