【摘 要】
:
目的 探讨三氯乙烯(TCE)的肝细胞毒性机制,以及筛选TCE相关的生物标志物。方法 运用2DE联合MALDI-TOF-TOF/MS鉴定差异蛋白,选择差异蛋白SET,联合使用Pull-Down串联亲和层析、液相色谱-串联质谱及Co-IP等,分离、鉴定并验证与SET相互作用的蛋白质。并运用血清蛋白质组学技术鉴定TCE致肝细胞损伤相关蛋白和TCE药疹样皮炎患者血清生物标志物。结果 发现TCE可诱导L-0
【机 构】
:
深圳市疾病预防控制中心现代毒理学重点实验室,广东深圳518055
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目的 探讨三氯乙烯(TCE)的肝细胞毒性机制,以及筛选TCE相关的生物标志物。方法 运用2DE联合MALDI-TOF-TOF/MS鉴定差异蛋白,选择差异蛋白SET,联合使用Pull-Down串联亲和层析、液相色谱-串联质谱及Co-IP等,分离、鉴定并验证与SET相互作用的蛋白质。并运用血清蛋白质组学技术鉴定TCE致肝细胞损伤相关蛋白和TCE药疹样皮炎患者血清生物标志物。结果 发现TCE可诱导L-02肝细胞SET蛋白表达上调和TCE诱导肝细胞具有低剂量适应性反应效应。为此,利用慢病毒介导的RNA干扰技术成功得到稳定干扰SET的肝细胞。随后,以正常肝细胞与SET缺陷肝细胞为受试细胞,探讨了SET改变与TCE刺激后细胞增殖、细胞凋亡、PP2A活性及胱天蛋白酶3活性等指标之间的相互关系,结果显示,慢病毒介导的SET的下调显著性降低了TCE在肝细胞中诱导的细胞毒性。丝切蛋白1,peroxiredoxin2和S100-A11可能在不同程度参与TCE毒作用下SET介导的肝细胞凋亡。同时,鉴定了62个与SET可能有相互作用的侯选蛋白,其中45个存在GO共注释;并验证了SET与eEF1A1/eEF1A2的相互作用以及TCE可改变其相互作用和亚细胞分布。鉴定了6个与TCE致肝损伤相关的抗原蛋白,并验证了NM23可作为TCE引起的自身免疫性疾病的潜在生物标志物。通过比较职业性三氯乙烯药疹样皮炎患者、三氯乙烯职业接触者和健康人群各自的血清肽谱,使用ClinProTools软件建立血清肽诊断模型。验证实验证明了诊断模型的高灵敏度和高特异性,并发现蛋白ABCA12和PRSS1在患者血清中出现明显的下调。结论 SET及其相关蛋白均可通过不同途径参与L-02细胞凋亡或增殖的进程,筛选了TCE毒性相关的可应用于诊断、治疗及预后监测的潜在生物标志物。
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