脾虚模型小鼠对白色念珠菌易感性的细胞免疫机制研究

发布时间：2018-05-21 06:38

本文选题：脾虚证 + 白色念珠菌　；参考：《辽宁中医药大学》2016年博士论文

【摘要】：目的:对正常及脾虚小鼠经口感染白色念珠菌,通过检测小鼠粪便中活菌数和小肠组织病理改变,观察脾虚小鼠对白色念珠菌的易感性;通过检测小肠黏膜固有层CD4+T和CD8+T细胞亚群分布,分析脾虚小鼠经口感染白色念珠菌对CD4+T和CD8+T细胞亚群分布的影响;通过检测小肠组织中IL-4、IL-10、IL-12、IFN-γm RNA及蛋白表达水平,推测脾虚小鼠感染白色念珠菌后Th1/Th2平衡的变化;通过检测小肠组织中穿孔素、颗粒酶基因及蛋白表达水平,阐述经口感染白色念珠菌小鼠CTL细胞通过穿孔素/颗粒酶途径杀伤靶细胞的机制。材料与方法:健康SPF级昆明种小白鼠60只,随机分为两组:空白组(30只)和脾虚造模组(30只)。对脾虚造模组小鼠采用饮食失节加劳倦过度的方法制备脾虚小鼠模型。模型制备成功后,再次将空白组小鼠随机均分为2组:正常对照组和正常+白色念珠菌组;将脾虚造模组小鼠随机均分为2组:脾虚模型组和脾虚+白色念珠菌组。实验第1天起,采用的饮食失节加劳倦过度复合因素造模法制备小鼠脾气虚泄泻模型。给脾虚造模组小鼠单日喂饲甘蓝,并强制性游泳至耐力极限(指小鼠游泳至无力游动,经驱赶仍不能继续游动,且出现身体向腹侧蜷曲、发抖、欲溺水等征象);双日施加猪油0.2m L/10g体重,灌胃,并正常进食,连续14天。正常对照组小鼠正常饲养。实验14天后,按照脾虚模型宏观诊断标准对脾虚造模组小鼠进行模型评价。脾虚模型制备成功后,次日对正常+白色念珠菌组和脾虚+白色念珠菌组小鼠经口感染白色念珠菌,白色念珠菌浓度为2×108/m L,剂量为0.2m L/10g体重,正常对照组和脾虚模型组小鼠给予等量生理盐水经口灌胃,染菌后,各组小鼠均正常饲养,至实验第35天,即染菌后的第21天,处死各组小鼠,进行相关指标检测。采用HE染色及电镜的方法,观察小鼠小肠黏膜的病理变化及超微结构改变;检测粪便中活菌数及念珠菌种类;采用流式细胞术检测各组小鼠小肠黏膜固有层中T淋巴细胞亚群的分布;采用western-blot法和RT-PCR法检测各组小鼠小肠组织中IL-4、IL-10、IL-12、IFN-γ蛋白及基因表达水平;采用western-blot法、免疫荧光法和RT-PCR法检测各组小鼠小肠组织中Perforin、Granzyme蛋白及基因表达水平。结果:1.各组小鼠小肠黏膜形态学改变:正常对照组小鼠小肠黏膜完整,绒毛排列整齐,肌层薄厚均匀适中。脾虚模型组小鼠与正常对照组比较,小肠黏膜比较完整,偶见小肠绒毛排列不整齐等改变。正常+白色念珠菌组和脾虚+白色念珠菌组小鼠均显示出不同程度的病变,尤以脾虚+白色念珠菌组小鼠病理改变最为明显,主要表现为绒毛缺失,排列不整齐,黏膜下层有炎性细胞浸润。2.各组小鼠小肠超微结构改变:正常对照组小鼠小肠微绒毛排列整齐,细胞内细胞器丰富,线粒体、内质网、核糖体清晰可见。脾虚模型组小鼠小肠微绒毛略短,但排列尚比较整齐。正常+白色念珠菌组小鼠小肠微绒毛稀疏,长短不一,细胞质内线粒体肿胀、偶见空泡样改变。脾虚+白色念珠菌组小鼠小肠微绒毛明显减少,排列紊乱,线粒体明显肿胀,嵴消失,呈空泡样改变,内质网扩张,表面核糖体脱落。3.各组小鼠粪便中活菌数检测结果显示:与正常对照组比较,脾虚模型组、脾虚+白色念珠菌组小鼠粪便中活菌数显著增高(P0.01或P0.05),有统计学差异,与脾虚模型组比较,脾虚+白色念珠菌组小鼠粪便中活菌数显著升高(P0.01),有统计学差异。4.各组小鼠粪便中念珠菌种类检测结果显示:正常对照组热带念珠菌和克柔念珠菌偶有生长,检出率均为10%。正常+白色念珠菌组小鼠粪便中以白念菌检出率最高,为50%,光滑念珠菌检出率为20%,克柔念珠菌检出率为10%。脾虚模型组小鼠以热带念珠菌检出率最高,为30%,其次为光滑念珠菌,为20%。脾虚+白色念珠菌组小鼠以白色念珠菌检出率最高,为40%,其次为光滑念珠菌(30%)和克柔念珠菌(20%)。5.各组小鼠肠黏膜固有层CD3+T细胞的检测结果显示:各组小鼠肠黏膜固有层CD3+T细胞组间比较无统计学差异。与正常对照组比较,正常+白色念珠菌组、脾虚模型组及脾虚+白色念珠菌组小鼠小肠黏膜固有层中CD4+T细胞比例均不同程度下降。与正常+白色念珠菌组比较,脾虚+白色念珠菌组小鼠小肠黏膜固有层CD4+T细胞百分比显著下降(P0.01)。与脾虚模型组比较,脾虚+白色念珠菌组小鼠小肠黏膜固有层CD4+T细胞百分比显著下降(P0.01)。正常对照组、正常+白色念珠菌组、脾虚模型组间比较,小鼠小肠黏膜固有层中CD8+T细胞比例无统计学差异,但均与脾虚+白色念珠菌组比较,有统计学差异(P0.05或P0.01)。与正常对照组比较,其余三组小鼠小肠黏膜固有层中CD4+T/CD8+T比值均有不同程度下降,有显著性差异(P0.01);正常+白色念珠菌组与脾虚+白色念珠菌组比较,有统计学差异(P0.01);脾虚模型组与脾虚+白色念珠菌组比较,有统计学差异(P0.01)。6.各组小鼠小肠组织中IFN-γm RNA及蛋白的表达水平显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中IFN-γm RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组IFN-γm RNA及蛋白表达水平显著降低(P0.01),而脾虚+白色念珠菌组IFN-γm RNA及蛋白表达水平显著升高(P0.01);与脾虚模型组比较,脾虚+白色念珠菌组IFN-γm RNA及蛋白表达水平显著升高(P0.05)。7.各组小鼠小肠组织中IL-4 m RNA及蛋白表达水平检测结果显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中IL-4 m RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组及脾虚+白色念珠菌组IL-4 m RNA及蛋白表达水平显著降低(P0.01);与脾虚模型组比较,脾虚+白色念珠菌组IL-4 m RNA及蛋白表达水平显著升高(P0.01)。8.各组小鼠小肠组织中IL-12 m RNA及蛋白表达水平检测结果显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中IL-12 m RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组IL-12 m RNA及蛋白表达水平显著降低(P0.01),而脾虚+白色念珠菌组IL-12 m RNA及蛋白表达水平显著升高(P0.05);与脾虚模型组比较,脾虚+白色念珠菌组IL-12 m RNA及蛋白表达水平显著升高(P0.01或0.05)。9.各组小鼠小肠组织中IL-10 m RNA及蛋白表达水平检测结果显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中IL-10 m RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组及脾虚+白色念珠菌组IL-10 m RNA及蛋白表达水平显著降低(P0.01);与脾虚模型组比较,脾虚+白色念珠菌组IL-10 m RNA及蛋白表达水平显著升高(P0.01或0.05)。10.各组小鼠小肠组织中Perforin m RNA及蛋白的表达水平结果显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中Perforin m RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组Perforin m RNA及蛋白表达水平显著降低(P0.01),而脾虚+白色念珠菌组Perforin m RNA及蛋白表达水平显著升高(P0.01);与脾虚模型组比较,脾虚+白色念珠菌组Perforin m RNA及蛋白表达水平显著升高(P0.01)。11.各组小鼠小肠组织中Granzyme B m RNA及蛋白表达水平检测结果显示:与正常对照组比较,正常+白色念珠菌组、脾虚模型组、脾虚+白色念珠菌组小鼠小肠组织中Granzyme B m RNA及蛋白表达水平显著升高(P0.01);与正常+白色念珠菌组比较,脾虚模型组Granzyme B m RNA及蛋白表达水平显著降低(P0.01或P0.05);与脾虚模型组比较,脾虚+白色念珠菌组Granzyme B m RNA及蛋白表达水平显著升高(P0.01)。结论:1.脾虚证小鼠肠道念珠菌群紊乱。经口感染白色念珠菌后,粪便活菌数明显增加,加重肠道的念珠菌群紊乱及小肠组织的病理改变。2.脾虚小鼠的CD4+T/CD8+T比例发生改变。经口感染白色念珠菌后,CD4+T、CD8+T亚群分布变化更为明显,机体的免疫功能受损更为明显。3.白色念珠菌感染时,机体的体液免疫及细胞免疫功能均发生变化,但以细胞免疫为主。4.经口感染白色念珠菌可引起小鼠小肠局部CTL的活化,导致Perforin和Granzyme表达水平的升高。脾虚加重了机体白色念珠菌感染病情,Perforin和Granzyme高表达水平可能是脾虚小鼠感染白色念珠菌的发生机制之一。
[Abstract]:Objective: To observe the oral Candida albicans infection in normal and spleen asthenia mice, by detecting the number of living bacteria in the feces and pathological changes of small intestine in mice, the susceptibility to Candida albicans in spleen asthenia mice was observed. By detecting the distribution of CD4+T and CD8+T cell subsets in the propria of the small intestinal mucosa, the oral Candida albicans infected by the spleen asthenia mice on CD4+T and CD8+T were analyzed. The influence of the distribution of cell subsets; by detecting the level of IL-4, IL-10, IL-12, IFN- gamma m RNA and protein expression in the small intestinal tissue, the changes of Th1/Th2 balance after infection with Candida albicans in spleen asthenia mice were estimated. By detecting the perforin, Granzyme gene and protein expression in small intestine tissues, the passage of the oral Candida albicans CTL cells through oral infection was introduced. The mechanism of perforin / granzyme pathway killing target cells. Materials and methods: 60 healthy Kunming mice were randomly divided into two groups: blank group (30) and spleen deficiency model (30 rats). The spleen asthenia mice model was prepared by diet loss and overfatigue in spleen deficiency model mice. After the model preparation was successful, the blank group was reduced to a small group. The rats were randomly divided into 2 groups: normal control group and normal + Candida albicans group. The spleen asthenia model mice were randomly divided into 2 groups: spleen deficiency model group and spleen asthenia + Candida albicans group. After the first day of experiment, the model of spleen deficiency and tiredness was used to produce spleen deficiency diarrhea model in mice. Brassica oleracea, and compulsory swimming to the endurance limit (refers to the mice swimming to the inability to swim, still can not continue to swim, and the body to the abdominal crouching, shivering, drowning and other signs); double day to apply the lard 0.2m L/10g weight, gavage, and normal feeding for 14 days, normal control group normal feeding. 14 days after the experiment, according to spleen asthenia model After the spleen deficiency model was successfully prepared, the normal + Candida albicans group and the spleen asthenia + Candida albicans group were infected with Candida albicans, the concentration of Candida albicans was 2 x 108/m L, and the dose was 0.2m L/10g, and the normal control group and the spleen deficiency model group were given the same amount. The normal saline was fed through the mouth. After dyeing the bacteria, all the mice were kept normally, and the mice were killed for thirty-fifth days, that is, twenty-first days after the infection, the mice were killed and the related indexes were detected. The pathological changes and ultrastructural changes of the small intestinal mucosa were observed by HE staining and electron microscopy, and the number of living bacteria and Candida species in the feces was detected. Flow formula was used. The distribution of T lymphocyte subsets in the small intestinal mucosa propria of each group was detected by cell operation, and the levels of IL-4, IL-10, IL-12, IFN- gamma protein and gene expression were detected by Western-blot and RT-PCR methods. Western-blot, immunofluorescence and RT-PCR methods were used to detect Perforin, Granzyme eggs in small intestine tissues of mice in each group. The expression level of Rhizoma Bletillae gene. Results: 1. the morphological changes of small intestinal mucosa of mice in each group: the small intestinal mucosa of the normal control group was complete, the villi were arranged well and the muscularis thickness was evenly and moderately. The small intestine mucosa was more complete in the spleen deficiency model group than the normal control group. The mice of the spleen deficiency + Candida albicans group showed different degrees of pathological changes, especially in the spleen deficiency and Candida albicans group, the most obvious pathological changes were the loss of the villus, the irregular arrangement, the small intestinal ultrastructure of the mice in each group of.2. groups with inflammatory cell infiltration in the submucosa: the small intestinal microvilli in the normal control group were arranged neatly and finely. The intracellular organelles were abundant, mitochondria, endoplasmic reticulum and ribosome were clearly visible. The small intestinal microvilli in the mice of the spleen deficiency model group were slightly shorter, but the small intestinal microvilli in the normal + Candida albicans group were sparse and different, the mitochondria were swollen in the cytoplasm, and the vacuoles like changes were seen in the cytoplasm. The results showed that the number of living bacteria in the spleen asthenia model group and the spleen asthenia + Candida albicans group increased significantly (P0.01 or P0.05) in the feces of the mice of the spleen asthenia and Candida albicans (P0.01 or P0.05). Compared with the spleen deficiency model group, the number of living bacteria in the feces of spleen asthenia + Candida albicans increased significantly (P0.01), and there was a statistical difference in the detection of Candida species in the feces of.4. mice: the normal control group of Candida tropicalis and Candida kuriuri were even growing, and the detection rates were all white in the feces of 10%. normal + Candida albicans. The detection rate of Candida albicans was the highest, the detection rate of Candida smooth Candida was 20%, the detection rate of Candida Kurus in 10%. spleen deficiency model group was the highest, 30%, followed by Candida albicans. The detection rate of Candida albicans in 20%. spleen asthenia + Candida albicans group was the highest, 40%, followed by Candida smooth Candida (30%) and krou. The detection of intestinal mucosa propria CD3+T cells in each group of.5..5. showed that there was no statistical difference between the groups of intestinal mucosa propria in each group. Compared with the normal control group, the proportion of CD4+T cells in the small intestinal mucosa of normal + Candida albicans group, spleen deficiency model group and spleen asthenia + Candida albicans group was different Compared with the normal + Candida albicans group, the percentage of CD4+T cells in the small intestinal mucosa propria of the spleen asthenia + Candida albicans group decreased significantly (P0.01). Compared with the spleen deficiency model group, the percentage of CD4+T cells in the small intestinal mucosa propria of spleen asthenia + Candida albicans decreased significantly (P0.01). Normal control group, normal + Candida albicans group, Compared with the spleen deficiency model group, the proportion of CD8+T cells in the small intestinal mucosa propria of mice was not statistically different, but compared with the spleen asthenia + Candida albicans group, there were statistical differences (P0.05 or P0.01). Compared with the normal control group, the ratio of CD4+T/CD8+ T in the propria of the small intestinal mucosa of the other three groups decreased in varying degrees, there was significant difference (P0.01 Compared with the spleen asthenia and Candida albicans group, the normal + Candida albicans group had statistical difference (P0.01), the spleen deficiency model group and the spleen asthenia + Candida albicans group had statistical difference (P0.01) the expression level of IFN- gamma m RNA and protein in the small intestinal tissues of.6. mice showed that the normal + Candida albicans group and the spleen deficiency model were compared with the normal control group. The expression level of IFN- gamma m RNA and protein in small intestinal tissue of spleen asthenia + Candida albicans group increased significantly (P0.01). Compared with normal + Candida albicans group, IFN- gamma m RNA and protein expression level in spleen deficiency model group decreased significantly (P0.01), but IFN- gamma m RNA and protein expression level in spleen asthenia + Candida albicans group increased significantly (P0.01), and spleen deficiency model. IFN- gamma m RNA and protein expression level in the group of spleen deficiency and Candida albicans increased significantly (P0.05) and the expression level of IL-4 m RNA and protein expression in small intestinal tissues of each group of.7. mice showed that: compared with the normal control group, normal + Candida albicans, spleen deficiency model group, spleen asthenia + Candida albicans group mice small intestinal tissue IL-4 m RNA and protein Compared with normal + Candida albicans group, the expression level of IL-4 m RNA and protein in spleen asthenia model group and spleen deficiency + Candida albicans group decreased significantly (P0.01), and the expression of IL-4 m RNA and protein in spleen asthenia + Candida albicans group was significantly higher than that in spleen asthenia model group (P0.01) and IL-12 m in small intestine tissues of mice in each group of spleen asthenia + Candida albicans (P0.01).8. group (P0.01) The results of RNA and protein expression showed that: compared with the normal control group, the expression level of IL-12 m RNA and protein in small intestinal tissues of normal + Candida albicans group, spleen deficiency model group and spleen deficiency + Candida albicans group increased significantly (P0.01), compared with normal + Candida albicans group, the expression level of IL-12 m RNA and protein in spleen deficiency model group decreased significantly. The level of IL-12 m RNA and protein expression in spleen asthenia and Candida albicans increased significantly (P0.05), and the expression level of IL-12 m RNA and protein expression in spleen asthenia + Candida albicans group was significantly increased (P0.01 or 0.05) in spleen asthenia + Candida albicans group (P0.01 or 0.05), IL-10 m and protein expression in small intestine tissues of mice in each group showed that the results showed that the normal control group was with the normal control group. Compared with normal + Candida albicans group, spleen deficiency model group, spleen asthenia + Candida albicans group, the expression level of IL-10 m RNA and protein increased significantly (P0.01). Compared with normal + Candida albicans group, the IL-10 m RNA and protein expression level of spleen asthenia model group and spleen deficiency + Candida albicans group decreased significantly (P0.01); compared with the spleen deficiency model group, the expression level of the spleen deficiency and Candida albicans group was significantly lower than that of the spleen deficiency model group. IL-10 m RNA and protein expression level of spleen asthenia + Candida albicans increased significantly (P0.01 or 0.05) the expression level of Perforin m RNA and protein in small intestine tissues of.10. mice showed that: compared with normal control group, normal + Candida albicans group, spleen deficiency model group, spleen deficiency + Candida albicans group mice small intestine tissue Perforin m RNA and The level of protein expression increased significantly (P0.01). Compared with the normal + Candida albicans group, the expression level of Perforin m RNA and protein in the spleen deficiency model group decreased significantly (P0.01), but the expression level of Perforin m RNA and protein in the spleen asthenia + Candida albicans group increased significantly (P0.01), and the spleen asthenia + Candida albicans group Perforin m RNA and protein compared with the spleen deficiency model group. The expression level of Granzyme B m RNA and protein expression in small intestine tissues of mice in each group was significantly increased (P0.01). The results showed that the Granzyme B m RNA and protein expression level in the small intestinal tissues of normal + Candida albicans, spleen deficiency model and spleen asthenia + Candida albicans increased significantly (P0.01) and normal + with normal control group. Compared with the group of Candida albicans, the expression level of Granzyme B m RNA and protein in the spleen deficiency model group was significantly decreased (P0.01 or P0.05). Compared with the spleen deficiency model group, the Granzyme B m RNA and protein expression level of the spleen asthenia + Candida albicans group increased significantly (P0.01). Conclusion: 1. spleen asthenia mice with intestinal candidiasis disorder. After oral Candida albicans infection, feces The number of living bacteria increased significantly, the disorder of Candida albicans in the intestine and the pathological changes of small intestinal tissue changed the CD4+T/CD8+T ratio in.2. spleen asthenia mice. After oral Candida albicans infection, the distribution of CD4+T and CD8+T subgroups was more obvious, and the immune function of the body was more obvious in.3. Candida albicans infection, body humoral immunity and The cellular immune function changes, but the oral Candida albicans infected with cellular immune.4. can cause the activation of local CTL in the small intestine of mice, which leads to the increase of the expression level of Perforin and Granzyme. Spleen deficiency aggravates the disease of Candida albicans, and the high level of Perforin and Granzyme may be the white Candida infection in mice with spleen deficiency. One of the mechanisms of bacteria.
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R519.3

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