高胰岛素诱导基质细胞分泌IGF促进前列腺增生的机制研究

Hyperinsulinemia is closely related to the progress of benign prostatic hyperplasia(BPH) in elderly men. We found that the prostate weight of experiment group was significantly higher than that of control group in hyperinsulinemia mouse model. However, the mechanism that hyperinsulinemia can promote prostatic hyperplasia prostate epithelial cell proliferation is still unclear. Preliminary results showed that the high expression of c-jun in stromal cell could promote secretion of insulin-like growth 1(IGF-1), which stimulates prostate epithelial cell proliferation through binding the IGF receptor. Herein, we hypothesize that hyperinsulinemia induce high expression of c-jun in stromal cell and the IGF-1, secreted by the stromal cell, can promote prostate epithelial cell proliferation and development of BPH. Based on the coculture of stromal cell and epithelial cell, we are going to use KO hyperinsulinemia mouse model , which treated with specific inhibitor, to prove that hyperinsulinemia promotes prostate epithelial cell proliferation. To defind possible key points and phosphorylation sites of hyperinsulinemia inducing c-jun expression in stromal cell, CRISPR/Cas9 gene editing system, co-immunoprecipitation and site-directed mutagenesis will be undertook, respectively. Promoter deletion and dual luciferase report system will also be utilized to demonstrate the transcriptional regulation of C-Jun on IGF-1. The scientific evidences above will help us better understand the mechanism about how hyperinsulinemia promoting BPH and will provide therapeutic strategy for BPH treatment.

高胰岛素血症与老年男性前列腺增生症的进展密切相关。我们在研究高胰岛素血症的小鼠模型中发现其前列腺重量显著增加。但高胰岛素促进前列腺增生的机制是什么？前期实验已证明c-jun高表达的间质细胞通过分泌IGF-1，作用于前列腺上皮IGF受体促进细胞的增殖。我们提出“高胰岛素诱导基质细胞表达c-jun并分泌胰岛素样生长因子1（IGF-1），介导腺上皮细胞增殖促进前列腺增生”的假设。拟进一步利用高胰岛素血症小鼠模型，结合基质细胞与上皮细胞共培养体系，协同针对性抑制剂处理，获得高胰岛素促进上皮细胞增殖的证据。通过CRISPR/Cas9基因敲除技术获得高胰岛素诱导基质细胞表达c-jun的关键节点，免疫共沉淀和点突变技术发现结合的磷酸化位点和作用机制，同时通过启动子缺失和双荧光报告实验获得c-jun对IGF-1直接转录调控的证据，揭示高胰岛素促进前列腺增生的机制。为前列腺增生的治疗提供一个可靠的实验依据。

高胰岛素血症与老年男性前列腺增生症的进展密切相关。众多的临床研究发现2型糖尿病患者的前列腺体积更大，且体积的大小与血清胰岛素水平相关。但高胰岛素促进前列腺增生的机制是什么？为揭示高胰岛素促进前列腺增生的相关机制，我们通过对C57BL/6小鼠喂养60%高脂饲料构建2型糖尿病小鼠模型，同时利用细胞共培养体系，以胰岛素为切入点，发现在高胰岛素刺激下间质细胞分泌IGF-1、HPGD及INHBE等相关因子，且在2型糖尿病患者及小鼠前列腺样本上高表达。此外，我们通过WB检测发现PRDX3在前列腺增生组织中高表达，且随着其表达的增高，细胞增殖越明显，ROS水平越低，LC3 II蛋白表达越低。这一功能可被PRDX3的siRNA所抵消。说明PRDX3通过调控自噬影响着前列腺上皮细胞增殖。同时，已有研究表明雌激素可调节胰岛素的敏感性，并对前列腺增生具有直接作用。我们利用Western印迹检测雌二醇(E2)、PER特异性激动剂G1、同时加入G1及GPER特异性拮抗剂G15对LC3蛋白表达量的影响，运用自噬检测试剂盒并再次观察上述药物对相同种类细胞中自噬活动的变化；通过CCK8检测加人上述药物对RWPE-1及BPH-1细胞活性的影响。证明了GPER的激活可导致自噬活动的抑制及前列腺上皮细胞增殖。通过上述研究，希望为前列腺增生的预防和治疗提供一些新的思路及研究基础。