土壤依赖型人肠道细菌阻止抗生素诱导肠微生态失衡的作用机制研究

The widespread use of antibiotics in clinical practice is an important inducement for the imbalance of intestinal microflora in modern population. Intestinal microecological imbalance often is the cause of a variety of chronic diseases and major diseases. Our previous studies showed that a variety of bacteria in the intestinal tract of mice lived on the soil in the diet, which was called soil-dependent bacteria of mouse intestine. These bacteria have the ability to prevent the broad-spectrum antibiotics from killing the intestinal probiotics and maintaining the intestinal micro ecological balance, as well as the prevention of enteritis and immune system disease for mice. Whether there is such a kind of bacteria in the human gut, their composition, structure and related function, has not been reported. In this study, we will collect the fecal samples of children from different regions with two kinds of cleanliness levels and the fecal samples of SPF mouse model we will perform high throughput sequencing of 16S rRNA gene and functional genomic, broad-spectrum antibiotics interference, molecular biological methods of RT-PCR to analyze the diversity and ecological function of the soil-dependent human intestinal bacteria, and to explore the molecular ecological mechanism on the prevention of intestinal microecological imbalance. Soil-dependent bacterium was also isolated from human feces by plate culture and then inoculated into the intestinal tract of mice to further verify the function of maintaining intestinal microecological imbalance. The results will provide experimental evidence for the prevention and treatment of intestinal microecological imbalance and for the development of drugs targeted intestinal bacteria.

抗生素在临床上的广泛使用是导致现代人群肠道微生态失衡的重要原因，肠微生态失衡又是多种慢性病和重大疾病发生原因。我们前期研究显示，小鼠肠道中含有多种细菌依赖于食入体内的土壤生存（土壤依赖型小鼠肠道细菌），这些细菌具有阻止广谱抗生素对肠道益生菌杀伤和维持肠道微生态平衡的作用，对小鼠肠炎和免疫系统疾病有显著预防效果。人肠道中是否也存在这类细菌，它们的结构组成与相关作用，目前尚未见报道。 该项申请拟通过对不同清洁度地区儿童肠道微生物与小鼠肠道微生物对照分析，采用16S rRNA基因和功能基因组高通量测序分析、广谱抗生素干扰、RT-PCR等手段，分析土壤依赖型人肠道细菌的多样性、生态功能与阻止广谱抗生素诱导肠微生态失衡的分子生态学机制；并采用平板分离与回接方式，验证该类细菌的生态功能与调控效果，为肠道微生态失衡症防治与靶向肠道细菌的药物研发提供实验依据。

该项研究为了证明 “土壤依赖型细菌具有阻止广谱抗生素对肠道益生菌杀伤和维持肠道微生态平衡的作用” 这一假说，我们依次完成以下工作：（1）抗生素处理前后小鼠生活环境中土壤成分与小鼠肠道微生物类型关系研究，目的是进一步了解土壤依赖型细菌多样性与生存条件；结果发现土壤成分对小鼠肠道微生态无显著影响；（2）服用抗生素前后，农村儿童与城市儿童肠道微生物结构类型比较分析。这一研究的目的是为了证明人类肠道微生态中也存在土壤依赖型细菌，其功能与小鼠相一致；结果发现偏远农村儿童体内也存在土壤依赖型细菌，这些细菌的功能与维持肠道微生态平衡有关；（3）分离培养土壤依赖型人肠道细菌，并将分离菌株的多种组合回接小鼠肠道。这一试验的目的是证明土壤依赖型细菌具有阻止广谱抗生素对肠道益生菌杀伤和维持肠道微生态平衡的作用。但结果发现，菌株组合没有“阻止广谱抗生素对肠道益生菌杀伤和维持肠道微生态平衡的作用”。研究到此，我们完成了该项研究的所有内容，但结论与我们提出假设完全不同。.课题组经过深入思考与讨论，提出新的假说：“环境中的矿物类物质通过营养肠道中多种类型的微生物，阻止广谱抗生素对肠道益生菌杀伤，达到维持肠道微生态平衡的作用”。接下来我们完成了（4）矿物类物质干预实验。实验结果证明“环境中的矿物类物质通过营养肠道中多种类型的微生物，阻止广谱抗生素对肠道益生菌杀伤和维持肠道微生态平衡的作用”假说是正确的！接下来，我们又完成了（5）矿物类中药用于小鼠过敏性疾病的治疗与（6）矿物类中药安全性评价实验，结果发现安全无毒性，对小鼠湿疹有较好的疗效。最后，我们（7）通过与中医合作，采用中药方剂对过敏性患者进行治疗，结果疗效显著。到目前为止，我们治疗过的过敏性患者（湿疹、荨麻疹、过敏性鼻炎、过敏性哮喘等）达上千人，治愈率每种疾病都在90%以上

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