rhTGF-β1及转染TGF-β1基因对兔角膜内皮细胞增殖的影响  

Effects of rhTGF-β_1 and TGF-β_1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro

作　　者：马慧香[1] 徐锦堂[1] 江振友[2] 张穗梅[3] 赵松滨[1] 陈建苏[1] 

机构地区：[1]暨南大学医学院眼科研究室,广东广州510632 [2]暨南大学医学院微生物学与免疫学教研室,广东广州510632 [3]暨南大学医学院病理生理教研室,广东广州510632

出　　处：《中国病理生理杂志》2008年第5期915-919,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30371519);广东省重大科研资助项目(No.2001A302020102)

摘　　要：目的:探讨不同浓度的重组人转化生长因子-β1(rhTGF-β1)及TGF-β1基因转染对体外培养兔角膜内皮细胞增殖的影响。方法:用MTT法检测不同浓度rhTGF-β1作用下角膜内皮细胞的增殖。用脂质体介导转染方法,将TGF-β1基因转移入培养的兔角膜内皮细胞,HE染色法观察细胞组织形态学变化;ELISA法检测转染细胞培养上清中TGF-β1表达量;流式细胞仪检测细胞生长周期变化;DNA电泳法检测转染细胞凋亡情况。结果:MTT检测示5-20μg/LrhTGF-β1抑制角膜内皮细胞增殖;0.5-1μg/L组对增殖无影响;0.05～0.1μg/L组促进细胞增殖。TGF-β1基因转染细胞形态无明显异常,细胞培养上清中TGF-β1的浓度约为(98±3)ng/L。流式细胞仪检测示,基因转染组S期和G2/M期细胞比例减少、PI值降低,但加入EGF后细胞生长基本正常。DNA电泳检测示基因转染组未见凋亡带。结论:rhTGF-β1对角膜内皮细胞增殖的影响具有剂量依赖性;TGF-β1基因转染影响角膜内皮细胞增殖、但不诱发凋亡,其抑制作用可被外源性EGF拮抗。AIM： To investigate the effects of rhTGF-β1 and TGF-β1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro. METHODS ： Cell growth induced by various concentrations of rhTGF-β1 was determined by MTT proliferation assay. Under the induction of liposomes, recombinant pSecTag2 - TGF - β1MP vectors were transferred into the corneal endothelial cells. Morphological changes of transfected cells were observed by HE staining. The expression levels of TGF - β1 were assessed by ELISA. Cell cycle analysis was assessed by flow cytometry. DNA fragment analysis was used to confirm the presence of apoptosis. RESULTS： rhTGF - β1 in concentrations of 5 -20μg/L showed a significant suppressive effect on the proliferation of corneal endothelial cells, 0. 5 - 1μg/L had no effect, 0. 05 - 0. 1μg/L facilitated cell growth, as compared with negative controls. The morphous of transfected corneal cells had no significant abnormality compared with normal cells. According to the result of ELISA, the concentration of TGF - β1 in the supematant was calculated to be （98-3） ng/L. Flow cytometry assay showed that S and G2/M phase of transfected cells decreased significantly compared with that of control group, but the cell cycle recovered normally after adding 10μg/ L EGF into the culture medium. Agarose electrophoresis didn＇t show marked ladders in transfected group. CONCLUSION： Effects of rhTGF -β1 on the proliferation of corneal endothelial cells are different with various concentrations. TGF-β1 gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells, but does not in- duce cell apoptosis. EGF is the antagonist of this suppressive effect.

关 键 词：转化生长因子Β 基因转染 角膜内皮细胞 细胞增殖 

分 类 号：R363[医药卫生—病理学]