MSC调控免疫微环境干预TGF-β/Smad信号通路治疗CD肠道纤维化的作用及机制研究

The intestinal fibrosis is a challenging problem in clinical treatment of Crohn's disease (CD). Our previous studies indicate that mesenchymal stem cell (MSC) can regulate intestinal local immune microenvironment, reduce the expression of fibrosis-related factors in the CD intestinal tract, and effectively reduce the shortening of intestine. TGF - beta/Smad signaling pathways is a critical step during the fibrosis in various tissues. It was found that MSC can inhibit TGF-beta/Smad signaling pathways to relieve glomerular sclerosis. Therefore, we speculate that MSC can affect intestinal fibrosis in CD through this pathway. To analyze the mechanism, this study will focus on the immune effect of MSC function in regulating the TGF-beta/Smad pathway: 1.With the method of tissue microarray, combined with clinical data, we will analyze the correlation between factors expression in immune microenvironment and those in TGF-beta/Smad pathway, finding out key immune factors that are clearly associated with the occurrence and development of intestinal fibrosis in CD; 2.Using animal experiment to demonstrates the effect of MSC on prevention and treatment of intestinal fibrosis in CD, and confirms that this action is related with the expression of TGF-beta/Smad pathway, which is influenced by immune microenvironment; 3.Through molecular and cellular experiments, we investigate the mechanism of MSC in prevention and treatment of fibrosis in CD. This research will clarify the role and molecular mechanism of MSC in intestinal fibrosis development in CD, providing new ideas for clinical therapeutics of intestinal fibrosis in CD patients.

肠道纤维化是CD临床治疗的难题。我们的前期研究显示，MSC可调节肠道局部免疫微环境、降低CD肠道中纤维化相关因子表达，并能有效减少病变肠道的短缩。大量研究证实TGF-β/Smad信号通路是参与纤维化的主要通路。有研究发现MSC可抑制该信号通路表达从而减轻肾小球硬化。因此我们推测MSC可能经过此通路影响CD肠道纤维化。为解析其机制，本研究将重点围绕MSC免疫调节功能对TGF-β/Smad通路的影响进行：1.组织芯片结合临床资料,分析免疫微环境因子与TGF-β/Smad通路表达的相关性，明确CD肠道纤维化发生发展的关键免疫因子；2.动物实验明确MSC对CD肠道纤维化的防治效果，并证实该作用与调控免疫微环境影响TGF-β/Smad通路表达相关；3.分子及细胞实验深入探讨MSC防治CD纤维化的机制。本项目将初步阐明MSC影响CD肠道纤维化发生发展的作用及机制，为CD肠道纤维化的临床治疗提供新思路。

肠道纤维化是克罗恩病（CD）临床治疗的难题，目前间充质干细胞（MSC）的相关研究和应用显示其对CD有一定的治疗作用，但其对纤维化的疗效和具体机制仍不明确。本研究主要研究内容为验证MSC对CD肠道纤维化的作用，并探讨其作用的机制。本研究通过三硝基苯磺酸（TNBS）反复灌肠7周构建CD肠道纤维化的小鼠模型，通过马松三色染色法验证肠道纤维化的程度；转录组测序和基因富集分析检测MSC治疗前后肠道纤维化相关基因表达的变化；免疫荧光分析模型小鼠肠道上皮间质转化的改变；实时荧光定量PCR和免疫组化分析，阐明MSC的抗纤维化特性与免疫微环境的关系；用Western blot验证潜在的信号通路的改变。结果显示，经TNBS诱导治疗后，模型小鼠的结肠组织中有纤维化蛋白积累和炎性细胞的浸润。预防性输注MSC可有效抑制结肠缩短，而治疗性输注MSC可减轻结肠重量。预防性输注MSC可抑制纤维化蛋白的表达和EMT的发生，而治疗性输注MSC能够达到逆转已经存在的肠纤维化的效果，同时也减少了EMT的发生。两种MSC输注方式均能够使促纤维化因子IL-1β、IL-6和IL-13的分泌均下降，而抗纤维化因子IL-10上升。两种MSC输注方式均能够有效抑制TNBS诱导的TGF-β的表达及Smad2和Smad3的磷酸化。综上所述，本研究证实MSC能够通过调节免疫微环境、抑制TGF-β/Smad信号通路和改善EMT来发挥抑制肠道纤维化的作用。本研究的完成初步阐明了MSC影响CD肠道纤维化发生发展的作用及机制，为CD肠道纤维化的临床治疗提供新思路。