基于PCR-DGGE技术分析中老年人肠道菌群结构与免疫功能的关系

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目的通过检测中老年人的各项免疫指标和肠道菌群组成,分析中老年人免疫功能与其肠道菌群结构的关系,探索高龄引起的肠道菌群结构变化对中老年人免疫功能可能的影响。方法选取50~75周岁,健康中老年人99人作为研究对象,抽取空腹静脉血,检测CD3~+T细胞数、CD4~+T细胞数、CD8~+T细胞数及血清中的IgA、IgM、IgG。收集各研究对象的粪便,提取粪便细菌基因组DNA,采用细菌16S rRNA V6~V8区通用引物进行PCR扩增,对扩增产物进行变性梯度凝胶电泳。采用聚类分析、主成分分析、方差分析等解析研究对象肠道菌群结构特征及与其免疫指标的相关性。结果研究对象肠道菌群组成表现出高度多态性和显著的个体差异,CD3~+T细胞和CD4~+/CD8~+水平与肠道菌群结构有相关性,CD4~+/CD8~+偏高的人群与CD4~+/CD8~+偏低和正常人群相比肠道菌群丰富度(S)偏低,但是未观察到IgA、IgM、IgG与肠道菌群结构存在明显相关。结论中老年人肠道菌群结构与免疫水平相关,肠道菌群多样性的减少可能会影响其机体CD8~+T细胞为中心的非特异性自然免疫功能。 Objective To detect the immune indicators of middle-aged and elderly people and the composition of intestinal flora, to analyze the relationship between immune function and the structure of intestinal microflora in middle-aged and elderly people, and to explore the immune function of elderly population Impact. Methods Fifty - 75 years of age and 99 healthy middle - aged and elderly people were selected as experimental subjects. Fasting venous blood was collected for detecting the number of CD3 ~ + T cells, CD4 ~ + T cells, CD8 ~ + T cells and serum IgA and IgM , IgG. The feces of each research object was collected, genomic DNA of fecal bacteria was extracted, and PCR amplification was carried out using general primers of bacterial 16S rRNA V6 ~ V8 region. The amplified products were subjected to denaturing gradient gel electrophoresis. The cluster analysis, principal component analysis and analysis of variance (ANOVA) were used to analyze the structural characteristics of intestinal microflora and its correlation with immune parameters. Results The intestinal flora of the study subjects showed highly polymorphism and significant individual differences. The levels of CD3 ~ + T cells and CD4 ~ + / CD8 ~ + were correlated with the intestinal microflora, CD4 ~ + / CD8 ~ + High population with low CD4 ~ + / CD8 ~ + intestinal flora compared to normal and low population richness (S), but did not observe the IgA, IgM, IgG and intestinal flora structure was significantly correlated . Conclusion The structure of intestinal flora in middle-aged and elderly people is related to the level of immunity, and the decrease of intestinal flora may affect the nonspecific natural immune function of CD8 + T cells in the center.
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