摘要目的 观察转化生长因子-β(TGF-β)在激光诱导脉络膜新生血管形成(CNV)中的作用.方法 6～8周龄雄性C57BL/6J小鼠80只随机分为正常对照组、光凝模型组、光凝加磷酸盐缓冲液(PBS)对照组(PBS对照组)、光凝加TGF-β受体抑制剂组(TGF-β受体抑制剂组),每组20只.光凝模型组、PBS对照组及TGF-β受体抑制剂组小鼠均采用激光光凝诱导CNV形成.激光光凝后1、2、3、4周,对正常对照组及光凝模型组小鼠行荧光素眼底血管造影(FFA)检查,动态观察小鼠模型CNV的形成情况;采用蛋白免疫印迹法(Western blot)检测正常对照组及光凝模型组小鼠视网膜内TGF-β的蛋白表达以及正常对照组、PBS对照组、TGF-β受体抑制剂组小鼠视网膜内VEGF和TNF-α的蛋白表达.激光光凝后2周,作视网膜色素上皮细胞/脉络膜组织铺片,采用荧光染色计算各组CNV面积.结果 FFA检查结果显示,正常对照组小鼠视网膜血管以视盘为中心呈放射状走行,脉络膜血管呈网状分布;激光光凝后1周,光凝模型组小鼠光凝区可见荧光素渗漏呈盘状强荧光团.Western blot检测结果显示,光凝模型组小鼠视网膜内TGF-β蛋白表达随激光光凝时间延长而逐渐升高.光凝模型组与正常对照组小鼠视网膜内TGF-β蛋白表达比较,差异有统计学意义(F=13.042,P＜0.05).激光光凝后1、2、3、4周,PBS对照组、TGF-β受体抑制剂组小鼠视网膜内TNF-α(F=14.721、17.509)、VEGF(F=18.890、11.251)蛋白表达均较正常对照组明显增加,差异均有统计学意义(P＜0.05).与PBS对照组比较,TGF-β受体抑制剂组小鼠视网膜内TNF-α、VEGF蛋白表达明显降低,差异均有统计学意义(F=21.321、16.160,P＜0.05).激光光凝后2周,TGF-β受体抑制剂组小鼠脉络膜内CNV面积明显小于PBS对照组及光凝模型组,差异均有统计学意义(F=4.482,P＜0.05).结论 TGF-β可能通过上调TNF-α和VEGF蛋白表达水平促进CNV形成,应用其抑制剂可以减少CNV形成.

abstractsObjective To investigate the effects of transforming growth factor-β (TGF-β) in choroidal neovascularization (CNV) induced by laser in mice.Methods Eighty male C57BL/6J mice at the age of 6-8 weeks old were randomly divided into the normal control,photocoagulation model,photocoagulation with phosphate buffered saline (PBS control group) and photocoagulation with TGF-β receptor inhibitor groups (TGF-β receptor inhibitor group),twenty mice of each group.Fundus argon laser photocoagulation was performed in the photocoagulation model group,PBS control group and TGF-β receptor inhibitor group to induce CNV.One week,two,three and four weeks after the laser procedure,fundus fluorescein angiography (FFA) was carried out in the normal control or photocoagulation model groups to observe CNV formation dynamically.Western blot was used to analyze the expressions of TGF-β in the retina from the mice of normal control or photocoagulation model groups,and VEGF or TNF-α in the retina of normal control,PBS control or TGF-β receptor inhibitor groups.The CNV areas of each group were evaluated by using fluorescein stain on retinal pigment epithelium (RPE)/choroid flat mounts after two weeks of photocoagulation.Results The FFA results showed the retinal vessels centered on the optic disc and arranged radially,while the choroidal vascular present network distribution in the normal control mice.Significant leakage of fluorescein showed discoid strong fluorophore in photocoagulation sites of retina at one week after photocoagulation.The quantitative analysis results of Western blot demonstrated that the TGF-β protein expression levels in retina of photocoagulation model mice gradually increased with time passing.The protein expression levels of TGF-β were significant differences in the photocoagulation model group comparing with the normal control group (F=13.042,P＜0.05).The protein expression levels of TNF-α (F=14.721,17.509) and VEGF (F=18.890,11.251) increased significantly in retina of PBS control or TGF-β receptor inhibitor groups when compared with that of normal control group at one week,two,three and four weeks after photocoagulation,and the differences were both statistically significant (P＜0.05).Compared with PBS control group,the protein levels of TNF-α and VEGF in retina from TGF-β receptor inhibitor group were significantly reduced,the differences was statistically significant (F=21.321,16.160,P＜0.05).Two weeks after laser photocoagulation,a distinct reduction in CNV lesion size in the TGF-β receptor inhibitor group mice when compared to PBS or normal control groups,the differences was statically significant (F=4.482,P＜0.05).Conclusion TGF β may promote CNV formation by up-regulating both TNF α and VEGF protein expressions,the application of its specific inhibitor is able to reduce CNV progression.