CAJ | 학술논문

目的分析三甲基氯化锡(trimethyltin chloride,TMT-Cl)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-Cl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-电喷雾线性离子阱质谱技术比较TMT-Cl染毒的vero细胞和正常vero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Annexin A1和α6-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Annexin AI和α6-Tubulin在mRNA水平的表达.结果比较TMT-Cl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-Cl染毒组α6-Tubulin蛋白质和mRNA表达水平均下调,差异有统计学意义(P＜0.01).与对照组比较,各染毒剂量组Annexin A1蛋白表达水平均上调,25和50 μmol/L组mRNA表达水平下调,100 μmol/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-Cl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.
목적 분석삼갑기록화석(trimethyltin chloride,TMT-Cl)유도적비주록후신세포(vero세포)화정상vero세포지간차이표체단백질,이심조상관적표지물,탐토TMT-Cl소치신손상적분자궤제.방법 통과쌍향응효전영화액상색보-전분무선성리자정질보기술비교TMT-Cl염독적vero세포화정상vero세포지간단백질표체보적차이,단백면역인적기술분석기중적2개차이단백Annexin A1화α6-Tubulin적표체이험증쌍향전영적결과,반전록형광정량취합매련식반응기술진일보분석Annexin AI화α6-Tubulin재mRNA수평적표체.결과 비교TMT-Cl염독적vero세포화정상vero세포단백질표체보,공획득15개차이단백점,9개통과질보분석득도감정,3개상조,6개하조.여대조조비교,각TMT-Cl염독조α6-Tubulin단백질화mRNA표체수평균하조,차이유통계학의의(P＜0.01).여대조조비교,각염독제량조Annexin A1단백표체수평균상조,25화50 μmol/L조mRNA표체수평하조,100 μmol/L조적mRNA표체수평상조,차이균유통계학의의(P＜0.01).결론 쌍향응효전영결합질보분석사선도적9개차이표체단백여TMT-Cl소치신세포독성밀절상관,가작위조기진단、치료급료효평개적후선생물표지물.
Objective To explore the biomarkers and mechanism of kidney toxicity induced by trimethyltin chloride (TMT-Cl) through analyzing the differences of protein expression profiles between vero cells and vero cells exposed to TMT-Cl.Methods The differences of protein expression levels of three paired samples of vero cells and vero cells exposed to TMT-Cl were compared by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-electrospray inonization-linear trap quadrupole (LC-ESI-LTQ).The differences of expression levels of Annexin A1 andα-Tubulin proteins were validated with western blot assay,and the differences of mRNA expression levels of Annexin A1 and-Tubulin genes were detected with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results Fifteen spots of differential expression in protein profiles between vero cells and vero cells exposed to TMT-Cl were found,and 9 of these spots were identified by LC-ESI-LTQ.The expression levels of 3 proteins (Annexin A1,similar to RAN protein and a hypothetical 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,Sadenosylhomocysteine hydrolase and a hypothetical protein) reduced.The expression levels of α-Tubulin protein and mRNA significantly decreased in vero cells exposed to TMT-Cl,as compared with vero cells (P＜0.01 ).The expression of Annexin A1 protein in all exposure groups was significantly up-regulated,the expression of Annexin A1 mRNA in the groups exposed to 25 and 50μmol/L TMT-Cl was significantly downregulated,and The expression of Annexin AI mRNA in the group exposed to 100 μmol/L TMT-Cl was significantly up-regulated (P＜0.01).Conclusions The results of present study suggest that 9 proteins with differential expression detected by LC-ESI-LTQ may be related to the kidney toxicity induced by TMT-Cl,which can serve as the biomarkers of early diagnosis and therapeutic effect for the kidney toxicity induced by TMT-Cl.