【作者】 张燕；

【导师】 赵琴平；

【作者基本信息】 武汉大学 ， 病原生物学， 2019， 硕士

【摘要】 目的血淋巴细胞是湖北钉螺固有免疫防御中的主要效应细胞,但目前对其形态和功能研究十分匮乏。本文对湖北钉螺血淋巴细胞进行形态学描述和分类,探讨血淋巴细胞各亚型在抗感染过程中的组成变化;并通过观察血淋巴细胞在抗感染过程中的吞噬能力和凋亡水平,及检测NADPH氧化酶(NADPH oxidase,NOX)和白介素17(Interleukin 17,IL 17)在钉螺软体和血淋巴细胞中表达水平的动态变化,探讨湖北钉螺血淋巴细胞在血吸虫感染过程中发挥的免疫防御功能。方法通过吉姆萨染色和电子显微镜,观察湖北钉螺血淋巴细胞在形态上的异质性。收集感染血吸虫前后0 h-8 d的钉螺血淋巴细胞,通过吉姆萨染色和流式细胞仪,检测血淋巴细胞各亚型含量的动态变化;通过磁珠吞噬实验,检测血淋巴细胞吞噬水平的动态变化;通过流式细胞仪,检测血淋巴细胞凋亡水平的动态变化;运用荧光定量PCR,检测血吸虫感染前后钉螺NOX和IL 17在软体和血淋巴细胞中的表达水平变化。结果根据细胞内部有无颗粒,可将钉螺血淋巴细胞分为颗粒细胞和透明细胞,分别为血淋巴细胞总数的6.93±0.40%和91.74±0.41%。根据颗粒性质,可进一步将颗粒细胞分为嗜酸性和嗜碱性颗粒细胞,分别占血淋巴细胞总数的2.86±0.12%和4.10±0.29%。根据细胞大小,可将透明细胞分为小透明细胞、中等透明细胞和大透明细胞,分别占血淋巴细胞总数的87.83±0.88%,4.65±1.13%和0.26±0.04%。血吸虫感染后,钉螺内血淋巴细胞数量明显上升,感染后6 h达最高。其中大透明细胞和颗粒细胞上升较为明显,在感染后6 h分别占总细胞数的0.29%和11.59%。吞噬细胞和凋亡细胞比例在感染后迅速升高,感染后6 h达最高,分别占总细胞数的60.7%和68.1%。q PCR结果显示:血淋巴细胞中NOX-1的表达在感染后逐渐上升,感染后48 h达最高,随后逐渐下降,感染后8 d降至最低;NOX-2在感染后逐渐上升,感染后24 h升至最高,随后逐渐下降,感染后4 d降至最低,8 d后再次升高,为0 h的24倍;IL 17-1在感染后上升,感染后12 h升至最高,随后逐渐下降,至感染后4 d降至最低,感染后8 d,表达量略高于正常水平;IL 17-2在感染后呈持续上升,感染后8 d的表达量最高,是对照组的60.88倍。结论湖北钉螺血淋巴细胞存在形态和功能上的异质性。根据细胞大小和颗粒的有无及其性质,将钉螺血淋巴细胞分为嗜酸性颗粒细胞、嗜碱性颗粒细胞、小透明细胞、中等透明细胞和大透明细胞;其中,颗粒细胞和大透明细胞在抗血吸虫感染中发挥主要作用。钉螺可通过增强血淋巴细胞吞噬细胞比例和调节其凋亡水平,抵抗病原体入侵;且感染后12 h内是钉螺抗感染的关键时期。血淋巴细胞中NOX和IL 17的表达在感染后6 h开始上升,在感染后不同时间点达最高水平,结合前期转录组分析结果,认为:钉螺体内存在依赖NOX和IL 17途径的信号通路,以调节血淋巴细胞吞噬和凋亡水平,协调发挥对血吸虫的免疫防御功能。更多还原

【Abstract】 Objective The hemocytes of Oncomelania hupensis is the main effector of immune defense against Schistosoma japonicum.The morphological and functional characterization of hemocytes remains unclear.In this study,the basic morphologic and classification were deeply investigated.The dynamic change of hemocytes after S.japonicum challenge was evaluated,including composition of subpopulations,the phagocytosis and apoptosis,and the expression profiles of NOX(NADPH oxidase)and IL 17(Interleukin 17)m RNA in hemocytes,to understand the role of hemocytes against S.japonicum infection.Methods The hemocytes were collected from 0 h to 8 d after S.japonicum infection.The morphology of normal haemocytes was observed under Giemsa staining and electronic microscope.The dynamic change in amount of haemocytes and subpopulations were evaluated after S.japonicum challenge by Giemsa staining and flow cytometry.In addition,the ability of hemocytes in phagocytosis by devouring the latex beads and apoptosis was checked by flow cytometry.The dynamic expression of NOX and IL 17 in hemocytes were estimated through real-time PCR.Results The hemocytes in Oncomelania hupensis were divided into granulocytes and hyalinocytes according to existence of granules,accounted for 6.93±0.40% and91.74±0.41% of total hemocytes,respectively.The granulocytes could be further divided into basophilic and eosinophilic granulocytes,accounted for 2.86±0.12% and4.10±0.29% of total hemocytes,respectively.While the hyalinocytes were divided into small hyalinocytes(SH),medium hyalinocytes(MH),and large hyalinocytes(LH)based on the size,accounted for 87.83±0.88%,4.65±1.13%,and 0.26±0.04% of total hemocytes,respectively.The total hemocytes count(THC)increased dramatically at 6h after challenge,and the granulocytes and large hyalinocytes increased most,accounted for 0.29% and 11.59% of total hemocytes at 6 h post-infection.The percentage of hemocytes in phagocytosis and apoptosis increased to highest at 6 h after challenge,accounted for 60.7% and 68.1% of total hemocytes,respectively.The expression of NOX-1 m RNA in hemocytes increased after challenge,and up to highest at 48 h post-infection and decreased to lowest at 8 d post-infection.The expression of NOX-2 increased to highest at 24 h post-infection and then decreased to lowest at 4 d post-infection followed by higher expression at 8 d post-infection.The expression of IL 17-1 increased to highest at 12 h post-infection and decreased to lowest at 4 d postinfection,but increased to higher at 8 d post-infection.The expression of IL 17-2continuously increased after challenge,and the expression was 60.88 times higher at 8d post-infection than 0 h.Conclusion The hemocytes in O.hupensis show the heterogeneity in morphology and function.The hemocytes can be divided into basophilic granulocytes,eosinophilic granulocytes,small hyalinocytes(SH),medium hyalinocytes(MH)and large hyalinocytes(LH)based on the granules and the size.Among them,the granulocytes and LH play key roles against S.japonicum infection.Phagocytosis and apoptosis may help hemocytes defense against S.japonicum,especially at the early 12 h after infection.The expression of NOX and IL 17 m RNA increased after infection and reached to the highest level at different times.The NOX and IL 17 related signal pathways were thought to play roles to adjust the ability of hemocytes in phagocytosis and apoptosis,to help snails defense against S.japonicum.更多还原