纳米山药多糖双歧杆菌合生元结肠靶向微生态调节剂及作用机制研究

Dysbacteriosis is a serious threat to the human health. The microecologic modulator as the treatment of dysbacteriosis mainly concentrates on probiotics, but some drawbacks, such as live bacteria intolerance stomach acid, low survival rate in intestine and bad biological effects, need to be solved urgently. Based on our previous work, we will purify CYP by the molecule gel column chromatography, get connection mode by the GC-MS, determine the structure by LC-MS, and prepare the synbiotics colon targeting microecological modulator based on nano CYP according to the principle of pH and adhesivity. Finally, we will carried out the targeting action studies byγ-ray scanning technology, the detecting number of intestinal flora and genetic change by the PCR-DGGE, the investigating the biological effects and mechanism to the treatment of the intestinal dysbacteriosis, the expoloring immunology effect and mechanism by the SYNERGY HT automatic multifunctional microplate reader, WALLAC liquid scintillation counter, FACSAria flow cytometry, and so on, and the studying the physiological effect and mechanism by radioimmunoassay and patch clamp technique. This project has an important significance to solve problems that low biological effects of living bacterium preparation, it will provide new ideas to reveal mechanism and targeting treatment of diarrhea, enteritis and diseases related with intestinal dysbacteriosis. It will provide new supplement to strengthening and restoring.Although the study of traditional Chinese medicine as microbial modulator has made some progress, this research will possess a positive meaning to the development and utilization of Traditional Chinese Medicine Resources.

菌群失调严重威胁人类生命与健康。国内外治疗菌群失调的微生态调节剂主要是益生菌制剂，存在活菌不耐受胃酸，进入肠道存活率低，生物效应差等瓶颈问题亟待解决。课题组在前期工作基础上以分子凝胶柱层析纯化山药多糖（CYP），GC-MS获取连接方式，LC-MS构型测定,依据pH值与黏附原理以纳米CYP为益生元与双歧杆菌制成合生元结肠靶向微生态调节剂，γ-扫描技术研究体内靶向性，PCR-DGGE检测肠道菌群及基因变化，研究治疗肠道菌群失调生物学效应及机制，采用SYNERGY HT全自动多功能酶标仪、WALLAC液闪仪、FACSAria流式细胞仪等研究免疫学效应及机制，放免法及膜片钳技术研究生理学效应及机制。本研究对解决活菌类制剂瓶颈问题有重要意义，对揭示结肠靶向微生态调节剂作用机理，为肠道菌群失调、肠炎及相关疾病靶向治疗提供新思路，为中药扶正固本理论提供新的补充，对我国中药资源开发利用具有十分积极的意义。

菌群失调时肠道优势菌数量减少，治病菌大量增加或菌群易位，人体免疫功能低下，引发急慢性腹泻、结肠炎、肿瘤等多种疾病。因此，维护人体肠道正常菌群、恢复其微生态平衡对疾病预防、治疗非常重要。 . 本研究用热水法成功提取了山药多糖，用凝胶色谱柱纯化得到两组均一山药多糖（CYP-1、CYP-2），CYP-1含有鼠李糖、阿拉伯糖、甘露糖、葡萄糖、半乳糖五种单糖；CYP-2由鼠李糖、阿拉伯糖、葡萄糖、半乳糖4种单糖组成，CYP-1和CYP-2异头碳构型为β型，CYP-2含有α型。并用湿磨法制备了纳米山药多糖，将纳米山药多糖（益生元）与双歧杆菌（益生菌）制成合生元制剂，制剂质量、稳定性符合药典要求；经X-射线、γ-扫描技术证实制剂具有体内结肠靶向性。首次对纳米山药多糖进行系统研究，明确了靶向制剂对菌群失调大鼠生物学、免疫学及生理学作用机制。生物学机制经DGGE及高通量实验从基因水平上证实靶向制剂具有扶植优势菌群生长，增加定植抗力，竞争性夺养，抑制致病菌生长，增加肠道菌群丰富度，减少菌群易位，肠道挥发性脂肪酸增加，降低肠道pH值，恢复肠道微生态平衡；其抗炎机制为减少肠道组织中MDA而提高SOD活力，减少因炎症产生的炎症介质如NO、MPO含量。免疫学机制为增加免疫器官指数，使NK、CTL细胞杀伤活性增强，脾脏、胸腺细胞内活性氧含量降低，血清中细胞因子IL-1α、IL-4、IL-17，IL-1β、IL-6、GM-CSF、TNF-α含量降低，IL-2和IFN-γ含量升高，外周血淋巴细胞、胸腺、脾脏淋巴细胞、结肠组织细胞凋亡率降低，上调免疫球蛋白IgA、IgM、IgG及其亚型含量，可使Bcl-2升高，Bax降低，促进脾脏、胸腺淋巴、外周血中性粒细胞增殖，可增加脾脏、胸腺淋巴细胞线粒体膜电位，提高机体特异性和非特异性免疫功能。生理学机制为，在细胞和分子水平上，靶向制剂可以增加模型大鼠结肠慢波频率与位移，恢复肠道蠕动；增加结肠神经节细胞和Cajal间质细胞数量及膜电流，对结肠组织有保护作用，在整体水平上，调节血清及结肠组织血管活性肠肽，胃动素，生长抑素和P物质的水平，使其上升接近正常水平，调解消化腺分泌和消化道运动，增加消化道组织代谢及生长，并调节其他激素释放。本实验制备的纳米山药多糖双歧杆菌合生元结肠靶向微生态调节剂是一种理想的结肠靶向微生态调节剂，为今后微生态调节剂研究提供了新思路