三甲基氯化锡诱导的非洲绿猴肾细胞蛋白质表达谱改变

The protein expression profiles induced by trimethyltin chloride in vero cells

目的分析三甲基氯化锡（trimethyhinchloride，TMT—C1）诱导的非洲绿猴肾细胞（vero细胞）和正常vero细胞之间差异表达蛋白质，以寻找相关的标志物，探讨TMT—C1所致肾损伤的分子机制。方法通过双向凝胶电泳和液相色谱一电喷雾线性离子阱质谱技术比较TMT．C1染毒的vem细胞和正常veto细胞之间蛋白质表达谱的差异，蛋白免疫印迹技术分析其中的2个差异蛋白AnnexinA1和et6-Tubulin的表达以验证双向电泳的结果，反转录荧光定量聚合酶链式反应技术进一步分析AnnexinAl和仅6一Tubulin在mRNA水平的表达。结果比较TMT—C1染毒的vero细胞和正常vero细胞蛋白质表达谱，共获得15个差异蛋白点，9个通过质谱分析得到鉴定，3个上调，6个下调。与对照组比较，各TMT．C1染毒组d6一Tubulin蛋白质和mRNA表达水平均下调，差异有统计学意义（P〈O．01）。与对照组比较，各染毒剂量组AnnexinAl蛋白表达水平均上调，25和50p．μmol／L组mRNA表达水平下调，100μmol／L组的mRNA表达水平上调，差异均有统计学意义（P〈0．01）。结论双向凝胶电泳结合质谱分析筛选到的9个差异表达蛋白与TMT．C1所致肾细胞毒性密切相关，可作为早期诊断、治疗及疗效评价的候选生物标志物。

Objective To explore the biomarkers and mechanism of kidney toxicity induced by trimethyltin chloride （TMT-C1） through analyzing the differences of protein expression profiles between vero cells and vero cells exposed to TMT-C1. Methods The differences of protein expression levels of three paired samples of vero cells and veto cells exposed to TMT-C1 were compared by two-dimensional gel electrophoresis （2-DE） and liquid chromatography-electrospray inonization-linear trap quadrupole （LC-ESI-LTQ）. The differ- ences of expression levels of Annexin Alandc^-Tubulin proteins were validated with western blot assay, and the differences of mRNA expression levels of Annexin Alandc^-Tubulin genes were detected with quantitative re- verse transcriptase-polymerase chain reaction （qRT-PCR）. Results Fifteen spots of differential expression in protein profiles between vero ceils and vero cells exposed to TMT-C1 were found, and 9 of these spots were i- dentified by LC-ESI-LTQ. The expression levels of 3 proteins （Annexin Al,similar to RAN protein and a hy- pothetical protein） increased and the expression levels of 6 proteins （growth factor receptor-bound protein 10, tubulin alpha 6,heterogeneous nuclear ribonucleoprotein, similar to elongation factor SIII p15 subunit, S- adenosylhomocysteine hydrolase and a hypothetical protein） reduced. The expression levels of c^-Tubulin pro- tein and mRNA significantly decreased in vero cells exposed to TMT-C1, as compared with vero cells （P〈 0.01 ） . The expression of Annexin A1 protein in all exposure groups was significantly up-regulated, the ex- pression of Annexin A1 mRNA in the groups exposed to 25 and 50p^mol/L TMT-C1 was significantly down- regulated, and The expression of Annexin A1 mRNA in the group exposed to 100 Ixmol/L TMT-C1 was signifi- cantly up-regulated （P〈0.Ol）. Conclusions The results of present study suggest that 9 proteins with differen- tial expression detected by LC-ESI-LTQ may be related to the kidney toxicity induced by TMT-C1, which can serve as the biomarkers of early diagnosis and theraoeutic effect for the kidney toxicity induced by TMT-C1.