紫外辐射诱导人晶状体上皮细胞凋亡机制中TGF-β2/Smad-4细胞信号传导通路的作用

Effect of TGF-β2 signaling pathway in ultraviolet B-induced cellular apoptosis of human lens epithelial cells

目的了解转化生长因子β2(TGF-β2)/Smad-4细胞信号传导通路是否参与B波段紫外线(UVB)辐射诱导人晶状体上皮细胞系(HLECs)的凋亡过程,探讨HLEC紫外线损伤的可能保护机制。方法HLECs预先在含0.01μg/ml AF-302-NA的培养基中孵育24h,行480mW/cm2的UVB照射。采用流式细胞仪(FCM)和TUNEL方法检测细胞凋亡,采用RT-PCR检测TGF-β受体(TβRs)和bax mRNA表达水平的变化,免疫荧光染色方法进行Smad-4蛋白定位。结果UVB照射组HLECs凋亡比例明显高于对照组;TβRs和凋亡促进子bax的mRNA表达水平增高,UVB照射后Smad-4表达量明显升高,并从HLECs细胞胞浆转移到细胞核内;AF-302-NA可以抑制UVB引起的Smad-4由细胞浆向细胞核的转移,部分抑制UVB照射引起的TβRs和bax mRNA表达量的增高,并可降低HLECs的凋亡比例。结论在UVB诱导HLECs凋亡机制中,凋亡信号通过TGF-β2/Smad-4信号传导通路传递,阻断TGF-β2/Smad-4信号传导通路可以在一定程度上抑制UVB照射所引起的HLECs凋亡。

Objective To investigate the signaling characteristics of transforming growth factor-β2 （TGF-β2） and Smads signal pathway in ultraviolet-B（UVB） irradiation-induced human lens epithelial cells （HLECs）apoptosis. Methods HLECs line was cultivated and passaged in DMEM medium containing 20% FBS and incubated for 24 hours in the medium with or without 0. 01 μg/ml AF-302-NA（a monoclonal anti-TGF-β2 neutralization antibody）prior to exposure to 480 mW/cm^2 UVB. HLECs in control group were not irradiated and stimulated by UVB and AF-302-NA. Cell apoptosis was assessed by immunocytochemistry using TUNEL and flow cytometer using Annexin V-FITC apoptosis detection. Gene expression was evaluated using RT-PCR. Smad-4 was localized by immunocytochemistry. Results In blank control and AF-302-NA groups, nearly no TUNEL positive HLECs were found;while in UVB group, a lots of apoptotic positive cells were seen. A few apoptotic cells were found in UVB ＋ AF-302-NA group. UVB-irradiation induced the accumulation of Smad-4 in HLECs nucleus and sequentially enhanced the expression of apoptotic gene,bax,in HLECs. Translocation of Smad-4 to cell nucleus was inhibited in UVB ＋ AF-302-NA group. TβRs mRNA was not expressed in blank control group but little expression in AF-302-NA group. After UVB irradiation,the expression of TI3Rs mRNA was increased and showed a higher level in UVB ＋ AF-302-NA group. However,expression of TβRⅢ mRNA was inhibited by AF302-NA. Expression of bax mRNA was significantly decreased in HLECs in UVB ＋ AF-302-NA group. Conclusion TGF-132 signal pathway partially transmits apoptotic signal and might be an initiator of cellular apoptosis of HLECs after UVB irradiation. Interdiction of TGF-β2 signal pathway could partially protect HLECs from apoptosis induced by UVB-irradiation in vitro.