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

Association between intestinal flora and immunity in middle-aged and aged people by PCR-DGGE

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 analyze whether the effects of the aging caused changes in intestinal flora to the immunity by evaluating the potent association between the intestinal flora and their immunity of middle-aged and aged healthy subjects based on profiling the composition of their fecal bacteria and testing their immune parameters. Methods Total 99 healthy volunteers aged 50- 75 were recruited. Blood samples collected from them were analyzed for CD3＋T cell,CD4＋T cell,CD8＋T cell,serum Ig A,Ig M and Ig G with flow cytometer（ FCM） and immunoturbidimetry. The fecal samples were also collected from them.The genomic DNA of fecal bacteria was extracted and amplified with the universal primers of bacterial 16S rRNA V6- V8 region by PCR. The DNA amplifications were tested with denaturing gradient gel electrophoresis（ DGGE）. Cluster analysis,principal component analysis（ PCA） and variance analysis were used to characterize fecal bacteria composition andanalyze the association with observed immune parameters. Results The fecal microbiota were highly polymorphic and greatly varied among the tested subjects. CD3＋T cell and CD4＋/CD8＋ levels correlated with intestinal flora structure. Intestinal flora richness（ S） of population with high CD4＋/ CD8＋ level was lower compared with population with normal or low CD4＋/ CD8＋ level. However,no apparent association was observed between serum Ig A,Ig M,Ig G levels and the fecal microbiota among the tested subjects. Conclusion The decrease in the diversity of intestinal flora might affect CD8＋T cell based innate immunity of host. The deep association could exist between intestinal flora and their immunity in middle-aged and aged people.