CLEC4M介导HCV感染人PBDC实验研究  

The in vitro study of HCV infected of human dendritic cells interfered by CLEC4M

作　　者：王媛媛[1] 聂青和[1] 高禄化[1] 

机构地区：[1]第四军医大学唐都医院传染病科,全军感染病诊疗中心,西安710038

出　　处：《肝脏》2013年第4期224-227,共4页Chinese Hepatology

基　　金：国家自然科学基金资助项目(81170390)

摘　　要：目的通过对表达CLEC4M的CD14细胞与HCV结合来证实CLEC4M是介导HCV感染的特异性受体。方法应用磁珠分选出健康人的CD14+细胞,检测表达CLEC4M后将细胞共分4组,阳性对照组:CLEC4M+HCV RNA+血清;干扰组:CLEC4M+鼠抗人CLEC4M抗体+HCV RNA+血清,每组均与收集到的3份HCV阳性患者血清作用,阴性对照组:CLEC4M+正常人血清;空白对照组:CLEC4M+5%DMEM。干扰组在加入3组HCV RNA+血清之前,先分别加入CLEC4M抗体进行干预,其余3组滴入同体积PBS。培养24h后利用RT-PCR检测细胞表面结合的HCV RNA含量,培养48h后用免疫荧光法检测细胞与病毒结合情况。结果流式结果表明分离出的CD14细胞中CLEC4M阳性率高达80%以上。RT-PCR结果显示干扰组HCV RNA含量明显低于阳性对照组。免疫荧光可直观看到阳性对照组有大量绿色荧光,而干扰组只有很少的部分,空白对照组和阴性对照组无荧光。结论表达CLEC4M的CD14细胞可以与HCV结合,而病毒的结合可以被CLEC4M抗体竞争性抑制。Objective To confirm CLEC4M is the specific receptor which can mediate HCV infection via binding HCV virus to CD14 cells which expressed CLEC4M. Methods The CD14 cells which were sorted from healthy subjects by using magnetic bead and divided into four groups according to CLEC4M expression. Three patients＇ serum with HCV- RNA＋ were added into CD14 cells for positive control group and interference group, respectively. Healthy subjects＇ serum was added into CD14 cells for negative control group and 5 % DMEM for blank group. Prior to being added three patients＇ serum with HCV RNA ＋ into interference group, the CLEC4M antibody should be added to intervene. Same volume of PBS was added into the rest of the three groups respectively. RT-PCR was used to measure the HCV RNA expression of cell surface after 24 hours and the immunofluorescence was used to observe the combining situation of cells and viruses after 48 hours. Results The results of flow cytometry showed that the rate of CLEC4M positive in CDl4 cells was as high as 80G. Compared to positive control group, the HCV RNA expression of interference group was signifieantly decreased. By using the method of immunofluorescence, a great quantity of green fluorescence could be observed in positive control group, but little fluorescence in interference group. No fluorescence could be observed in both gap control group and negative control group. Conclusion The CD14 cells which expressed CLEC4M can combine with HCV virus and it can be competitive inhibited by CLEC4M antibody.

关 键 词：CLEC4M CD14细胞 树突状细胞 外周血树突状细胞 丙型肝炎病毒 免疫荧光 

分 类 号：R512.63[医药卫生—内科学]