NGF对人视网膜血管内皮细胞体外成管作用的影响  

NGF on canaliculization of human retinal vascular endothelial cells in vitro

作　　者：王丁丁[1] 于强[2] 陈子林[1] 柳夏林[2] 

机构地区：[1]广东省惠州市中心人民医院眼科,516001 [2]中山大学中山眼科中心,广东省广州市510060

出　　处：《眼科新进展》2009年第12期886-889,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30500554;30740078)~~

摘　　要：目的探讨神经生长因子(nerve growth factor,NGF)对人视网膜血管内皮细胞(human retinal vascular endothelial cells,HRVEC)体外形成毛细血管管腔的影响。方法20g.L-1胰蛋白酶消化人眼球视网膜组织,内皮细胞培养液培养HRVEC,第Ⅷ因子相关抗原抗体DAB染色法及荧光染色法进行HRVEC的组织化学鉴定。取第3代HRVEC接种于铺有基质胶(Matrigel)的24孔板(每孔200×103个细胞),分为100μg.L-1NGF、100μg.L-1NGF+200nmol.L-1K252a、50μg.L-1碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)、50μg.L-1bFGF+200nmol.L-1K252a和对照组(人内皮细胞培养液)共5组,1h后各组依次添加各种培养液200μL培养12h后观察照相,分析比较5组新生毛细血管结构及面积。结果接种后8d左右梭形HRVEC细胞铺满培养瓶,第Ⅷ因子相关抗原鉴定阳性;测量5组新生毛细血管面积依次为:(2.76±0.31)mm2、(1.79±0.62)mm2、(2.99±0.58)mm2、(2.86±0.61)mm2、(1.41±0.69)mm2;100μg.L-1NGF、50μg.L-1bFGF组HRVEC均形成完整的管状结构,小管面积均明显大于对照组(均为P<0.05);100μg.L-1NGF+200nmol.L-1K252a组小管结构完整性被打破,小管面积小于100μg.L-1NGF组(P<0.05);50μg.L-1bFGF+200nmol.L-1K252a组小管面积、结构与50μg.L-1bFGF组比较无明显改变(P>0.05)。结论NGF可在体外促进HRVEC形成新生毛细血管管腔,K252a可抑制NGF促进HRVEC形成新生毛细血管管腔的作用,但k252a不能阻断bFGF的作用。Objective To investigate the effects of nerve growth factor（NGF）on capillary canaliculization of human retinal vascular endothelial cells（HRVEC）in vitro.Methods Retinal tissues of human eyeballs were treated with 20 g·L-1 trypsin,HRVEC was cultured with endothelial cells culture fluid and histochemically identified with Ⅷ factor related antigen-antibody DAB staining and fluorescent staining.HRVEC at the 3rd generation was inoculated at 24 hole plate（200×10^3 cells at each hole）with matrigel,and divided into 100μg·L^-1 NGF group,100 μg·L^-1 NGF combined with 200 nmol·L^-1 K252a group,50 μg·L^-1 basic fibroblast growth factor（ bFGF） group,50 μg·L^-1 bFGF combined with 200 nmol·L^-1 K252a group and control group （human endothelial cells culture fluid）. One hour later, 200μL culture fluid was added to each group for 12 hours and photograph was taken. Structure and area of new blood capillaries were analyzed and compared among five groups. Results At 8 days after inoculation, fusiform shaped HRVEC spread the whole culture flask and the VIH factor related antigen was positive. The areas of new blood capillaries were （2.76± 0.31 ） mm2,（1.79±0.62）mm2, （2.99±0. 58）mm2, （2.86±0.61 ）mm2 and （ 1.41±0.69） mm2,respectively,in each group;In 100μg·L^-1 NGF group and 50μg·L^-1 bFGF group, HRVEC formed a complete canaliculization and the area of canaliculus was significantly more than that in control group （ both P 〈 0.05 ） ; The integrity of the structure of canaliculus was broken in 100 μg·L^-1 NGF combined with 200 nmol·L^-1 K252a group and the area of canaliculus was less than that in 100μg·L^-1 NGF group （P 〈0.05） ;No significant difference was found in structure and area of canaliculus between 50μg·L^-1 bFGF combined with 200 nmol·L^-1 K252a group and 50μg·L^-1 bF- GF group（P 〉 0. 05 ）. Conelusions NGF can promote the canaliculization of new blood capillary of HRVEC in vitro. K252a can inhibit the effects of NGF on HRVEC,but not inhib

关 键 词：神经生长因子 人视网膜血管内皮细胞 基质胶 K252A 

分 类 号：R776[医药卫生—眼科]