2型糖尿病及其微血管并发症患者血清microRNA表达谱筛选及临床价值研究

发布时间：2018-06-24 11:13

  本文选题：2型糖尿病 + 微血管并发症　； 参考：《南京师范大学》2015年硕士论文

【摘要】：目的：筛选2型糖尿病(Type 2 diabetes mellitus, T2DM)及其微血管并发症(Type 2 diabetes mellitus microvascular complications,T2DMC)患者特异变化血清微小核糖核酸(microRNA,miRNA)表达谱,评价其对T2DM及T2DMC的诊断、鉴别诊断和病情监控临床价值。方法：收集2013年9月～2014年5月间南京军区总院内分泌科收治的新发或一个月内未经任何治疗的T2DM及T2DMC各87例,同时收集同期在医院体检中心年龄、性别完全匹配的健康体检者87例做为正常对照。首先采用高通量、高灵敏度的低密度芯片技术(TaqMan Low Density Array, TLDA)对T2DM.T2DMC及健康对照各25例样本分别组成的混合血清中768种miRNA的表达水平进行检测,初筛T2DM和T2DMC特异变化的一组血清miRNA;然后运用准确的实时荧光定量PCR技术(quantitative real-time polymerase chain reaction, qRT-PCR)对初筛miRNA在TLDA所使用的同一批样本中进行复筛(复筛组)；进一步运用qRT-PCR技术在另外三组T2DM.T2DMC和健康对照各62例样本中对复筛结果进行验证(验证组),筛选T2DM及T2DMC患者血清中稳定变化的一组miRNA。同时,运用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定全部患者及对照血清样本中氧化低密度脂蛋白(ox-LDL).氧化脂蛋白(a) [ox-Lp(a)]、β2糖蛋白氧化低密度脂蛋白复合物(p2-GPI-ox-LDL)和β2糖蛋白脂蛋白(a)复合物[β2-GPI-Lp(a)]勺水平,运用Spearman秩相关分析和逻辑回归分析患者特异变化血清miRNA与上述脂蛋白、临床指标相关性及是否为T2DM及T2DMC独立危险因素。结果：TLDA结果显示,T2DM及T2DMC患者血清miRNA表达谱与健康对照相比具有明显差异,53种miRNA在T2DM及T2DMC患者血清中较对照组表达上升,117种miRNA在患者表达下降。结合TLDA初筛及文献调查结果,我们选取了12种miRNA在复筛组样本中进行复筛,发现8种miRNA,包括miR-571、miR-661、 miR-1303、miR-152、miR-1323、miR-892b、miR-770-5p和miR-886-3p水平在两组患者血清中显著上升,其中在T2DMC患者中变化更为显著(变化倍数2,P0.05)；8种miRNA在验证组中变化趋势与复筛组一致(变化倍数2,P0.05)；ROC曲线分析显示,8种血清miRNA对T2DM诊断的ROC曲线下面积(AUC)在0.774～0.877之间,对T2DMC诊断的ROC曲线下面积在0.823～0.946之间,对T2DM和T2DMC鉴别诊断的ROC曲线下面积在0.611～0.675之间。根据选定的cutoff值,8种血清miRNA对T2DM诊断的灵敏性和特异性分别在68.2%～88.1%和63.0%～84.5%之间；对T2DMC诊断的灵敏性和特异性分别在64.8%～84.7%和80.6%～98.6%之间；进一步分析上述miRNA的鉴别诊断价值显示,3种miRNA对T2DM和T2DMC的鉴别诊断具一定临床价值,其AUC、灵敏性和特异性分别为：miR-886-3p(AUC=0.675,灵敏性=0.651,特异性=0.628)、miR-892b(AUC=0.639,灵敏性=0.662,特异性=0.622)和miR-571(AUC=0.611,灵敏性=0.603,特异性=0.608)。ELISA结果显示,T2DM和T2DMC患者ox-LDL、ox-Lp(a)、β2-GPI-ox-LDL、β2-GPI-Lp(a)水平亦显著高于健康对照组(P均0.05)。Spearman秩相关分析显示,T2DM和T2DMC患者变化的8种血清miRNA与空腹血糖(fasting blood-glucose, FPG)、ox-LDL、ox-Lp(a)呈显著正相关(P均0.05)。单因素逻辑回归分析显示,8种血清miRNA、氧化脂蛋白及其复合物均为预测T2DM和T2DMC患者发病的独立危险因素；进一步多因素分析结果显示,在排除性别、年龄等影响因素后,miR-770、miR-661和甘油三酯仍是两组患者独立危险因素。结论：8种血清包括miR-571、miR-661、miR-1303、miR-152、miR-1323、miR-892b、 miR-770-5p和miR-886-3p在T2DM和T2DMC患者患者中显著上调,有望作为T2DM及T2DMC的辅助诊断标志物。
[Abstract]:Objective : To investigate the expression profiles of microribonucleic acid ( microRNA , miRNA ) in patients with type 2 diabetes mellitus ( T2DM ) and its microvascular complications ( Type 2 diabetes mellitus complicated complications , T2DMC ) .
Serum levels of low density lipoprotein ( ox - LDL ) in sera of patients with T2DM and T2DMC were determined by using the enzyme - linked immunosorbent assay ( ELISA ) . Results : The results showed that the levels of serum miRNA in patients with T2DM and T2DMC were significantly higher than those in healthy controls . Results : The results showed that the levels of serum miRNA in patients with T2DM and T2DMC were significantly higher than those in healthy controls .
The change trend of 8 kinds of miRNA in the validation group was consistent with that of the compound sieve group ( changing multiple 2 , P0.05 ) .
ROC curve analysis showed that the area under ROC curve ( AUC ) of 8 kinds of serum miRNA in the diagnosis of T2DM was between 0.784 and 0.877 , the area under ROC curve of T2DMC diagnosis was between 0.823 and 0.694 , and the area under ROC curve for the differential diagnosis of T2DM and T2DMC was 0.611 - 0.675 . According to the selected cutoff value , the sensitivity and specificity of 8 kinds of serum miRNA to the diagnosis of T2DM were 68.2 % - 88.1 % and 63.0 % - 84.5 % , respectively ;
The sensitivity and specificity of T2DMC diagnosis were 64.8 % 锝，

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