TLR2、TLR4在乙型肝炎病毒转染的HepG2细胞株的表达

Expression of TLR2 and TLR4 of HepG2 strain transfected in hepatits B virus.

目的探讨乙肝病毒全基因组瞬时转染HepG2细胞后,对HBV抗原分泌,病原识别受体Toll样受体2、4(TLR2、4)的表达及细胞增殖的影响。方法已构建pBlueScript-HBV质粒并经测序,转化宿主菌,菌后提取质粒,用Sapl酶切,获得HBV3.2kb基因组,采用脂质体瞬时转染肝癌细胞株HepG2,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TLR2、4在细胞株上表达的阳性细胞率,并与空白对照绗对比。结果HBsAg利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,差异均有显著性意义(P<0.05)。TLR2、4在转染后均有上调,TLR4在各组间差异显著(P<0.05),但TLR2只在最大剂量转染组时差异有显著性(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TLR2、4的表达呈显著正相关(P<0.01)。结论研究结果初步表明乙肝病毒可能直接引起HepG2细胞TLR2、4的上调,而且TLR的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 - llke rectors of 2,4（TLR2,4） on HepG2 cells after tranfection with hepatitis B virus genome, also including secretion of HBV antigen and proliferation of cells. Methods To crate pBluScript - HBVplasmid, sequencing, trandform bacteria, extract plasmid, and cut with enzyme Sap 1, obtain 3.2kb genome, and trand- ct HeG2 cells with lipo- etamine 2000, IMX examine HBsAg, HBeAg and Trypan blue staining examine the sum of living cells. FCM examine the present of positive cells percent ecpressing TLR2 and TLR4 in contrast to the normal group. Results HbsAg and HbeAg expression increased significantly with the transfection dosage excluding the expresion of HbsAge in 4μg and 6μg. The intensity of TLR4 expression in HepG2 cells was significantly higher than that of the contrl group （P 〈 0.0 % ）, but TLR2 was higher significantly in the 8μg group. The sum of living cells reduced with the increasing of transection dosage. The expression of HBsAg and HbeAge and the the living cells were notably positive correlated with the intensity of TLR2,4. Conclusion HBV may result in the dirtect expression of TLR2,4, it may make cell apoptosis or necrosis directly, which may be not dependent on immune hurt.