摘要
目的:探讨血小板源性生长因子受体α(PDGFRα)对过氧化氢(H_2O_2)诱导的黑素细胞凋亡的影响。方法:以黑素细胞PIGI为研究对象,分别用不同浓度的H_2O_2作用后,用MTT法检测细胞活力,并计算半数抑制浓度。经H_2O_2处理的PIGI细胞转染空载体p CMV6或PDGFRα过表达载体p CMV6-PDGFRα,以不做转染的细胞为空白对照,RT-q PCR和Western blot检测转染效率。用半数抑制浓度的H_2O_2作用于过表达PDGFRα的PIGI细胞,MTT法检测细胞活力,绘制细胞生长曲线;流式细胞术检测细胞凋亡情况;Western blot检测细胞中p38、磷酸化p38(p-p38)和cleaved caspase-3的蛋白水平;二氯二氢荧光素二乙酸酯(DCDHF-DA)法检测细胞中活性氧簇(ROS)水平。结果:H_2O_2作用后PIGI细胞生长减慢,细胞凋亡率升高,细胞中ROS水平、p-p38和cleaved caspase-3蛋白水平升高(P<0.05),H_2O_2的半数抑制浓度为0.7 mmol/L。PDGFRα过表达载体能够成功上调PIGI细胞中PDGFRα的mRNA和蛋白水平(P<0.05)。过表达PDGFRα后经过H_2O_2处理的PIGI细胞存活率升高(P<0.05),细胞凋亡率下降(P<0.05),细胞中的ROS水平下降(P<0.05),p-p38和cleaved caspase-3蛋白水平降低(P<0.05)。结论:PDGFRα能够抑制H_2O_2诱导的黑素细胞凋亡,部分逆转H_2O_2对黑素细胞增殖的抑制作用,降低细胞中的ROS水平,其作用机制可能与p-p38和cleaved caspase-3蛋白水平的变化有关。
AIM：To investigate the effects of platelet-derived growth factor receptorα（PDGFRα）on melanocyte apoptosis induced by hydrogen peroxide（H2O2）.METHODS：Melanocyte PIGI was used as the research object.After exposed to H2O2at different concentrations,the cell viability was detected by MTT assay.The PIGI cells were transfected with empty vector p CMV6 or PDGFRαover-expression vector p CMV6-PDGFRα.The transfection efficiency was determined by RT-q PCR and Western blot.The effect of H2O2on the viability of the PIGI cells after over-expression of PDGFRαwas measured by MTT assay.The cell apoptosis was analyzed by flow cytometry.The protein levels of p38,p-p38 and cleaved caspase-3 in the cells were detected by Western blot.DCDHF-DA was used to estemate the generation of reactive oxygen species（ROS）in the cells.RESULTS：The viability of PIGI cells decreased after exposed to H2O2（P〈0.05）,and the half maximal inhibitory concentration of H2O2was 0.7 mmol/L.Transfection with PDGFRαover-expression vector successfully induced high expression of PDGFRαat mRNA and protein levels in the PIGI cells,and increased the viability of the cells with H2O2treatment（P〈0.05）.Over-expression of PDGFRαdecreased the apoptotic rate of PIGI cells treated with H2O2（P〈0.05）,and the level of ROS in the cells（P〈0.05）.The protein levels of cleaved caspase-3 and pp38 were also decreased（P〈0.05）.CONCLUSION：PDGFRαinhibits the apoptosis of melanocytes induced by H2O2,partially reverses the growth inhibition of melanocytes by H2O2,and decreases the ROS level.The mechanism may be related to regulating the protein levels of p-p38 and cleaved caspase-3 in the cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第11期2060-2066,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81500173)
