Study On The Cellular Immune Mechanism Of Susceptibility To Candida Albicans In Mice Model Of Spleen Deficiency

Purpose: The susceptibility to candida albicans was evaluated through counting the albicans content in feces and observing small intestine tissue pathological changes; To analysis the distribution of CD4+T and CD8+T cell subpopulations induced by the spleen deficiency mice which were infected by oral candida albicans; to evaluated the balance of Th1/Th2 induced by the spleen deficiency mice which were infected by oral candida albicans, the IL- 4, IL-10, IL-12, IFN-γm RNAlevels of small intestine tissues were detected; The mechanism of killing target cells through perforin/particle enzymepathway by CTL cells were explored through determined the perforin, granzyme gene and protein expression level of small intestine tissues.Methods: Health SPF kunming mice were randomly divided into two groups: the blank group and model group(spleen deficient), 30 mice each group. The mouse model was established through fatigue integrated with imbalance diet. After the model established successfully, blank group mice were randomly divided into 2 groups: normal control group(15) and candida albicans infected group(15); model tmice were randomly divided into 2 groups: model group(15) andmodel with candida albicaninfected group(15). The mice were only fed with cabbage, and exhaustive weight-loading swimming exercise was performed in the first day, then, the mice were intragastricadminstration with Lard and received a normal diet in the second day; the experiment was last for 14 days. Then the model mice were evaluated by macroscopic diagnostic criteria of the spleen deficiency model. And the next day the mice in candida albicansinfected and model with candida albicans infected groups were oral infected with2×108/m Lcandida albicans(0.2 m L / 10 g), the mice in normal control group and model group were i.gsaline. The mice were sacrificed and the indices were detected in the 21 day after infection. The intestinal mucosa pathological and ultrastructure changes were observed by the method of HE staining and electron microscopy; The candidaalbicans content in feces was counted and candida species were detected; The distribution of T lymphocytesubsets in small intestinal lamina propriawere analyzed through flow cytometry;The IL-4、IL-10、IL-12 and IFN-γexpression levels of small intestine tissuewere detected by Western blot and RT-PCR methods; The perforin, granzyme protein and gene expression levels in small intestine tissues of mice were detected by Western-blot, immunefluorescence and RT-PCR methods.Results: 1.The changes of small intestinal mucosa in each group: Mice in normal control group exhibitedintact intestinal mucosa, fluff was arranged in neat rows, muscular layer of uniform thickness was moderate. Compared with normal control group, the small intestine villi arranged in irregular change was observed in model group. Mice in candida albicans infected group and showed pathological changes in different degree, especially mice in model withcandida albicans infectedgroupwhich showed loss of villi and irregular arrangement, submucosa with infiltration of inflammatory cells. 2.The changes of small intestine ultrastructure in each group: Mice in normal control group showed intestinal microvilli arranged in neat rows, abundant organelles in the cells, mitochondria, endoplasmic reticulum, ribosomes clearly visible. The intestinal microvillus slightly shorter, but the arrangement is neat of mice in model group. Mice incandida albicansshowedsparse intestinal microvilli, length, swelling of mitochondria in the cytoplasm, occasionally vacuolar changes. Mice in model with candidaalbicans infected group showed decreased intestinal microvilli, arranged in disorder, swollen mitochondria cristae, vacuolar changes, dilatation of the endoplasmic reticulum, the surface of the ribosome shedding 3.The result indicated that candidaalbicans contents are significantly increased in model and model with candidaalbicans infected groups than normal control group(P< 0.01 or P< 0.05); candidaalbicans contents were significantly increased in model with candida albicans infected group than model group(P< 0.01). 4.The results of candida species in feces showed: candida tropicalis and candida krusei occasionally growth in normal control group, detection rate was 10%. White read bacteria detection rate is the highest in candida albicans infected group, detection ratewas 50%, detection rate ofcandida glabrata was 20%, and detection rate of candida kruseiwas 10%. The detection rateofcandida tropicaliswas highest for 30% in the model group, followed by candida glabrata for 20%. The detection rate of candida albicanswas the highest for 40% in model with candida albicans infected group, followed by smooth candida(30%) and candida krusei(20%). 5.The results of CD3 + T cells test showed that there is no significant difference among groups. Compared with normal control group, The CD4 + T cell ratiosin the small intestinal mucosa lamina propria of mice were decreased with different degrees in candida albicans infected group, model group and model with candida albicansinfected group. Compared with candida albicans infected group, the CD4 + T cell ratioin the small intestinal mucosa lamina propria of mice wassignificant decreasedin model with candida albicansinfected group(P< 0.01). Compared with model group, the CD4 + T cell ratioin the small intestinal mucosa lamina propria of mice was significant decreased in model with candida albicansinfected group(P< 0.01). there was no significant difference of CD8 + T cell ratios among normal control, candida albicans infected and model groups, but had significant difference compared with model with candida albicansinfected group(P< 0.05 or P< 0.01). Compared with normal control group, the CD4 + T/CD8 +T ratiosin other groups were significant decreased(P< 0.01); there was significant difference of CD4 + T/CD8 +T ratiosbetween candida albicansinfected and model with candida albicans infected groups(P< 0.01);there was significant difference of CD4 + T/CD8 +T ratiosbetween model and model with candida albicans infected groups(P< 0.01). 6.The expression levels of IFN-γ m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels of IFN-γ m RNA and protein were significantly decreased in model group(P< 0.01),but significantly increased in model with candida albicans infected group(P< 0.01); Compared with model group, the expression levels of IFN-γ m RNA and protein were significantlyincreased in model with candida albicans infected group(P< 0.05). 7.The expression levels of IL-4 m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels of IL-4 m RNA and protein were significantly decreased in modelandmodel with candida albicans infected groups(P< 0.01); Compared with model group, the expression levels of IL-4 m RNA and protein were significantlyincreased in model with candida albicans infected group(P< 0.05). 8.The expression levels of IL-12 m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels of IL-12 m RNA and protein were significantly decreased in model group(P< 0.01), but significantly increased in model with candida albicans infected group(P< 0.01); Compared with model group, the expression levels of IL-12 m RNA and protein were significantly increased in model with candida albicans infected group(P< 0.05 or P< 0.01). 9.The expression levels of IL-10 m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels of IL-10 m RNA and protein were significantly decreased in modeland model with candida albicans infected groups(P< 0.01); Compared with model group, the expression levels of IL-10 m RNA and protein were significantly increased in model with candida albicans infected group(P< 0.05 or P< 0.01). 10.The expression levels of perforin m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels of perforin m RNA and protein were significantly decreased in modelgroup(P< 0.01), but significantly increased in model with candida albicans infected groups(P< 0.01); Compared with model group, the expression levels of perforin m RNA and protein were significantly increased in model with candida albicans infected group(P< 0.01). 11.The expression levels of granzyme B m RNA and protein were significantly increased in candida albicans infected, model and model with candida albicans infected groups than normal control group(P< 0.01); Compared with candida albicans infected group, the expression levels ofgranzyme B m RNA and protein were significantly decreased in modelgroup(P< 0.01); Compared with model group, the expression levels of granzyme B m RNA and protein were significantly increased in model with candida albicans infected group(P< 0.01).Conclusion: 1.The spleen deficiency mice showed disordersof intestinal candida albicans. After oral infected withcandida albicans, the candida albicans content in feces were increased and aggravated the disorders of theintestinal candida albicans and pathological changes of the intestinal tissue. 2.The proportion of CD4+T/CD8+T was changedin mice with spleen deficiency. After oral infection, the changes in distribution of CD4+T, CD8+T subgroup was more obvious, and the two indices had synergistic effect, which caused the damage of immune function more obvious. 3.The humoral and cellular immune function of the organism was changed when infected with candida albicans, but the cellular immunity was the main one. 4.Candida albicans infection can activatelocal CTLcells in small intestine, resulting in the increased expression of perforin and granzyme. Spleen deficiency aggravated the condition of Candida albicans infection, high expression level of perforin and Granzyme may be the mechanism of spleen deficiency of mice infectedwithcandida albicans.