pcDNA3-HBV瞬时转染对巨噬细胞活性的影响  

Effects on macrophages activity transfected transiently with pcDNA3-HBV

作　　者：高立芬[1] 孙汶生[1] 马春红[1] 张利宁[1] 曹英林[1] 刘素侠[1] 梁晓红[1] 刘华[1] 

机构地区：[1]山东大学医学院免疫学研究所,济南250012

出　　处：《中华传染病杂志》2005年第2期95-98,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金委海外青年学者合作研究基金资助项目(30128023);国家自然科学基金资助项目(30070341)

摘　　要：目的利用HBV全基因真核细胞表达载体pcDNA3-HBV研究HBV感染后对巨噬细胞活性的影响,探讨慢性乙型肝炎患者免疫耐受发生的机制。方法常规制备小鼠腹腔巨噬细胞,分别将pcDNA3-HBV、pcDNA3质粒DNA与绿色荧光蛋白(GFP)的质粒DNApEGFPN1以9∶1混合,利用脂质体转染试剂盒,共转染小鼠腹腔巨噬细胞。转染后72h,半定量逆转录聚合酶链反应(RTPCR)检测HBV前S1、TNFα、白介素(IL)-1βmRNA的表达,流式细胞术测GFP的表达强度、检测核因子κB(NF-κB)蛋白的表达,利用Griess反应检测转染前后培养上清液中NO的水平。结果流式细胞术结果表明,pcDNA3-HBV与pcDNA3转染的巨噬细胞中GFP的表达率无明显差异,pcDNA3-HBV转染的巨噬细胞中检测到前S1mRNA的表达。通过与βactin相比,pcDNA3-HBV转染的巨噬细胞中TNF-α、IL-1βmRNA的表达量显著低于空载体对照组(P<0.01);pcDNA3HBV转染的巨噬细胞表达NFκ-BRelA蛋白的百分数与空载体对照组相当,但平均荧光强度显著低于空载体对照组(pcDNA3HBV转染组为125.05;pcDNA3转染组为203.88);pcDNA3-HBV转染组巨噬细胞产生NO的水平显著低于pcDNA3转染组(分别为15.0±0.6,45.0±1.3,P<0.01)。结论pcDNA3HBV转染后使巨噬细胞NFκB活性显著降低,TNF-α、IL-1βmRNA的表达及NO的产生水平明显下降。Objective To explore the mechanism of immune tolerance in chronic B hepatitis, the effects of HBV infection on the functions of macrophages and related mechanism of signal transduction by transfection in vitro were studied. Methods Peritoneal macrophages of mice were isolated regularly and cultured, transfected transiently with pcDNA3-HBV or pcDNA3 plasmid DNA and cultured under the stimulation of LPS. After 72 h, RT-PCR was performed to detect the expression of PreS1, TNF-α or IL-1β mRNA. To detect the expression of NF-κB RelA protein by FCM, and the level of nitric oxide in cultural supernatant was measured with Griess reaction. Results After being transfected with pcDNA3-HBV,peritoneal macrophages had the expression of PreS1 mRNA, but have lower level of TNF-α、IL-1β mRNA and transcriptional factor NF-κB, compared with pcDNA3-transfected control group; the level of nitric oxide in pcDNA3-HBV group was also decreased. Conclusions Transient transfection of pcDNA3-HBV could decrease the function of macrophages directly by inhibiting NF-κB activity and effector molecules production, which may be one of the mechanisms of immune tolerance in chronic B hepatitis.

关 键 词：巨噬细胞活性 瞬时转染 半定量逆转录聚合酶链反应 慢性乙型肝炎患者 小鼠腹腔巨噬细胞 PCDNA3 HBV转染 真核细胞表达载体 β-actin 流式细胞术 IL-1β mRNA HBV全基因 绿色荧光蛋白 平均荧光强度 免疫耐受 质粒DNA 脂质体转染 

分 类 号：R512.62[医药卫生—内科学]