基于iTRAQ联合2D LC-MS/MS分析PM2.5对人类胎盘滋养细胞的影响

【摘 要】 目的：利用蛋白组学方法初步筛选PM2.5通过影响人类胎盘滋养细胞造成不良妊娠率增加可能涉及的蛋白分子和生物进程。方法：用0、30、60、120和200 μg/mL的PM2.5分别染毒HTR-8/SVneo细胞24 h与48 h后观察细胞增殖情况，最终确定以120 μg/mL PM2.5染毒细胞24 h和48 h进行后续实验。提取胞内蛋白，酶解消化后，同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation，iTRAQ）标记联合二维液相色谱串联质谱（2D LC-MS/MS）技术分析肽段，鉴定HTR-8/SVneo细胞内的差异表达蛋白，并进行生物信息学分析。结果：120 μg/mL PM2.5染毒24 h和48 h后的存活率分别为86.09%和52.36%，与对照组（0 μg/mL PM2.5染毒组）相比，细胞存活率显著下降（P＜0.05）。120 μg/mL PM2.5 24 h染毒组和48 h染毒组细胞中分别鉴定出182个和486个差异蛋白，涉及细胞生物进程22条和217条。结论：PM2.5对HTR-8/SVneo细胞的影响涉及大量细胞生物进程的改变；iTRAQ联合2D LC-MS/MS技术是一种对染毒后细胞内差异表达蛋白高敏感度、高通量筛选的有效途径，为今后对PM2.5增加不良妊娠结局风险的深入机制研究奠定基础。

Effects of PM2.5 on Human Placental Trophoblast Cells by iTRAQ Combined with 2D LC-MS/MS

Objective:To preliminarily screened the protein molecules and biological processes that may be involved in PM2.5 increases the rate of adverse pregnancy by affecting the placental trophoblast cells using proteomic technology.Methods:HTR-8/SVneo cells were exposed to 0,30,60,120 and 200 μg/mL PM2.5 for 24 h and 48 h,respectively,to observe the cell proliferation;finally,we selected 120 μg/mL PM2.5 treated with HTR-8/SVneo cells for 24 h and 48 h for follow-up experiments.After extraction of intracellular protein and enzyme digestion,peptides and differentially expressed proteins were analyzed and identified by isobaric tags for relative and absolute quantitation (iTRAQ) combined with two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) technology.Finally,bioinformatics analysis of the data was carried out.Results:The survival rates of cells treated with 120 μg/mL PM2.5 for 24 h and 48 h were 86.09％ and 52.36％,respectively,which were also remarkably decreased compared with that of control cells (P＜0.05).There were 182 differentially expressed proteins in the cells exposure to 120 μg/mL PM2.5 for 24 h,involved in 22 cell biological processes (BP);meanwhile,cells exposed for 48 h had 486 differential proteins,involving 217 BP.Conclusions:PM2.5 can affect a large number of BP in HTR-8/SVneo cells.iTRAQ with LC-MS/MS 2D technology is an effective way for high sensitivity and throughput screening of differentially expressed proteins in cells after exposure to PM2.5,which provides a basis for the future research on the mechanism of PM2.5 increased the risk of adverse pregnancy outcomes.