金属硫蛋白MTs在参环毛蚓平喘作用中的地位及其机制的研究

"Guang Di Long", a traditional Chinese medicine,is recognized high-quality products in the domestic and foreign medicine markets with effect of clearing away heat, arresting convulsiom, smooth wheezing and diuresis. The medicine is made of fresh animal Pheretima aspergillum, which mainly produced in Guangdong, Guangxi provinces of China. However, as the deepening of the quality study,our team had found that metallothothioneins (MTs) was the key protein, which conducted the physiological tolerance and enrichment of heavy metals in Pheretima aspergillum, and the reason why the heavy metal exceeded the limit standards in "Guang Di Long". We were going to systematic study and assess the impact of earthworm's extract that caused by silent MT gene to the inflammatory markers on asthmatic mice, keep the track of proteins and correlative components in Pheretima aspergillum' samples, determine role of MTs in the action of Pheretima aspergillum's antiasthma, explore its active material and mechanism to provide a strategy of dealing with excessive heavy metals in Pheretima aspergillum under full funding. Due to the limit of fund support and time, our team will adjust appropriately the content of research and focus on how to get Pheretima aspergillum that are targeted knocked out MT gene. The resolution of that issue is not only beneficial to surmount key technical difficulties in original research program to proactively explore for achievement the set target, but also to provide experimental basis for the ultimate goal.

“广地龙”因其清热定惊，通络，平喘，利尿的功效显著而被誉为地龙中的优质产品，其药材是由新鲜动物参环毛蚓加工而成，是主产于广东和广西的著名道地中药材。但随着质量研究不断地深入，本团队已发现MTs是介导参环毛蚓生理性耐受和富集的关键蛋白，也是导致广地龙药材重金属超标的主要原因。原本计划在全额资助条件下系统研究和比较参环毛蚓MT表达缺失的提取物对哮喘小鼠炎症标志物水平的影响、跟踪由目标基因表达沉默引起的参环毛蚓样本间蛋白质组和其它相关成分的变化、确定MTs在平喘作用中所占的地位、探索参环毛蚓发挥平喘治疗作用的物质基础及机制、以便制定解决广地龙药材重金属超标问题的策略。由于目前所获得的资助额度和时间限制，本团队将适当调整研究内容，集中解决如何利用RNAi技术获得靶向MTs编码基因敲除的活体参环毛蚓，该问题的解决既可以攻克原有研究方案中的关键技术难点，为实现既定目标进行前瞻性探索；又可以为实现原有目标提供实验依据。

摘要：参环毛蚓的药材习称为“广地龙”，是国内外药材市场中公认的地龙优质产品。本团队先期已确定金属硫蛋白MTs是介导参环毛蚓生理性耐受和富集的关键蛋白，也是导致广地龙重金属超标的主要原因。欲从源头上解决重金属超标问题，却受困于MTs在广地龙功效中的关联性不明确。据此，本项目以地龙最具特色的功效“平喘”为突破口，首先优化MTs提取纯化方法，获得了纯度较高的MTs，为MTs 的药效评价提供了不可或缺的实验材料。然后分别考察了MTs对哮喘小鼠气道炎症和气道高反应的影响，实验结果排除了MTs对平喘功效的贡献，为今后利用RNAi技术持久性阻断MT基因的表达提供科学依据。同时，本项目还采用siRNA序列进行体外转染实验，探讨了RNAi技术应用在参环毛蚓肠上皮细胞中的可行性。研究结果虽未能转染成功，但本课题组为究其原因，专项考察了参环毛蚓肠上皮细胞（IEC）的生物学特性，不仅获得与IEC细胞形态结构、代谢、增殖等方面有价值的实验数据，而且发现该细胞的生长周期长，处于G0期的细胞较多，DNA准备期较长是RNAi技术未能成功应用的重要原因。这些实验数据为后续RNAi的应用研究积累了经验，也对后续其他相关研究的开展具有重要的参考意义。