游离泛素（free UB）通过CXCR4跨膜受体通路在放射损伤中的作用机制研究

During cancer radiotherapy and nuclear accidents, ionizing radiation is likely to cause injuries to normal tissues. Our previous results showed that: (1) the levels of extracellular free ubiquitin are closely related to radiation-induced injury tissue radiosensitivity. and (2) free ubiquitin has the funcation to lighten radiation-induced injury. CXCR4, a transmembrane signal receptor, has the ability to transmit extracellular signal to intracellular. In this project, we will investigate the mechanism that how free ubiquitin exerts its radio-protective effect via CXCR4 transmembrane receptor. We will analysize skin, intestinal and lung tissues and derived cells, which have important biological functions but different radiosensitivities. The radio-protective mechanism of free ubiquitin will be explored by changing the levels of free ubiquitin and CXCR4 inhibition. The CXCR4 signaling pathway will be studied by chemical inhibitor or siRNAs to illustrate its role in mediating free ubiquitin-induced intracellular signal transduction. In addition, C57 wild-type and intestinal epithelia CXCR4 knock-out mice will be used to investigate the radio-protective role of free ubiquitin in vivo. The survival experiment and primary cells from skin, intestinal and lung tissues will be used to compare the radio-protective role of free ubiquitin in different tissues, confirming the role of CXCR4 pathway. This project aims to illustrate the radio-protective role and mechanism of free ubiquitin against ionizing radiation via CXCR4 signaling pathway.

我们的研究发现，细胞外游离泛素不仅与放射损伤和组织放射敏感性密切相关，且具有减轻放射损伤的作用。作为细胞外环境的信号受体，CXCR4跨膜受体蛋白能穿越细胞膜，将胞外信号传递到细胞内。本项目，我们将放射敏感性不同且具有重要生物效应的皮肤、肺和肠组织/细胞作为研究对象，通过提高/降低胞外游离泛素水平，观察其对放射敏感性不同的组织/细胞的放射损伤的影响；通过抑制/下调CXCR4表达以及抑制MAPK/ERK和/或PI3K/AKt信号通路，研究CXCR4及信号通路在胞外游离泛素对放射损伤影响中的机理；采用正常和肠上皮细胞CXCR4基因敲除小鼠，研究外源性游离泛素对CXCR4功能正常/缺失小鼠的放射防护作用；采用两种小鼠和它们的皮肤、肺、肠组织原代细胞，研究游离泛素对这些组织放射损伤的影响，验证CXCR4的作用机理。本项目旨在阐明细胞外游离泛素通过CXCR4跨膜受体及通路在放射损伤防护中的作用机理。

针对皮肤、胃肠道等电离辐射敏感的组织器官的辐射防护，本项目系统的进行了以下研究：（一）游离泛素的肠道辐射防护作用的研究。1．Free Ub预处理肠IEC-6细胞可提高细胞活力。2．Free Ub预处理可减少LDH由IEC-6细胞胞内向胞外漏出，从而减轻电离辐射照射下细胞损伤。3．Free Ub预处理可在辐照后12h促进IEC-6细胞数目增加（P<0.05），在辐照后24h明显促进IEC-6细胞数目增加（P<0.01）。4．Free Ub预处理可有效促进辐照后IEC-6细胞的迁移。5．Free Ub预处理后，降低电离辐射对IEC-6细胞细胞凋亡率。6．Free Ub预处理对辐照后线粒体的功能具有一定的保护作用。7. Free Ub预处理可以促进X射线照后IEC-6细胞FGF2蛋白的表达增加。8.Free Ub预处理可促使IEC-6细胞分泌较多的成纤维细胞生长因子FGF2。9.Free Ub预处理引起胞内FGF2蛋白表达增多，胞外分泌较多的FGF2，从而对细胞损伤进行修复，FGF2的表达及分泌可能是电离辐射后机体为抵御放射毒性产生的保护机制。（二）SDF-1/CXCR4跨膜受体通路对皮肤辐射防护作用的研究。1.在受照射的皮肤组织中SDF-1/ CXCR4轴的表达水平增加。2.表皮层特异性敲除CXCR4减轻了电离辐射诱导的皮肤损伤。3.SDF-1/ CXCR4轴抑制剂AMD3100在大鼠的放射性皮肤模型中减轻电离辐射导致的皮肤损伤。4.SDF-1通过PI3K-MAPK信号传导促进Smad2的核转移。5.AMD3100抑制人皮肤成纤维细胞中由于电离辐射激活的TGF-β/ Smad通路的活性。本项目共发表SCI论文10篇，中文核心期刊一篇，获授权专利3项，培养博士研究生8名，硕士研究生12名。