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

摘要/Abstract

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 × 10⁵/孔）和0.5 ml TLR4抗体（终浓度为1 μg/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χ²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.

关键词: 微小隐孢子虫, 树突状细胞, Toll样受体4, 抗体, 成熟度, 黏附

Abstract: Objective To analyze the functions of mouse dendritic cells infected with Cryptosporidium parvum in the presence of Toll-like receptor 4 (TLR4). Methods Cryptosporidium cysts were purified and excysted, then labeled by 5'(6)-carboxyldiacetic acid fluorescein succinimidyl ester. Its species was identified by PCR. Mouse bone marrow dendritic cells were prepared and cultured. On day 7 of culture, 1 ml of dendritic cell culture was transferred to a 6-well plate, and 1 ml of labeled excysted Cryptosporidium parvum was added (2 × 10⁵/well), together with 0.5 ml of TLR4 antibody (at a final concentration of 1 μg/ml) (assigned into the TLR4 group) or 0.5 ml RPMI 1640 (infection group). Cells in the control group were incubated with 1.5 ml RPMI 1640 only. Each group had 3 replicates. After 2-hour incubation, flow cytometry was performed to detect the relative expression level of CD11c. Flowjo software was used to compare the maturation state of dendritic cells. After 24 h of infection, adhesion of Cryptosporidium sporozoites to dendritic cells was observed under a fluorescence microscope. The difference in the expression level of CD11c was analyzed with chi-square test. Results PCR amplification on the purified Cryptosporidium generated a band of 830 bp, which was identified to be Cryptosporidium parvum. Flow cytometry showed that the relative expression level of CD11c on dendritic cells was 67.67 ± 1.80 in the TLR4 antibody group and 83.37 ± 3.73 in the infection group, which were significantly different (P < 0.05), and both had significant difference with the control group (7.06 ± 0.02) (P < 0.05). Flowjo software analysis showed that the peak of CD11c expression in the TLR4 antibody group was lower than that in the infection group, implying a lower maturity of dendritic cells in the TLR4 antibody group than in the infection group. Fluorescence microscopy showed adhesion of Cryptosporidium sporozoites to dendritic cells in the TLR4 group and the infection group. Conclusion TLR4 can induce maturation of dendritic cells after infection with Cryptosporidium parvum.

Key words: Cryptosporidium parvum, Dendritic cell, TLR4, Antibody, Degree of ripeness, Adhesion

中图分类号:

R382.3

方芬, 刘喆, 王桂军, 徐前明. 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[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 265-269.

Fen FANG, Zhe LIU, Gui-jun WANG, Qian-ming XU. In vivo characterization of mouse dendritic cells infected with Cryptosporidium parvum in the presence of Toll-like receptor 4[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(3): 265-269.

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