Tiki修饰的Wnt信号通路在大鼠移植肾中的作用机制研究

The development of interstitial fibrosis and tubular atrophy with ongoing inflammation is a major risk for progressive graft dysfunction eventually leading to the failure of the majority of renal transplants. Although multiple approaches are available for preventing or at least blunting immune responses, there is currently no effective treatment for the development and progression of interstitial fibrosis and tubular atrophy. The Wnt signaling pathway is involved in a myriad of biological processes, including embryonic development, stem cell regulation and human disease. Activation of Wnt signaling which could preserve renal allograft function, has been observed in renal transplants developing interstitial fibrosis and tubular atrophy. A membrane-tethered metalloprotease named Tiki has been identified recently, which antagonizes Wnt function in embryos and human cells via a Tiki homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Tiki has been found in mice and rat kidneys in our lab. To understand the contribution of Tiki on tubular atrophy and interstitial fibrosis, we are going to utilize an acute tubular injury model, a chronic renal injury model in mice and a rat allograft renal transplant model to perform this project.

移植肾的间质纤维化和肾小管萎缩引起的移植肾失功，目前仍无有效的治疗方法。Wnt信号通路参与许多生物学过程中，包括胚胎发育、干细胞调节以及癌症等人类的许多疾病。我们此前的研究已经发现在移植肾的间质纤维化和肾小管萎缩过程中，Wnt信号通路得以活化并具有保护肾功能的作用。Tiki作为一种金属蛋白酶，具有高度的保守性，被发现表达于非洲爪蟾蜍的胚胎发育过程中以及斑马鱼、线虫类、海葵、海绵动物和人类HEK293T细胞中，并参与调节Wnt信号通路。目前已发现多种物质参与调节Wnt信号通路，而Tiki作为一种新发现的蛋白酶，在肾脏损伤及移植肾间质纤维化和肾小管萎缩的作用机制尚无报道。我们已发现Tiki表达于大鼠和小鼠正常肾脏中。在此，我们拟利用小鼠急慢性肾损伤模型和大鼠同种异体肾移植模型，研究Tiki参与修饰的Wnt信号通路在肾脏损伤和移植肾中的作用机制，进一步明确Wnt信号通路在移植肾失功中的作用机制。

TIKI2是新近发现的Wnt蛋白的拮抗剂。既往的研究表明很多Wnt蛋白拮抗剂在肾细胞癌中发挥着重要的作用，但是TIKI2在肾细胞癌中的作用尚无相关研究。我们应用Oncomine数据库发现TIKI2在肾癌中高表达。随后应用qPCR检测了临床肾癌标本及其相应的癌旁正常肾组织中、肾癌细胞株及人近端肾小管上皮细胞HK-2细胞株中TIKI2 mRNA的表达水平。进而我们应用细胞增殖试验、克隆形成试验、侵袭试验等肿瘤细胞功能试验检测了在肾癌A498细胞株过表达TIKI2以及在肾癌769-P细胞株中敲减TIKI2的表达对肿瘤细胞的功能的影响。进一步，我们通过动物模型，研究TIKI2在体内对肾癌的增殖能力的影响。.本研究发现：.1.与癌旁正常肾组织相比，TIKI2 mRNA在肾癌组织中高表达。在大多数肾癌细胞株中，TIKI2 mRNA的表达水平均高于HK-2细胞株。.2.在TIKI2 mRNA表达水平最低的A498细胞株中过表达TIKI2，促进肾癌细胞的增殖、侵袭及克隆形成能力。.3.在TIKI2 mRNA表达水平最高的769-P细胞株中敲减TIKI2，减弱肾癌细胞的增殖、侵袭及克隆形成能力。.4.体内实验也证实TIKI2具有促进肾癌细胞增殖的能力。.5.Wnt/β-catenin信号通路未在TIKI2促进肾癌细胞增殖方面发挥作用。