利用转基因大鼠模型研究人组织激肽释放酶1对阴茎勃起功能的影响及作用机制

Erectile dysfunction (ED) refers to a sexual disorder with cavernosum smooth muscle and endothelial dysfunction. Nowadays, the therapeutic target of ED is mostly focused on activating NO-cGMP pathway, which shows a certain therapeutic effect on ED. However, it can only improve penile erectile function (EF) and fails to reverse corpus cavernosum lesion. Therefore, exploring a more ideal and effective therapeutic target becomes increasingly an urgent demand for ED treatment. It has been well known that human tissue kallikrein1 (hKLK1), as a pleiotropic agent, plays an important role in vasodilation, endothelial cell protection, anti-tissue fibrosis, and angiogenesis promoting effect, etc. Our previous studies showed that hKLK1 not only strongly relaxed cavernosum smooth muscle, but also significantly improved EF and induced anti-fibrotic effects on corpus cavernosum in transgenic rats harboring hKLK1gene (TGR (hKLK1)), which indicates that hKLK1 may also play multiple regulatory roles in cavernous tissue as well as in cardiovascular system. The purpose of this study is to explore the possible effects of hKLK1 on NO-cGMP and PGI2-cAMP signaling pathways, calcium and potassium channels, tissue fibrosis, and pathological mechanisms on ED at cellular and molecular level in corpus cavernosum of TGR(hKLK1) model in Vitro and in Vivo. To further elucidate the mechanism, we also intend to establish diabetic and senile TGR (hKLK1) models, which would contribute to finding a new target for the drug development and gene therapy of ED.

目前勃起功能障碍（ED）的基因治疗靶点主要集中于激活NO-cGMP通路，但其未能充分修复病变海绵体组织。人组织激肽释放酶1(hKLK1)在心血管系统中具有舒张血管、保护血管内皮功能、抗组织纤维化和促进血管生成等"多效剂"保护作用。申请者前期研究显示，hKLK1对大鼠离体海绵体平滑肌具有强力的舒张效应，并显著增强hKLK1转基因大鼠(TGR(hKLK1))勃起功能和抑制海绵体纤维化。本项目拟在前期研究基础上，利用TGR(hKLK1)模型，在离体、在体、细胞和分子水平研究hKLK1对海绵体NO-cGMP和PGI2-cAMP信号通路、Ca2+和K+通道以及纤维化水平的影响，探索其在勃起功能生理调控中的机制，并通过TGR(hKLK1)老龄和糖尿病模型，探讨其在病理条件下对勃起功能的保护作用和机制，从而阐明hKLK1对勃起功能的影响和作用机制。本研究有望为ED的基因和药物治疗寻找到更有效的新靶点。

目前勃起功能障碍（ ED）的基因治疗靶点主要集中于激活 NO-cGMP 通路，但其未能充分修复病变海绵体组织。人组织激肽释放酶 1(hKLK1)在心血管系统中具有舒张血管、保护血管内皮功能、抗组织纤维化和促进血管生成等“多效剂”保护作用。本项目在前期研究基础上， 利用 TGR(hKLK1)模型，在离体、在体、细胞和分子水平研究 hKLK1 对海绵体 NO-cGMP 和 PGI2-cAMP 信号通路、Ca2+和 K+通道以及纤维化水平的影响， 探索其在勃起功能生理调控中的机制，并通过TGR(hKLK1)老龄和糖尿病模型， 探讨其在病理条件下对勃起功能的保护作用和机制，从而阐明 hKLK1 对勃起功能的影响和作用机制。.本课题建立了可稳定遗传的转基因大鼠模型(TGR)，从整体水平和分子水平对hKLK1基因在老龄大鼠ED模型和糖尿病ED大鼠模型改善大鼠勃起功能的机制进行相关研究。得出以下结果：(1)成功建立老龄大鼠ED动物模型；(2)hKLK1基因通过DDAH-NOS-NO-cGMP通路、RhoA-ROCK-LIMK-cofilin通路、COX2-PGE-cAMP通路、改善阴茎海绵体纤维化、抑制阴茎海绵体内细胞凋亡等机制改善老龄ED大鼠勃起功能；(3)成功建立糖尿病大鼠ED动物模型；(4)hKLK1基因通过PI3K-AKT-NO-cGMP通路、降低阴茎海绵体内氧化应激水平、抑制阴茎海绵体组织内细胞凋亡、抑制TGFβ1- smad2/3- CTGF-collagen IV通路以保存海绵体平滑肌含量等机制改善糖尿病ED大鼠勃起功能；(5)成功培养大鼠主动脉内皮细胞及阴茎海绵体平滑肌细胞；(6)hKLK1可激活内皮细胞内PI3K-AKT-eNOS通路，引起平滑肌细胞内cGMP含量增高及钙离子浓度降低，从而促进平滑肌舒张；(7)hKLK1对细胞氧化应激损伤及凋亡具有保护作用。.本研究利用转基因大鼠模型研究hKLK1在老龄及糖尿病ED模型中对大鼠勃起功能的保护作用及机制，从体内体外水平证实hKLK1主要通过激活NO-cGMP、COX2-cAMP通路，抑制RhoA/ROCK通路，保护内皮功能，抑制阴茎海绵体氧化应激、凋亡及纤维化等机制保护两种生理病理条件下ED的发生发展，从而为ED的预防或治疗找到一种卓有成效的新方法。