CD8+T细胞调控良性前列腺增生上皮细胞AR信号通路的作用及其分子机制研究

Benign prostatic hyperplasia (BPH) is one of the most common diseases in aging men, but the underlying mechanism is far from completely understanding. More and more evidence showed that inflammation could be a key component in BPH pathogenesis. CD8+ T cells are the mainly infiltrated inflammatory cells found in BPH prostate tissues. However, the consequences of CD8+ T cells towards prostate tissues remained unclear. Recently, we have shown that low intra-prostatic dihydrotestosterone (DHT) induced the BPH epithelial cells to recruit CD8+ T cells (data from 64 cases of BPH patients with/without androgen suppression treatment).Infiltrating CD8+ T cells could promote the proliferation of BPH epithelial cells, and this effect seems to be affected by androgen receptor (AR) signaling. Therefore, we hypothesized that infiltrating CD8+ T cells could promote the proliferation of prostate epithelial cells via AR signaling that is involved in the development and progression of BPH. In this project, we will investigate the following aspects:⑴Clinical study: in BPH patients, we will study the CD8+ T cells infiltration and functional status（proliferation/apoptosis）in prostate tissues; we will detect the difference of cytokines, AR and androgen responsive genes.⑵Cytology study：in the co-culture system between prostate epithelial cells (human primary prostate epithelial cells and prostate epithelial cell lines, respectively) and CD8+ T cells, we will study the influence of CD8+ T cells on the proliferation and apoptosis of prostate epithelial cells; and examine the change of cytokines in condition medium, AR and AR responsive genes on prostate epithelial cells.⑶Molecular mechanism research: by knocking-in AR and knocking-out AR in prostate epithelial cells, we will study the influence of the different cytokines on the proliferation and apoptosis of prostate epithelial cells to see which cytokines could impact on AR signaling and androgen responsive genes. We will illuminate the influence of CD8+ T cells on AR signaling in BPH pathogenesis.

良性前列腺增生(BPH)是老年男性常见疾病，免疫炎症参与BPH发生发展，但T细胞亚型对BPH的作用及机制尚不明确。我们前期发现，抗雄激素治疗后BPH前列腺上皮周围CD8+T细胞浸润增多；CD8+T细胞可促进前列腺上皮细胞增殖。故假设，CD8+T细胞通过调控上皮细胞雄激素受体(AR)信号通路促进BPH发生发展。本课题拟：⑴临床研究：研究BPH前列腺CD8+T细胞浸润与细胞因子、AR的关系；⑵细胞学研究：利用我们已建立的原代和BPH前列腺上皮细胞系，与CD8+T细胞共培养,研究CD8+T细胞对上皮细胞增殖/凋亡的影响，以及CD8+T细胞相关细胞因子和上皮细胞AR及雄激素相关基因的变化；⑶分子机制研究：加入或阻断差异细胞因子研究其对AR敲除和过表达的上皮细胞增殖/凋亡的影响，确定与AR形成信号通路的效应细胞因子及雄激素相关基因。本研究拟阐明CD8+T调控AR信号通路影响BPH发病的机制。

良性前列腺增生（ benign prostatic hyperplasia, BPH）是老年男性的常见疾病，老龄男性和具有功能性的睾丸是BPH发生发展的两个必要条件；性相关激素、代谢因素、免疫炎症因素、生长因子等也均在 BPH 的发生发展中起到重要作用。5α还原酶能够将睾酮转变为生物活性更强的双氢睾酮，之后双氢睾酮与雄激素受体（AR） 特异性结合进入细胞核，激活AR信号通路并启动和调控一系列带有AR反应元件的雄激素相关基因的转录。我们前期研究发现，服用非那雄胺（5α-还原酶抑制剂）的患者BPH前列腺组织中浸润的CD8 + T炎症细胞明显增多，并且在低雄激素条件下，CD8 + T细胞通过分泌CCL5促进BPH上皮细胞（BEC）的增殖，但是作用机制尚不清楚。.基于此，我们拟用前列增生细胞株BPH1为模型，采取免疫组化、细胞共培养实验、CCK8、流式细胞周期、 western blot、荧光定量 PCR等技术，进一步验证CD8 + T细胞通过分泌CCL5促进BPH上皮细胞（BEC）的增殖的作用机制；同时，探究cGMP/ PKG信号通路可能的作用。.结果表明：结果表明：CD8 + T细胞可能通过分泌CCL5促进STAT5的磷酸化和CCND1在BEC中的表达增高；通过CCL5中和抗体或STAT5抑制剂（Pimozide）减弱了CCL5 / STAT5信号引起的前列腺上皮增殖作用。非那雄胺治疗患者BPH组织的IHC表明，BECs中PCNA的表达与CD8 + T细胞浸润水平和CCL5的表达高度相关。同时，BPH组织的浸润CD8 + T细胞与CCL5，细胞周期蛋白D1和PDE5的表达呈正相关，而在HFD诱导的前列腺增生大鼠模型中，PDE5-1激活cGMP / PKG信号可以抑制 CD8 + T细胞浸润以及CCL5和细胞周期蛋白D1表达。此外，cGMP / PKG途径的激活通过下调NFκB p65磷酸化而抑制了CD8 + T细胞分泌CCL5，从而通过CCL5 / STAT5 / CCND1途径抑制BPH-1的生长。.这些研究可以进一步丰富和完善对BPH发病机制的认识，并提示联合使用5α还原酶抑制剂降低雄激素水平和PDE5-Is治疗BPH提供一种新颖、有效的治疗方法。