HBV X蛋白即时高表达上调巨噬细胞分泌TNF-α和IL-1β

HBV X protein upregulates secretion of TNF-α and IL-1β by macrophages

目的:将乙型肝炎病毒(HBV)X蛋白真核表达载体pcDNA3.1(+)-HBx转染巨噬细胞,观察X蛋白即时高表达对巨噬细胞分泌细胞因子TNF-α、IL-1β的影响,为进一步研究HBVX蛋白的致病机制奠定实验基础。方法:将真核表达载体pcDNA3.1(+)-HBx通过SuperFectTM脂转染试剂转染巨噬细胞24小时后,用LPS刺激巨噬细胞,利用Westernblot鉴定X蛋白在巨噬细胞中的表达,并用酶联免疫吸附试验(ELISA)检测pcDNA3.1(+)-HBx转染的巨噬细胞不同时间(12、24、48小时)培养上清液中细胞因子TNF-α、IL-1β的含量,统计软件SPSS13.0分析所得数据。结果:将真核表达载体pcDNA3.1(+)-HBx转染巨噬细胞,Westernblot结果显示pcDNA3.1(+)-HBx能在巨噬细胞中表达约17kD的目的蛋白,即X蛋白;ELISA法测定细胞上清液中TNF-α、IL-1β的含量,LPS刺激组及pcDNA3.1(+)组与空白对照组有显著性差异(P<0.01),pcDNA3.1(+)-HBx组与LPS刺激组及pcDNA3.1(+)组有显著性差异(P<0.01),而LPS刺激组与pcDNA3.1(+)组无显著性差异(P>0.05),即LPS可活化巨噬细胞分泌细胞因子,且X蛋白即时高表达可引起LPS诱导的巨噬细胞分泌TNF-α和IL-1β明显增加。结论:乙型肝炎病毒X蛋白即时高表达上调LPS刺激的巨噬细胞分泌TNF-α和IL-1β。

Objective：To provide evidence for further studying the pathogenic mechanism of Hepatitis B virus （HBV） X protein （ HBx）, HBx effect on cytokine secretion by macrophages was studied. Methods ： Reconstructed plasmid pcDNA3. 1 （ ＋ ） -HBx was transfected into macrophages. Expression of X gene were assayed in macrophages lysate by Westem blot, and the concentration of TNF- ot or IL-113 was detected respectively by ELISA. All of data were analyzed by SPSS13.0. Results： In THP-1 cells, the pcDNA3. 1 （ ＋ ） -HBx plasmid expressed X protein with molecular weight of 17KD by Westem blot. The secretory volume of TNF-α or IL-1β from macrophages was determined by ELISA, the resuhs from analysis of all groups showed as following： control group was different from LPS group and pcDNA3.1 （ ＋ ） group （ P 〈 0.01 ）, and so was pcDNA3.1 （ ＋ ） -HBx group ; but there was no obvious difference between pcDNA3.1 （ ＋ ） group and LPS group（ P 〉 0.05 ）, all of which indicated that transient overexpression of HBX enhanced LPS-induced product of TNF-α and IL-1β by macrophages. Conclusion：Transient overexpression of HBx up regulated LPS-induced TNF-α and IL-1βsecretion in macrophages.