肝脓肿患者细胞角蛋白-19表达及其调控肝细胞DNA损伤的机制研究

Study on the expression of cytokeratin-19 in patients with liver abscess and its mechanism of regulating hepatocyte DNA damage

目的检测细胞角蛋白-19(CK19)在肝脓肿患者外周血中的表达,及其对肝细胞损伤的影响及机制研究。方法电化学发光法和酶联免疫吸附法检测肝脓肿患者外周血中细胞角蛋白19片段21-1(CYFRA21-1)与CK19的表达水平,运用RNA干扰技术在人WRL68肝细胞中特异性沉默CK19基因,通过荧光定量PCR(qRT-PCR)和蛋白质印迹(w estern blot)检测转染效果,CCK-8法和Annexin V-FITC/PI双染法分别检测不同处理下WR1I68细胞存活率和凋亡率,单细胞凝胶电泳技术分析乙醇诱导DNA损伤程度;试剂盒检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)水平。结果与健康人比较,肝脓肿患者血清中CYFRA21-1和CK19水平显著升高(P<0.05);转染siRNA-CK19的WR168细胞中CK19mRNA与蛋白相对表达水平显著下降(P<0.05);在不同浓度乙醇处理下WRL68细胞存活率显著下降(P<0.05);沉默CK19表达的WRL68细胞在乙醇处理后存活率有所上升(P<0.05),细胞凋亡率下降(P<0.05),同时减少了乙醇诱导的DNA损伤(P<0.05);此外,沉默CK19表达后可逆转乙醇诱导的WRL68细胞内SOD和GSH-Px活性降低以及MDA含量的升高(P<0.05)。结论在WRI68细胞中沉默CK19表达能减少乙醇诱导的DNA损伤,这种作用可能与改善细胞SOD、MDA和GSH-Px水平相关。

Objective To detect the expression of cytokeratin-19(CK19)in the peripheral blood of patients with liver ab-scess,and its effect on hepatocyte injury and its mechanism.Methods Electrochemiluminescence and enzyme-linked immunosorbent assay were used to detect the levels of eytokeratin 19 fragment 21-1(CYFRA21-1)and CK19 in the peripheral blood of patients with liver abscess,RNA interference was conducted to speeifically silence the CK19 gene in human WRL68 hepatocytes,,fluorescence quan-titative PCR(qRT-PCR)and Western blot were performed to detect the transfection ffect,CCK-8 method and Annexin V-FTTC/PI double staining method were used to deteet the survival rate and apoptosis rate of WRL68 cells under diferent treatments,single cell gel electrophoresis was utilized to analyze ethanol-induced DNA damage,activities of superoxide dismutase(SOD),glutathione peroxi-dase(GSH-Px)and contentofmalondialdehyde(MDA)were evaluated by the kis.Results Compared with healthy people,the serum levels of CYFRA21-1 and CK19 in patients with liver abscess were significantly increased(P<0.05);The relative expression levels of CK19 mRNA and protein in WRL68 cells transfected with siRNA-CK19 was decreased significantly(P<005);The survival rate of WRL68 cells was decreased significantly under different concentrations of ethanol(P<0.05);After ethanol treatment,siRNA-CK19 increased the survival rate of WRL68 cells(P<0.05),decreased the apoptosis rate(P<0.05),and reducedthe ethanol-induced DNA damage(P<0.05);In addition,silencing CK19 reversed ethanol-induced reduction of SOD and GSH Px activity and ethanol-in-ducedincrease of MDA content in WRL68 cells(P<0.05).Conclusion Silencing CK19 can reduce ethanol-induced DNA damage of WRL68 cells.This efet may be related to the improvementof SOD,MDA and GSH-Px.