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刘迪文,卫振,陈雁虹,谭海明.不同 FMDV 易感性豚鼠 PBLC 对模拟 FMDV 感染细胞的杀伤作用[J].中国实验动物学报,2020,28(2):0.
不同 FMDV 易感性豚鼠 PBLC 对模拟 FMDV 感染细胞的杀伤作用
Peripheral blood lymphocytes from food-and-mouth disease virus resistant guinea pigs show increased cytotoxicity to cells with simulative viral infection
投稿时间:2019-08-11  
DOI:10. 3969 / j.issn.1005-4847. 2020. 02. 008
中文关键词:  FMDV 抗性  豚鼠  细胞免疫  天然免疫
英文关键词:FMDV resistance  guinea pig  cytoimmunity  natural immunity
基金项目:
作者单位E-mail
刘迪文 浙江大学实验动物中心,杭州 310013 liudiwen2004@ 163.com 
卫振 浙江大学实验动物中心,杭州 310013  
陈雁虹 浙江大学实验动物中心,杭州 310013  
谭海明 浙江大学实验动物中心,杭州 310013  
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中文摘要:
      目的 探究豚鼠对口蹄疫病毒(FMDV)抗性的遗传学和免疫学机制。 阐明 FMDV 假病毒感染的细胞用于研究 FMDV 发病机制的可行性。 方法 通过含有 FMDV-VP1 片段的慢病毒载体侵染豚鼠原代肾细胞,构建 FMDV 假病毒感染的靶细胞。分离 FMDV 易感性(Zmu-1:DHP 品系)和抵抗性(英国种)的两个品系豚鼠外周血淋巴细胞(PBLC,未经抗原处理),与假病毒侵染的靶细胞共培养,用细胞毒性实验测定 PBLC 的杀伤率,并使用 ELISA 检测细胞培养上清中细胞因子的含量。通过流式细胞分选测定两个品系豚鼠 PBLC 中 CD32+ / CD4+及 CD3+ / CD8+细胞的比例。 结果 对于两个品系豚鼠而言,实验组 PBLC 的细胞毒性均显著大于各自的对照组( P < 0. 01);抵抗性品系豚鼠 PBLC 对靶细胞的细胞毒性显著大于易感性品系豚鼠(P < 0. 01);抵抗性品系豚鼠 PBLC 分泌的细胞杀伤因子 PF、GRA、GRB 及细胞表面 Fas 表达量显著大于易感性品系豚鼠( P < 0. 01) ,但分泌的 FasL 表达量无显著差异(P > 0. 05) ;抵抗性品系豚鼠 PBLC 分泌的细胞因子 IFN-γ、TNF-α、IL-2 及 IL-12 含量显 著大于易感品系(P < 0. 05) ,细胞表面 MHC I 及 MHC II 差异不显著(P > 0. 05) ;含有 FMDV-VP1 片段的慢病毒载体侵染豚鼠原代肾细胞,可使细胞表面表达 FMDV-VP1 蛋白;抵抗性品系豚鼠 PBLC 中 CD4+细胞数量显著少于易感性品系(P < 0. 05) ,抵抗性品系豚鼠 CD8+细胞数量显著高于易感性品系(P < 0. 05) 。 结论 FMDV-VP1 片段显著激发了效应细胞的免疫反应,增强效应细胞对靶细胞的细胞毒性;抵抗性豚鼠 PBLC 对 FMDV 感染细胞具有较强的天然细胞毒性;效应细胞对靶细胞产生细胞毒性的过程中,效应细胞分泌的细胞免疫因子参与了相关过程;通过含有 FMDV-VP1 片段的慢病毒载体侵染豚鼠原代肾细胞,构建 FMDV 假病毒感染的靶细胞有效模拟 FMDV 对靶细胞的感染,可提升后续研究工作的安全性。不同品系豚鼠的遗传特性决定了其对 FMDV 易感 性的差异。
英文摘要:
      Objective To investigate the genetic and immunologic mechanisms of resistance to foot-and-mouth disease virus ( FMDV) in guinea pigs and to assess the possibility of using pseudo-FMDV in the pathogenetic study of FMVD. Methods FMDV-VP1 peptide was expressed in primary renal cells derived from two different strains of guinea pigs using lentivirus. Peripheral blood lymphocytes (PBLC, without antigen processing) derived from two different strains of guinea pigs were isolated and cultured with lentivirus infected primary renal cells. PBLC cytotoxicity was determined using the lactate dehydrogenase ( LDH ) assay, and supernatant cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Moreover, the proportions of CD3+ / CD4+ and CD3+ / CD8+ cells were measured via flow cytometry. Results The cytotoxicity of PBLCs in experimental groups was significantly higher than in corresponding control groups (P < 0. 01). The cytotoxicity of PBLCs to target cells was significantly higher in the FMDV-resistant strain than in the FMDV-sensitive strain (P < 0. 01). Levels of PF, GRA, GRB, and Fas in serum derived from the FMDV-resistant strain were higher than in serum derived from the FMDV-sensitive strain (P < 0. 01). However, the expression of FasL showed no significant difference (P > 0. 05). Similarly, levels of IFN-γ, TNF-α, IL-2, and IL-12 were also higher (P < 0. 05). However, the expression of cell-surface MHC class I and II showed no significant difference (P > 0. 05). The FMDV-VP1 peptide was expressed on the surfaces of primary renal cells derived from guinea pigs when cells were infected by lentiviral-vector containing the DNA sequence of the FMDV-VP1 fragment. The quantity of CD4+ cells in PBLCs derived from the FMDV-resistant strain was significantly lower than in PBLCs derived from the FMDV-sensitive strain (P < 0. 05). The opposite trend was seen for the quantity of CD8+ cells ( P < 0. 05). Conclusions FMDV-VP1 peptides stimulate immunoreactions of effector cells and enhance the cytotoxicity of effector cells to target cells. PBLCs derived from the FMDV-resistant strain exerted strong cytotoxicity to FMDV infected cells. Cytokines secreted by effector cells participate in the cytotoxicity reaction. Primary renal cells derived from guinea pigs infected by lentiviral-vector containing the DNA sequence of the FMDV-VP1 fragment can be used to simulate FMDV infected cells, which could help to reduce risks associated with FMDV research. Finally, the genetic characteristics of different strains of guinea pigs determine their sensitivities to FMDV.
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