RhoA/ROCK1调控腹膜间皮细胞线粒体分裂与细胞骨架重构介导腹膜纤维化的机制研究

The Epithelial-to-Mesenchymal Transition (EMT) of peritoneal mesothelial cells is a key process of peritoneal fibrosis, but the onset mechanism of EMT remains unclear. We have found that the activation of RhoA/ROCK1 pathway could increase cell migration and induce EMT through regulating cytoskeleton remodeling. Our further experiment showed that Drp1 was activated and mitochondrial fission increased in the EMT model. Recently it was reported that ROCK1 is closely linked to Drp1-mediated mitochondrial fission. It is speculated that RhoA/ROCK1 pathway can also induce Drp1-mediated mitochondrial fission, thereby leading to EMT. This study will use RNA interference and gene transfection to regulate the expression of ROCK1 or Drp1 in peritoneal mesothelial cells, to observe the changes of cytoskeleton, mitochondrial fission and related regulatory molecules; and to construct peritoneal fibrosis model using ROCK1 targeted transfection knockdown mice, observing the effects of corresponding gene knockdown on cytoskeleton, mitochondrial fission and fibrosis progression. Thus, this study will confirm the mechanism of RhoA/ROCK1 pathway regulating peritoneal fibrosis and provide new ideas of clinical prevention and treatment of peritoneal dialysis.

腹膜间皮细胞上皮间充质转分化（EMT）是导致腹膜纤维化的中心环节，但启动机制未明。课题组前期研究发现RhoA/ROCK1通路的激活可调控细胞骨架重构增加细胞迁移，介导EMT发生。课题组进一步预实验表明，EMT模型中Drp1活化，线粒体分裂增加。近期文献报道ROCK1与Drp1介导的线粒体分裂密切相关。由此推测RhoA/ROCK1也可通过介导Drp1调控线粒体分裂，导致EMT。本研究将进一步采用RNA干扰、基因转染等单独或联合调控ROCK1、Drp1，观察腹膜间皮细胞细胞骨架、线粒体分裂及相关调控分子的变化；并利用腹膜定向转染敲低ROCK1小鼠建立腹膜纤维化模型，观察相应基因敲低对细胞骨架、线粒体分裂及纤维化程度的影响。由此，将明确RhoA/ROCK1通路对腹膜纤维化的作用及调控机制，为腹膜纤维化防治提供新思路。

腹膜间皮细胞上皮间充质转分化（EMT）是导致腹膜纤维化的中心环节，但启动机制未明。基于课题组前期研究、预实验及相关文献报道，本课题推测，在腹膜间皮细胞中，高糖激活RhoA/ROCK1通路，一方面通过活化Drp1促进线粒体的分裂，并再分布至迁移细胞的前体，提供供能基础，另一方面通过调控细胞骨架重构，提供结构基础，进而导致腹膜间皮细胞迁移增加及EMT。本研究利用腹膜间皮细胞及ROCK1抑制小鼠建立腹膜纤维化模型，采用RNA干扰、激动剂、抑制剂等处理，研究调控RhoA/ROCK1通路对高糖诱导EMT的作用，并通过联合干预RhoA、ROCK1，验证了RhoA、ROCK1对EMT及纤维化程度的影响，明确了RhoA/ROCK1通路的确立及其在腹膜间皮细胞EMT发生发展中的作用；进一步研究RhoA/ROCK1通路对myosinII的活性及细胞骨架重构的作用，并在体外及体内试验，通过联合干预ROCK1、myosinII，验证了myosinII为ROCK1的下游作用靶点，明确了RhoA/ROCK1通路对细胞骨架相关蛋白myosinII、actin的作用及调控机制；再进一步研究RhoA/ROCK1通路对Drp1的活性及线粒体分裂、再分布的作用，并在体外及体内试验，通过联合干预ROCK1、Drp1，验证了Drp1为ROCK1的下游作用靶点，明确了RhoA/ROCK1通路对Drp1活性、线粒体分裂的作用及调控机制。由此本课题明确了RhoA/ROCK1通路对腹膜纤维化的作用及调控机制，为临床干预治疗提供新靶点，潜在的靶点包括RhoA、ROCK1、myosinII以及Drp1等，为腹膜纤维化防治提供实验支持，对改善腹膜透析病人的长期预后具有潜在的重要价值。