缺氧对猴视网膜脉络膜微血管内皮细胞增生及p21表达的影响

Influence of hypoxia on cell proliferation and expression of p21 in rhesus retinal vascular endothelial cells

背景视网膜新生血管性疾病是临床常见的致盲性眼病，主要与血管内皮细胞增生有关，因此抑制血管内皮细胞的增生成为防治视网膜新生血管性疾病的研究重点。p21参与调控细胞在G1／S期的转变，抑制细胞增生，但p21在视网膜新生血管性疾病中的表达与血管内皮细胞增生的关系有待研究。目的探讨缺氧条件下体外培养猴视网膜脉络膜微血管内皮细胞（RF／6A）增生情况及其与p21表达变化的关系。方法体外培养RF／6A细胞株，复苏传代后计数稀释，接种于培养瓶中，细胞贴壁后分为常氧对照组（体积分数5％CO2＋体积分数95％O2培养）和缺氧实验组（1％O2＋5％CO2＋体积分数94％N2培养），缺氧实验组在缺氧培养箱中分别持续培养1、3、6、12h。用流式细胞仪检测常氧对照组和缺氧实验组RF／6A细胞周期的分布，MTT比色法检测并比较常氧对照组和缺氧实验组的细胞增生情况，Westernblot检测p21在常氧对照组和缺氧实验组RF／6A细胞中的表达。结果流式细胞仪检测结果显示，缺氧实验组G。／G，期细胞比例先降低后升高，各培养组间G0／G1期细胞比例的差异有统计学意义（F=20．083，P=0．000），S期和G2／M期细胞比例均先增高后降低，各组的总体差异均有统计学意义（F=7．861，P=0．001；F=10．305，P=0．003）。缺氧实验不同时间组G0／G1期细胞比例均明显低于常氧对照组，而S期和G2／M期细胞比例明显高于常氧对照组，差异均有统计学意义（P〈0．05）。MTT比色法检测结果显示，缺氧实验组细胞增生能力（A。值）先增强后减弱，各组的总体差异有统计学意义（F=7．768，P=0．001），缺氧实验3h组和6h组A570值分别为0．315±0．062和0．365±0．064，均明显高于常氧对照组的0．205±0．063，差异均有统计学意义（P〈0．05）。Westernblot检测结果显示，p21在缺氧实验组的表达先降低后升高，各组的总体比较差异有统计学意义（F=16．738，P=0．000），缺氧实验组各时间点细胞中p21相对表达量均明显低于常氧对照组，差异均有统计学意义（P〈0．05）。结论缺氧早期p21表达下调并诱导RF／6A细胞增生，但随着缺氧时间的延长，p21的表达上调，同时细胞增生将受到抑制。

Background Retinal neovascularization disease is a common cause of blinding. Retinal neovascularization is related to enhancing proliferation of vascular endothelial cells. So how to inhibit proliferation of vascular endothelial cells is a hot burning issue, p21 is known to be involved in the regulation of cell cycle and therefore inhibit the cell proliferation. However,the relationship of p21 and the proliferation of vascular endothelial cells in retinal neovascularization disease is for further study. Objective The aim of this experiment was to study the proliferation of rhesus retinal vascular endothelial cells（RF/6A） and expression of p21 in RF/6A cells under the hypoxia condition,and discuss their association. Methods The RF/6A cells were cultured and passaged in vitro, then they were randomly divided into normoxia culture group （5% CO2 ＋ 95% O2 ） and bypoxia for 1 hour, 3,6,12 hours group（ 1% 02＋5% CO2 ＋94% N2 ）. Flow cytometer（FCM） was used to check the distribution of RF/6A cell cycle in the normoxia culture group and hypoxia for 1 hour,3,6,12 hours groups. MTT assay was used to detect and compare the cell proliferation（As70 ）among the various groups. The expression of p21 in the cells was analyzed by Western blot. Results FCM showed that the cells proportion of G0/G1 stage was reduced initially and then increased afterward in hypoxia for 1 hour and 3,6,12 hours groups, showing a significant difference among 5 groups （F= 20. 083 ,P = 0. 000） , and the cells proportion of S stage and G2/M stage were increased firstly and then declined in different hypoxia groups with statistical significances （ F = 7. 861, P = 0. 001 ; F = 10. 305, P = 0. 003 ）. Compared with normoxia culture group,cells proportion of G0/G~ stage was declined and that of S stage and Gz/M stage were raised after hypoxia culture, showing statistically signifcant differences（ P〈0.05 ）. MTT showed that cell multiplication capacity（A570 value）strengthened firstly and then weakened in hypoxia groups with time prolongation, showing a significant difference among all the groups（ F= 7. 768 ,P = 0. 001 ） , and A570 value in hypoxia for 3 hours and 6 hours groups（0.315 ±0. 062,0. 365± 0. 064 ） was significantly higher than that of the normoxia group （0. 205 ± 0. 063 ） , respectively（P〈0. 05）. Western blot showed that the expression of p21 in the cells down-regulated at the beginning and then up-regulated with the increase of hypoxia time, and there was statistical significance （F = 16.738, P= 0. 000）. The p21 relative levels in different hypoxia groups were reduced in comparison with the normoxia group, showing statistical signifcances（P〈0.05）. Conclusions Short-term hypoxia could reduce the expression of the p21 in RF/6A and induce cell proliferation initially, then p21 increases and cell proliferation is inhibited with the prolongation of hypoxia time.