C5a/C5aR信号在微小隐孢子虫感染中的免疫调节作用研究

旨在探究C5a/C5aR信号在微小隐孢子虫感染过程中对宿主CD4⁺ T细胞免疫反应的调控作用。本研究以微小隐孢子虫（Cryptosporidium parvum）为研究对象，以BALB/c乳鼠和C5aR抑制BALB/c乳鼠为感染模型，应用实时荧光定量PCR和免疫组织化学技术检测了C.parvum感染前后乳鼠回肠组织中C5aR的表达变化，利用实时荧光定量PCR检测了隐孢子虫HSP70基因和CD4⁺ T细胞亚群（Th1、Th2、Th17细胞和Treg细胞）主效应细胞因子（IFN-γ、IL-4、IL-17和TGF-β）的转录变化，并通过病理组织切片观察乳鼠回肠黏膜的损伤情况。结果显示：与对照组乳鼠相比，C.parvum感染可以引起乳鼠回肠组织中C5aR的mRNA和蛋白表达水平显著上调（P<0.05），以及IFN-γ表达水平显著上调（P<0.05）；与C.parvum感染组乳鼠相比，C5aR抑制剂处理可引起C.parvum感染乳鼠回肠组织中Th1细胞、Th2细胞和Treg细胞的主效应细胞因子IFN-γ、IL-4和TGF-β显著下调表达（P<0.05），以及Th17细胞主效应细胞因子IL-17显著上调表达（P<0.05）。病理学观察发现，抑制C5aR能显著改善C.parvum感染引起的乳鼠回肠组织的绒毛直径和黏膜厚度变化（P<0.05），但不能改善绒毛长度、绒毛长度与隐窝深度比值。隐孢子虫HSP70基因的mRNA水平检测发现，抑制C5aR能显著影响C.parvum在回肠组织中的增殖（P<0.05）。C5a/C5aR信号可能通过动态调节CD4⁺ T细胞亚群主效应细胞因子的表达来参与宿主与隐孢子虫相互作用的过程，为深入理解隐孢子虫与宿主的互作机制提供了参考。

Immunomodulatory Effect of C5a/C5aR Signal during Cryptosporidium parvum Infection

The aim of this study was to explore the regulation of C5a/C5aR signal in the immune response of host CD4⁺ T cell during Cryptosporidium parvum infection. We have utilized C. parvum as the research object, and selected BALB/c suckling mice and C5aR inhibited BALB/c suckling mice as infection models. The expression level of C5aR in the ileum tissues of BALB/c suckling mice was detected by using real-time PCR and immunohistochemistry. The expression patterns of C. parvum HSP70 gene and main effector cytokines (IFN-γ, IL-4, IL-17 and TGF-β) of CD4⁺ T cell subsets (Th1, Th2, Th17 cells and Treg cells) were analyzed by real-time PCR. The injury of ileal mucosa in suckling mice was observed by histopathology analysis. Compared with mice in the control group, C. parvum infection could significantly increase the expression levels of C5aR mRNA and protein (P<0.05), as well as IFN-γ in the ileum tissues of suckling mice (P<0.05). Compared with mice infected with C. parvum, the expression levels of main effector cytokines of Th1, Th2 and Treg cells, namely IFN-γ, IL-4 and TGF-β, significantly reduced in C5aR inhibited BALB/C suckling mice infected with C. parvum (P<0.05), and the expression level of IL-17, the main effector cytokine of Th17 cells, significantly increased in suckling mice treated with C5aR inhibitor (P<0.05). Histopathology analysis of ileal mucosa in suckling mice indicated inhibition of C5aR could significantly improve the changes of villus diameter and mucosal thickness of ileum caused by C. parvum infection (P<0.05), but not for the villus length and the ratio of villus length to crypt depth. Analysis of the mRNA level for Cryptosporidium HSP70 gene showed that inhibition of C5aR could significantly affect the proliferation of C. parvum in ileum (P<0.05). C5a/C5aR signal may participate in the process of interaction between host and Cryptosporidium by dynamically regulating the expression of major effector cytokines for CD4⁺ T cell subsets, providing reference basis for further understanding the interaction mechanism between Cryptosporidium spp. and host.