Fractalkine/CX3CR1信号通路调控良性前列腺增生组织炎症发生与细胞增殖的机制研究

Many other studies and our previous study proved that benign prostatic hyperplasia (BPH) was often combined with tissue inflammation, and inflammation was supposed to be an important factor in BPH occurrence and development. However, the exact mechanism that how the inflammatory cytokines are released, how the inflammatory cells infiltrate, and how the prostatic cells proliferate, is still obscure. The pair of Fractalkine/CX3CR1 is a special couple of cytokine involved, exerting both chemotaxis and adhesion functions, and participating in inflammatory process. Our previous research found that Fractalkine was not expressed in normal prostate epithelial cells, and the Fractalkine was up-regulated significantly after intervention of inflammatory cells, almost as high as the BPH epithelial cells. The up-regulated Fractalkine could activate the Fractalkine/CX3CR1 signal pathway. Therefore, we suspect that Fractalkine/CX3CR1 may be an important pathway in secretion of inflammatory and adhesive cytokines, infiltration of lymphocytes, and cell proliferation. In the present project, we plan to use multiple techniques in molecular biology to prove that Fractalkine/CX3CR1 is an important pathway in prostate to induce infiltration of lymphocytes and the hyperplasia of prostate.

良性前列腺增生症(BPH)标本中组织炎症的发生率高，但BPH炎症因子表达、炎症细胞浸润、细胞增殖的具体信号途径并不明确。Fractalkine/CX3CR1是一对独特的趋化因子，对炎症细胞起趋化和黏附的双重作用，参与了炎症过程。我们前期研究发现正常前列腺上皮细胞Fractalkine呈低表达，炎症细胞干预后Fractalkine表达显著上调，上调Fractalkine后具有增强Fractalkine/CX3CR1通路效应的作用。因此，课题组首次提出Fractalkine/CX3CR1通路的过度激活，可诱导炎症因子释放、炎症细胞浸润并促进前列腺细胞增殖的假说。本项目拟通过多种分子生物学技术，证实Fractalkine/CX3CR1信号通路是诱导前列腺组织中炎症因子表达、黏附因子释放、炎症细胞浸润和前列腺细胞增殖的重要通路，并阐明该通路的具体分子调控机制，最终可能为BPH防治提供新的思路。

在国自然科学基金的资助下，本课题从2014年开始进行研究于2017年2月正式结题。为证实Fractalkine/CX3CR1信号通路是诱导前列腺组织中炎症因子表达、黏附因子释放、炎症细胞浸润和前列腺细胞增殖的重要通路，并阐明该通路的具体分子调控机制，课题组进行了系列分子实验及体外细胞实验，具体研究方法包括：HE染色、免疫组化染色、免疫荧光、RT-PCR、Western Blot、液相芯片、蛋白芯片、ELISA、SiRNA、Transwell、细胞培养等。课题组共收集66例前列腺增生组织标本和73例前列腺液标本，检测结果发现FKN蛋白（膜结合型，mFKN）主要表达于BPH组织腺上皮细胞和部分基质细胞胞膜，其受体CX3CR1弱表达或不表达于前列腺组织；前列腺增生组织中伴有炎症比例约93.94%，炎症程度的高低与FKN的表达正相关；FKN、ADAM10/17及下游炎症信号分子如NF-κB（p65和p50）、IL-1、IFN-γ、TGF-β、BCL-2等均随着炎症程度的增加而不同程度的升高。体外细胞学研究发现400ng/L LPS+10ng/L TNF-α刺激下BPH-1细胞表达并分泌炎症因子显著升高且其增殖活性不低于空白对照组。采用上述条件进行后续研究，结果发现FKN表达量也明显升高，然而干扰FKN表达后其下游炎症信号分子如NF-κB等的变化却无明显差异。通过腺病毒构建FKN过表达和低表达BPH-1细胞系重复上述研究，课题组同样发现低表达FKN时其下游炎症信号分子的表达不受明显影响，反之过表达FKN能促进下游炎症信号分子表达升高，同时过表达FKN能够显著提高对外周单核细胞(PBMC)的趋化作用。ADAM10/17是人体内重要的金属酶，课题组通过同时阻断ADAM10/17后发现mFKN的表达量有轻微升高，而游离FKN的量显著降低，研究证实AM10/17是剪切mFKN成为游离FKN的关键酶。综上，课题组通过系列实验阐明了Fractalkine/CX3CR1信号通路的具体分子调控机制并证实该通路的激活在前列腺增生相关组织炎症中发挥明显作用，通过FKN表达升高趋化外周单核细胞进入前列腺组织进一步放大组织炎症，最终导致前列腺增生。该信号通路的具体分子调控机制的阐明，最终可能为BPH 防治提供新的思路。

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