腺病毒介导Tum5重组基因对高糖刺激下恒河猴视网膜血管内皮细胞增生、迁移及管腔形成的影响

The effect of adenovirus-mediated recombinant Tum5 gene expression on Rhesus retinal vascular endothelial cells under high glucose

目的观察腺病毒介导Turn5重组基因对高糖刺激下恒河猴视网膜血管内皮细胞（RF／6A细胞）增生、迁移及管腔形成的影响。方法构建空载体病毒rAd-绿色荧光蛋白（GFP）和携带Tum5基因的重组腺病毒表达载体（rAd—Tum5）。分别感染RF／6A细胞，流式细胞仪检测感染效率。细胞感染48h后，采用蛋白免疫印迹法（Westernblot）检测Tum5重组基因体外表达。将RF／6A细胞分为正常对照组、高糖刺激对照组（HG组），空载体对照组（HG＋rAd—GFP组），Tum5基因组（HG＋rAd—Tum5组）。采用细胞计数试剂盒（CCK-8）、侵袭小室（Transwell）实验及基质胶（Matrigel）实验分析Tum5重组基因对高糖刺激下RF／6A细胞增生、迁移及管腔形成的影响。结果流式细胞仪检测结果显示，tAd—GFP、rA＆Tum5感染细胞的效率分别为55．13％、50．31％。Westernblot检测结果显示，HG＋rA＆Tum5组在相对分子质量14×10^3左右处可见Tum5蛋白表达条带；正常对照组、HG组、HG＋rAd—GFP组均未见Tum5蛋白表达条带。CCK-8、Transwell实验及Matrigel实验结果显示，正常组、HG组、HG＋rAd-GFP组、HG＋rAd-Tum5组4组间细胞增生数量、迁移数量及成形管腔数量比较，差异均有统计学意义（F-44．484、772．666、137．696，P（0．05）。组间细胞增生数量、迁移数量及成形管腔数量两两比较结果显示，HG组较正常对照组增加，差异有统计学意义（P〈0．05）；HG组与HG＋rAd—Tum5组之间无明显差异（P〉0．05）；HG＋rAd—Tum5组较HG组、HG＋rAd—GFP组明显减少，差异有统计学意义（P〈0．05）。结论腺病毒介导Tum5重组基因可抑制高糖刺激下RF／6A细胞的增生、迁移及管腔形成。

Objective To observe the expression in vitro and the influence of adenovirus-mediated recombinant Turn5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. Methods To construct the adenovirus vector of recombinant Turn5 gene （tAd-TurnS）, and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose （HG）-control group （HG group）, empty expression vector group （HG＋rAd-GFP）, and HG＋ tAd-Turn5 group. Western blot was used to detect the expression of Turn5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. Results The adenovirus vector of recombinant Turn5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Turn5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differencesbetween all groups in proliferation, migration and tubing of the RF/6A cell （F=44. 484,772. 666,137. 6962 P〈0.05）. The comparison of each group indicated that the HG group was higher than normal group （P〈0.05）. There were no statistical differences between HG group and HG＋ rAd-GFP group （P〉0.05）. However, the HG＋rAd-Tum5 group was less than HG group （P〈0.05）, and the same to HG＋rAd-GFP （P〈0.05）. Conclusion The adenovirus vector of recombinant Turn5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.