内皮祖细胞参与大鼠脉络膜新生血管生成的可能机制

Possible mechanism of endothelial progenitor cells in the development of rat choroidal neovascularization

目的探讨内皮祖细胞（EPC）在脉络膜新生血管（CNV）生成中的作用及其可能机制。方法实验研究。用532nm倍频激光光凝视网膜制备BN大鼠CNV模型，每只大鼠1只眼为实验眼，另1只眼为正常对照眼。24只大鼠随机分为3组，分别在激光光凝后3、7、14d经组织病理学观察CNV的形态学变化，通过免疫荧光多标记法观察光凝局部EPC的募集及光凝局部EPC表达分泌的可能促血管生成因子。三组间均数比较采用方差分析，进一步两两比较采用LSD—t检验。结果组织病理学和免疫荧光染色结果显示，大鼠实验眼光凝后3d光凝局部增生、移行的细胞自破损的Bruch膜长入视网膜下腔，光凝后7d光凝局部有新生血管管腔样结构形成，至光凝后14d，CNV趋于稳定，其内管腔样结构进一步增多并连接成网。大鼠正常对照眼视网膜中未见EPC，实验眼激光光凝后3d即有大量EPC出现在光凝局部，占CNV中内皮细胞的（79．29±11．27）％；光凝后7dEPC见于光凝局部管腔样结构中，CNV内EPC占内皮细胞的（47．13±5．78）％；至光凝后14d，CNV内EPC数量显著下降，占内皮细胞的（10．83±2．79）％；3组问差异有统计学意义（F=104．623，P〈0．05）。进一步行两两比较，差异亦均有统计学意义（P值均〈0．05）。免疫荧光三标记染色结果显示，大鼠实验眼CNV局部的EPC表达血管内皮生长因子、白细胞介素-10、碱性成纤维细胞生长因子和基质金属蛋白酶9。结论EPC不仅可通过募集、整合的方式直接参与CNV的生成，还可以通过分泌血管生成因子的间接方式参与CNV的生成。

Objective To investigate the role and possible mechanism of endothelial progenitor cell （EPC） in the development of choroidal neovascularization （CNV）. Methods Experimental study. Twenty- four BN rats were divided into 3 groups. One eye of each animal was induced by laser photocoagulation with 532 nm laser and the contralateral eye was taken as control. Three, seven and fourteen days after photogoagulation the formation of CNV was observed by histopathological study and the recruitment of EPC and the possible pro-angiogenic growth factors released by EPC during the development of CNV were examined by multi-labeled immunofluorescence staining. The difference among the 3 groups was analyzed by ANOVA and the comparison between any 2 groups was further checked by LSD-t test. Results Both the histopathological study and the immunofluorescence staining indicated that within the lasered lesions proliferated and migrated ceils grew into the subretinal space through the broken Bruch membrane 3 days after photocoagulation,7 days after photocoagulation lumen-like structures were observed and CNV became stable until 14 days after photocoagulation. No EPC was observed in the normal retina whereas EPC were recruited into the lasered lesions 3 days after photocoagulation, comprising （ 79.29 ±11.27 ） % of the total endothelial cell population within CNV. At 7-day EPC constituted new vessels within CNV area and theproportion decreased to （47. 13 ± 5.78 ）%. Then its number decreased dramatically 14 days after photocoagulation contributing to （ 10. 83 ±2. 79） % of the endothelial cells in CNV. The differences either among the 3 groups （ F = 104. 623, P 〈 0. 05 ） or between any 2 groups （ P 〈 0. 05 ） were statistically significant. Moreover, triple labelled immunofluorescence staining showed that the EPC within CNV area could also secret pro-angiogenic factors such as vascular endothelial growth factor, IL-10, bFGF and MMP9. Conclusion EPC involves in the development of CNV not only through participating in the formation of new vessels within the CNV area but also through secreting pro-angiogenic factors.