急性肠损伤时补体C3胞内激活上调Paneth细胞并促进肠黏膜再生的机制研究

Acute gastrointestinal injury (AGI) is mainly complicated from intra-abdominal infection. The promptly repair of intestinal mucosa in early stage of AGI can effectively improve its outcomes. Recent studies indicate that Paneth cells (PCs) in Lieberkühn are related to the regulation of epithelial regeneration of intestinal mucosa, however, the concrete mechanism remains obscured. Our preliminary studies suggest that exogenous complement C3 supplementation could effectively reverse complement C3 depletion, enhance adaptive immunity and promote the regeneration of intestinal mucosa. Therefore, we hypothesize that complement C3 may enhance intestinal regeneration by up-regulating mTORC1 signal pathway of intestinal stem cells (ISCs) through intracellular activation. The purpose of this study is two-fold: first, to investigate the effect of exogenous complement C3 on the regulation of PCs during the process of AGI; and second, to explore the potential mechanism of improved intestinal epithelium regeneration after exogenous complement C3 therapy.. The current study contains in vitro and in vivo parts. For the in vivo part, the pathological changes and bacterial loads of vital organs in abdomen will be evaluated after complement C3 therapy. Afterwards, the activities of PCs and ISCs would be examined by IHC, WB, PCR and other approaches. Besides, the intracellular activation of complement C3 in PCs would be evaluated via IHC, WB, IF, FCM. Meanwhile, the secreting factors from PCs, such as Wnt3 and EGF, will be tested by FISH method, and the mTORC1 kinase and subsequent signal substrates would be detected by using WB and other means. For the in vitro part, the Lieberkühn crypts from ileum segments would be cultured in vitro with the persistent stimulation from exogenous C3 protein. To unravel the potential pathway, the siRNA of Bst1, a specific ectoenzyme from mTORC1 kinase, will be used to inhibit the whole signal pathway of mTORC1, and the role of exogenous C3 would be re-assessed by blocking the CTSL function.. This study will further investigate the mechanism of intestinal regeneration during AGI, and confirm the role of complement based therapy in treatment of intestinal injuries.

急性肠损伤（AGI）可由腹腔感染进展诱发。AGI早期行肠黏膜修复干预可有效改善其预后。新近研究表明肠Lieberkühn隐窝内Paneth Cells（PCs）参与调控肠黏膜上皮再生，但机制未明。我们前期研究新发现：AGI早期给予外源性补体C3可逆转C3耗竭，增强细胞免疫应答，促肠上皮再生。因而认为C3可能通过胞内激活途径上调PC活性进而影响肠黏膜再生。课题拟用小鼠升结肠腹膜炎模型诱导AGI，观察AGI早期PCs胞内C3激活，探索C3胞内激活是否上调mTORC1信号通路调节下游肠干细胞（ISCs），促肠上皮再生。研究分两部分：体内评价AGI后肠损伤病理，检测PCs、ISCs活性及PCs胞内C3激活效应分子（C3a/b、CTSL）水平，检测mTORC1激酶及下游Wnt3、EGF水平；体外培养回肠隐窝观察C3对PCs、ISCs影响，拟抑制CTSL、mTORC验证C3胞内激活是调控关键通路。本研究将进一步探索肠损伤修复再生的可能机制，诠释补体系统在肠功能损伤修复调控中的重要作用。

急性肠损伤（AGI）可由腹腔感染进展诱发，是临床外科治疗中面临的重要挑战。AGI早期行肠黏膜修复干预可有效改善其预后。新近研究表明肠上皮下方的Lieberkuhn隐窝内潘氏细胞（Paneth Cells, PCs）参与了肠黏膜上皮损伤的修复再生过程，但具体机制不明。而课题组前期研究发现AGI早期给予外源性补体C3治疗不仅可以逆转感染后期出期的补体耗竭及免疫抑制，还发现肠上皮再生能力得到增强。课题组提出了C3可能通过胞内活化途径影响PCs功能进行参与肠黏膜的损伤修复的研究假说。为验证该研究假说，我们进行了针对性的体内和体外实验，得到了如下有意义的研究结果：. 1、再次验证外源性补体C3补充可有效改善AGI小鼠模型的48小时存活率（13.3% vs 37.0%, P<0.01）。这与AGI时多脏器缺血性损伤的明显减弱是相关的，尤其是肠黏膜屏障的相对完整。. 2、证实补体C3直接参与了AGI时肠黏膜上皮的损伤修复过程。研究发现肠上皮基底存在大量的C3免疫复合物沉积，外源性C3补充可强化这一局部聚集反应，而局部PCs的增殖及活性也同时显著增强（48小时后流式检测PCs数量比：7.6% vs 12.6%, P=0.003），说明外源性C3的胞内活化的确上调了PCs的功能。. 3、发现C3外源性补充可上调肠上皮隐窝内肠干细胞（Intestinal Stem Cells, ISCs）的活性，而PCs的体外培养干预实验表明PCs可通过活化mTORC1底物蛋白S6及磷酸化增强胞外酶Bst1的表达，进一步影响下游cADPR的表达并最终影响ISCs的增殖活化。课题组通过Bst1的SiRNA转染及CTSL阻断实验进一步证实上述调控过程同样受到了外源性C3在PCs胞内活化的调控。. 通过上述系列研究，课题组较完整的验证了本研究假说，揭示了固有免疫重要成员——补体C3通过PCs胞内活化途径（CTSL→C3aR→mTORC1→【S6→p-S6→Bst1】→cADPR）调控肠黏膜内ISCs进而干预肠黏膜损伤后的修复与再生过程。该项研究发掘了补体系统在免疫调理、凝血障碍调节之外的新功能，填补了国内外补体研究的一项空白。更为重要的是，该项研究为后续肠功能障碍的研究与治疗提供了一种新兴的干预途径，具有较好的临床意义。. 学术成果：截止目前，受该项目资助，已共发表7篇论文。

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