TGF-β_2体外抑制牛角膜内皮细胞增殖的研究  

Inhibitory effect of transforming growth factor-β_2 on bovine corneal endothelial cells in vitro

作　　者：陆秀兰[1] 张明昌[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院眼科,中国湖北省武汉市430022

出　　处：《国际眼科杂志》2009年第6期1058-1060,共3页International Eye Science

摘　　要：目的:观察体外不同浓度的转化生长因子β2(transforming growth factor-β2,TGF-β2)对牛角膜内皮细胞(bovine corneal endothelial cells,bCECs)的抑制作用,探讨其可能的分子机制。方法:培养新生小牛的角膜内皮细胞,将第3代内皮细胞转移至96孔培养板,实验分6组,每组设定4个复孔。1,2组分别为只含有培养液不含细胞和TGF-β2的空白对照组,和只含细胞和培养液不含TGF-β2的阴性对照组,3～6组分别加入含0.1,1,10,100ng/LTGF-β2的培养液。作用24,48,72h后用MTT检测法测定各孔吸光度A值。作用48h后,用流式细胞检测仪对各组细胞周期进行检测。RT-PCR法测定不同浓度的TGF-β2组p27Kip1基因的表达情况。结果:TGF-β2浓度为0.1～1ng/L,作用24～72h,能明显的使bCECs的吸光度发生改变,作用48h抑制效果最明显。0.1和1ng/L浓度组吸光度与阴性对照组相比有显著差异性,作用48h可使bCECs的G0/G1周期细胞所占比例与阴性组对比有显著差异,其p27Kip1表达较其他组明显增强。结论:0.1～1ng/L TGF-β2作用48h对bCECs具有显著增殖抑制作用,其抑制作用可能是通过上调细胞周期蛋白p27来实现的。AIM： To observe the inhibitory effect of transforming growth factor-β2 （ TGF-β2 ） on the bovine corneal endothelial cells （bCECs） in vitro and investigate its possible molecular mechanism. METHODS： MTT assay： the 3nd passage bCECs were treated with different concentrations of TGF-132 （0.1,1,10, 100ng/L）. The cells were cultured in three 96-wells plates for 24 ,48 ,72 hours respectively. There were 6 groups including a vacant control group and a negative control group in each plate. The inhibitory effect of TGF-β2 on the bCECs growth was measured by the value of absorption of 3-（ 4, 5-dimethvlthiazozyl ）-2, 5-diphenyl tetrazodium bromide（MTT） 24, 48, 72 hours after the treatment. Flow cytometer（FCM）： The cell cycle of each group were tested by FCM 48 hours after the treatment. RT-PCR： the bCECs were cultured in vitro and incubated for 48 hours , then collected for RNA extraction and reserve transcription. The primer was designed for bovine p27Kip1 based on its gene sequence. The expression of gene p27Kip1 was detected by RT-PCR. RESULTS： The 0.1-1 ng/L group represented signifi-cant difference compared with the negative control group and the other groups at the same testing time. FCM ： the 0.1 and lng/L group represented significant difference of the G0/G1 phase cells ration compared with the negative control group. The 1ng/L group represented the significant difference with the 10 and 100ng/L group, while there was no significant difference between the 0.1 and lng/L group. The expression of p27Kip1 is stronger in all the TGF-β2 groups , while the 0.1 and 1ng/L group show the significant difference compared with the negative control group. CONCLUSION：TGF-β2 at the concentration of 0.1-1 ng/L can inhibit the proliferation of bCECs significantly after 48h incubation ,and it might be associated with the upgrading expression of p27Kip1.

关 键 词：转化生长因子Β2 牛角膜内皮细胞 抑制 P27KIP1 

分 类 号：R772[医药卫生—眼科]