肠道白念珠菌感染对脾虚小鼠小肠组织中IFN-γ和IL-10 mRNA及蛋白表达水平的实验研究
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摘要
目的:观察脾虚小鼠感染白念珠菌后小肠组织中IFN-γ和IL-10 mRNA和蛋白表达水平的变化,探讨脾虚小鼠在白念珠菌感染过程中的部分免疫机制,为临床诊治白念珠菌感染及脾虚证提供一定的参考依据。方法:采用饮食失节加劳倦过度的复合因素制备小鼠脾虚证模型,经肠道感染白念珠菌14 d后,检测粪便中的活菌数,应用RT-PCR方法检测肠组织局部γ干扰素(IFN-γ)和白细胞介素10(IL-10)mRNA的表达水平,应用Westornblot方法检测肠组织中IFN-γ和IL-10的蛋白表达水平。结果:与正常对照组比较,其他三组小鼠粪便中活菌数、肠组织中IFN-γ和IL-10 mRNA及其蛋白的表达水平均显著增高(P<0.01);与正常白念感染组比较,脾虚模型组小鼠小肠组织中IFN-γ、IL-10 mRNA及蛋白表达水平显著降低(P<0.01),而脾虚白念感染组显著升高(P<0.05或0.01);与脾虚模型组比较,脾虚白念感染组小鼠粪便中活菌数、小肠组织中IFN-γ和IL-10 mRNA及蛋白表达水平也显著升高(P<0.01或P<0.05)。结论:小鼠脾虚后可发生肠道菌群紊乱,比正常小鼠对白念珠菌的易感性增强,在肠道局部抗白念珠菌的作用主要以细胞免疫为主,Th1类细胞因子发挥主要作用。
Objective: The present study observed the changes occuring to the expression levels of Interferongamma( IFN-γ),Interleukin-10( IL-10) mRNA and protein detected from the small intestine tissue of spleen deficiency mice which were infected by Candida albicans. It aimed to probe into the partial immune mechanism when mice of spleen deficiency were infected by Candida albicans,so as to provide the evidence for diagnosing and treating infection of Candida albicans and spleen deficiency. Methods: This research applied irregular diet and excessive fatigue to make spleen deficiency model mice and the bacteria in the faeces were monitored by viable count on the 15 thday after the infection of Candida albicans. The present study applied RT-PCR and adopted Western blot to detect the level of mRNA and protein of both IFN-γand IL-10 in the intestine tissue of mice. Results: Compared with the normal group,the number of living bacteria in the faeces and the expression levels of IFN-γ,IL-10 mRNA and protein in other three groupswere increased significantly( P < 0. 01). Compared to the group of normal mice with infection of Candida albicans,the expression levels of IFN-γ,IL-10 mRNA and protein in the spleen deficiency group were significantly reduced( P < 0. 01),while they were significantly increased( P < 0. 05 or P < 0. 01) in the group of spleen deficiency accompanied with infection of Candida albicans. The number of living bacteria in the faeces and the expression levels of IFN-γ,IL-10 mRNA and protein in the intestine tissue was increased significantly( P < 0. 01 or P < 0. 05) as compared with that in the spleen deficiency model group. Conclusion: The results of the present study demonstrated that dysbacteriosis may occur when mice have spleen deficiency syndromeand they are more susceptible to Candida albicans than normal mice. Cellular immunity plays the leading role in anti-infection of Candida albicans in local instetineand in which cytokines of Th1 type play the main role.
Objective: The present study observed the changes occuring to the expression levels of Interferongamma( IFN-γ),Interleukin-10( IL-10) mRNA and protein detected from the small intestine tissue of spleen deficiency mice which were infected by Candida albicans. It aimed to probe into the partial immune mechanism when mice of spleen deficiency were infected by Candida albicans,so as to provide the evidence for diagnosing and treating infection of Candida albicans and spleen deficiency. Methods: This research applied irregular diet and excessive fatigue to make spleen deficiency model mice and the bacteria in the faeces were monitored by viable count on the 15 thday after the infection of Candida albicans. The present study applied RT-PCR and adopted Western blot to detect the level of mRNA and protein of both IFN-γand IL-10 in the intestine tissue of mice. Results: Compared with the normal group,the number of living bacteria in the faeces and the expression levels of IFN-γ,IL-10 mRNA and protein in other three groupswere increased significantly( P < 0. 01). Compared to the group of normal mice with infection of Candida albicans,the expression levels of IFN-γ,IL-10 mRNA and protein in the spleen deficiency group were significantly reduced( P < 0. 01),while they were significantly increased( P < 0. 05 or P < 0. 01) in the group of spleen deficiency accompanied with infection of Candida albicans. The number of living bacteria in the faeces and the expression levels of IFN-γ,IL-10 mRNA and protein in the intestine tissue was increased significantly( P < 0. 01 or P < 0. 05) as compared with that in the spleen deficiency model group. Conclusion: The results of the present study demonstrated that dysbacteriosis may occur when mice have spleen deficiency syndromeand they are more susceptible to Candida albicans than normal mice. Cellular immunity plays the leading role in anti-infection of Candida albicans in local instetineand in which cytokines of Th1 type play the main role.
引文
[1]Sandrine Albac,Antonin Schmitz,Carolina Lopez-Alayon,et al.Candidaalbicans is able to use M cells as a portal of entry across the intestinal barrier in vitro[J].Cellular Microbiology,2016,18(2):195-210.
[2]The yeast Candida albicansevades human complement attack by secretion of aspartic proteases[J].Molecular Immunology,2009,47(2-3):465-475.
[3]王守岩,孙宏伟,马贤德,等.白色念珠菌感染对脾虚小鼠小肠病理及超微结构的影响[J].辽宁中医药大学学报,2016,18(8):24-26.
[4]韩晓伟,马贤德,孙宏伟,等.脾虚小鼠肠道感染白色念珠菌的局部黏膜免疫机制研究[J].世界中西医结合杂志,2016,11(8):1037-1039,1048.
[5]黄柄山,毛翼楷,范隆昌,等.饮食失节所致的脾虚动物模型及中药治疗观察[J].中西医结合杂志,1983,3(5):295-299.
[6]陈小野,周永生,樊雅莉,等.脾气虚证动物模型规范化的初步研究[J].中国医药学报,2001,16(4):52-58.
[7]商安全,潘红超,张康见,等.白念珠菌感染的临床分布药敏及产生物膜分析[J].现代预防医学,2016,43(6):1130-1133.
[8]覃羽华,易雪丽,许桂丹,等.758株条件致病性真菌的临床分布及耐药性分析[J].现代预防医学,2015,42(15):2779-2781.
[9]Yiqing Tong,Jianguo Tang.Candidaalbicans infection and intestinal immunity[J].Microbiological Research,2017,198:27.
[10]王频佳,王娴,谢成彬,等.双歧杆菌脂磷壁酸对深部白色念珠菌感染小鼠细胞免疫的影响[J].重庆医学,2016,45(11):1467-1469.
[11]熊延靖,董群.干扰素-γ在抗系统性白念珠菌感染中的作用机制[J].热带病与寄生虫学,2008,6(2):113-116.
[12]安金刚,李巍,刘玉峰.真菌感染与天然免疫[J].中国真菌学杂志,2007,2(3):182-185.
[13]章梅,夏天,张仲海,等.四君子汤对脾虚患者血浆细胞因子的影响[J].第四军医大学学报,2000,21(4):411-413.
[14]马贤德,孙杨,孙宏伟,等.白色念珠菌肠道感染对脾虚模型小鼠小肠组织中IL-10和IL-12影响的实验研究[J].辽宁中医杂志,2015,42(11):2226-2229.
[15]AR Khosravi,HShokri,SDarvishi Altered.Immune responses in patients with chronic mucocutaneous candidiasis[J].Journal De Mycologie Medicale,2014,24(2):135-140.
[16]吴呈霖,骆雪萍,吴晔,等.大鼠侵袭性肺部热处理光滑念珠菌感染肺组织Dectin-1和IL-10的表达[J].广东医学,2014,35(22):3468-3470.
[17]L Romani,PPuccetti.Protective tolerance to fungi:the role of IL-10 and tryptophan catabolism[J].Trends in Microbiology,2006,14(4):183-189.
[2]The yeast Candida albicansevades human complement attack by secretion of aspartic proteases[J].Molecular Immunology,2009,47(2-3):465-475.
[3]王守岩,孙宏伟,马贤德,等.白色念珠菌感染对脾虚小鼠小肠病理及超微结构的影响[J].辽宁中医药大学学报,2016,18(8):24-26.
[4]韩晓伟,马贤德,孙宏伟,等.脾虚小鼠肠道感染白色念珠菌的局部黏膜免疫机制研究[J].世界中西医结合杂志,2016,11(8):1037-1039,1048.
[5]黄柄山,毛翼楷,范隆昌,等.饮食失节所致的脾虚动物模型及中药治疗观察[J].中西医结合杂志,1983,3(5):295-299.
[6]陈小野,周永生,樊雅莉,等.脾气虚证动物模型规范化的初步研究[J].中国医药学报,2001,16(4):52-58.
[7]商安全,潘红超,张康见,等.白念珠菌感染的临床分布药敏及产生物膜分析[J].现代预防医学,2016,43(6):1130-1133.
[8]覃羽华,易雪丽,许桂丹,等.758株条件致病性真菌的临床分布及耐药性分析[J].现代预防医学,2015,42(15):2779-2781.
[9]Yiqing Tong,Jianguo Tang.Candidaalbicans infection and intestinal immunity[J].Microbiological Research,2017,198:27.
[10]王频佳,王娴,谢成彬,等.双歧杆菌脂磷壁酸对深部白色念珠菌感染小鼠细胞免疫的影响[J].重庆医学,2016,45(11):1467-1469.
[11]熊延靖,董群.干扰素-γ在抗系统性白念珠菌感染中的作用机制[J].热带病与寄生虫学,2008,6(2):113-116.
[12]安金刚,李巍,刘玉峰.真菌感染与天然免疫[J].中国真菌学杂志,2007,2(3):182-185.
[13]章梅,夏天,张仲海,等.四君子汤对脾虚患者血浆细胞因子的影响[J].第四军医大学学报,2000,21(4):411-413.
[14]马贤德,孙杨,孙宏伟,等.白色念珠菌肠道感染对脾虚模型小鼠小肠组织中IL-10和IL-12影响的实验研究[J].辽宁中医杂志,2015,42(11):2226-2229.
[15]AR Khosravi,HShokri,SDarvishi Altered.Immune responses in patients with chronic mucocutaneous candidiasis[J].Journal De Mycologie Medicale,2014,24(2):135-140.
[16]吴呈霖,骆雪萍,吴晔,等.大鼠侵袭性肺部热处理光滑念珠菌感染肺组织Dectin-1和IL-10的表达[J].广东医学,2014,35(22):3468-3470.
[17]L Romani,PPuccetti.Protective tolerance to fungi:the role of IL-10 and tryptophan catabolism[J].Trends in Microbiology,2006,14(4):183-189.