体外扩增的Vγ9Vδ2T细胞抗H1N1流感病毒效应的研究

The antiviral activity against H1N1 of expanded human Vγ9Vδ2 T cells in vitro

目的探讨帕米磷酸钠（PAM）体外扩增培养的人外周血Vγ9Vδ2 T细胞对感染流感病毒H1N1的人肺上皮细胞A549的细胞毒效应。方法 PAM和IL-2联合刺激培养健康成人外周血单个核细胞（PBMC）10~14 d,免疫磁珠分选法纯化得到Vγ9Vδ2 T细胞,再将Vγ9Vδ2 T细胞与感染流感病毒的A549细胞按不同的效靶比（E∶T）接触共育或按E∶T=20∶1非接触共育6 h。流式细胞分析术检测A549的存活率及Vγ9Vδ2 T细胞穿孔素（perforin）的表达情况,收集培养上清检测流感病毒的滴度,提取培养上清及A549细胞的总RNA,实时荧光定量检测流感病毒基质蛋白M1基因的表达情况。结果 PAM在体外能有效活化并扩增Vγ9Vδ2 T细胞。Vγ9Vδ2 T细胞对感染流感病毒的A549细胞具有较强的杀伤作用,且随E∶T的升高而增大,在E∶T=20∶1时,Vγ9Vδ2 T细胞能杀伤60%的感染了流感病毒的A549细胞。接触共育组的A549细胞杀伤率明显高于非接触共育组（P〈0.05）。Vγ9Vδ2 T细胞经流感病毒感染后Perforin的表达明显上调。接触共育组的病毒复制也明显受到抑制（P〈0.05）。结论 PAM刺激扩增的Vγ9Vδ2 T细胞能有效杀伤感染流感病毒的人肺上皮细胞A549,其效应机制依赖于两者细胞间的接触。

To explore the cytotoxicity of pamidronate（PAM）-expanded Vγ9Vδ2 T cells against influenza virusinfected-human lung alveolar epithelial cells（A549）,peripheral blood mononuclear cells（PBMC） were isolated fromblood of healthy donors by density gradient centrifugation,and then cultured in RPMI-1640 with PAM and IL-2.After 10-14 day-culture,Vγ9Vδ2 T cells were harvested and purified to cultured with infected A549 s at differentE∶T ratio by physical contact or at E∶T=20∶1 by Transwell system for 6 h.Survival situation of A549 s and theprotein expression of perforin of virus-infected Vγ9Vδ2 T cell were detected by flow cytometry.Culturesupernatants were collected for virus titration.Total RNA of culture supernatants and A549 s were extracted,andthen the detection of gene expression of influenza virus matrix protein M1 was performed by Real-time PCR.Datashowed that Vγ9Vδ2 T cells were efficiently expanded by PAM in vitro,and the PAM-expanded Vγ9Vδ2 T cellsefficiently killed influenza virus-infected A549 s in a dose dependent manner.The cytolysis of virus-infected A549 sby Vγ9Vδ2 T cells was much higher in the physical contact coculture system than that of non-physical contact one.Much more production of cytolytic granule-perforin by Vγ9Vδ2 T cells was detected following virus infection.Viralreplication was also significantly inhibited in physical contact coculture system.In conclusion,PAM-expanded Vγ9Vδ2 T cells display potent cytotoxicity against influenza virus infected-A549 s depending on physical contact.