过表达TLR2分子的B淋巴细胞系模型的建立  

Establishment of B lymphocyte model overexpressing humanTLR2/1 and TLR2

作　　者：徐菁[1] 何柳[2] 周芳婷 潘勤[2] 陈姗 罗靓 XU Jing;HE Liu;ZHOU Fangting;PAN Qin;CHEN Shan;LUO Liang(Department of Pharmaceutical Analysis,School of Pharmacy Faculty,Hubei University of Chinese Medicine,Wuhan 430071,China;Department of Anatomy,Wuhan University TaiKang Medical School(School of BasicMedical Sciences),Wuhan 430071,China;Department of Clinical Laboratory,Wuhan Caidian District People’s Hospital,Wuhan 430100,China)

机构地区：[1]湖北中医药大学、药学院、药物分析教研室,武汉430071 [2]武汉大学泰康医学院(基础医学院)人体解剖学与组织胚胎学系,武汉430071 [3]武汉市蔡甸区人民医院检验科,武汉430100

出　　处：《生物学杂志》2024年第4期24-29,共6页Journal of Biology

基　　金：湖北省教育厅科学研究计划指导性项目(B2021115)。

摘　　要：为构建稳定表达人源TLR2分子的B淋巴细胞系模型,将编码人TLR1、TLR2和TLR6的基因分别克隆到pCDH-CMV-MCS-EF1-GFP-puro慢病毒载体上,测序正确后经293T细胞包装成完整具备感染性的病毒颗粒,转染Nalm-6细胞(TLR2^(-)),嘌呤霉素筛选获得稳定表达TLR2分子的Nalm-6细胞(TLR2^(+))。利用倒置荧光显微镜观察细胞状态和绿色荧光表达情况,通过蛋白质免疫印迹法(Western Blot)检测相关蛋白表达水平,采用CCK-8以及流式细胞术检测细胞存活率和细胞增殖水平。结果显示:倒置荧光显微镜下可见成功转染慢病毒的Nalm-6细胞表达绿色荧光和嘌呤霉素抗性。Western Blot检测到TLR2在Nalm-6细胞中成功表达,且TLR2信号的激活明显增强PI3K-AKT信号轴分子的磷酸化水平。CCK-8和流式细胞术发现TLR2活化可提升细胞存活率,促进细胞增殖。研究成功构建了过表达TLR2分子的B淋巴细胞系模型,并在此基础上验证TLR2活化不仅能够增强B细胞PI3K-AKT信号通路传导,还可促进细胞增殖。此模型为进一步探究TLR2通路对B细胞抗感染免疫功能的影响奠定基础。To construct a B lymphocyte model stably expressing human TLR2,the coding sequences of human TLR1,TLR2 and TLR6 were cloned and inserted into the pCDH-CMV-MCS-EF1-GFP-puro lentiviral vector.After verification of correct insertion by sequencing,these plasmids were packaged into complete infectious virus particles by transfection into 293T cells and transduced into Nalm-6 cells(TLR2^(-)),and puromycin was used to select Nalm-6 cells that stably expressed TLR2(TLR2^(+)).An inverted fluorescence microscope was used to observe the cell status and green fluorescent protein expression.Western blott was used to measure the expression levels of related proteins.CCK-8 and flow cytometry assays were used to determine the cell viability and evaluate cell proliferation.The results of inverted fluorescence microscopy showed that Nalm-6 cells were successfully infected with lentiviruses with green fluorescent protein expression and puromycin resistance.Western Blot analysis showed that TLR2 was successfully expressed in Nalm-6 cells and that activation of TLR2 signaling significantly increased the levels of phosphorylated PI3K-AKT signaling axis proteins.The CCK-8 and flow cytometry assays showed that TLR2 activation could increase the cell viability and promote cell proliferation.In summary,in this study,a B lymphocyte line model overexpressing TLR2 was successfully constructed and was used to verify that TLR2 activation can upregulate the PI3K-AKT signaling pathway in B cells and promote their proliferation.This model lays the foundation for further exploring the impact of the TLR2 pathway on the anti-infective immune function of B cells.

关 键 词：TLR2 慢病毒载体 B淋巴细胞 PI3K-AKT信号通路 细胞增殖 

分 类 号：Q786[生物学—分子生物学]