不同细胞唾液酸受体类型及唾液酸转移酶与流感病毒敏感性的研究

Study on the types of sialic acid receptors and sialyltransferases of different cells and their sensitivity to influenza virus

为研究人/禽流感病毒对细胞的敏感性差异,本研究选取9种常用细胞,分别以105 TCID50的人源甲型流感病毒(IAV)CA04株与禽流感病毒(AIV)Y280株感染不同细胞,检测不同细胞中流感病毒的增殖水平;利用免疫荧光试验检测细胞表面唾液酸(SA)受体类型及其与流感病毒的结合能力;采用流式细胞术定量检测每种细胞表面的SA类型及含量;通过RT-PCR方法检测唾液酸转移酶(ST)(ST3GAL4、ST6GAL1)基因mRNA的转录水平。结果显示,感染CA04株后,MDCK、16HBE、DF-1和A549检测到了病毒滴度,但病毒低度较低为:3.2×10^2 TCID50/0.1 mL^0.95×10^1 TCID50/0.1mL。感染Y280株后,病毒滴度从高到低依次为MDCK、DF-1、Vero、293T、CHO-k1、Hep-2、A549、16HBE和BHK,病毒滴度为6.3×103 TCID50/0.1 mL^9.9×10^1 TCID50/0.1mL;免疫荧光试验结果显示,MDCK、16HBE、DF-1和A549细胞表面存在大量SAα(2-6)Gal结构的糖链,BHK细胞表面该糖链最少,MDCK、DF-1和16HBE细胞表面比其它细胞含有更多的SAα(2-3)Gal结构的糖链,因此MDCK、DF-1和A549细胞对人源流感病毒CA04株的结合能力最强,MDCK、DF-1则对AIV Y280株结合能力较强;流式细胞术定量分析结果显示,9种细胞中MDCK和DF-1细胞表面SAα(2-6)Gal糖链结构含量最高,其结合人流感病毒CA04株的能力较强;而不同细胞表面SAα(2-3)Gal糖链结构含量差异不大,其结合AIV Y280株的能力无显著差异;RT-PCR检测结果显示,MDCK、DF-1细胞的ST相关基因(ST3GAL4、ST6GAL1)mRNA转录水平高于其它细胞,BHK细胞的ST3GAL4、ST6GAL1 mRNA转录水平则最低。因此,MDCK、DF-1细胞与流感病毒的结合能力较强,而BHK细胞与流感病毒的结合能力较低。上述结果表明,SA含量、ST转录水平较高的细胞,其结合流感病毒量也较多,对流感病毒的敏感性也高;反之,则对流感病毒敏感性较低。以上结果表明,细胞表面SA含量、ST基因转录水平与结合的流感病毒量均呈正相关。本研究为进一步探究SA及ST在流感病毒感染中的作用机制奠定了基础,同时为研究人/禽流感病毒中宿主细胞选择提供了参考依据。

In order to study the sensitivity differences between human and avian influenza viruses to different cells,9 commonly used cell lines were selected in this study.We infected different cells with 105 TCID50 of human influenza A virus(IAV)CA04 strain or avian influenza virus(AIV)Y280 strain,and detected the replication levels of influenza viruses in different cells.We applied immunofluorescence to detect the sialic acid(SA)receptor types in the cell surface and the irabilities to bind to influenza virus.We used flow cytometry to quantitatively detect the types and the relative amounts of SA on the surface of each cell line.The transcription level of ST(ST3GAL4,ST6GAL1)mRNAs was detected by RT-PCR method.The results showed that virus titers were detected in cell lines including MDCK,16HBE,DF-1 and A549 after infection with the CA04 strain,although the virus titers were low(3.2×10^2 TCID50/0.1 mL-0.95×10^1 TCID50/0.1 mL).After infection with the Y280 strain,the virus titers in the cell lines with an order from high to low were MDCK,DF-1,Vero,293 T,CHO-k1,Hep-2,A549,16HBE and BHK,with virus titers of 6.3×103 TCID50/0.1 mL-9.9×10^1 TCID50/0.1 mL.Immunofluorescence test results showed a large number of SAα(2-6)Gal on the surface of MDCK,16HBE,DF-1 and A549 cells,with the least in BHK and the most in MDCK,DF-1 and 16HBE cells.SAα(2-3)Gal in MDCK,DF-1 and A549 cells have the strongest binding abilities to human CA04 strain,while MDCK and DF-1 have strong binding ability to AIV Y280 strain.The results of quantitative analysis by flow cytometry showed that the amount of surface SAα(2-6)Gal in MDCK and DF-1 cells were the most among the 9 types of cells,and their binding to human influenza virus CA04 were stronger,while the amouts of surface SAα(2-3)Gal in the surface of these cell lines were comparable,and there is no significant difference in the irabilities to bind to AIV Y280 strain.RT-PCR results showed that the mRNA transcription levels of ST-related genes(ST3GAL4,ST6GAL1)in MDCK and DF-1 cells were higher than those in other cells,while the transcription levels of ST3GAL4 and ST6GAL1 mRNAs in BHK cells were the lowest.The above results indicate that cells with more SA in the surface and higher ST transcription level have larger amounts of binding influenza virus and thus are more sensitive to influenza virus,and vice versa.This study lays a foundation for further exploring the mechanism of SA and ST in influenza virus infection,and provides a reference for the study of host cell selection in human/avian influenza viruses.