囊肿上皮细胞MAPK信号通路上游调控机制的研究

It is well known that VEGF/VEGFR signal pathway is very important for the regeneration of Autosomal Dominant Polycystic Kidney Disease （ADPKD） . Cyclooxygenase-2 inhibitor celecoxib (CXB) can inhibited VEGFR from a variety of tumor cells. Cyst-lining epithelial cells has the similar characteristic of high proliferation as tumor cells. Our findings provide the first direct evidence that CXB suppresses renal cyst-lining growth in ADPKD, which are chieved by the blockade of binding between VEGF and VEGFR-2.It is still unclear how to regulate upstream of MAPK signal pathway VEGFR-2 expression. Which has becoming the center point of controling signaling pathway.We suppose that CXB could effect transcription factors concerning of VEGFR-2 and interfer VEGF/VEGFR/Raf/ERK/MAPK signaling pathway. By gene chip technology we screened out suitable transcription factors：HLF，PBX1，Smad3，STAT3 and NF-κB1.In front of four transcription factors were detected inhibition of RNA content by realtime fluorescence quantitative PCR. Transcription factors and VEGFR-2 expression would be determined by tests in vitro and in vivo. All these data showed the potent and critically beneficial therapy target on ADPKD.

VEGF与其受体结合诱导的信号通路在常染色体显性多囊肾病（ADPKD）发生发展中居重要地位。环氧酶-2抑制剂塞来昔布（CXB）可抑制肿瘤细胞VEGFR表达。囊肿上皮细胞具有类肿瘤生长特性。申请人发现CXB减少VEGFR2表达，干扰MAPK通路，通过非COX-2依赖途径抑制增殖。但信号通路的上游如何被调控尚不清楚。我们推测："CXB调控VEGFR2相关转录因子表达，通过上游阻断VEGF/Raf/MAPK通路抑制细胞增殖。" 前期实验采用基因芯片技术已筛选出与VEGFR2密切相关的转录因子HLF、PBX1、Smad3、STAT3和NF-κB1。荧光定量PCR证实CXB能抑制前4种转录因子RNA含量。下一步拟通过体内、外实验进一步观察在CXB处理后转录因子表达量和活性的改变以及与VEGFR2的相互作用。进一步揭示囊肿上皮细胞MAPK信号通路上游VEGFR2的调控机制，发现ADPKD的治疗新靶点。

VEGF信号转导通路在常染色体显性多囊肾病（ADPKD）的发生发展中处核心地位。基因芯片筛查出多囊肾与正常肾差异基因包括氯离子通道蛋白（ANO）家族，其中在多囊肾组织ANO1，3，4高表达，ANO5低表达。体外实验在多囊肾病人和PKD1RC/RC小鼠模型肾组织通过real-time PCR、免疫组化和Western blot 从RNA水平到蛋白水平均证明上述结果。已知多囊蛋白2（PKD2）作为钙离子通道蛋白在纤毛中与PKD1共转运是多囊肾的发病主要机制。ANO1,3,4是否影响PKD2的纤毛转运对多囊肾的发生尤为重要。体内实验在人肾小管上皮细胞（RCTE）的3D免疫荧光共聚焦技术显示ANO1在纤毛基底呈环状分布，而ANO3，4进入纤毛体内，与PKD2共表达。说明ANO3，4作为氯离子通道蛋白与PKD2钙离子通道蛋白功能协同。ANO1特异性抑制剂T16Ainh-A01可抑制STAT3和AKT信号通路，提示：ANO1通过激活STAT3和AKT信号通路，参与多囊肾病的发生、发展。人多囊肾小管细胞（GANAB）纤毛变长，PKD2、ANO3，4表达减弱，说明ANO3，4对纤毛转运通路的影响促进多囊肾病的发生发展。课题组第一次发现ANO家族参与多囊肾发生、发展，并初步阐明ANO1蛋白功能。为寻找多囊肾治疗新靶点提供了理论依据。课题组将该项研究在ASN会议上报道并已投稿。