【作者】 李萍；

【导师】 阎春生； 哈小琴；

【作者基本信息】 兰州大学 ， 公共卫生与预防医学·营养与食品卫生学， 2021， 硕士

【摘要】 目的通过病例对照研究和动物模型实验,探讨Sestrin2蛋白,微量五元素(铜、锌、钙、镁和铁)以及血栓相关标志物在2型糖尿病及2型糖尿病合并血栓中的表达水平及相关性,找出对研究疾病具有高特异性、高敏感性的诊断预测指标,从而为2型糖尿病血栓的预防和诊断提供参考。方法1.收集兰州市某三甲医院2017年6月～2019年12年住院的2型糖尿病患者信息,根据并发症类型分为2型糖尿病无血管并发症患者(A组),2型糖尿病微血管并发症患者(B组),2型糖尿病大血管并发症患者(C组)和2型糖尿病微+大血管并发症患者(D组);根据是否合并血栓性疾病又分为2型糖尿病不合并血栓组(T2DM组)和2型糖尿病合并血栓组(T2DM+T组)。利用卡方检验,方差分析/非参检验分析各组间一般资料,血糖,生化和凝血指标的差异性,用Logistic回归分析疾病影响因素;用受试者工作特征曲线(ROC)分析影响因素对疾病的诊断预测价值。2.选取兰州市某三甲医院基础医学实验室动物中心SPF级成年wistar雄性大鼠50只,采用高脂饲料联合链脲佐菌素的方法建立T2DM模型(T2DM组),在此基础上用角叉菜胶诱导建立2型糖尿病合并血栓模型(T2DM+T组),同时设立对照组(Control组)。用全自动分析仪检测大鼠的血糖,血脂和凝血指标,利用双抗体夹心酶联免疫吸附试验(ELISA)检测Sestrin2蛋白,内皮素-1(ET-1),血管内皮生长因子(VEGF),P选择素(P-selectin),血栓调节蛋白(TM),凝血酶抗凝血酶复合物(TAT),纤溶酶-a2抗纤溶酶复合物(PIC)和组织型纤溶酶原激活物抑制剂复合物(t-PAIC)。采用方差分析/非参检验比较上述指标在各组间的差异,利用相关性分析Sestrin2与其他研究指标间的相关关系。用HE染色法进行大鼠尾部病理切片制作。3.收集兰州市某三甲医院2020年7月～2020年11月住院T2DM患者信息。分为2型糖尿病组(T2DM组)和2型糖尿病合并血栓组(T2DM+T组),以同期住院的其他患者作为对照组(Control组)。采用全自动分析仪检测血糖,生化和凝血指标,用双抗体夹心ELISA法检测血清中Sestrin2、ET-1、VEGF和Pselectin,用化学发光免疫法检测血浆中TM、TAT、PIC和t-PAIC,用原子吸收法检测血清微量五元素的含量。利用卡方检验,方差分析/非参检验分析上述指标在各组间的差异,用Logistic回归分析疾病影响因素;用ROC曲线分析影响因素对疾病的诊断预测价值。结果1.T2DM各并发症组以及T2DM是否合并血栓组之间均在年龄、病程和是否患高血压上之间的差异具有统计学意义(P<0.05)。T2DM有血管并发症者较无血管并发症者TG、TC、FIB、FDP和D-Dimer升高,SOD降低,APTT、PT和TT缩短;T2DM+T组较T2DM组在TG、TC、LDL、HCY、FIB、FDP和D-Dimer的表达升高,HDL和SOD降低,PT和TT缩短(P<0.05)。Logistics回归分析发现,以A组为参考时,B组的危险因素仅是病程(OR=1.086),C组的危险因素仅为年龄(OR=1.073),D组的影响因素是年龄、病程和PT,OR值分别为1.060,1.072和0.691;与T2DM组相比,T2DM+T组的危险因素是年龄、患高血压、HCY和PT(P<0.05),保护因素是FDP和SOD(P<0.05)。对T2DM+T的影响因素进行ROC分析,诊断价值由高到低依次是年龄、PT、患高血压、SOD、HCY和FDP,AUC依次为0.742、0.738、0.689、0.681、0.666和0.616。2.动物模型实验中,与Control组相比,T2DM和T2DM+T组FPG、GSP、TG、TC、LDL、HCY升高,Sestrin2、HDL和SOD降低(P<0.05),APTT、FIB、D-Dimer、TM、TAT、PIC和t-PAIC三组间比较存在差异性(P<0.05),而ET-1、VEGF和P-selectin组间差异无统计学意义(P>0.05)。相关性分析发现,Sestrin2与FPG、GSP、TG、LDL和HCY呈负相关,与TM和VEGF呈正相关(P<0.05)。3.在此部分病例对照研究中,Control组、T2DM和T2DM+T组在年龄、病程和患高血压方面的差异具有统计学意义(P<0.05)。与Control组相比,T2DM和T2DM+T组FPG、GSP、TG、TC、HDL、HCY、FIB、FDP、D-Dimer、Sestrin2、Ca、Cu和Fe水平升高(P<0.05),LDL、SOD、Mg和Zn降低(P<0.05),APTT和PT缩短(P<0.05);T2DM+T组TM、TAT、PIC、t-PAIC和ET-1的水平均高于T2DM和Control组(P<0.05);Logistics回归分析显示,与Control组相比,T2DM的危险因素有年龄、TG、PIC和Ca(P<0.001),T2DM+T的危险因素是年龄、TC、FIB和Sestrin2,而SOD和Mg是其保护因素(P<0.05)。与T2DM相比,T2DM+T的危险因素则是病程、PIC和Sestrin2,Zn是保护因素(P<0.05)。经ROC分析显示,Sestrin2与年龄、TG、PIC和Ca联合诊断T2DM的能力较高,曲线下面积AUC=0.953,敏感度为89.9%,特异度为87.8%;Sestrin2单独诊断T2DM+T的价值较低(AUC=0.651),而与SOD、Mg和FIB联合诊断T2DM+T的能力增强,AUC为0.986,敏感度和特异度均达90%以上;与T2DM相比,Sestrin2与病程、PIC和Zn联合诊断T2DM+T的能力最强,AUC为0.907。结论1.人群病例对照研究中,年龄、病程和是否合并高血压是影响血栓发生的重要因素,且具有一定诊断价值,在临床诊疗中应予以重视;2.人群病例对照研究中所选的14项临床实验室指标中仅有HCY、PT、FDP和SOD对2型糖尿病血栓具有一定诊断能力,但诊断价值有限;3.Sestrin2可能与内皮损伤和修复能力相关,且其与年龄、SOD、TC、FIB和Mg元素联合诊断2型糖尿病血栓的价值最高。更多还原

【Abstract】 Objective To analyze the expression level and correlation of Sestrin2 protein,five trace elements(copper,zinc,calcium,magnesium and iron)and thrombus-related markers in type 2 diabetes and type 2 diabetes with thrombus through case-control studies and animal model experiments.At the same time,to find out the diagnostic predictors with high specificity and sensitivity for the researched diseases,so as to provide reference for the prevention and diagnosis of type 2 diabetes thrombosis.Methods 1.Collect information about type 2 diabetes patients who were hospitalized in a tertiary A hospital in Lanzhou from June 2017 to December 2019.According to the type of complications,they were divided into patients with type 2diabetes without vascular complications(group A),and type 2 diabetes with microvascular complications Patients with type 2 diabetes(group B),type 2 diabetes with macrovascular complications(group C),and type 2 diabetes with micro+macrovascular complications(group D);according to whether they are combined with thrombotic diseases,they are divided into type 2 diabetes without thrombosis Group(T2DM group)and type 2 diabetes with thrombosis group(T2DM+T group).Use chi-square test,analysis of variance/non-parametric test to analyze the differences in general data,blood glucose,biochemical and coagulation indicators between groups,use Logistic regression to analyze influencing factors of disease;use receiver operating characteristic curve(ROC)curve analyze the predictive value of influencing factors for the diagnosis of diseases.2.Select 50 SPF adult wistar male rats from the Laboratory Animal Center of Basic Medicine in a tertiary hospital in Lanzhou,and use high-fat diet combined with streptozotocin to establish a T2 DM model(T2DM group),and use carrageenan on this basis Induce and establish a type 2 diabetes mellitus with thrombosis model(T2DM+T group),meantime,establish a control group(Control group).Use an automatic analyzer to detect blood glucose,blood lipids and coagulation indicators in rats,and use double antibody sandwich enzyme-linked immunosorbent assay(ELISA)to detect Sestrin2 protein,endothelin-1(ET-1),vascular endothelial growth factor(VEGF),P-selectin(Pselectin),thrombomodulin(TM),thrombin antithrombin complex(TAT),plasmin-a2 antiplasmin complex(PIC)and tissue-type plasminogen activation Bio-inhibitor complex(t-PAIC).Analysis of variance/non-parametric test was used to compare the differences between the above indicators in each group,Use correlation to analyze the correlation between Sestrin2 and other research indicators.The pathological section of rat tail was made by HE staining method.3.Collect information about T2 DM patients hospitalized from July 2020 to November 2020 in a tertiary hospital in Lanzhou.Divided into type 2 diabetes group(T2DM group)and type 2 diabetes combined with thrombosis group(T2DM+T group),at the same time,patients with other diseases as the control group(Control group).Use automatic analyzer to detect blood glucose,biochemical and coagulation indicators,use double antibody sandwich ELISA method to detect serum Sestrin2,ET-1,VEGF and P-selectin,use chemiluminescence immunoassay to detect plasma TM,TAT,PIC and tPAIC,using atomic absorption method to detect the content of trace five elements in serum.The chi-square test,analysis of variance/non-parametric test were used to analyze the differences of the above indicators among the groups,logistic regression was used to analyze the influencing factors of the disease;ROC curve was used to analyze the predictive value of the influencing factors in the diagnosis of the disease.Results 1.There were statistical differences in age,course of disease,and hypertension between the complication groups of T2 DM and whether T2 DM combined with thrombosis(P<0.05).Compared with those without vascular complications in T2 DM,TG,TC,FIB,FDP and D-Dimer increased,SOD decreased,APTT,PT,and TT shortened;compared with T2 DM group,the T2DM+T group had higher levels of TG,TC,LDL,The expression of HCY,FIB,FDP and D-Dimer increased,HDL and SOD decreased,and PT and TT shortened(P<0.05).Logistics regression analysis found that when group A was used as a reference,the risk factor of group B was only the course of disease(OR=1.086),the risk factor of group C was only age(OR=1.073),and the influencing factors of group D were age,course of disease and The PT and OR values were 1.060,1.072 and 0.691,respectively.Compared with the T2 DM group,the risk factors of the T2DM+T group were age,hypertension,HCY and PT(P<0.05),and the protective factors were FDP and SOD(P<0.05).The ROC analysis of the influencing factors of T2DM+T showed that the diagnostic value in descending order was age,PT,hypertension,SOD,HCY and FDP,and the AUC was 0.742,0.738,0.689,0.681,0.666 and 0.616.2.In animal model experiments,compared with the Control group,FPG,GSP,TG,TC,LDL and HCY were increased in the T2 DM and T2DM+T groups,Sestrin2,HDL and SOD were decreased(P<0.05),APTT,FIB,D-Dimer,TM,TAT,PIC and t-PAIC were different among the three groups(P<0.05),but ET-1,VEGF and P-selectin were no significant difference among groups(P>0.05).Correlation analysis found that Sestrin2 was negatively correlated with FPG,GSP,TG,LDL and HCY,and positively correlated with TM and VEGF(P<0.05).3.In this part of the case-control study,the Control group,T2 DM and T2DM+T group had statistically significant differences in age,course of disease and hypertension(P<0.05).Compared with the Control group,the levels of FPG,GSP,TG,TC,HDL,HCY,FIB,FDP,D-Dimer,Sestrin2,Ca,Cu and Fe were increased in the T2 DM and T2DM+T groups(P<0.05).LDL,SOD,Mg and Zn were decreased(P<0.05),APTT and PT were shortened(P<0.05);the levels of TM,TAT,PIC,t-PAIC and ET-1 in the T2DM+T group were higher than in the T2 DM and Control groups(P<0.05);Logistic regression analysis showed that compared with the Control group,the risk factors for T2 DM were age,TG,PIC and Ca(P<0.001).The risk factors for T2DM+T were age,TC,FIB and Sestrin2,while SOD And Mg were its protective factors(P<0.05).Compared with T2 DM,the risk factors of T2DM+T were the course of disease,PIC and Sestrin2,Zn were the protective factor(P<0.05).ROC analysis showed that Sestrin2 combined with age,TG,PIC and Ca has a higher ability to diagnose T2 DM.The area under the curve AUC=0.953,the sensitivity is 89.9%,and the specificity is 87.8%;Sestrin2 alone is more valuable in diagnosing T2DM+T Low(AUC=0.651),and the combined diagnosis of T2DM+T is enhanced with SOD,Mg and FIB,AUC=0.986,sensitivity and specificity are both over 90%;compared with T2 DM,Sestrin2 is associated with disease course,PIC and Zn The combined diagnosis of T2DM+T has the strongest ability,AUC=0.907.Conclusions 1.In the population case-control study,age,course of disease,and whether or not hypertension is an important factors affecting the occurrence of thrombosis,and has a certain diagnostic value,which should be paid attention to in clinical diagnosis and treatment;2.14 indexs selected in the population case-control study Among the clinical laboratory indicators,only HCY,PT,FDP and SOD have a certain diagnostic ability for type 2 diabetes thrombus,but the diagnostic value is limited;3.Sestrin2 may be related to endothelial damage and repair ability,and it is combined to age,SOD,TC,FIB and Mg elements has the highest value in diagnosing type 2 diabetes thrombus.更多还原