基于2D LC-MS/MS技术的蛋白组学方法iTRAQ筛选2型糖尿病合并大血管病变的血清生物标记物及预测因子

Type 2 diabetic macroangiopathy as a global health problem is becoming more and more seriously. In our previous work, we took long-term primary prevention of coronary heart disease with T2DM in short duration without macroangiopathy. Seven years later, the incidence of those patients with subclinical macroangiopathy reduced effectively （Diabetes Care 2012）.During the following up, however, we found that some patients with well-controlled biochemical index still turned to have macroangiopathy. We believe, outside current treatment strategies, there may be some new protein therapeutic targets that should be looking for as new biomarkers. The emerging proteomics research can be used to compare protein expression differences in tissue or organ with various disease states to find disease-related proteins as biomarkers. It is an initial application that we use proteomics method iTRAQ to analysis serum of the patients with diabetic macroangiopathy. We expect to filter biomarkers of diabetic macroangiopathy as monitoring indicators provide new therapeutic targets. At the same time, we want to identify serum of the patients 10 years ago before joining the group who currently have clinical/subclinical macroangiopathy .We expect to find incidence predictors of type 2 diabetic macroangiopathy, in the hope of making early warning for the prevention of type 2 diabetic macroangiopathy as well as obtaining innovative achievements.

T2DM大血管病变是全球面临的日益严重的健康问题。我们前期对不合并大血管病变短病程T2DM患者进行长期冠心病一级预防，7年后亚临床大血管病变发生率有效降低（Diabetes Care 2012），但随访中发现部分生化指标控制良好的患者仍出现大血管病变，我们认为在目前治疗策略外，可能存在其他的蛋白治疗靶点，应寻找新的生物标志物。新兴的蛋白组学研究用于比较组织或器官在不同疾病状态下蛋白质表达差异，找到疾病相关的蛋白质作为生物标志物。我们首次采用iTRAQ蛋白组学方法，解析T2DM大血管病变患者血清，预期筛选出糖尿病大血管病变生物标志物，作为监测指标，提供诊断依据及新的治疗靶点；我们同时鉴定目前已发生临床/亚临床大血管病变的患者10年前入组时的血清，预期发现T2DM大血管并发症的发病预测因子，以期为T2DM大血管病变防治预警，可望取得创新性成果。

背景：筛选2型糖尿病（Type 2 diabetes mellitus，T2DM）合并下肢血管病变的血清生物标志物。.内容：根据其有无T2DM及是否合并下肢血管病变，分别纳入无糖尿病无大血管病变组（Non-diabetes & non-macrovascular group，NDNM）、T2DM不合并大血管病变组(Diabetes & non-macrovascular group，DNM）、T2DM合并亚临床大血管病变组（Diabetes & subclinical macrovascular group，DSM）及T2DM合并临床下肢血管病变组（Diabetes & peripheral arterial disease group，DPA），共4组，每组30例。应用同位素相对标记与绝对定量（isobaric Tags for Relative and Absolute Quantitation，iTRAQ）肽段标记联合高效液相色谱-质谱技术对血清进行蛋白质鉴定，筛选差异蛋白质参与的最主要的代谢/信号转导通路。应用ELISA对差异蛋白质定量检测。.结果：T2DM发生亚临床大血管病变的危险因素为年龄，OR值为1.111。T2DM发生临床下肢血管病变的危险因素为年龄、T2DM病程及性别，OR值分别为1.225、1.575及0.014。四组血清样品鉴定到888个蛋白质。T2DM合并亚临床大血管病变有34个差异蛋白质，其中4个蛋白质主要参与补体及凝血级联反应通路，ELISA结果表明α2-巨球蛋白（Alpha 2 macroglobulin，A2M）及补体7（Complement 7，C 7）表达与iTRAQ结果一致，均上调，A2M组间差异有统计学意义；T2DM合并临床下肢血管病变有52个差异蛋白质。.意义：补体及凝血级联反应通路与糖尿病下肢血管病变密切相关；A2M可能为糖尿病下肢血管病变的预警血清标志物并指导患者的个体化治疗。