转甲状腺素蛋白对RPE细胞及视网膜微血管内皮细胞生物学行为的影响

摘要

目的 观察转甲状腺素蛋白(TTR)对视网膜色素上皮细胞(RPEC)增生及视网膜微血管内皮细胞(RMVEC)增生、迁移的影响并考察其与VEGF mRNA表达的关系,方法 通过加入外源性TTR考察TTR对RPEC增生的影响;利用小干扰RNA (siRNA)沉默视网膜色素上皮自身表达的TTR,使用Western blot检测沉默效果;RT-PCR法考察沉默后VEGFmRNA的变化;通过transwell共培养体系,观察TTR沉默前后RMVEC增生、迁移行为的改变.两组间比较行t检验,三组间两两比较用SNK法.结果 MTT比色法显示,4μmol/L组吸光度(A)值(0.36±0.01)低于0μmol/L组A值(0.72±0.02),差异有统计学意义(t=18.08,P＜0.0001),TTR对RPEC的增生有抑制作用;Western blot法检测TTR的表达抑制,实验组与对照组间差异有统计学意义(P＞0.05),TTR的表达被有效沉默;沉默TTR表达后检测VEGF mRNA表达,空白对照组、阴性对照组、实验组3个组间两两比较,差异均无统计学意义(P＞0.05),TTR的沉默表达并未在mRNA水平上引起VEGF的表达改变;48 h RPEC-RMVEC共培养细胞增生实验结果显示,RMVEC增生数NCsiRNA组[(362.8±17.7)个]和TTR siRNA组[(252.4±13.92)个]间差异有统计学意义(t=4.901,P=0.0012),沉默TTR表达会抑制RMVEC的增生能力.48 hRPEC-RMVEC共培养细胞迁移实验结果显示,RMVEC向外迁移数NCsiRNA组[(229.0±14.9)个]和TTR siRNA组[(147.8±12.2)个]比较,差异有统计学意义(t=4.213,P=0.0029).沉默TTR表达会抑制RMVEC的迁移能力.结论 TTR能抑制RPEC增生、促进RMVEC增生和迁移,这一现象并非通过VEGF实现,其机制值得进一步研究.%Objective To explore the effects of transthyretin (TTR) on biological behavior of retinal pigment epithelial cells (RPECs) and retinal microvascular epithelial cells (RMVECs).Methods RPECs were cultured with exogenous TTR to explore the effect of TTR on the proliferation of RPECs.The expression of TTR of RPECs was silenced by TTR specific small interfering RNA and the expression of TTR was detected by using Western blotting to identify the efficacy of TTR silence.The level of vascular endothelial growth factor (VEGF) massage RNA was detected by using RT-PCR to identify the interaction between VEGF and TTR.The different proliferation and migration abilities of RMVECs with different expressions of TTR were measured by transwell system.Results MTT assay showed that RPECs with 0 μmol/L TTR glowed faster than with 4 μmol/L TTR (t=18.08,P＜0.0001).The expression level of TTR was decreased in the small interfering RNA group as compared with the negative control group (P＜0.05).RT-PCR assay showed no differential expression of VEGF after the silencing of TTR (P＞0.05).The transwell assay showed RMVECs with the silence of TTR proliferated more slowly than RMVECs without the treatment (t=4.901,P=0.0012),and also migrated more slowly (t=4.213,P=0.0029).Conclusions TTR can inhibit the proliferation of RPECs and promote the proliferation and migration of RMVECs without the help of VEGF,the mechanism of which may be worth further study.