口腔和阴道黏膜上皮细胞与白念珠菌相互作用的免疫学机制比较研究

发布时间：2018-06-08 21:49

本文选题：白念珠菌 + 口腔上皮细胞　；参考：《北京协和医学院》2016年博士论文

【摘要】：念珠菌是临床常见的机会性致病真菌,其中白念珠菌是引起人体口腔及阴道等部位黏膜感染的主要念珠菌。众所周知,口腔及阴道部位的黏膜上皮细胞是抵抗念珠菌感染的“第一道防线”。前期研究发现,上皮细胞抵抗白念珠菌感染主要通过激活炎性信号通路(如ERK/JUK/p38等信号通路)、释放炎性细胞因子等发挥免疫功能。鉴于临床上口腔念珠菌病更易发生在免疫低下的病人、更易造成系统播散感染,但阴道念珠菌病更易发生在免疫正常女性、少见系统播散感染,故不同部位的上皮细胞可能存在不同的抗白念珠菌感染的机制。白念珠菌胞壁成分ALS3及SSA1与黏附功能有关,在诱导上皮细胞产生抗念珠菌免疫的过程中起到重要的作用。前期已有研究证明敲除ALS3可降低诱导口腔上皮细胞分泌产生炎性细胞因子及炎性信号通路蛋白的表达。但却很少有实验研究ALS3敲除对阴道上皮细胞诱导产生炎性细胞因子的影响及可能涉及到的炎性通路。也很少有实验研究SSAl敲除对上皮细胞产生炎性细胞因子的影响及可能涉及到的炎性通路。本实验主要研究白念珠菌感染口腔上皮细胞及阴道上皮细胞诱导炎症细胞因子的产生及ERK信号通路蛋白激活的分子机制的差异,同时探讨白念珠菌胞壁成分AIs3蛋白、Ssa1蛋白在此免疫过程中存在的可能作用机制。第一章口腔和阴道上皮细胞抗白念珠菌感染的免疫差异目的：探索口腔上皮细胞及阴道上皮细胞体外感染白念珠菌免疫反应的异同性。方法：口腔上皮细胞(leuk-1)、阴道上皮细胞(VK2/E6E7)进行体外培养,给予白念珠菌(SC5314)不同菌量的感染。观察不同部位来源的上皮细胞在非感染及白念珠菌感染状态下免疫效应分子及通路蛋白表达的异同,检测白念珠菌感染不同上皮细胞时,其细胞毒性、炎性细胞因子分泌及p-ERK1/2、p-MKP1蛋白表达的差异。结果：口腔和阴道上皮细胞,在体外培养的形态上存在很多共性：倒置显微镜下见铺路石样外观,多角形,大小不一,形态不规则,细胞核呈圆形或卵圆形。细胞经过瑞氏-吉姆萨复合染液染色后,都可呈现出较为典型的细胞质粉红色,细胞核蓝(黑)色的表现。生长曲线测定结果,口腔leuk-1细胞与阴道VK2/E6E7细胞的生长增殖过程存在差异,Leuk-1细胞的增殖速度较VK2/E6E7细胞快；两组细胞在生长增殖的过程中细胞直径呈现出类似“抛物线”形状的变化图；但leuk-1细胞直径整体仍较VK2/E6E7细胞偏大。白念珠菌感染状态下,死亡细胞沿菌丝分布。细胞损伤实验结果显示,同一种细胞的损伤度与白念珠菌感染的菌量及感染时间有关,即损伤度与白念珠菌菌量、时间成正比。两组细胞对比,白念珠菌对不同上皮细胞的损伤具有特异性：同一菌量感染状态下,白念珠菌对阴道上皮细胞VK2/E6E7的损伤较重,而对口腔上皮细胞leuk-1的损伤较轻。炎性细胞因子的检测显示：白念珠菌在口腔上皮细胞与阴道上皮细胞中均可刺激诱导GM-CSF、G-CSF、IL-1α、IL-1β、RANTES、MIP-3α、IL-8的分泌量增多,但亦存在一定差异,口腔上皮细胞在未感染状态时可产生多种细胞因子,在低剂量白念珠菌感染时即可产生较多的分泌量；而阴道上皮细胞在未感染时,多数因子分泌量极少,即使在白念珠菌感染后,多数细胞因子的分泌量仍增幅不大。两种细胞出现细胞因子最大诱导量的白念珠菌刺激菌量不同。在口腔感染过程中, IL-8、GM-CSF、MIP-3α及RANTES的分泌高峰出现在白念珠菌感染菌量／细胞量(MOI)=0.01时,G-CSF、IL-1β的分泌高峰出现在MOI=0.1时,IL-1 α的分泌高峰出现在MOI=1时。而阴道上皮细胞在感染过程中,IL-8βGM-CSF的分泌高峰出现在MOI=0.01时,G-CSF的分泌高峰出现在MOI=0.1,而IL-1α、IL-1β及MIP-3α的分泌高峰出现在MOI=1时。IL-4及IL-12两种细胞因子,未显示明显的白念珠菌剂量依赖性或是规律性的趋势。在白念珠菌感染口腔和阴道上皮细胞2h后,分别检测其p-ERK1/2及p-MKP1蛋白的表达情况,显示白念SC5314感染组的p-ERK1/2及p-MKP1蛋白的表达都较对照组高,差异有统计学意义(p0.05)。结论：白念珠菌感染口腔和阴道这两种不同部位上皮细胞所产生的细胞损伤及炎性细胞因子的含量并不相同,上皮细胞抗白念珠菌的免疫应答存在部位差异性。第二章白念珠菌ALS3敲除株感染口腔和阴道上皮细胞后免疫学机制的比较研究目的：探索白念珠菌ALS3基因在白念珠菌刺激口腔上皮细胞及阴道上皮细胞产生抗白念珠菌免疫反应的过程中的作用,及其存在的可能的免疫机制差异。方法：体外分别培养口腔上皮细胞和阴道上皮细胞,给予白念珠菌SC5314(WT株)及白念珠菌ALS3敲除株(als3△)不同菌量的感染。测定白念珠菌WT株及als3△株分别感染不同上皮细胞时,其细胞毒性、炎性细胞因子及ERK1/2、MKP1通路蛋白的表达,对比不同菌量、不同菌株、不同来源细胞的免疫反应的差异。结果：白念珠菌ALS3基因敲除后,其菌丝的生长不受影响,与SC5314野生株比较,无统计学差异(p0.05)。但对比WT及als3△感染上皮细胞的结果,als3△对细胞的损伤明显降低(p0.05),且其细胞损伤程度呈剂量依赖型,即MOI值越大,损伤也越大。als3△感染口腔上皮细胞后,GM-CSF、G-CSF、,IL-1α、IL-1β、MIP-3α和IL-8细胞因子的产生量均较WT感染组降低(p0.05)。als3△感染阴道上皮细胞后,除了RANTES及MIP-3 a外的其他细胞因子,都较WT组降低(p0.05)。对比分析WT株分别感染两种不同来源上皮细胞诱导产生G-CSF、RANTES、MIP-3 α、IL-8的结果,发现口腔上皮细胞leuk-1组这些细胞因子的产量高于阴道细胞VK2/E6E7组(p0.05)；而als3△感染时,两组细胞间上述细胞因子的产量并无统计学差异(p0.05)。口腔上皮细胞分别感染白念珠菌WT与als3△ 2h后,WT感染组与als3△感染组中p-ERK1/2蛋白的表达结果无统计学差异(p0.05);但WT感染组中p-MKP1蛋白的表达量高于als3△感染组的表达量,差异有统计学意义(p0.05)。在阴道上皮细胞VK2/E6E7分别感染白念珠菌WT与als3△的蛋白检测中,WT感染组p-ERK1/2蛋白及p-MKP1蛋白的表达量均较als3△感染组的表达量高,差异有统计学意义(p0.05)。结论：白念珠菌ALS3基因的表达可以诱导口腔上皮细胞和阴道上皮细胞产生多种细胞因子。且als3△刺激两组细胞产生某些炎性细胞因子的能力有一定的差异。在白念珠菌感染口腔上皮细胞诱导免疫反应的过程中,A1s3蛋白不完全通过激活ERK免疫信号通路增强p-MKP1蛋白的表达来参与免疫反应,而在白念珠菌感染阴道上皮细胞的过程中,Als3蛋白通过激活ERK通路诱导p-MKP1蛋白的表达增强来参与免疫反应。表明口腔上皮细胞及阴道上皮细胞在应对白念珠菌Als3蛋白诱导的免疫反应中,存在部位性差异。第三章白念珠菌SSA1敲除株感染口腔和阴道上皮细胞后免疫学机制的比较研究目的：探讨白念珠菌SSAl基因表达在刺激口腔上皮细胞及阴道上皮细胞产生抗白念珠菌免疫效应过程中的作用。方法：体外培养口腔、阴道上皮细胞,分别用白念珠菌SC5314(WT)及白念珠菌SSAl基因敲除株(ssa1△)进行刺激。测定不同菌量刺激两种不同上皮细胞时在细胞损伤、炎性细胞因子分泌、通路蛋白p-ERK1/2及p-MKP1表达的变化。对比不同组别间的差异,分析可能存在的机制。结果：体外培养白念珠菌SSAl敲除株,2h后测量菌丝长度较WT株明显缩短(p0.05)。ssa1△在感染上皮细胞24h后无法形成菌落,只有散在的菌丝,经台盼蓝进行细胞染色后,可见死亡细胞沿菌丝分布。对比WT感染上皮细胞的结果,ssa1△损伤细胞的能力降低,但也可通过提高感染菌量而加大损伤。WT与ssal△分别感染口腔上皮细胞,ssal△感染组的GM-CSF、IL-1α、IL-1β、 RANTES、MIP-3α、IL-8的分泌量均较WT感染组降低(p0.05)。WT与ssal△分别感染阴道上皮细胞时,ssa1△感染组在G-CSF、IL-1 α、MIP-3 α、IL-8细胞因子的产生量降低,与WT感染组相比有统计学差异(p0.05)。白念珠菌WT株感染两种不同来源的上皮细胞时,口腔上皮细胞组IL-1β、RANTES、 MIP-3 α、IL-8的分泌量比阴道上皮细胞组高(p0.05)。而在ssa1△感染两种细胞时,两组细胞上述细胞因子产生量的对比无统计学差异(p0.05)。口腔上皮细胞分别感染白念珠菌WT与ssa1△株2h后检测通路蛋白,WT感染组p-ERK12及p-MKP1蛋白表达量较ssa1A感染组高,差异有统计学意义(p0.05)。在阴道上皮细胞分别感染WT与ssal△的蛋白检测中,白念珠菌WT感染组p-ERK12蛋白及p-MKP1蛋白的表达量均较ssa1△感染后的表达量高,差异有统计学意义(p0.05)。结论：白念珠菌SSA1基因与白念珠菌菌丝延长的功能有关。SSA1基因的敲除,降低了白念珠菌对上皮细胞的损伤,也降低了白念珠菌对口腔上皮细胞及阴道上皮细胞炎性细胞因子的产生及信号通路的激活。口腔上皮细胞及阴道上皮细胞在应对白念珠菌Ssa1蛋白诱导的免疫反应中,存在部位性差异。小结：白念珠菌感染口腔上皮细胞及阴道上皮细胞时,两种不同来源的上皮细胞激发的免疫效应并不相同,其所分泌炎症效应因子及ERK炎症通路蛋白表达方面存在差异,这可能是口腔、阴道两部位抗白念珠菌结果差异的机制之一。通过敲除白念珠菌胞壁成分基因ALS3、SSA1的菌株,分别感染口腔上皮细胞及阴道上皮细胞,发现敲除后的菌株感染可以降低细胞产生的炎症细胞因子及通路蛋白表达,还可消除两种细胞存在的部分差异,故白念珠菌ALS3、SSA1基因表达对诱导口腔及阴道上皮细胞抗白念珠菌感染免疫反应的发生,有重要作用；对口腔、阴道两部位抗白念珠菌感染免疫差异的发生也有重要作用,此实验结果可为口腔念珠菌感染及阴道念珠菌感染的防治工作研究提供更多的理论依据。
[Abstract]:Candida is a common opportunistic pathogenic fungus, in which Candida albicans is the main Candida albicans causing mucous infection in the oral and vaginal parts of the human body. It is known that mucous epithelial cells in the oral and vaginal sites are the first line of defense against Candida infection. Earlier studies found that epithelial cells resisted Candida albicans infection. It is necessary to activate inflammatory cell factors by activating inflammatory signaling pathway (such as ERK/JUK/p38 signaling pathway) and releasing inflammatory cytokines. In the light of clinical oral candidiasis, it is easier to cause systemic dissemination of infection in immunocompromised patients, but vaginal candidiasis is more likely to occur in the immune normal women. The epithelial cells in the same site may have different mechanisms for anti Candida albicans infection. The cell wall composition of Candida albicans, ALS3 and SSA1, is related to adhesion function. It plays an important role in the induction of immunization of Candida albicans in epithelial cells. Earlier studies have shown that knockout ALS3 can lower the secretion of oral epithelial cells to produce inflammation. The expression of cytokines and inflammatory signaling pathway proteins. However, there are few experimental studies on the effects of ALS3 knockout on the induced inflammatory cytokines induced by vaginal epithelial cells and the possible inflammatory pathways involved. There are few experimental studies on the effects of SSAl knockout on the inflammatory cytokines produced by epithelial cells and the possible inflammatory pathways involved. The experiment mainly studies the production of inflammatory cytokines induced by oral Candida albicans infection in oral epithelial cells and vaginal epithelial cells and the difference in molecular mechanism of ERK signaling protein activation. The possible mechanism of AIs3 protein in Candida albicans and the possible mechanism of Ssa1 protein in this immunization process. Immunological differences of Candida albicans against Candida albicans in vitro: To explore the similarities and differences between oral epithelial cells and vaginal epithelial cells in vitro infection of Candida albicans. Methods: oral epithelial cells (leuk-1), vaginal epithelial cells (VK2/E6E7) were cultured in vitro, and the infection of different strains of albicans (SC5314) was given. Differences in the expression of immune effector and pathway protein in non infected and Candida albicans infected epithelial cells. The cytotoxicity, inflammatory cytokine secretion and the difference of p-ERK1/2, p-MKP1 protein expression in Candida albicans infected with different epithelial cells. Results: oral and vaginal epithelial cells, in vitro culture form There are a lot of common features: the appearance of paving stone under inverted microscope, polygon, different size, irregular shape, round or oval nuclei. After dyed by the Rayleigh GIM composite dye, the cell can show a more typical cytoplasm pink, nuclear blue (black) color. Growth curve determination results, oral leuk- The growth and proliferation of 1 cells and vaginal VK2/E6E7 cells were different, and the proliferation rate of Leuk-1 cells was faster than that of VK2/E6E7 cells. The diameter of cells in the two groups was similar to the shape of "parabola" in the process of growth and proliferation, but the diameter of leuk-1 cells was still larger than that of VK2/ E6E7 cells. The cell damage test showed that the damage degree of the same cell was related to the amount of Candida albicans infection and the time of infection, that is, the damage degree was proportional to the amount of Candida albicans, and the two groups of cells were specific to the damage of different epithelial cells: in the condition of the same amount of infection, white Candida albicans The damage of Candida to the VK2/E6E7 of vaginal epithelial cells was heavy, but the damage to the leuk-1 of oral epithelial cells was lighter. The detection of inflammatory cytokines showed that Candida albicans in oral epithelial cells and vaginal epithelial cells stimulated GM-CSF, G-CSF, IL-1 a, IL-1 beta, RANTES, MIP-3 a, and IL-8 increased, but there was also a certain difference. The oral epithelial cells can produce a variety of cytokines in the uninfected state and produce more secretory quantities at low doses of Candida albicans, while most factors secrete most of the vaginal epithelial cells when they are not infected. Even after Candida albicans infection, the secretion of most cytokines is still small. Two kinds of cells appear. In the process of oral infection, the peak of IL-8, GM-CSF, MIP-3 alpha and RANTES appeared during the oral infection process, when the peak of Candida albicans infection / cell volume (MOI) =0.01, the peak of the secretion peak of G-CSF, IL-1 beta appeared at MOI=0.1, and the peak of IL-1 a appeared in MOI=1. And the vaginal epithelial cells were at the peak. During the infection process, when the peak of IL-8 beta GM-CSF secreted at MOI=0.01, the peak of G-CSF secreted at MOI=0.1, and the peak of IL-1 a, IL-1 beta and MIP-3 alpha appeared at MOI=1.IL-4 and IL-12 two cytokines, which did not show a significant dose dependence or regular trend of Candida albicans. In Candida albicans infection oral and vaginal After 2h, the expression of p-ERK1/2 and p-MKP1 protein were detected respectively. The expression of p-ERK1/2 and p-MKP1 protein in the white memory SC5314 infection group was higher than that of the control group. The difference was statistically significant (P0.05). Conclusion: Candida albicans infection in the oral and vaginal two different parts of the epithelial cells caused by cell damage and inflammation The immune response of Candida albicans against Candida albicans is different. Second the comparative study of the immunological mechanism of Candida albicans ALS3 knockout strains infected with oral and vaginal epithelial cells: To explore the production of Candida albicans ALS3 gene in Candida albicans to stimulate oral epithelial cells and vaginal epithelial cells The effect of anti Candida albicans immune response and the possible differences in immune mechanism. Methods: oral epithelial cells and vaginal epithelial cells were cultured in vitro, and the infection of Candida albicans SC5314 (WT strain) and Candida albicans ALS3 knockout (als3 delta) strains were given. The infection of Candida albicans and als3 delta strains were determined respectively. The cytotoxicity, inflammatory cytokines and the expression of ERK1/2, MKP1 pathway protein in different epithelial cells, compared with different strains, different strains, different sources of cell immune response. Results: the growth of the mycelium of Candida albicans ALS3 gene was not affected, and compared with the wild SC5314, there was no statistical difference (P0.05). But the comparison was compared with that of the wild SC5314 (P0.05). The results of WT and als3 delta infection of epithelial cells showed that the damage of als3 delta to cells was significantly reduced (P0.05), and the degree of cell damage was dose-dependent, that is, the greater the MOI value, the greater the damage to.Als3 delta infection of oral epithelial cells, GM-CSF, G-CSF, and the production of IL-1 alpha, IL-1 beta, MIP-3 alpha and IL-8 cell factors were lower than those of the infected group. After infection of vaginal epithelial cells, all the other cytokines except RANTES and MIP-3 a were lower than those in the WT group (P0.05). The results of WT strains respectively infected by two different sources of epithelial cells induced G-CSF, RANTES, MIP-3 alpha and IL-8, respectively, found that the production of these cytokines in the leuk-1 group of oral epithelial cells was higher than that of the vaginal cell VK2/E6E7 group. .05), while als3 delta infection, there was no statistical difference between the two groups of cell factors (P0.05). The expression of p-ERK1/2 protein in WT infection group and als3 delta infection group had no statistical difference (P0.05) after infection of Candida albicans WT and als3 delta 2H respectively, but the expression of p-MKP1 protein in WT infection group was higher than that of those in WT infection group. The expression of delta infection group was significant (P0.05). In the protein detection of VK2/E6E7 infection of Candida albicans WT and als3 Delta in vaginal epithelial cells, the expression of p-ERK1/2 protein and p-MKP1 protein in WT infection group were higher than that of als3 delta infection group, and the difference has the significance of unified planning (P0.05). Conclusion: ALS3 gene of Candida albicans The expression can induce a variety of cytokines in oral epithelial cells and vaginal epithelial cells. And the ability of als3 delta to stimulate two groups of cells to produce some inflammatory cytokines is different. In the process of inducing immune response in oral Candida albicans infected oral epithelial cells, A1s3 protein does not enhance the p-M by activating the ERK immune signal pathway. The expression of KP1 protein is involved in the immune response, and in the process of Candida albicans infected with vaginal epithelial cells, Als3 protein induces the enhancement of the expression of p-MKP1 protein by activating the ERK pathway to participate in the immune response. It shows that oral epithelial cells and vaginal epithelial cells exist in the immune response induced by Als3 protein of Candida albicans. A comparative study of the immunological mechanism of Candida albicans SSA1 knockout strains infected with oral and vaginal epithelial cells in Chapter third: To explore the role of the SSAl gene expression in oral Candida albicans and vaginal epithelial cells to stimulate the immune effect of Candida albicans in oral epithelial cells and vaginal epithelial cells. Using Candida albicans SC5314 (WT) and Candida albicans SSAl gene knockout strain (SSA1 delta), the changes in cell injury, inflammatory cytokine secretion, and pathway protein p-ERK1/2 and p-MKP1 expression in two different epithelial cells were measured respectively. The possible mechanisms were analyzed in contrast to the differences between different groups. The strain of Candida albicans SSAl knockout strain was cultured. After 2h, the length of mycelium was significantly shorter than that of WT strain (P0.05).Ssa1 Delta. After infection of epithelial cells 24h, the colony could not be formed. Only the scattered mycelium, after trypan blue was stained, the death cells were distributed along the mycelium. Compared with the result of the WT infected skin cells, the ability of SSA1 delta damaged cells was reduced. But by increasing the amount of infection bacteria, the damage of.WT and SSAL delta infection in oral epithelial cells, GM-CSF, IL-1 a, IL-1 beta, RANTES, MIP-3 a, IL-8 in SSAL delta infection group are lower than those of WT infection group (P0.05). The amount of production decreased, compared with the WT infection group (P0.05). When the WT strain of Candida albicans infected with two different sources of epithelial cells, the secretion of IL-1 beta, RANTES, MIP-3 a, IL-8 was higher than that of the vaginal epithelial cell group (P0.05) when the WT strain of Candida albicans was infected. The amount of cytokines produced above in the two groups was in the SSA1 delta infection of the two cells. There was no statistical difference (P0.05). Oral epithelial cells were infected with Candida albicans WT and SSA1 delta 2H respectively, and p-ERK12 and p-MKP1 protein expression in WT infection group were higher than that of ssa1A infection group, and the difference was statistically significant (P0.05). In the protein detection of WT and SSAL delta infection in vaginal epithelial cells, Candida albicans WT infection group The expression of K12 protein and p-MKP1 protein was higher than that of SSA1 delta infection. The difference was statistically significant (P0.05). Conclusion: the deletion of.SSA1 gene related to the function of SSA1 gene of Candida albicans and the extension of Candida albicans hyphae decreased the damage of Candida albicans to epithelial cells, and also reduced Candida albicans to oral epithelial cells and Yin. The production of inflammatory cytokines in the epithelial cells and the activation of the signal pathway. There is a regional difference between oral epithelial cells and vaginal epithelial cells in response to the immune response induced by Ssa1 protein of Candida albicans. Summary: when Candida albicans infected with oral epithelial cells and vaginal epithelial cells, two different sources of epithelial cells excite the epithelial cells. The effect of pestilence is not the same. There is a difference in the expression of inflammatory effect factor and ERK inflammatory pathway, which may be one of the mechanisms of oral and vaginal two parts of Candida albicans. By knocking out the strains of Candida albicans cell wall component gene ALS3 and SSA1, the infection of oral epithelial cells and vaginal epithelial cells, respectively, is found. The strain infection after knockout can reduce the expression of inflammatory cytokines and pathway proteins produced by the cells, and also eliminate the partial differences in the presence of two kinds of cells. Therefore, the expression of ALS3 and SSA1 gene of Candida albicans plays an important role in inducing the immune response of oral and vaginal epithelial cells against Candida albicans infection. Two parts of the oral cavity and vagina are resistant to the immune response to Candida albicans infection. Candida albicans infection also plays an important role in the occurrence of immune differences. The results can be used to prevent Candida infection and vaginal Candida infection.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R519.3

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