三氯乙烯诱导人L-02肝细胞适应性反应的差异蛋白质组学分析

Differential proteomic analysis of adaptive response induced by trichloroethylene in human L-02 hepatocytes

目的研究三氯乙烯(TCE)诱导L-02肝细胞的适应性反应及蛋白质表达改变。方法利用噻唑蓝(MTT)比色法,以0.5%二甲基亚砜(DMSO)作为溶剂对照组,确定对细胞增殖无明显作用的1μmo.lL-1TCE作为TCE诱导L-02肝细胞适应性反应的预刺激浓度,预刺激时间12 h,预刺激后再刺激浓度(适应组)为30μmo.lL-1TCE,刺激时间24 h。然后选取0,1,30和1+30μmo.lL-1TCE(1μmo.lL-1TCE预刺激12 h后,再次接受30μmo.lL-1TCE刺激24 h)4组平行进行双向凝胶电泳分离细胞总蛋白,银染显色,图像分析后,挑选差异蛋白进行基质辅助激光解吸电离飞行时间串联质谱鉴定。结果1μmo.lL-1TCE预刺激L-02肝细胞后,能降低30μmo.lL-1TCE攻击产生的细胞毒性。与单独用30μmo.lL-1TCE攻击相比,细胞增殖能力增加明显,细胞死亡减少。分析比较4组二维电泳图谱,发现15个蛋白质斑点表达发生改变,对其中5个差异较明显的蛋白点进行质谱分析鉴定,分别为Ku 86自体抗原相关蛋白1、透明质酸蛋白(hyaluronan)介导细胞运动受体、谷胱甘肽转移酶ω1、白细胞弹性蛋白酶抑制剂和空泡ATP合酶亚单位B。结论TCE可诱导L-02肝细胞产生适应性反应,引起L-02肝细胞蛋白表达改变,差异蛋白大多参与机体保护。

AIM To study the adaptive response and the proteome expression induced by trichloroethylene （ TCE ） in L-02 liver cells. METHODS Using MTT test to investigate the cytotoxicity of TCE in L-02 liver cells. 0.5% DMSO was used as solvent control. According to the concentration-time response relation of TCE in L-02 liver cells, 1μmol·L^-1 TCE was choosed as the preconditioning concentration for 12 h, and 30μmol·L^-1 TCE as the stimulating concentration for 24 h. Then four groups （treated L-02 with 0, 1, 30, 1 ＋ 30μmol·L^-1 TCE, respectively） were set for further study. The total cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining. Digital images were analyzed using ImageMaster 2D Platinum 5. 0 software. The differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry （ MALDI-TOF-TOF-MS ）. RESULTS Comparing with 30μmol·L^-1 TCE stimulation alone, 1μmol·L^-1 TCE preconditioning could reduce the cytotoxicity induced by 30 μmol·L^-1 TCE. Fifteen protein spots were found to express differentially among four groups, and 5 remarkably different expressed proteins were identified as KARP-1- binding protein, hyaluronan mediated motility receptor, glutathione transferase omega 1, leukocyte elastase inhibitor and vacuolar ATP synthase subunit B. CONCLUSION Adaptive response can be induced by low concentration TCE, it can change the proteome expression of L-02 liver cell. Most of the identified proteins participate in protecting human body.