念珠菌病细胞免疫机制的研究

发布时间：2018-05-23 18:07

  本文选题：动物 + 念珠菌　； 参考：《华中科技大学》2007年博士论文

【摘要】： 第一部分趋化因子及其受体在阴道念珠菌病小鼠模型中的表达及其意义 【目的】检测阴道念珠菌病小鼠模型中趋化因子及其受体的表达,探讨趋化因子及其受体在小鼠阴道念珠菌病发病中的作用。 【方法】将试验小鼠随机分为A组(雌激素处理感染组)、B组(雌激素处理未感染组)、C组(未用雌激素处理感染组)及正常组,前3组又分为2d、4d、7d、14d及21d组,每组各5只。建立阴道念珠菌病小鼠模型,从微生物学及组织病理学角度对小鼠的感染状态进行评估,继之以RT-PCR、Real-time RT-PCR、免疫组织化学染色SABC法及ELISA方法检测接种白念珠菌后各时间点小鼠阴道粘膜及腰淋巴结中趋化因子及其受体的表达。 【结果】(1)阴道真菌载量:A组小鼠阴道内为持续感染,其真菌载量持续维持在高水平,菌丝评分与感染的严重程度有关;B组小鼠整个21d观察不到任何真菌菌落;C组小鼠早期(2~7d)真菌载量较A组低(P0.05),第14d真菌载量迅速下降,第21d基本观察不到真菌菌落。(2)阴道粘膜病理学改变:A组小鼠白念珠菌接种后第2d,阴道腔内即可见少量紫红色线状菌丝,有PMNs浸润;第4d阴道腔内可见成团紫红色线状菌丝及卵圆形孢子,同时见大量PMNs;第7d不仅阴道腔内可见成团菌丝及孢子,亦可见菌丝及孢子由粘膜表面向角质上皮穿透,阴道腔内、阴道粘膜及粘膜下可见大量的以PMNs为主的炎性细胞浸润;第14d阴道腔内可见部分菌丝断裂;第21d阴道腔内断裂菌丝较前增多。C组小鼠接种后第2~7d病理改变基本同A组,第14d时阴道腔内仅见少量菌丝,21d时未见明显菌丝及孢子。B组小鼠阴道腔及阴道粘膜未见菌丝及孢子。(3)雌激素对阴道粘膜及淋巴结趋化因子及其受体的mRNA表达无明显影响。所有感染组小鼠的阴道粘膜趋化因子及其受体的mRNA水平均明显升高(P0.05),其中A组小鼠阴道趋化因子水平持续维持在较高水平,C组小鼠在感染消退时(第14d和21d时)其阴道趋化因子的产生亦降至基础水平;而相应的趋化因子受体在A组和C组小鼠阴道粘膜均见明显升高(P0.05)。与之不同的是,在腰引流淋巴结,感染组小鼠MCP-1、MCP-2、IP-10及MIG均未见明显改变(P0.05),SDF-1则在A组小鼠感染后第2d, 7~21d明显升高(P0.05),C组无明显变化(P0.05)。趋化因子受体CCR2在A组小鼠感染后第7~21d明显升高(P0.05),C组无明显变化(P0.05);CXCR3分别在A、C组小鼠感染后第7d、第4~14d明显升高;CXCR4分别在A、C组小鼠感染后第2~21d, 7~14d明显升高(P0.05)。(4)对A组小鼠阴道粘膜MCP-1、CCR2及IP-10 mRNA水平进一步行Real-time RT-PCR定量分析:MCP-1 mRNA在感染后各时间点均明显升高(P0.05),第14d达高峰(升高9.254倍);CCR2在感染后第7~21d明显升高(P0.05),第7d达高峰(升高5.278倍);IP-10在感染后第7~21d明显升高(P0.05),第14d达高峰(升高7.013倍)。以MCP-1升高最为明显。(5)免疫组化结果显示A组小鼠阴道粘膜中趋化因子MCP-1、SDF-1及CXCR4主要在角质形成细胞上表达,MCP-1位于胞核及胞浆,SDF-1及CXCR4则主要位于胞浆及胞膜。此外,固有层中浸润的部分炎性细胞及血管内皮细胞的胞浆亦可见MCP-1、SDF-1及CXCR4表达。B组及正常组小鼠阴道粘膜未见MCP-1、SDF-1及CXCR4的表达。半定量分析结果表明:A组小鼠在接种白念珠菌后各时间点MCP-1、SDF-1及CXCR4表达均明显增加(P0.05)。其中MCP-1在接种后第2d即见表达,2周时达高峰并持续到第3周。SDF-1及CXCR4的表达在接种后第2d即开始增加,第7d时达高峰,第14d和21d开始下降。(6)对小鼠阴道粘膜及淋巴结MCP-1蛋白水平行ELISA检测,可见MCP-1在A组小鼠感染后各时间点均明显升高,C组小鼠感染后第7d明显升高(P0.05);而腰淋巴结MCP-1在感染前后无明显改变(P0.05)。(7)阴道灌洗液涂片后染色显示,PMNs、巨噬细胞、淋巴细胞和其他类型白细胞所占比例在白念珠菌感染或雌激素造成的假发情状态下无明显改变(P0.05)。 【结论】趋化因子及其受体可能在局部阴道黏膜白念珠菌感染中起一定的作用。此外,SDF-1及趋化因子受体亦可能参与淋巴结对白念珠菌的防御。 第二部分白念珠菌与树突状细胞的相互作用及其机理 【目的】研究白念珠菌与树突状细胞(dendritic cell, DC)相互作用后,白念珠菌对DC吞噬功能、表面标志及其电生理学特性的影响,体外水平探讨DC在机体抵御白念珠菌感染中可能的作用。 【方法】(1)将白念珠菌加入DC2.4细胞培养基中,动态观察二者共孵育后30min, 1h及2h后DC2.4的吞噬功能。(2)应用流式细胞计量术检测与白念珠菌共孵育前、孵育后2h及4h时DC2.4表面MHCⅡ及共刺激分子CD80、CD86的表达。(3)应用膜片钳全细胞记录模式观察白念珠菌对DC Ik(delayed rectifier potassium current,延迟整流钾电流)的影响及伊曲康唑注射液(斯皮仁诺)对白念珠菌致DC Ik电流增加的影响。(4)应用激光扫描共聚焦显微镜观察白念珠菌对DC2.4细胞内钙浓度(intracellular calcium concentration, [Ca2+]i)的影响及伊曲康唑、氟康唑注射液(大扶康)分别对静息状态下DC[Ca2+]i及白念珠菌致DC[Ca2+]i升高的影响。 【结果】(1) DC2.4与白念珠菌共孵育30min, 1h及2h后,吞噬百分率分别为74.76±1.04%, 76.42±1.13%及90.99±1.42%;吞噬指数分别为1.328±0.023, 2.386±0.041, 3.844±0.037。(2)与孵育前相比,孵育后2h DC2.4细胞上CD80分子的表达显著上升,细胞阳性比率由孵育前41.22%升至73.91%,平均荧光强度也由61.34升至81.94(P0.05);孵育后4h CD80分子的表达有所下降,细胞阳性比率50.85%,平均荧光强度57.52;但较孵育前仍升高(P0.05)。CD86在孵育前已高表达,细胞阳性比率98.92%,故孵育后表达变化不明显,2h和4h分别为96.93%和93.57%(P0.05)。MHCⅡ在孵育前仅1.13%,荧光强度48.36;孵育后2h升至3.79%,荧光强度56.1(3P0.05);4h降为0.64%,荧光强度43.17,较孵育前无明显改变(P0.05)。(3)白念珠菌使DC2.4细胞Ik峰值可逆性升高(30.03±5.76)%,用标准细胞外液洗脱白念珠菌后Ik电流可部分恢复。伊曲康唑本身对DC2.4细胞Ik峰值无明显影响(P0.05),其与DC2.4预孵育10min后可使白念珠菌致DC Ik电流增加的幅度减小(P0.05)。此外,白念珠菌可使DC2.4 Ik的I-V曲线升高。(4)白念珠菌使DC2.4细胞[Ca2+]i荧光强度增加,且呈浓度依赖性。伊曲康唑本身即可使DC2.4细胞[Ca2+]i荧光强度降低,与DC2.4预孵育后可使白念珠菌致DC [Ca2+]i升高的幅度减小(P0.05)。氟康唑对DC[Ca2+]i及白念珠菌致DC[Ca2+]i的升高无明显影响(P0.05)。 【结论】(1)白念珠菌可能通过升高DC Ik电流及[Ca2+]i而促进其表面CD80及MHCⅡ表达升高,吞噬功能增强,进而影响DC的免疫功能状态。(2)抗真菌药物伊曲康唑可能通过降低DC [Ca2+]i及白念珠菌致DC Ik电流及[Ca2+]i升高的幅度进而抑制DC的功能,对宿主的免疫系统起负性调节作用;而氟康唑对DC的功能可能无明显影响。
[Abstract]:Expression and significance of chemokine and its receptor in vaginal candidiasis in mice
[Objective] to investigate the expression of chemokine and its receptor in the mouse model of vaginal candidiasis, and to explore the role of chemokine and its receptor in the pathogenesis of vaginal candidiasis in mice.
[Methods] the experimental mice were randomly divided into A group (estrogen treated infection group), group B (estrogenic treatment group), group C (no estrogen treatment group) and normal group. The first 3 groups were divided into 2D, 4D, 7d, 14d and 21d group, each group was 5. The mice model of vaginal candidiasis was established, and the infection of mice from microbiology and histopathology The status was evaluated by RT-PCR, Real-time RT-PCR, immunohistochemical staining SABC and ELISA methods to detect the expression of chemokine and its receptor in the vagina mucosa and the lumbar lymph nodes at each time point after inoculation of Candida albicans.
[results] (1) vaginal fungal load: the A mice were infected with persistent infection in the vagina, and its fungal load remained at a high level. The mycelial score was related to the severity of the infection; the whole 21d in group B mice did not observe any fungus colony; the early (2~7d) fungal load of group C mice was lower than that of the A group (P0.05), the 14d fungal load declined rapidly, and 21d basic. Fungal colonies were not observed. (2) pathological changes in vaginal mucosa: 2D in A mice after inoculation of Candida albicans, a small number of purple red linear mycelium in the vagina cavity, PMNs infiltration, 4D vaginal mycelial mycelium and oval spore in the vagina cavity, and a large number of PMNs; 7d not only vagina intravaginal mycelium and spores, but also vaginal intravaginal mycelium and spores, also found in the vagina It can be seen that the mycelium and spores are penetrated from the mucous surface to the corneous epithelium, in the vagina cavity, in the vagina mucosa and under the mucous membrane, a large number of inflammatory cells are infiltrated with PMNs mainly; in the 14d vagina, some of the mycelium can be seen in the vagina, and the 21d vagina breaks the mycelium of the.C group and the 2~7d pathological changes are basically the same as the A group, and the vagina at the time of 14d. Only a small amount of mycelium was found in the cavity, and no mycelium and spores were not seen in the vagina and vaginal mucosa of group.B mice. (3) there was no obvious effect of estrogen on the mRNA expression of the vagina mucosa and lymph node chemokines and their receptors in the vagina mucosa and the mRNA levels of the vagina chemokines and their receptors in all the infected mice (P0. 05), in group A, the level of vagina chemokines was maintained at a high level. In group C mice, the production of the vagina chemokines was also reduced to the base level when the infection subsided (at 14d and 21d); the corresponding chemokine receptors were significantly elevated in the A and C mice (P0.05). There was no significant change in MCP-1, MCP-2, IP-10 and MIG in the infected mice (P0.05), and SDF-1 in A group after infection, and 7~21d obviously increased (P0.05), but there was no obvious change in C group (P0.05). 4~14d increased significantly; CXCR4 was in A and C mice were significantly higher (P0.05) after infection (P0.05). (4) quantitative analysis of MCP-1, CCR2 and IP-10 mRNA levels in the vaginal mucosa of A mice. There was a significant increase (P0.05), the peak of 7D reached a peak (5.278 times higher), IP-10 was significantly elevated in 7~21d after infection (P0.05), 14d reached its peak (7.013 times). (5) MCP-1 was the most obvious. (5) the immunohistochemical results showed that the chemotactic factor MCP-1, SDF-1 and CXCR4 in the vaginal mucosa of A mice were mainly expressed on the keratinocytes, MCP-1 lies in the nucleus and cell of the cell. The plasma, SDF-1 and CXCR4 were mainly located in the cytoplasm and the cytoplasm. In addition, the cytoplasm of some inflammatory cells and vascular endothelial cells in the lamina propria was also MCP-1. The expression of MCP-1, SDF-1 and CXCR4 in the SDF-1 and CXCR4 expression.B group and the normal group of vaginal mucous membrane were not found. The semi quantitative analysis showed that the A group mice were inoculated with Candida albicans at every time. The expression of point MCP-1, SDF-1 and CXCR4 increased significantly (P0.05). Among them, MCP-1 was expressed at 2D after inoculation. The expression of.SDF-1 and CXCR4 at the peak of 2 weeks and the expression of CXCR4 began to increase after inoculation, the peak at 7d, 14d and 21d began to decline. (6) the levels of vaginal mucous membrane and lymph node protein in mice were detected. It was found that MCP-1 was significantly increased at each time point after infection in group A mice, and the 7d increased significantly in C group after infection (P0.05), but there was no significant change in the lumbar lymph node MCP-1 before and after infection (P0.05). (7) after smear of vaginal lavage fluid, the proportion of PMNs, macrophage, lymphocyte and other types of white blood cells was found in Candida albicans infection or estrogen There was no significant change in the condition of the wig condition (P0.05).
[Conclusion] chemokines and their receptors may play a role in the infection of Candida albicans in local vaginal mucosa. In addition, SDF-1 and chemokine receptors may also be involved in the defense of Candida albicans in lymph nodes.
The second part is the interaction between Candida albicans and dendritic cells and its mechanism.
[Objective] to study the effect of Candida albicans on the phagocytic function, surface markers and electrophysiological characteristics of DC after the interaction of Candida albicans with dendritic cell (DC), and the possible role of DC in the body against Candida albicans infection in vitro.
[method] (1) Candida albicans were added to the DC2.4 cell culture medium to dynamically observe the phagocytic function of DC2.4 after 30min, 1H and 2h after incubation. (2) before incubating with Candida albicans by flow cytometry, MHC II on the DC2.4 surface and CD80, CD86 expression of CO stimulators at 2h and 4h after incubation. (3) full cell recording of patch clamp (3). The effect of Candida albicans on DC Ik (delayed rectifier potassium current, delayed rectifier potassium current) and the effect of Itraconazole Injection (spen) on the increase of DC Ik current induced by Candida albicans. (4) observation of calcium concentration in DC2.4 cells by Candida albicans by laser scanning confocal microscopy (intracellular calcium concentrat) The effects of ion, [Ca2+]i and itraconazole and Fluconazole Injection on the increase of DC[Ca2+]i in resting state DC[Ca2+]i and Candida albicans were observed.
[results] (1) DC2.4 and Candida albicans were incubated with 30min, and the percentage of phagocytic percentage was 74.76 + 1.04%, 76.42 + 1.13% and 90.99 + 1.42%, respectively. The phagocytic index was 1.328 + 0.023, 2.386 + 0.041, 3.844 + 0.037. (2), compared with before incubation, the expression of CD80 molecules on 2H DC2.4 increased significantly after incubation, and the positive ratio of cells was incubated. The first 41.22% rose to 73.91%, the average fluorescence intensity increased from 61.34 to 81.94 (P0.05), and the expression of 4h CD80 molecules decreased after incubation, the positive ratio of cells was 50.85%, and the average fluorescence intensity was 57.52, but the expression of.CD86 was higher than before incubation (P0.05) before incubation, and the positive ratio of cell Yang was 98.92%, so that the expression changes were not obvious after incubation, 2h and 4H were respectively 96.93% and 93.57% (P0.05).MHC II were only 1.13% before incubation, the fluorescence intensity was 48.36, 2h increased to 3.79%, fluorescence intensity 56.1 (3P0.05), 4H decreased to 0.64%, and fluorescence intensity was 43.17, no significant change before incubation (P0.05). (3) Candida albicans increased the Ik peak reversibility (30.03 + 5.76)% of DC2.4 cells (30.03 + 5.76). Ik electricity was eluted from Candida albicans with standard extracellular fluid Itraconazole itself had no significant effect on the Ik peak of DC2.4 cells (P0.05), and the amplitude of DC Ik current induced by Candida albicans decreased (P0.05) after 10min incubation with DC2.4. In addition, Candida albicans could increase the I-V curve of DC2.4 Ik. (4) Candida albicans increased the fluorescence intensity of DC2.4 cells and showed a concentration dependence. Itraconazole itself can reduce the fluorescence intensity of [Ca2+]i in DC2.4 cells and reduce the increase of DC [Ca2+]i induced by Candida albicans (P0.05) after preincubation with DC2.4. Fluconazole has no significant effect on the increase of DC[Ca2+]i in DC[Ca2+]i and Candida albicans (P0.05).
[Conclusion] (1) Candida albicans may increase the expression of CD80 and MHC II by increasing the DC Ik current and [Ca2+]i, and increase the expression of CD80 and MHC II, increase the phagocytic function, and then affect the immune function of DC. () antifungal drug itraconazole may inhibit the power of DC by decreasing the amplitude of DC Ik current and [Ca2+]i increase induced by DC [Ca2+]i and Candida albicans. It can play a negative role in regulating the immune system of the host, while fluconazole may have no significant effect on the function of DC.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392;R519
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