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学科主题: 泌尿外科学
题名:
树突状细胞在慢性附睾炎及无精子症中的表达及其对精子的免疫损伤
作者: 郑文忠
答辩日期: 2016-05-13
导师: 李贤新
专业: 泌尿外科学
授予单位: 北京大学
授予地点: 北京大学深圳医院
学位: 硕士
关键词: 树突状细胞 ; 辅助性T淋巴细胞 ; 慢性附睾炎 ; 无精子症 ; 免疫损伤
其他题名: Phenotypic analysis of dendritic cell subsets in onically inflamed human epididymis and azoospermia and its immune destruction on sperm
分类号: R698
摘要:

 目的  探讨树突状细胞(Dendritic cell, DC)在慢性附睾炎及无精子症中的表达及其对精子的免疫损伤。方法  研究对象为正常及慢性附睾炎组织石蜡标本各10例,无精子症患者睾丸25例及正常睾丸8例。免疫组化检测DC标记物(CD11c, CD123、CD1a)、巨噬细胞标记物(CD163, CD68)及Th细胞标记物(CD4)在正常及慢性附睾炎组织的表达与分布;同时检测DC在无精子症及正常睾丸组织中的表达丰度;通过免疫荧光染色分析慢性附睾炎CD11c+ 髓系树突状细胞(Myeloid dendritic cell, mDC)、CD68+ 或CD163+ 巨噬细胞及CD4+ T淋巴细胞胞内因子特性;结合免疫组化及荧光染色技术观察部分附睾尾部管腔内DC及巨噬细胞对精子的捕获损伤作用。采用酶联免疫吸附实验(Enzyme linked immunosorbent assay, ELISA)对比男性慢性生殖道炎症(Chronic inflammation of genital tract, CIGT)及正常生育男性精液IL-23p19,IL-17,IL-6等DC及Th17细胞相关炎症因子表达;通过免疫荧光技术检测IL-17R在精子中的表达及定位;并设立不同浓度梯度IL-17与精子进行体外共培养后,通过计算机辅助精液分析(Computer-aided semen analysis, CASA)、流式细胞术、染色质扩散法分析精子活力、线粒体膜电位、DNA损伤及凋亡率改变。 结果 无精子症患者睾丸组织较正常CD11c及CD123表达水平显著增高;慢性附睾炎较正常附睾CD11c, CD123, CD1a, CD163, CD68, HLA-DR及CD4表达水平显著增高;免疫荧光染色结果显示:CD11c+ mDC, CD68+及CD163+巨噬细胞表达炎症因子IL-23p19及IL-6;Th细胞可表达IL-17及IFN-γ;部分慢性附睾炎尾部管腔内可见CD11c+ DCs及巨噬细胞捕获精子;CIGT患者精液IL-17、IL-23p19及IL-6表达水平较正常显著增高;免疫荧光结果显示IL-17R定位于精子颈部,进一步体外共培养实验表明随IL-17浓度升高及培养时间延长精子活力显著降低,而精子线粒体膜电位、DNA损伤及凋亡率则显著升高。结论  DC在慢性附睾炎及无精子症发生发展中起重要作用,且附睾炎中CD11c+IL-23+/IL-6+ mDC及CD68+IL23+/IL-6+巨噬细胞参与构成Th17细胞持续激活微环境。此外,CD11c+ mDC及巨噬细胞在慢性附睾炎管腔通过捕获作用直接损伤精子,而Th17细胞则通过分泌IL-17对精子产生免疫损伤。

英文摘要:

Objective To investigate the acteristic phenotype of dendritic cells (DCs) in epididymis tissues of onically inflamed epididymis and azoospermia patients by using morphology stainig assay, then to study the immune damage of spermatozoa directed by DCs and Th (T helper) lymphocytes of onic epididymitis. Methods Objects were patients in Peking Universtiy Shenzhen Hospital and The First Affiliated Hospital of Shenzhen University urology department between December 2008 and July 2014. Firstly, we compared the expression of DCs specific marker (CD11c, CD123, CD1a, HLA-DR), macrophages (CD163, CD68) and Th lymphocytes in 10 epididymitis and 10 normal epididymal tissues by using immunohistochemical staining assay. Then, the cytokine profiles of CD11c+ myeloid dendritic cells (mDCs), CD68+ or CD163+ macrophages and CD4+ Th cells were identified by immunoflurescence double staining assay. In addition, the relationship between DCs, macrophages and spermatozoa in onically inflamed epididymis were analysis by immunohistochemical and immunoflurescence staining assay. Secondly, the expression intensity of DC and Th lymphocyte associated inflammatory cytokines such as IL-23p19, IL-6 and IL-17 in the semen of 28 normal volunteer candidates and 36 patients with onic inflammation of genital tract (CIGT) by using enzyme linked immuno-sorbent assay (ELISA). The location of IL-17 receptor (IL-17R) in human sperm was identified by immunoflurescence single staining assay and the expression intensity of IL-17R between normal and CIGT sperm were analyzed by flow cytometry. The vitality, DNA damage, mitochondria potential and apoptosis rate of the sperm which obtained from healthy donors and cocultured with gradient IL-17 were evaluted by comptuer-aid semen analysis (CASA), flow cytometry and omatin diffusion assay. Results The expression of CD11c, CD123, CD163, CD68, CD83, HLA-DR and CD4 were significantly increased in onically inflamed epididymis tissues compared with its control (P<0.05). CD11c and CD123 were significantly increased in azoospermia patients. The results of immunoflurescence double staining showed that both CD11c+ mDCs and CD68+ or CD163+ macrophages in onic inflamed epididymis expressed inflammatory cytokines such as IL-23p19 and IL-6. Furthermore, CD4+ Th cells in onic epididymitis tissues expressed cytokines IL-17 and IFN-γ which represent Th17 and Th1 lymphocytes respectively. Both immunohistochemical and immunoflurescence studies suggested that DCs (CD11c+, CD209+) and macrophages (CD68+, CD163+) capture spermatozoa in the lumen of onic cauda inflamed epidisymis. The expression of IL-17, IL23p19 and IL-6 were significantly evaluated in the semen of CIGT patients compared with its control (P<0.05). IL-17R were specific locate in the neck compartment of sperm and the expression intensity were significantly increased in CIGT patients (P<0.05). In addition, the mitochondria potential, DNA damage and apoptosis rateof cocultured sperm were positively correlated with the concentration of IL-17 and coculture time (P<0.05). Conclusion The expression of DCs and macrophages associated with an inflammatory cytokine profile were significantly increased in onic inflamed epididymis, which contributed to the continuously activation of Th17 cells and may play an important role in the development of epididymitis progression. Furthermore, CD11c+ mDCs and macrophages destructing spermatozoa by a directly capture way, meanwhile, Th17 cells impair sperm by producing IL-17 which specific combine with IL-17R in the neck compartment of human sperm.Objective To investigate the acteristic phenotype of dendritic cells (DCs) in epididymis tissues of onically inflamed epididymis and azoospermia patients by using morphology stainig assay, then to study the immune damage of spermatozoa directed by DCs and Th (T helper) lymphocytes of onic epididymitis. Methods Objects were patients in Peking Universtiy Shenzhen Hospital and The First Affiliated Hospital of Shenzhen University urology department between December 2008 and July 2014. Firstly, we compared the expression of DCs specific marker (CD11c, CD123, CD1a, HLA-DR), macrophages (CD163, CD68) and Th lymphocytes in 10 epididymitis and 10 normal epididymal tissues by using immunohistochemical staining assay. Then, the cytokine profiles of CD11c+ myeloid dendritic cells (mDCs), CD68+ or CD163+ macrophages and CD4+ Th cells were identified by immunoflurescence double staining assay. In addition, the relationship between DCs, macrophages and spermatozoa in onically inflamed epididymis were analysis by immunohistochemical and immunoflurescence staining assay. Secondly, the expression intensity of DC and Th lymphocyte associated inflammatory cytokines such as IL-23p19, IL-6 and IL-17 in the semen of 28 normal volunteer candidates and 36 patients with onic inflammation of genital tract (CIGT) by using enzyme linked immuno-sorbent assay (ELISA). The location of IL-17 receptor (IL-17R) in human sperm was identified by immunoflurescence single staining assay and the expression intensity of IL-17R between normal and CIGT sperm were analyzed by flow cytometry. The vitality, DNA damage, mitochondria potential and apoptosis rate of the sperm which obtained from healthy donors and cocultured with gradient IL-17 were evaluted by comptuer-aid semen analysis (CASA), flow cytometry and omatin diffusion assay. Results The expression of CD11c, CD123, CD163, CD68, CD83, HLA-DR and CD4 were significantly increased in onically inflamed epididymis tissues compared with its control (P<0.05). CD11c and CD123 were significantly increased in azoospermia patients. The results of immunoflurescence double staining showed that both CD11c+ mDCs and CD68+ or CD163+ macrophages in onic inflamed epididymis expressed inflammatory cytokines such as IL-23p19 and IL-6. Furthermore, CD4+ Th cells in onic epididymitis tissues expressed cytokines IL-17 and IFN-γ which represent Th17 and Th1 lymphocytes respectively. Both immunohistochemical and immunoflurescence studies suggested that DCs (CD11c+, CD209+) and macrophages (CD68+, CD163+) capture spermatozoa in the lumen of onic cauda inflamed epidisymis. The expression of IL-17, IL23p19 and IL-6 were significantly evaluated in the semen of CIGT patients compared with its control (P<0.05). IL-17R were specific locate in the neck compartment of sperm and the expression intensity were significantly increased in CIGT patients (P<0.05). In addition, the mitochondria potential, DNA damage and apoptosis rateof cocultured sperm were positively correlated with the concentration of IL-17 and coculture time (P<0.05). Conclusion The expression of DCs and macrophages associated with an inflammatory cytokine profile were significantly increased in onic inflamed epididymis, which contributed to the continuously activation of Th17 cells and may play an important role in the development of epididymitis progression. Furthermore, CD11c+ mDCs and macrophages destructing spermatozoa by a directly capture way, meanwhile, Th17 cells impair sperm by producing IL-17 which specific combine with IL-17R in the neck compartment of human sperm.Objective To investigate the acteristic phenotype of dendritic cells (DCs) in epididymis tissues of onically inflamed epididymis and azoospermia patients by using morphology stainig assay, then to study the immune damage of spermatozoa directed by DCs and Th (T helper) lymphocytes of onic epididymitis. Methods Objects were patients in Peking Universtiy Shenzhen Hospital and The First Affiliated Hospital of Shenzhen University urology department between December 2008 and July 2014. Firstly, we compared the expression of DCs specific marker (CD11c, CD123, CD1a, HLA-DR), macrophages (CD163, CD68) and Th lymphocytes in 10 epididymitis and 10 normal epididymal tissues by using immunohistochemical staining assay. Then, the cytokine profiles of CD11c+ myeloid dendritic cells (mDCs), CD68+ or CD163+ macrophages and CD4+ Th cells were identified by immunoflurescence double staining assay. In addition, the relationship between DCs, macrophages and spermatozoa in onically inflamed epididymis were analysis by immunohistochemical and immunoflurescence staining assay. Secondly, the expression intensity of DC and Th lymphocyte associated inflammatory cytokines such as IL-23p19, IL-6 and IL-17 in the semen of 28 normal volunteer candidates and 36 patients with onic inflammation of genital tract (CIGT) by using enzyme linked immuno-sorbent assay (ELISA). The location of IL-17 receptor (IL-17R) in human sperm was identified by immunoflurescence single staining assay and the expression intensity of IL-17R between normal and CIGT sperm were analyzed by flow cytometry. The vitality, DNA damage, mitochondria potential and apoptosis rate of the sperm which obtained from healthy donors and cocultured with gradient IL-17 were evaluted by comptuer-aid semen analysis (CASA), flow cytometry and omatin diffusion assay. Results The expression of CD11c, CD123, CD163, CD68, CD83, HLA-DR and CD4 were significantly increased in onically inflamed epididymis tissues compared with its control (P<0.05). CD11c and CD123 were significantly increased in azoospermia patients. The results of immunoflurescence double staining showed that both CD11c+ mDCs and CD68+ or CD163+ macrophages in onic inflamed epididymis expressed inflammatory cytokines such as IL-23p19 and IL-6. Furthermore, CD4+ Th cells in onic epididymitis tissues expressed cytokines IL-17 and IFN-γ which represent Th17 and Th1 lymphocytes respectively. Both immunohistochemical and immunoflurescence studies suggested that DCs (CD11c+, CD209+) and macrophages (CD68+, CD163+) capture spermatozoa in the lumen of onic cauda inflamed epidisymis. The expression of IL-17, IL23p19 and IL-6 were significantly evaluated in the semen of CIGT patients compared with its control (P<0.05). IL-17R were specific locate in the neck compartment of sperm and the expression intensity were significantly increased in CIGT patients (P<0.05). In addition, the mitochondria potential, DNA damage and apoptosis rateof cocultured sperm were positively correlated with the concentration of IL-17 and coculture time (P<0.05). Conclusion The expression of DCs and macrophages associated with an inflammatory cytokine profile were significantly increased in onic inflamed epididymis, which contributed to the continuously activation of Th17 cells and may play an important role in the development of epididymitis progression. Furthermore, CD11c+ mDCs and macrophages destructing spermatozoa by a directly capture way, meanwhile, Th17 cells impair sperm by producing IL-17 which specific combine with IL-17R in the neck compartment of human sperm.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125369
Appears in Collections:北京大学深圳医院_学位论文

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作者单位: 北京大学深圳医院

Recommended Citation:
郑文忠. 树突状细胞在慢性附睾炎及无精子症中的表达及其对精子的免疫损伤[D]. 北京大学深圳医院. 北京大学. 2016.
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