Toll样受体4诱导感染微小隐孢子虫的小鼠树突状细胞功能的体外研究

发布时间：2018-04-27 03:25

本文选题：微小隐孢子虫 + 树突状细胞　；参考：《中国寄生虫学与寄生虫病杂志》2017年03期

【摘要】：目的体外分析Toll样受体(TLR)4诱导感染微小隐孢子虫(Cryptosporidium parvum)小鼠的树突状细胞(DC)功能。方法纯化脱囊隐孢子虫经5(6)-羧基二乙酸荧光素琥珀酰亚胺酯标记隐孢子虫子孢子,PCR鉴定隐孢子虫种。制备小鼠骨髓源树突状细胞,培养至第7天,于培养板各孔中分别加入1 ml DC,TLR4抗体组加入1 ml脱囊标记的隐孢子虫子孢子(2×10~5/孔)和0.5 ml TLR4抗体(终浓度为1μg/ml),感染组加入脱囊标记的隐孢子虫子孢子1 ml和0.5 ml RPMI 1640培养基,空白对照组加入1.5 ml RPMI 1640培养基。每组各3孔。培养2 h后,流式细胞术检测各组的CD11c~+相对表达水平,Flowjo软件比较各组树突状细胞的成熟情况。感染后24 h,荧光显微镜观察各组隐孢子虫子孢子与树突状细胞的黏附情况。各组CD11c~+相对表达水平间的差异采用χ~2检验。结果纯化后的隐孢子虫经PCR扩增,获得长度为830 bp的条带,鉴定为微小隐孢子虫。流式细胞术检测结果显示,TLR4抗体组和感染组的树突状细胞CD11c~+相对表达水平分别为67.67±1.80和83.37±3.73,与空白对照组(7.06±0.02)比较,差异均有统计学意义(P0.05);TLR4抗体组与感染组间差异也有统计学意义(P0.05)。Flowjo软件分析结果显示,TLR4抗体组的CD11c+相对表达水平的峰值低于感染组,表明TLR4抗体组DC的成熟度低于感染组。荧光显微镜观察发现,TLR4组和感染组的隐孢子虫子孢子均可与树突状细胞发生黏附现象。结论 TLR4可诱导感染隐孢子虫小鼠的DC成熟。
[Abstract]:Objective to study the dendritic cell (DC) function of Cryptosporidium parvum infected by Toll like receptor TLR4 in vitro. Methods Cryptosporidium demystis were purified and identified by PCR. Mouse bone marrow-derived dendritic cells were prepared and cultured until the 7th day. 1 ml DDC TLR4 antibody group was added to the culture plate, and 1 ml demystified Cryptosporidium spores 2 脳 10 5 / well and 0. 5 ml TLR4 antibody (final concentration 1 渭 g / ml) were added to the culture plate, respectively. The infected group added 1 ml and 0. 5 ml RPMI 1640 culture medium of demystified Cryptosporidium spores, respectively. 1. 5 ml RPMI 1640 medium was added to the blank control group. Each group had 3 holes. After cultured for 2 h, the relative expression level of CD11c- ~ was detected by flow cytometry. Flowjo software was used to compare the maturation of dendritic cells in each group. The adhesion of Cryptosporidium spores to dendritic cells was observed by fluorescence microscope 24 hours after infection. The difference of CD11c- relative expression level in each group was tested by 蠂 ~ 2 test. Results the purified Cryptosporidium was amplified by PCR and identified as Cryptosporidium minima. The expression of CD11c~ in dendritic cells in TLR4 antibody group and infected group was 67.67 卤1.80 and 83.37 卤3.73respectively, which was compared with that in control group (7.06 卤0.02). There was significant difference between the antibody group and infected group. The results showed that the peak level of CD11c expression in the antibody group was lower than that in the infected group, indicating that the maturity of DC in the TLR4 antibody group was lower than that in the infected group. Fluorescence microscopy showed that Cryptosporidium spore of TLR4 group and infected group could adhere to dendritic cells. Conclusion TLR4 can induce DC maturation in Cryptosporidium mice.
【作者单位】：安徽农业大学动物科技学院;
【基金】：安徽省自然科学基金(No.1308085MC49)~~
【分类号】：R382.3

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