衰老标记蛋白30高表达对紫外线诱导人晶状体上皮细胞凋亡的影响

Effects of SMP-30 overexpression on apoptosis of human lens epithelial cells induced by ultraviolet B irradiation

目的 探讨衰老标记蛋白30（SMP-30）高表达对于紫外线B（UVB）诱导人晶状体上皮细胞凋亡的影响.方法 实验研究.以人晶状体上皮细胞系（HLE-B3）为模型,构建真核质粒pRFP-N1-SMP-30,利用脂质体转染的方法将其导入不同分组的细胞中,并以UVB刺激,用MTT法检测高表达的SMP-30对UVB诱导的HLE-B3细胞生存力的影响;利用细胞凋亡检测试剂盒测定高表达的SMP-30对UVB诱导的HLE-B3细胞凋亡的作用;并用免疫印迹法验证高表达的SMP-30对UVB诱导的凋亡相关蛋白表达的影响,通过活性氧自由基水平的检测分析高表达的SMP-30对UVB诱导的HLE-B3细胞活性氧表达的作用.数据的分析选择方差分析联合Dunnett的统计学方法.结果DNA测序证实插入的序列正确,证明pRFP-N1-SMP-30质粒构建成功.UVB照射后SMP-30高表达组HLE-B3细胞生存率为0.90±0.14,细胞的凋亡率为0.43±0.06,活性氧的水平0.52±0.02,与对照组相比差异均有统计学意义（t=5.830,9.934,12.19;P〈0.05）.UVB处理后,SMP-30高表达可显著下调Bax和cleav-caspase-3的表达,同时上调Bcl-2和Pro-caspase-3的表达.结论 高表达的SMP-30通过调控凋亡相关蛋白的表达及抑制活性氧的产生来缓解UVB照射导致的人晶状体上皮细胞的凋亡,从而发挥对晶状体上皮细胞的保护作用.

Objective This study was to observe the effect of SMP-30 on ultraviolet B （UVB）-induced apoptosis of human lens epithelial cells（HLE-B3） in vitro. Methods Experimental study. The SMP-30 cDNA was amplified by PCR and inserted into the pRFP-N1 expressing vector which had been double digested by XhoI/HindIII. HLE-B3 cells were cultured and divided into three groups：normal group, pRFP-N1 vector plasmid group and pRFP-N1-SMP-30 plasmid group （SMP-30）. Then cells were exposed to UVB and the survival rate of cells was detected by MTT assay. The effects of SMP-30 on UVB-induced HLE-B3 apoptosis were measured by the Cell Death Detection ELISA kit. Meanwhile, the influence of SMP-30 on UVB-induced apoptosis-relative protein expression in HLE-B3 cells was tested by Western blots. Moreover, 2′, 7′-Dichlorofluorescin diacetate staining was performed to monitor the protective effects of SMP-30 on UVB-induced HLE-B3 reactive oxygen species（ROS）. One-way analysis of variance combined with Dunnett＇s statistical method were performed to analyze the data. Results The full length of PSF cDNA fragment was correctly inserted into the pRFP-N1 vector, which was confirmed by DNA sequencing. The SMP-30 fragment was inserted to the plasmid pRFP-N1 correctly, which was also confirmed by DNA sequencing. The PRFP-N1-SMP-30 plasmid was transfected into HLE-B3 successfully. SMP-30 expression was up-regulated in the transfection group, compared with the control group. Data showed that the survival rate of HLE-B3 after the pRFP-N1-SMP-30 plasmid transfection was 0.90±0.14, while the apoptosis rate was 0.43 ± 0.06 and the ROS production was 0.52 ± 0.02, showing significant difference in comparison with the vector plasmid group and the normal group（t=5.830, 9.934, 12.19, P〈0.05）. In the meantime, SMP-30 overexpression down-regulated the levels of Bax and cleav-caspase-3, but up-regulated the Bcl-2 and Pro-caspase-3 expression levels under UVB irradiation. Conclusion SMP-30 overexpression plays a protective role in UVB-induced apoptosis via regulating the expression of apoptosis-related proteins and inhibiting the production of ROS in HLE-B3 cells.