鼠视网膜新生血管发生过程中Twist基因的表达

目的 探讨视网膜新生血管发生过程中Twist基因的表达情况.方法 对照实验研究.将40只新生C57/BL小鼠分为两组:(1)对照组,正常空气环境中饲养;(2)高氧组,在高氧环境中制作小鼠缺氧性视网膜新生血管动物模型.将出生第7天的小鼠放人氧箱,在75%浓度的高氧环境中饲养5 d后,取出氧箱.待出氧箱第12天和第17天(视网膜新生血管发生高峰时间)分别处死小鼠,制作视网膜铺片和切片,观察小鼠视网膜新生血管发生情况.应用免疫组织化学染色检测小鼠视网膜血管内皮细胞(VE)-钙黏蛋白、波形蛋白及Twist基因表达变化情况;提取全视网膜RNA,应用逆转录聚合酶链反应(RT-PCR)法检测VE-钙黏蛋白、波形蛋白及Twist基因表达变化情况.应用SPSS 11.5统计学软件进行数据处理,对高氧组与对照组小鼠出生后不同时间的VE-钙黏蛋白、波形蛋白、Twist基因表达的灰度值比较,采用两因素析因设计的方差分析,以P<0.05作为差异有统计学意义.结果 免疫组织化学检测结果显示,出生后第17天的小鼠,高氧组VE-钙黏蛋白表达的灰度值(65.19±8.39)与对照组(75.36±7.04)相比明显减少(F=8.616,P=0.009),波形蛋白表达的灰度值(95.09±14.13)与对照组(82.14±6.32)相比明显升高(F=6.999,P=0.016),Twist基因表达的灰度值(119.48±7.90)与对照组(93.30±6.37)相比亦明显升高(F=66.557,P=0.000).RT-PCR检测结果显示,不同处理组间波形蛋白、Twist基因表达的灰度值与检查时间存在交互作用(F=5.508,P=0.032;F=17.760,P=0.001).结论 在小鼠视网膜新生血管形成过程中,细胞转型调控的Twist基因发挥着重要作用,其作用机制可能是通过下调血管内皮细胞间的紧密连接蛋白,促进内皮细胞转型实现.

The mechauism of twist gene regulation during the retina angiogenesis

Objective To study the mechanisms of the Twist gene and cell transition during the angiogenesis in a mouse model of oxygen induced retinopathy. Methods It was a experimental study. 40new born C57/BL mice were divided into two groups;the control group, which were fed in the room air, and the study group, which were fed 7 days in the normal environment, followed by exposure to hyperoxia (75% O2) for 5 days. The mice were sacrificed at post born 12 day and post born 17 day. The Twist and VE-cedherin expression were detected with immunohistochemistry. The total retinal RNA was extracted and the expression level of the VE-cadherin, vimentin and twist were examined with RT-PCR method. Results Immunohistochemistry proved there was no significant difference at post born 12 day. At post born 17 day,compared with the control group (75.36±7.04), VE-cadherin expression of hyperoxia group( 65.19±8.39 )decreased ( F = 8.616, P = 0.009). Compared with the control group ( 82.14±6.32), Vimentin expression of hyperoxia group (95.09±14.13 ) increased, compared with the control group (93.30±6.37 ), Twist expression of hyperoxia group ( 119.48±7.90) increased ( F = 66.557, P = 0.000 ) significantly. RT-PCR examination revealed there exist the interaction between Vimentin, Twist expression and the detection time (F = 5.508, P = 0.032;F = 17.760, P = 0.001 ;respectively). At post born 12 day, compared with the control group ( 0.77±0.10 ), VE-cadherin expression of hyperexia group ( 0.64±0.09 ) decreased ( P =0.047 ). Compared with the control group ( 0. 24±0.05 ), Vimentin expression of hyperoxia group (0.39±0.09)inereased(P =0.033). At post born 17 day, compared with the control group(0.75±0.12), VE-cadherin expression of hyperoxia group(0.51±0.07) decreased more obviously( P = 0.002), compared with the control group ( 0.36±0.06 ), Vimentin expression of hyperoxia group (0.70±0.14 ) increased ( P =0.000). Compared with the control group(0.89±0.11 ), Twist expression of hyperoxia group( 1.24 ±0.15 )increased( P = 0.003 ). Conclusion Twist, as a cell transition gene, participated in the angiogenesis of the oxygen induced retinopathy, and that the Twist induced endothelium-mesenchymal transition may be one of the main reasons.