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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(P<0.05);TLR4抗体组与感染组间差异也有统计学意义(P<0.05)。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。
Objective To analyze the role of Toll-like receptor (TLR) 4 in inducing dendritic cell (DC) infection in Cryptosporidium parvum mice in vitro. Methods Cryptosporidium sporozoite was purified by Cryptosporidium parvum and labeled with 5 (6) - fluorescein diacetate succinimidyl ester. The Cryptosporidium species were identified by PCR. Mouse bone marrow-derived dendritic cells were prepared and cultured to day 7, 1 ml DC was added to each well of the culture plate, and 1 ml of the decellularized cryptosporidium sporozoites (2 × 10 -5 / well) was added into the TLR4 antibody group ) And 0.5 ml of TLR4 antibody (final concentration of 1 μg / ml). The infected group was added with 1 ml of decellularized Cryptosporidium sporozoites and 0.5 ml of RPMI 1640 medium. The blank control group was added with 1.5 ml of RPMI 1640 medium. Each group of 3 holes. After cultured for 2 h, the relative expression levels of CD11c ~ + in each group were detected by flow cytometry. Flowjo software was used to compare the maturation status of dendritic cells in each group. 24 h after infection, the adhesion of Cryptosporidium sporozoite and dendritic cells in each group was observed by fluorescence microscopy. The differences of relative expression levels of CD11c ~ + in each group were tested byχ ~ 2 test. Results The purified Cryptosporidium was amplified by PCR, and a band of 830 bp was obtained and identified as Cryptosporidium parvum. The results of flow cytometry showed that the relative expression levels of CD11c ~ + in dendritic cells of TLR4 antibody group and infected group were 67.67 ± 1.80 and 83.37 ± 3.73, respectively, which were statistically different from those of the blank control group (7.06 ± 0.02) (P <0.05). The difference between TLR4 antibody group and infection group was also statistically significant (P <0.05). Flowjo software analysis showed that the relative peak level of CD11c + expression in TLR4 antibody group was lower than that in infected group, indicating that the maturation of DC in TLR4 antibody group was lower than that in infected group. Fluorescence microscopy showed that Cryptosporidium sporozoites in both TLR4 and infected groups could adhere to dendritic cells. Conclusion TLR4 can induce DC maturation in mice infected with Cryptosporidium.