摘要
目的乙肝病毒全基因组体外扩增,构建质粒并测序,酶切后瞬时转染HepG2细胞,观察对HBV抗原分泌,病原识别受体Toll样受体3(TLR3)的表达及细胞增殖的影响。方法长片段PCR法扩增HBVDNA全长,构建pBlueScript-HBV质粒并转化宿主菌,摇菌后提取质粒经测序,用SapⅠ酶切,获得HBV3.2kb基因组,采用脂质体瞬时转染HepG2细胞株,IMX检测HBV所分泌抗原,台盼蓝计数检测细胞增殖活性,直接免疫荧光流式细胞术(FCM)检测表达TLR3的阳性细胞百分率,并与空白对照组对比。结果HBsAg和HBeAg的分泌随着转染HBVDNA量的增加而升高,除4μg和6μg组两组间比较的表面抗原外,差异均有显著性(P<0.05)。TLR3在转染后均有上调,除4μg与6μg组间比较外,各组间差异有显著性(P<0.05)。台盼蓝拒染法示细胞存活随转染剂量的升高而减少(P<0.05),但4μg和6μg两组间比较差异无显著意义(P>0.05)。而各转染剂量组HBsAg和e抗原的分泌及细胞的存活数均与TLR3的表达呈显著正相关(P<0.01)。结论本研究初步表明乙肝病毒可能直接引起HepG2细胞TLR3的上调,而且上调可能启动了细胞的凋亡或坏死机制,成为HBV损伤细胞的非免疫细胞介导机制。
Objective To investigate the expression of Toll -like receptor 3 （TLR3） in HepG2 cells after transfection with hepatitis B virus genonle secretion of HBV antigen and the proliferation of cells after infection with HBV. Methods The experiments included creating pBluScript - HBV plasmid, sequencing, transforming bacteria, extracting plasmid, and cutting with enzyme Sap I, obtaining 3.2 kb genome, and transfecting HepG2 cells with lipofectamine 2000, detection of HBsAg and HBeAg, counting living cells. Trapan blue staining and examintion tile expression of TLR3 by FCM. Results HBsAg and HBeAg expression increased significantly with the transfection dosage except the expression of HBsAg in the 4 μg and 6 μg groups. The intensity of TLR3 expression in HepG2 cells was significantly higher than that of control group （ P 〈 0.05 ）. The number of living cells was reduced with increasing transfection dosage. The expression of HBsAg and HBeAg and the number of living cells were positively correlated with intensity of TLR3. Conclusion HBV may result in TLR3 expression directly. It may result in cell apoptosis or necrosis directly via a non - immune mechanism.
出处
《广东医学》
CAS
CSCD
北大核心
2007年第12期1905-1907,共3页
Guangdong Medical Journal
基金
教育部新世纪优秀人才支持计划(编号:NCET-04-0797)
广东省肝脏疾病研究重点实验室启动项目(编号:2005B60148)
