目的:研究转化生长因子TGF-β2对体外培养人角膜内皮细胞(HCEC)增殖的影响机制。方法:体外培养经血清饥饿法获得同步化的HCEC,用不同浓度TGF-β2对HCEC进行不同时间的处理,采用细胞计数、MTT染色以及流式细胞仪(FCM)和RealtimePCR检测TGF-β2对HCEC的增殖、细胞周期及细胞中p27kip1和p21cip1表达的影响。结果:TGF-β21～15μg/L对HCEC有显著的增殖抑制作用,具有浓度和时间依赖性,9μg/L是TGF-β2抑制HCEC细胞增殖的峰浓度。9μg/LTGF-β2处理后,处于G1/G0期HCEC数量显著增加,并具有时间依赖性。9μg/LTGF-β2处理24h后,p27kip1表达量显著增加,48h后p27kip1和p21cip1的表达量均显著增加,也具有时间依赖性。结论:TGF-β2对HCEC具有显著的浓度和时间依赖性增殖抑制作用,可能通过先后诱导p27kip1和p21cip1表达量的增加将细胞拘留在G1/G0期来实现的。

·AIM:To investigate the inhibitory effect of transforming growth factor(TGF)-β2 on the proliferation of human corneal endothelial cells(HCEC)in vitro,and its cellular and molecular mechanisms.·METHODS:Synchronized HCEC,prepared from in vitro cultured HCEC line by serum-starvation,were treated with TGF-β2 at different concentration for different time duration,respectively.After HCEC treated with TGF-β2,the inhibitory effect of proliferation,phase status in cell cycle,and expression of p27kip1 and p21cip1 of HCEC was examined by methods of cell number counting,MTT staining,flow cytometry(FCM)and realtime PCR,respectively.·RESULTS:TGF-β2 at the concentration of 1-15μg/L had obvious inhibitory effects on HCEC proliferation in dose-and time-dependent manner,9μg/L was the peak concentration of TGF-β2 for inhibiting HCEC cell proliferation.After treated with 9μg/L TGF-β2,the population of HCEC in G1/G0 phase increased obviously in time-dependent manner.After HCEC treated with 9μg/L TGF-β2,the expression of p27kip1 increased obviously after 24 hours,and expression of p27kip1 and p21cip1 increased obviously 48 hours later in time-dependent manner.·CONCLUSION:TGF-β2 has obvious inhibitory effect on HCEC proliferation,and the inhibition is in dose-and time-dependent manner.The mechanism of TGF-β2 inhibition is most probably accomplished by inducing the in turn expression of p27kip1 and p21cip1 which keep HCEC rest on G1/G0 phase.·

R770.2

中国国家高技术研究发展863计划资助项目(No.2006AA02A132)~~