党参皂苷对跨H9N2 AIV感染肺微血管内皮侵袭细胞功能的影响  

Effects of Codonopsis saponins on T cells invasion assay across H9N2 AIV infected pulmonary microvascular endothelium

作　　者：乔畅 刘翔 冯波[2] 穆祥[2] 张涛[2] 董虹[2] 胡格[1] 张倩[1] QIAO Chang;LIU Xiang;FENG Bo;MU Xiang;ZHANG Tao;DONG Hong;HU Ge;ZHANG Qian(College of Animal Science and Technology,Beijing Agricultural College,Beijing 102206,China;Beijing Key Laboratory of Veterinary Medicine(Chinese Medicine),Beijing Agricultural College,Beijing 102206,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京农学院兽医学(中医药)北京市重点实验室,北京102206

出　　处：《中国兽医学报》2024年第8期1800-1806,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(2033202073/001)。

摘　　要：为探讨党参皂苷对H9N2亚型禽流感病毒(avian influenza virus,AIV)感染引起机体免疫抑制的调控作用,首先应用不同质量浓度(5、10、20 mg/L)党参皂苷孵育大鼠肺微血管内皮细胞(rat pulmonary microvascular endothelial cells,RPMECs),采用RT-PCR和流式细胞术检测PD-L1表达水平变化,并以ELISA试剂盒检测上清液中TNF-α、IFN-γ、IL-10含量及空斑法检测上清液中H9N2 AIV的滴度。然后利用8μm孔径Transwell板建立RPMECs和T细胞共培养体系,以模仿循环T细胞跨过微血管迁移到病毒感染部位的过程。在Transwell上室中培养RPMECs,用H9N2 AIV接种RPMECs,1 h后加入含20 mg/L党参皂苷的培养基,36 h后加入T细胞。作用8 h后,收集下室中的T细胞,用流式细胞仪检测CD4^(+)T细胞和CD8^(+)T细胞的比例,用ELISA试剂盒检测上清液中IL-2、IFN-γ和Granzyme B的表达水平,流式细胞分选技术检测Perforin-1阳性T细胞的比例,MTT细胞增殖及细胞毒性检测试剂盒检测T细胞的增殖活性,Annexin V-FITC/PI对T细胞染色后用流式细胞仪检测凋亡细胞的百分比。结果显示,党参皂苷可显著降低H9N2 AIV感染诱导的RPMECs中PD-L1的表达水平、IL-10及TNF-α表达量,降低T细胞凋亡率,但可显著提高跨内皮迁移T细胞中IL-2、IFN-γ、Perforin-1和Granzyme B的表达量及T细胞的增殖活性。结果表明,党参皂苷可显著抑制RPMECs中H9N2 AIV诱导的PD-L1的表达,增强跨内皮细胞层迁移T细胞的抗病毒功能,有利于增强宿主对H9N2 AIV抵抗能力。In order to investigate the regulatory effect of Codonopsis saponins on the immunosuppression caused by H9N2 subtype avian influenza virus(AIV)infection,rat pulmonary microvascular endothelial cells(RPMECs)were incubated with different concentrations of Codonopsis saponins(5,10 and 20 mg/L).The expression level of PD-L1 was detected by RT-PCR and flow cytometry,and the contents of TNF-α,IFN-γand IL-10 in supernatant were detected by ELISA kit.The titer of H9N2 AIV in supernatant was detected by plaque method.Then,a co-culture system of RPMECs and T cells was established using a Transwell plate with an aperture of 8μm to mimic the migration of circulating T cells across microvessels to the site of viral infection.RPMECs were cultured in the upper chamber of Transwell,inoculated with H9N2 AIV,supplemented with 20 mg/L Codonopsis saponins 1 h later,and T cells 36 h later.After 8 h of treatment,T cells in the lower compartment were collected and the proportions of CD4^(+)T cells and CD8^(+)T cells were detected by flow cytometry,the expression levels of IL-2,IFN-γand granzyme B in the supernatant were detected by ELISA,and the proportions of perforin-1positive T cells were detected by flow cytometry.The proliferation activity of T cells was detected with the MTT cell proliferation and cytotoxicity assay kit,and the percentage of apoptotic cells was detected by flow cytometry after staining of T cells with Annexin V-FITC/PI.The experimental results showed that Codonopsis saponins could significantly reduce the expression level of PD-L1,IL-10and TNF-αin RPMECs induced by H9N2AIV infection,and reduce the apoptosis rate of T cells.However,the expression levels of IL-2,IFN-γ,perforin-1and granzyme B in transendothelial migration T cells and the proliferation activity of T cells were significantly increased.In this study,Codonopsis saponins can significantly inhibit the expression of H9N2AIV-induced PD-L1in RPMECs,enhance the antiviral function of T cells migrating across the endothelial layer,and enhance the resista

关 键 词：党参皂苷 微血管内皮细胞 PD-L1 T细胞 H9N2 AIV 

分 类 号：S853.7[农业科学—临床兽医学] S852.65[农业科学—兽医学]