本文選題：動(dòng)物 + 念珠菌　； 參考：《華中科技大學(xué)》2007年博士論文

【摘要】： 第一部分趨化因子及其受體在陰道念珠菌病小鼠模型中的表達(dá)及其意義 【目的】檢測(cè)陰道念珠菌病小鼠模型中趨化因子及其受體的表達(dá),探討趨化因子及其受體在小鼠陰道念珠菌病發(fā)病中的作用。 【方法】將試驗(yàn)小鼠隨機(jī)分為A組(雌激素處理感染組)、B組(雌激素處理未感染組)、C組(未用雌激素處理感染組)及正常組,前3組又分為2d、4d、7d、14d及21d組,每組各5只。建立陰道念珠菌病小鼠模型,從微生物學(xué)及組織病理學(xué)角度對(duì)小鼠的感染狀態(tài)進(jìn)行評(píng)估,繼之以RT-PCR、Real-time RT-PCR、免疫組織化學(xué)染色SABC法及ELISA方法檢測(cè)接種白念珠菌后各時(shí)間點(diǎn)小鼠陰道粘膜及腰淋巴結(jié)中趨化因子及其受體的表達(dá)。 【結(jié)果】(1)陰道真菌載量:A組小鼠陰道內(nèi)為持續(xù)感染,其真菌載量持續(xù)維持在高水平,菌絲評(píng)分與感染的嚴(yán)重程度有關(guān);B組小鼠整個(gè)21d觀察不到任何真菌菌落;C組小鼠早期(2~7d)真菌載量較A組低(P0.05),第14d真菌載量迅速下降,第21d基本觀察不到真菌菌落。(2)陰道粘膜病理學(xué)改變:A組小鼠白念珠菌接種后第2d,陰道腔內(nèi)即可見少量紫紅色線狀菌絲,有PMNs浸潤(rùn);第4d陰道腔內(nèi)可見成團(tuán)紫紅色線狀菌絲及卵圓形孢子,同時(shí)見大量PMNs;第7d不僅陰道腔內(nèi)可見成團(tuán)菌絲及孢子,亦可見菌絲及孢子由粘膜表面向角質(zhì)上皮穿透,陰道腔內(nèi)、陰道粘膜及粘膜下可見大量的以PMNs為主的炎性細(xì)胞浸潤(rùn);第14d陰道腔內(nèi)可見部分菌絲斷裂;第21d陰道腔內(nèi)斷裂菌絲較前增多。C組小鼠接種后第2~7d病理改變基本同A組,第14d時(shí)陰道腔內(nèi)僅見少量菌絲,21d時(shí)未見明顯菌絲及孢子。B組小鼠陰道腔及陰道粘膜未見菌絲及孢子。(3)雌激素對(duì)陰道粘膜及淋巴結(jié)趨化因子及其受體的mRNA表達(dá)無明顯影響。所有感染組小鼠的陰道粘膜趨化因子及其受體的mRNA水平均明顯升高(P0.05),其中A組小鼠陰道趨化因子水平持續(xù)維持在較高水平,C組小鼠在感染消退時(shí)(第14d和21d時(shí))其陰道趨化因子的產(chǎn)生亦降至基礎(chǔ)水平;而相應(yīng)的趨化因子受體在A組和C組小鼠陰道粘膜均見明顯升高(P0.05)。與之不同的是,在腰引流淋巴結(jié),感染組小鼠MCP-1、MCP-2、IP-10及MIG均未見明顯改變(P0.05),SDF-1則在A組小鼠感染后第2d, 7~21d明顯升高(P0.05),C組無明顯變化(P0.05)。趨化因子受體CCR2在A組小鼠感染后第7~21d明顯升高(P0.05),C組無明顯變化(P0.05);CXCR3分別在A、C組小鼠感染后第7d、第4~14d明顯升高;CXCR4分別在A、C組小鼠感染后第2~21d, 7~14d明顯升高(P0.05)。(4)對(duì)A組小鼠陰道粘膜MCP-1、CCR2及IP-10 mRNA水平進(jìn)一步行Real-time RT-PCR定量分析:MCP-1 mRNA在感染后各時(shí)間點(diǎn)均明顯升高(P0.05),第14d達(dá)高峰(升高9.254倍);CCR2在感染后第7~21d明顯升高(P0.05),第7d達(dá)高峰(升高5.278倍);IP-10在感染后第7~21d明顯升高(P0.05),第14d達(dá)高峰(升高7.013倍)。以MCP-1升高最為明顯。(5)免疫組化結(jié)果顯示A組小鼠陰道粘膜中趨化因子MCP-1、SDF-1及CXCR4主要在角質(zhì)形成細(xì)胞上表達(dá),MCP-1位于胞核及胞漿,SDF-1及CXCR4則主要位于胞漿及胞膜。此外,固有層中浸潤(rùn)的部分炎性細(xì)胞及血管內(nèi)皮細(xì)胞的胞漿亦可見MCP-1、SDF-1及CXCR4表達(dá)。B組及正常組小鼠陰道粘膜未見MCP-1、SDF-1及CXCR4的表達(dá)。半定量分析結(jié)果表明:A組小鼠在接種白念珠菌后各時(shí)間點(diǎn)MCP-1、SDF-1及CXCR4表達(dá)均明顯增加(P0.05)。其中MCP-1在接種后第2d即見表達(dá),2周時(shí)達(dá)高峰并持續(xù)到第3周。SDF-1及CXCR4的表達(dá)在接種后第2d即開始增加,第7d時(shí)達(dá)高峰,第14d和21d開始下降。(6)對(duì)小鼠陰道粘膜及淋巴結(jié)MCP-1蛋白水平行ELISA檢測(cè),可見MCP-1在A組小鼠感染后各時(shí)間點(diǎn)均明顯升高,C組小鼠感染后第7d明顯升高(P0.05);而腰淋巴結(jié)MCP-1在感染前后無明顯改變(P0.05)。(7)陰道灌洗液涂片后染色顯示,PMNs、巨噬細(xì)胞、淋巴細(xì)胞和其他類型白細(xì)胞所占比例在白念珠菌感染或雌激素造成的假發(fā)情狀態(tài)下無明顯改變(P0.05)。 【結(jié)論】趨化因子及其受體可能在局部陰道黏膜白念珠菌感染中起一定的作用。此外,SDF-1及趨化因子受體亦可能參與淋巴結(jié)對(duì)白念珠菌的防御。 第二部分白念珠菌與樹突狀細(xì)胞的相互作用及其機(jī)理 【目的】研究白念珠菌與樹突狀細(xì)胞(dendritic cell, DC)相互作用后,白念珠菌對(duì)DC吞噬功能、表面標(biāo)志及其電生理學(xué)特性的影響,體外水平探討DC在機(jī)體抵御白念珠菌感染中可能的作用。 【方法】(1)將白念珠菌加入DC2.4細(xì)胞培養(yǎng)基中,動(dòng)態(tài)觀察二者共孵育后30min, 1h及2h后DC2.4的吞噬功能。(2)應(yīng)用流式細(xì)胞計(jì)量術(shù)檢測(cè)與白念珠菌共孵育前、孵育后2h及4h時(shí)DC2.4表面MHCⅡ及共刺激分子CD80、CD86的表達(dá)。(3)應(yīng)用膜片鉗全細(xì)胞記錄模式觀察白念珠菌對(duì)DC Ik(delayed rectifier potassium current,延遲整流鉀電流)的影響及伊曲康唑注射液(斯皮仁諾)對(duì)白念珠菌致DC Ik電流增加的影響。(4)應(yīng)用激光掃描共聚焦顯微鏡觀察白念珠菌對(duì)DC2.4細(xì)胞內(nèi)鈣濃度(intracellular calcium concentration, [Ca2+]i)的影響及伊曲康唑、氟康唑注射液(大扶康)分別對(duì)靜息狀態(tài)下DC[Ca2+]i及白念珠菌致DC[Ca2+]i升高的影響。 【結(jié)果】(1) DC2.4與白念珠菌共孵育30min, 1h及2h后,吞噬百分率分別為74.76±1.04%, 76.42±1.13%及90.99±1.42%;吞噬指數(shù)分別為1.328±0.023, 2.386±0.041, 3.844±0.037。(2)與孵育前相比,孵育后2h DC2.4細(xì)胞上CD80分子的表達(dá)顯著上升,細(xì)胞陽性比率由孵育前41.22%升至73.91%,平均熒光強(qiáng)度也由61.34升至81.94(P0.05);孵育后4h CD80分子的表達(dá)有所下降,細(xì)胞陽性比率50.85%,平均熒光強(qiáng)度57.52;但較孵育前仍升高(P0.05)。CD86在孵育前已高表達(dá),細(xì)胞陽性比率98.92%,故孵育后表達(dá)變化不明顯,2h和4h分別為96.93%和93.57%(P0.05)。MHCⅡ在孵育前僅1.13%,熒光強(qiáng)度48.36;孵育后2h升至3.79%,熒光強(qiáng)度56.1(3P0.05);4h降為0.64%,熒光強(qiáng)度43.17,較孵育前無明顯改變(P0.05)。(3)白念珠菌使DC2.4細(xì)胞Ik峰值可逆性升高(30.03±5.76)%,用標(biāo)準(zhǔn)細(xì)胞外液洗脫白念珠菌后Ik電流可部分恢復(fù)。伊曲康唑本身對(duì)DC2.4細(xì)胞Ik峰值無明顯影響(P0.05),其與DC2.4預(yù)孵育10min后可使白念珠菌致DC Ik電流增加的幅度減小(P0.05)。此外,白念珠菌可使DC2.4 Ik的I-V曲線升高。(4)白念珠菌使DC2.4細(xì)胞[Ca2+]i熒光強(qiáng)度增加,且呈濃度依賴性。伊曲康唑本身即可使DC2.4細(xì)胞[Ca2+]i熒光強(qiáng)度降低,與DC2.4預(yù)孵育后可使白念珠菌致DC [Ca2+]i升高的幅度減小(P0.05)。氟康唑?qū)C[Ca2+]i及白念珠菌致DC[Ca2+]i的升高無明顯影響(P0.05)。 【結(jié)論】(1)白念珠菌可能通過升高DC Ik電流及[Ca2+]i而促進(jìn)其表面CD80及MHCⅡ表達(dá)升高,吞噬功能增強(qiáng),進(jìn)而影響DC的免疫功能狀態(tài)。(2)抗真菌藥物伊曲康唑可能通過降低DC [Ca2+]i及白念珠菌致DC Ik電流及[Ca2+]i升高的幅度進(jìn)而抑制DC的功能,對(duì)宿主的免疫系統(tǒng)起負(fù)性調(diào)節(jié)作用;而氟康唑?qū)C的功能可能無明顯影響。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392;R519
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本文編號(hào)：1925785