玻连蛋白（Vitronectin）糖基化修饰调控放射性肺损伤的机制研究

Radiation-induced lung toxicity (RILT) is the key dose-limiting factor in increasing radiation dose for thoracic radiotherapy. We have previously identified and reported glycoprotein Vitronectin (VTN) as a potential biomarker for RILT in patients with lung cancer receiving radiotherapy by proteomics. Then we showed VTN plays an important role in RILF by in vitro and in vivo studies. Higher levels of VTN and glycosylated VTN were correlated with more severe RILT. So in this study we’ll perform systematic investigation of VTN glycosylation and in-depth analysis of site-specific glycoforms of VTN in RILT. .Firstly, we have set up a cellular model with radiation-induced damage after irradiation and we’ll make a profile study of the disease-related site-specific glycoforms of VTN in radiation-induced damage. Then, we’ll compare the VTN glycosylation profile in serum of patients with and without RILT after radiotherapy by glycoproteomics, to identify the RILT-related differential glycoforms and differential glycan abundance of VTN. While the 2 arms will be balanced with respect to age, general, cancer stage, histological type and dose volume histogram (DVH). Finally, we’ll verify the findings and study the mechanism of differential glycoforms in vitro. We’ll set up 5 groups of WI-38 cell: group A, normal cell without irradiation; group B, normal cell with irradiation; group C, cell infected by empty lentivirus; group D, cell infected by VTN-overexpressing lentivirus; group E, cell infected by VTN-siRNA lentivirus. 48 hours after 8 Gy irradiation, we will compare the differential glycoforms and differential glycan abundance of VTN in the 5 groups, and study the mechanism of differences..We hope to make it clear whether the alteration of VTN glycosylation could modulate the incidence of RILT, and if yes, how does it work? If we can resolve these questions in this study, we may have the potency to predict and even prevent the incidence of RILT.

放射性肺损伤是制约胸部放疗剂量提高的限制因素，我们前期研究证实玻连蛋白（Vitronectin，VTN）具有调控放射性肺损伤发生发展的作用，该作用可能是通过VTN某些糖基化位点、糖基化水平的改变实现的。因此本项目将通过一系列糖蛋白质组学方法研究VTN糖基化修饰调控放射性肺损伤的机制。先在细胞水平明确细胞放射损伤模型中VTN糖基化修饰的全面信息（糖基化位点、各位点糖型）；随后比较发生与未发生放射性肺损伤患者外周血中VTN糖基化位点糖型及表达水平的差异，找出与调控放射性肺损伤可能相关的糖基化位点糖型及表达量；最后在细胞水平比较经不同干预方式处理导致VTN不同表达水平组（共5组）之间VTN糖基化位点糖型和糖型表达水平的差异，与前一部分结果相验证，探索与调控放射性损伤相关的VTN糖基化位点糖型及表达水平，试图阐释VTN糖基化修饰调控放射性肺损伤的机制，为放射性肺损伤的防治及个体化放疗实施奠定基础。

放射性肺损伤是制约胸部放疗剂量提高的一个关键限制性因素，我们前期研究证实玻连蛋白（Vitronectin，VTN）具有调控放射性肺损伤发生、发展的作用，该作用可能是通过VTN某些糖基化位点、糖基化水平的改变实现的。因此我们首先在细胞水平明确细胞放射损伤模型中VTN糖基化修饰的全面信息（糖基化位点、各位点糖型），通过外源性表达系统高表达VTN，通过质谱研究细胞照射前后VTN糖基化修饰的信息。结果发现辐照并没有增加VTN的糖基化位点，甚至减少了糖基化位点。因此我们将研究方向调整为研究VTN诱导放射性肺损伤及上皮间质转化（EMT）的初步机制，体外细胞实验结果显示，敲减VTN后凋亡信后增强（Bax、BCL2、Caspase3上调），细胞转移能力减弱（E-cadherin、N-cadherin上调），炎症信号增强（NF-κB）。目前我们正在进行体内动物实验研究。同时通过对比接受胸部放疗后发生与未发生放射性肺损伤患者外周血VTN蛋白表达及SNP位点差异，进一步明确VTN诱导放射性肺损伤机制。结果显示VTN在≥2级放射性肺炎患者中表达显著增加（p<0.001），并且随着肺炎等级的增加而增加，VTN基因SNP位点rs2227721的AA/AC突变，rs704的GG/GA特定突变能显著降低放射性肺炎的发病风险（p=0.041和0.008）。后续将继续VTN调控放射性肺损伤发生发展作用机制的体外动物实验。