本文選題：急性心肌梗死 + miR-486��； 參考：《鄭州大學》2016年博士論文

【摘要】：背景和目的心血管疾病因其逐年增長的發(fā)病率和死亡率,一直以來受到各界廣泛關注。盡管目前已有廣泛可用的診斷治療途徑,心血管疾病的流行、死亡率和治療花費在發(fā)達國家及發(fā)展中國家仍呈上升趨勢。急性心肌梗死(acute myocardial infarction,AMI)是世界范圍內最常見的心血管疾病之一,為有效控制AMI的發(fā)生和發(fā)展,尋求新的診斷和治療的指標和方法十分必要。近年以來,研究表明mi RNA在疾病發(fā)生發(fā)展中可通過靶向調節(jié)其下游靶基因發(fā)揮作用。在多個研究領域都得到廣泛重視,包括腫瘤、心血管疾病等等。已發(fā)現多種mi RNA在急性心肌梗死患者血液和血漿中發(fā)生改變,如mi R-1、mi R-133a,mi R-208b,mi R-499,和mi R-328,提示循環(huán)mi RNA在AMI早期診斷中具有價值。本實驗的目的是篩選AMI發(fā)生時血液循環(huán)中異常表達的mi RNA;分析異常表達mi RNA表達水平變化,并判斷其作為輔助診斷的價值;初步探索AMI發(fā)生時異常表達mi RNA發(fā)揮調控作用的可能途徑。第一部分急性心肌梗死發(fā)生時血液循環(huán)中異常表達mi RNA篩選和鑒定方法1.收集30例AMI患者和30例健康成人志愿者的血漿樣本。2.采用Agilent mi RNA芯片分別檢測隨機選取3例AMI患者和3例健康成年志愿者血漿中mi RNA的表達情況,篩選有顯著差異表達的mi RNA。3.應用q RT-PCR的方法檢測30例AMI患者和30例健康成人志愿者的血漿樣本中mi RNA芯片篩選出的有差異表達的mi RNA的表達情況,對mi RNA芯片的結果進行驗證。4.應用統計學軟件SPSS21.0對實驗數據進行統計學分析,檢驗標準α=0.05。結果1.通過mi RNA芯片檢測出共有36種mi RNA在AMI患者血漿中出現異常表達,其中有23種mi RNA表達水平顯著升高(P0.05),13種mi RNA表達水平明顯下降(P0.05)。2.q RT-PCR驗證的結果表明30例AMI患者血漿中除mi R-361-5p,mi R-182-5p,mi R-497-5p,mi R-20a-5p四種mi RNA表達水平與正常對照組相比無顯著性差異(P0.05),其余32種mi RNA表達水平較正常對照組相比出現顯著性變化(P0.05),其中上調的mi RNA有20種,下調的mi RNA有12種。第二部分AMI發(fā)生時血液循環(huán)中mi R-486,mi R-150和mi R-26a表達變化分析及作為輔助診斷的價值方法1.收集110例AMI和110例健康成人志愿者的血漿樣本,110例AMI患者中分別有45例NSTEMI患者和65例STEMI患者,采集入院之后發(fā)病4h以內,24h,48h,72h的血漿樣本。2.q RT-PCR法檢測mi R-486,mi R-150和mi R-26a在AMI各類型各時間段血漿樣本中的表達水平。3.應用受試者工作特性曲線(ROC)反映靈敏度與特異度,應用ROC曲線下面積(AUCROC)比較mi R-486,mi R-150和mi R-26a對AMI的輔助診斷價值。4.應用統計學軟件SPSS21.0處理所有數據,檢驗標準α=0.05。結果1.AMI發(fā)作4h以內,mi R-486和mi R-150表達量分別為9.655±1.427和7.420±0.911,與正常對照組相比表達水平均顯著上調(P0.05);mi R-26a表達量為0.092±0.104,與正常對照組相比表達水平顯著下調(P0.05)。2.AMI發(fā)作4h以內,24h,48h,72h,mi R-486和mi R-150表達水平均隨時間延長逐漸降低,至72h時與正常對照組無顯著性差異(P0.05);mi R-26a表達水平隨時間延長逐漸升高,至72h時與正常對照組無顯著性差異(P0.05)。3.AMI發(fā)作4h以內,mi R-486,mi R-150和mi R-26a的AUC曲線下面積分別為0.731(P0.05),0.678(P0.05),0.763(P0.05),表明這三種mi RNA的表達水平檢測對檢測AMI的發(fā)生有一定的價值。這三種mi RNA聯合檢測的AUC曲線下面積為0.792(P0.05),表明具有更高的診斷價值。4.AMI發(fā)作4h以內,mi R-486,mi R-150和mi R-26a表達在AMI的兩種類型STEMI和NSTEMI中存在顯著性差異。mi R-486,mi R-150和mi R-26a在STEMI中的AUC曲線下面積分別為0.695(P0.05),0.639(P0.05),0.750(P0.05),表明這三種mi RNA的表達水平檢測對檢測STEMI的發(fā)生有一定的價值;mi R-486,mi R-150和mi R-26a在NSTEMI中的AUC曲線下面積分別為0.782(P0.05),0.734(P0.05),0.782(P0.05),表明這三種mi RNA的表達水平檢測對檢測NSTEMI的發(fā)生有一定的價值;在STEMI和NSTEMI時這三種mi RNA聯合檢測的AUC曲線下面積分別為0.765(P0.05),0.833(P0.05),說明三者聯合檢測具有更高的靈敏度和特異度,且在NSTEMI時具有更高的診斷價值。第三部分mi R-486,mi R-150在AMI發(fā)生時的作用機制的初步探索方法1.通過Target Scan和mi Randa尋找可能與mi R-486,mi R-150相關的靶基因2.用Overlap PCR的方法擴增突變型CDKN1B 3’UTR和ALDH2 3’UTR片段,用普通PCR來擴增野生型CDKN1B 3’UTR和ALDH2 3’UTR片段,將擴增出來的片段與pmir GLO雙粘載體進行重組,從而分別構建野生型和突變型CDKN1B 3’UTR和ALDH2 3’UTR的重組雙熒光素酶報告載體。3.將野生型和突變型CDKN1B 3’UTR重組報告載體分別與mi R-150 mimics或者mi R-150 scramble共同轉染到HEK 293T細胞中;將野生型和突變型ALDH2 3’UTR重組報告載體分別與mi R-486 mimics或者mi R-486 scramble共同轉染到HEK 293T細胞中。通過雙熒光素酶報告實驗驗證CDKN1B是否為mi R-150的靶基因,ALDH2是否為mi R-486的靶基因。4.應用統計學軟件SPSS21.0處理所有數據,檢驗標準α=0.05。結果1.生物信息學檢測發(fā)現基因CDKN1B的3’UTR區(qū)可能是mi R-150的一個下游直接作用靶點,ALDH2的3’UTR區(qū)可能是mi R-486的一個下游直接作用靶點。2.經酶切驗證,PCR驗證和測序結果驗證,成功構建了野生型和突變型CDKN1B 3’UTR和ALDH2 3’UTR的重組雙熒光素酶報告載體。3.雙熒光素報告酶實驗顯示,mi R-150 mimics和WT-pmir GLO-CDKN1B共轉染組熒光素酶活性為0.54±0.065,顯著低于其他三個共轉染組(P0.05),表明CDKN1B是mi R-150作用的靶基因;mi R-486 mimics和WT-pmir GLOALDH2共轉染組熒光素酶活性為0.62±0.045,顯著低于其他三個共轉染組(P0.05),表明ALDH2是mi R-486作用的靶基因。結論1.急性心肌梗死患者循環(huán)血液中,存在32種mi RNA出現表達異常,20種mi RNA表達明顯上調,12種mi RNA表達明顯下調。2.急性心肌梗死發(fā)作4h以內,mi R-486和mi R-150表達水平明顯上調,mi R-26a表達水平明顯下調,且隨時間延長變化趨勢越不明顯。mi R-486,mi R-150和mi R-26a聯合檢測有望成為急性心肌梗死的輔助診斷指標,尤其適用于NSTEMI的輔助診斷。3.mi R-150和mi R-486分別負向調節(jié)CDKN1B和ALDH2的表達,可能在急性心肌梗死發(fā)生發(fā)展中發(fā)揮一定作用。
[Abstract]:Background and objective cardiovascular disease has been widely concerned because of its increasing incidence and mortality year by year. Despite the widespread available diagnostic methods, the prevalence of cardiovascular disease, mortality and treatment costs are still rising in developed and developing countries. Acute myocardial infarction (acute myocardial) Infarction, AMI) is one of the most common cardiovascular diseases in the world. In order to effectively control the occurrence and development of AMI, it is necessary to seek new diagnostic and therapeutic targets and methods. In recent years, studies have shown that MI RNA can play a role in regulating the target gene by targeting the target gene in the development of the disease. In many fields, it has been obtained. A variety of MI RNA have been found in the blood and plasma of patients with acute myocardial infarction, such as mi R-1, MI R-133a, MI R-208b, MI R-499, and MI R-328. Mi RNA; analyze abnormal expression of MI RNA expression level, and judge its value as auxiliary diagnosis; preliminary explore the possible way of abnormal expression of MI RNA when AMI occurs. In part 1, abnormal expression of MI RNA screening and identification in blood circulation during the occurrence of acute myocardial infarction 1., 30 cases of AMI patients and 30 healthy adult cases were collected. The plasma sample.2. of human volunteers was selected by Agilent mi RNA chip to detect the expression of MI RNA in plasma of 3 AMI patients and 3 healthy adult volunteers, and to screen the MI RNA.3. application Q RT-PCR method with significant differential expression for the detection of 30 cases of AMI patients and 30 healthy adult volunteers. The expression of the differentially expressed mi RNA, the results of the MI RNA chip were verified by the.4. application statistics software SPSS21.0 for statistical analysis of the experimental data. The test standard alpha =0.05. result 1. detected a total of 36 mi RNA in the AMI patients' plasma by Mi RNA chip, and there were 23 kinds of expression levels. (P0.05), the expression level of 13 kinds of MI RNA decreased significantly (P0.05).2.q RT-PCR verification. The results showed that there were no significant differences in the levels of MI R-361-5p, MI R-182-5p, MI, and four kinds of expressions in the plasma of the AMI patients compared with the normal control group, and the other 32 kinds of expressions were compared with the normal control group. Significant changes (P0.05), among which there were 20 kinds of MI RNA, 12 down regulated mi RNA, MI R-486, MI R-150 and MI R-26a in the blood circulation during the occurrence of AMI, and the value method of auxiliary diagnosis. 1. the plasma samples of 110 cases and 110 healthy adult volunteers were collected, and 45 of 110 patients were respectively. Patients and 65 patients with STEMI were collected after admission to 4h, 24h, 48h, and 72h plasma samples were detected by.2.q RT-PCR method for the detection of MI R-486, MI R-150 and Mi expressions in each type of plasma samples. The auxiliary diagnostic value of R-486, MI R-150 and MI R-26a for AMI.4. applied statistics software SPSS21.0 to process all the data, and to test the standard alpha =0.05. results within 1.AMI 4h, 9.655 + 1.427 and 7.420 + 0.911 respectively, compared with the normal control group. The expression level of 24h, 48h, 72h, MI R-486 and MI R-150 decreased gradually with time, and there was no significant difference between the normal control group and the normal control group. There was no significant difference between the normal control group and the normal control group. There was no significant difference between the normal control group and the normal control group. The expression level of 24h, 48h, 72h, MI R-486 and MI R-150 increased gradually. The area under the AUC curve of MI R-486, MI R-150 and MI R-26a is 0.731 (P0.05), 0.678 (P0.05) and 0.763 (0.763), indicating that the three kinds of expression levels have a certain value for the occurrence of the detection. The area under the three combined detection curves is 0.792, indicating a higher level of 4H. The diagnostic value of.4.AMI is within 4h, MI R-486, MI R-150 and MI R-26a have significant differences in the two types of STEMI and NSTEMI AMI. The area under the curve is 0.695, 0.639, 0.750. The values of MI R-486, MI R-150 and MI R-26a under AUC curves in NSTEMI are 0.782 (P0.05), 0.734 (P0.05), and 0.782 (P0.05). P0.05), 0.833 (P0.05), indicating that the combined detection of the three has a higher sensitivity and specificity, and has a higher diagnostic value at NSTEMI. Third part of the MI R-486, MI R-150 is a preliminary exploration of the mechanism of the occurrence of AMI. 1. through Target Scan and MI Randa. The mutant CDKN1B 3 'UTR and ALDH2 3' UTR fragment were amplified by R, and the wild type CDKN1B 3 'UTR and ALDH2 3' UTR fragments were amplified by ordinary PCR, and the amplified fragments were reorganized with the pmir GLO double adhered carrier to construct the recombinant double luciferase reporter vector of wild type and mutant 3 '3' and 3 'respectively. The wild type and mutant CDKN1B 3 'UTR recombinant report vectors were transfected to HEK 293T cells, respectively, with MI R-150 mimics or MI R-150 scramble, respectively. The experiment verifies whether CDKN1B is the target gene of MI R-150, and whether ALDH2 is the target gene of MI R-486.4. applied statistics software SPSS21.0 to deal with all data and test the standard alpha =0.05. result 1. bioinformatics detection found that the 3 'UTR region of the gene CDKN1B may be a downstream direct target of MI. A direct targeting target of downstream.2. was verified by enzyme digestion and verified by PCR and sequencing results. The recombinant double luciferase reporter of wild type and mutant CDKN1B 3 'UTR and ALDH2 3' UTR was successfully constructed,.3. double luciferase reporter experiment showed that the luciferase activity of MI R-150 mimics and WT-pmir GLO-CDKN1B co transfected group was 0.54 + 0.. 065, significantly lower than the other three co transfection groups (P0.05), indicating that CDKN1B was the target gene for MI R-150, and the luciferase activity of MI R-486 mimics and WT-pmir GLOALDH2 co transfection group was 0.62 + 0.045, significantly lower than the other three co transfected groups (P0.05), indicating that ALDH2 was the target gene of MI. The expression of 32 kinds of MI RNA was abnormal, the expression of the 20 mi RNA was obviously up-regulated, the expression of the 12 mi RNA was obviously down regulated within 4H of the acute myocardial infarction, and the expression level of MI R-486 and MI R-150 was obviously down regulated. It is expected to be an auxiliary diagnostic indicator for acute myocardial infarction, especially for the auxiliary diagnosis of NSTEMI,.3.mi R-150 and MI R-486, which can negatively regulate the expression of CDKN1B and ALDH2, and may play a role in the development of acute myocardial infarction.

【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R542.22

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相關期刊論文 前1條

1 Hua Zhou;Xiao-yan He;Shao-wei Zhuang;Juan Wang;Yan Lai;Wei-gang Qi;Yi-an Yao;Xue-bo Liu;;Clinical and procedural predictors of no-ref low in patients with acute myocardial infarction after primary percutaneous coronary intervention[J];World Journal of Emergency Medicine;2014年02期

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本文編號：1839881