TLR4-miR-351-IL-11通路在辐射防护中的作用和机制研究

The study of radioprotection for medical and bio-defense applications are of greatest importance. To develop effective therapeutic drug requires the elucidation of the underlying mechanisms of radioprotection..Our previous study proved that TLR4 played an important role in radioprotection. We found that mice deficient in TLR4 were more susceptible to IR-induced mortality and morbidity. LPS, which activated TLR4 in vivo, induced radio-resistance. Moreover, TLR4 in vivo ligands are required for basal radio-resistance. However, the underlying molecular mechanism of TLR4 mediated radioprotection is not clear yet. In this project, we try to study the underlying molecular mechanism in depth. We have constructed the TLR4 mediated radioprotection model in vitro and in vivo by using TLR4 overexpression cell lines and TLR4 KO mice. To identify the potential molecules involved in TLR4 mediated radiation resistance, we have compared the cytokines and miRNAs profiles before and after radiation in TLR4 KO mice and WT mice. We found that IL-11 was up-leveled by TLR4 mostly and miR-351 was inhibited by TLR4 mostly. Significantly, we found miR-351 could inhibit IL-11 expression by 3'UTR dependent way, and inhibited STAT3 and NF-κB, the transcriptional factors of IL-11..Based on previous data, we proposed our hypothesis: TLR4-miR-351-IL-11 pathway played a critical role in TLR4 mediated radioprotection. .To completely confirm this hypothesis, we intended down or up-regulated miR-351 in vivo or in vitro by plasmid transfection, adenovirus vector and miR-351 KO mice to identify the role of miR-351. Similarly, we tried to study the role of IL-11 by using recombination IL-11, IL-11 over- and down-expression vector and IL-11 KO mice. As we have proved that miR-351 could target IL-11, we next tried to study the regulation effect of miR-351 to the IL-11 signal pathway, and by which way miR-351 and IL-11 participated in radiation resistance..Applicant has been focusing on the radioprotection effect of TLR4 and its underlying molecular mechanism for several year and have a lot of experience in radiobiology study. This is a probe study and is the first time to study the role of miR-351 in radioprotection, and to construct the miR-351 KO mice. We believe that, if funded, with our professional efforts and functioned-well lab, this project would elucidate the underlying mechanism of TLR4-miR-351-IL-11 mediated radioprotection. If successful, our study would contribute a better understanding in radiobiology, and favor the development of novel radioprotective drugs.

研究电离辐射损伤防治具有重要理论和实用科学价值。我们前期研究证明TLR4具有显著的辐射防护作用，然而目前关于TLR4辐射防护作用的分子机制仍不十分清楚,制约了其向应用研究的发展。课题组在TLR4辐射防护作用分子机制方面进行了探索研究，筛选出辐射条件下TLR4 KO小鼠相关差异最显著的miRNA miR-351和细胞因子IL-11，初步发现miR-351可靶向抑制IL-11信号通路。在此基础上提出TLR4→miR-351→IL-11通路可能在TLR4辐射防护中发挥关键作用的科学假设。本项目拟从分子、细胞、组织和体内多层次进行系统实验研究，揭示上述三个重要分子之间的相互关系，探讨它们与TLR4辐射防护作用之间的内在联系；阐明TLR4-miR-351-IL-11通路在辐射防护中的具体作用及其分子机制，从而证实我们的假说，为电离辐射损伤防治研究提供新的理论和实验基础，为辐射防护寻找新靶点和新途径。

研究电离辐射损伤防治具有重要理论和实用科学价值。该领域目前面临的主要科学问题：一是缺乏作用机理明确的辐射防护新靶点，二是缺乏高效低毒的辐射防护剂。目前国内外针对大剂量照射导致的重度以上骨髓型急性放射病等电离辐射损伤还没有有效的治疗手段。.我们前期研究提示调控TLR4信号途径在放射损伤防治中具有重要作用。但是对其确切效应和分子机理仍未见系统研究。本课题在国家自然科学基金资助下，根据原计划书内容，成功构建小鼠放射损伤模型，进一步明确了TLR4信号通路的辐射防护作用；完成了TLR4辐射防护作用相关重要靶分子IL-11，IL-6和miR-351的筛选和锁定，明确了IL-11和miR-351在TLR4辐射防护中的具体作用；并探讨了TLR4-miR-351-IL-11通路参与TLR4辐射防护作用的机制。在项目执行过程中，我们也开展了一些原计划没有列入的工作；比如Zymosan-A和FG-4592防治辐射损伤新技术研究等。研究发现：1. TLR4 KO显著加重电离辐射损伤，而激活TLR4防护电离辐射损伤的效果好，具有较强的应用前景。2. 成功筛选出与TLR4下游相关重要基因及效应分子IL-11和miR-351；并发现IL-11和miR-351抑制剂对辐射损伤也具有显著的防治作用并解析了其中的分子机制；3. 此外我们也发现Zymosan-A和FG-4592对辐射损伤具有显著的防治作用。以上结果表明调控TLR4信号途径尤其是TLR4-miR-351-IL-11可以作为防护电离辐射损伤的新途径，为防治电离辐射损伤的提供了新的理论基础。进一步采用体内实验证实了LPS，Zymosan-A，MPLA，FG-4592，IL-11和miR-351抑制剂等具有重要的理论价值和应用前景。.本项目总体执行情况良好，标注本基金已发表论文10篇，其中第一标注4篇，第二标注4篇，第三标注1篇，第四标注1篇，其中影响因子5分的SCI论文3篇，总影响因子40.1；后续已经成文待发表SCI论文1-2篇；本项目组3名中级职称成员2人晋升为副教授；1人晋升为高级实验师；培养和协助培养博士研究生3名；培养硕士研究生2名。并有望继续培养博士、硕士研究生各1名。获得上海市科技进步一等奖1项等奖励；受邀在国内外学术做大会报告或分会报告 5次，顺利圆满地完成本项目。

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