CRISPR/Cas9系统编辑敲除腹膜间皮细胞PKCα改善高糖透析液诱导的腹膜纤维化的可行性及机制研究

High glucose dialysate induced peritoneal fibrosis is the most important cause of the failure of peritoneal dialysis. Our previous experiments based on different PKC isoforms gene knockout mice have proven for the first time across the world that PKCα subtypes play an important role in controlling multiple signal transduction pathway leading to peritoneal fibrosis. Blocking PKCα can improve high glucose induced peritoneal fibrosis. However, pharmacology cannot develop simple PKC subtype inhibitor. In our research we innovatively propose to inject AAV plasmid into peritoneal cavity through peritoneal catheter in order to knockout PKCα of peritoneal mesothelium cell by CRISPR/Cas9 edit system. First we are trying to use immunohistochemical technology, proteomics and flow cytometry to prove that CRISPR/Cas9 mediated PKC α knockout can block TGF-β1-EMT signaling pathways, decrease inflammatory factor expression such as MCP–1 and VEGF, ameliorate macrophage chemotaxis. We also will prove it in the co-culture system to modify abdominal endo- environment. Then we will inject AAV plasmid into mouse peritoneal cavity through peritoneal catheter in order to knockout PKCα peritoneal mesothelium cell by CRISPR/Cas9 system. We will prove that CRISPR/Cas9 mediated PKCα knockout can inhibit intra-abdominal network signal activation and prevent peritoneal fibrosis in start phase by using the mouse peritoneal dialysis model in vivo. And provides new strategies for the clinical treatment.

高糖透析液诱导的腹膜纤维化是导致腹膜透析失败的最重要原因。本课题组前期实验已在国际上首次证实PKCα亚型直接调控多条经典通路的上游“总开关”，阻断PKCα可改善高糖透析液诱导的腹膜纤维化。目前药理学无法研制单纯的PKC亚型抑制剂而无法应用于临床。本课题创新性地提出应用CRISPR/Cas9技术通过腹膜透析管注入慢病毒质粒编辑腹膜间皮细胞已达到精准敲除PKCα的假设。首先拟以细胞共培养的形式模拟腹腔内环境，利用免疫组化、蛋白质组学及流式技术，证实此方法阻断PKCα可以下调MCP-1,VEGF等多种炎症因子表达，阻断TGF-β1—EMT信号通路，减少炎症细胞等的趋化。然后制备小鼠的CRISPR慢病毒质粒，以在体实验方式在小鼠腹透模型中证实此方法可以敲除腹膜间皮细胞PKCα并有效改善高糖透析液诱导的腹膜纤维化并揭示其具体机制，为最终实现临床治疗打下坚实基础。

高糖透析液诱导的腹膜纤维化是导致腹膜透析失败的最重要原因。本课题组前期实验已在国际上首次证实PKCα亚型是直接调控多条经典通路的上游“总开关”。阻断PKCα可改善高糖透折液诱导的腹膜纤维化。本课题组成功制备 CRISPR 慢病毒载体，感染人腹膜间皮细胞并完成相应检测，证实敲除PKCα可以减轻高糖刺激引起的腹膜纤维化。本课题组新增益生菌干酪乳杆菌张调节小鼠肠道菌群并抑制腹膜纤维化的研究，国际上首次发现干酪乳杆菌张可干预改善小鼠肠道菌群构成，增加产短链脂肪酸菌丰度，短链脂肪酸抑制巨噬细胞增殖及促炎表型，从而抑制高糖诱导的腹膜纤维化。新增槐杞黄调节小鼠腹膜透析相关纤维化的研究，证实槐杞黄可抑制腹透小鼠外周血及腹腔局部炎症因子水平，降低外周血巨噬细胞及CD4+T细胞占比，从而改善高糖透析液诱导小鼠腹膜纤维化。