血管内皮生长因子和转化生长因子-β2对人视网膜色素上皮细胞分化及其介导胶原凝胶收缩的机制研究

Critical role and involvement of vascular endothelial growth factor and transforming growth factor-β2 in collagen gel contraction induced by retinal pigment epithelial cells

目的研究血管内皮生长因子（VEGF）与转化生长因子（TGF）-β2对人视网膜色素上皮（RPE）细胞分化及其介导胶原凝胶收缩的机制。方法实验研究。原代培养人RPE细胞，将其与I型胶原凝胶混合，观察细胞在胶原中的收缩情况。实验分为3组，VEGF（50μg/L）组、TGF—β2（5μg/L）组和对照组，比较3组凝胶收缩性状的变化。同时，对人RPE细胞的增殖、形态和分化演变趋势进行分析。3组检测数据的多重比较采用单因素方差分析，进一步两两比较采用SNK—q检验。结果与I型胶原凝胶混合后，VEGF组和TGF—β2组的凝胶收缩率均表现出时间相关性，于24、48、72h分别达到（34．7±3．1）％、（44．3±6．0）％、（44．0±7．2）％和（29．3±3．1）％、（31．7±3．5）％、（29．0±3．6）％。与对照组[（20．0±0．5）％、（17．3±3．6）％、（19．1±0．8）％]相比，各时间点凝胶收缩率差异均有统计学意义（24h：F=26．220，P=0．001；48h：F=26．796，P=0．001；72h：F=21．522，P=0．002）；3组间各时间点两两比较差异亦有统计学意义（SNK—q检验，P〈0．05）。免疫印迹法检测两个实验组内各时点人RPE细胞内α-平滑肌肌动蛋白（α-SMA）表达水平，差异均有统计学意义（TGF-p2组：F=1．134，P：0．000；各时间点：SNK—q检验，P〈0．05；VEGF组：F=279．179，P=0．000；各时间点：SNK—q检验，P〈0．05）。进一步两两比较，TGF-β2组胶原凝胶收缩效应较VEGF组显著（6h：F=3．646，P=0．000；24h：F=18．706，P=0．003；48h：F=124．195，P=0．000；72h：F=76．811，P=0．000）。倒置显微镜与激光扫描共焦显微镜下观察可见，TGF-β2组和VEGF组人RPE细胞形态均发生了成纤维细胞样转化。结论VEGF和TGF-β2均可诱导人RPE细胞发生成纤维细胞样转化，从而引起胶原凝胶的收缩性状改变，这可能是玻璃体视网膜界面性疾病发生、发展的机制之一.

Objective To investigate the influence of vascular endothelial growth factor （VEGF） and transforming growth factor （TGF）-β2 to human retinal pigment epithelium（RPE） cell differentiation,and the mechanism of collagen gel contraction mediated by RPE cells. Methods Experiment study. An in vitro collagen gel contraction assay was performed to evaluate the effect of cultured human RPE in addition of VEGF and TGF-β2 at indicated time points （24 h,48 h and 72 h）. Three groups were established in the experiment ：control group,50 μg/L VEGF group and 5 μg/L TGF-132 group. The effects of both cytokines on the collagen gel contraction were analyzed by the reduced diameter of the collagen gel. And the changes of cell morphology and their transdifferentiation were assessed to estimate the possible connection between RPE transdifferentiation and collagen gel contraction. One-way ANOVA was used in conjunction with SNK-q test to assess statistical significance at different time periods within groups. Results There were differences on collagen gel contraction rates among VEGF group [ （ 34.7 ± 5.1 ） %, （ 44. 3 ± 6.0 ） %, （ 44.0 ± 7.2 ） % ], TGF-β2 group [ （29. 3 ± 3. 1 ） %, （ 31.7 ± 3.5 ） %, （ 29. 0 ±3.6 ） % ] and control group [ （ 20. 0 ± 0. 5 ） %, （ 17.3 ± 3.6） %, （ 19. 1 ± 0. 8 ） % ] at 24 h,48 h and 72 h after cultured （ 24 h ： F = 26. 220, P = 0. 001 ; 48 h ：F = 26. 796 ,P = 0. 001 ;72 h：F = 21. 522, P = 0. 002）, and on each time point two two comparison in the three groups （ SNK-q test, P 〈 0.05 ）. There were differences on protein expression level of α-smooth muscle actin（α-SMA） in 50 μg/L VEGF group and 5 μg/L TGF-β2 group at difference time points, respectively （ TGF-β2 group ： F = 1.134, P = 0, 000 ; each time point ： SNK-q test, P 〈 0.05 ; VEGF group ： F = 279. 179 ,P = 0. 000 ; each time point ： SNK-q test, P 〈 0.05 ）. Moreover, TGF-132 （ 5 p.g/L） demonstrated stronger and more permanent gel contraction than VEGF（50 μg/L）（6 h：F- 3. 646,P =0. 000;24 h：F = 18. 706,P =0. 003;48 h：F= 124. 195,P =0. 000;72 h：F =76. 811 ,P =0. 000）. RPE cells＇ form happened fibroblasts sample transformation in both VEGF group and TGF-132 group . Conclusions Both VEGF and TGF-β2 can induce the collagen gel contraction, partly by means of inducing the expression of α-SMA and RPE contraction, which may thus contribute to the explanations of vitro-retinal diseases.