本文選題：糖尿病心肌病 + GLP-1��； 參考：《天津醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:糖尿病(DM)是由遺傳及環(huán)境在內(nèi)的多種因素共同引起的一組以慢性血糖水平升高為特征的代謝性疾病。心血管疾病是DM的主要并發(fā)癥之一。近年來(lái)研究證實(shí),糖尿病心肌病(DCM)與DM患者心血管疾病的高發(fā)病率和高死亡率密切相關(guān)。而心肌細(xì)胞凋亡是DCM的主要發(fā)病機(jī)制之一。利拉魯肽(LR)是人工合成的胰高血糖素樣肽-1(GLP-l)受體激動(dòng)劑,不僅能發(fā)揮降糖作用,還具有心血管系統(tǒng)保護(hù)作用。微小RNA(miRNA)是一類保守的內(nèi)源性非編碼單鏈小分子RNA,其在心血管疾病的發(fā)生、發(fā)展中扮演著重要角色。miR-27a是目前研究較多的與細(xì)胞凋亡密切相關(guān)的miRNA。而我們的前期研究發(fā)現(xiàn),GLP-1可通過激活A(yù)MPK抑制心肌細(xì)胞miR-27a的表達(dá)。因此本研究旨在探討GLP-1受體激動(dòng)劑LR是否通過抑制心肌細(xì)胞miR-27a的表達(dá),發(fā)揮抗心肌細(xì)胞凋亡的作用,從而為其在防治DCM、降低DM患者心血管疾病死亡率中的應(yīng)用提供理論依據(jù)。方法:1.低糖培養(yǎng)H9c2心肌細(xì)胞,使用終濃度為150pmol和300pmol的miR-27a mimics以及終濃度為150pmol和300pmol的miR-27a inhibitor分別轉(zhuǎn)染細(xì)胞48小時(shí),并設(shè)置正常對(duì)照組,用熒光定量PCR方法檢測(cè)各組miR-27a的表達(dá)水平,從而檢測(cè)轉(zhuǎn)染效率。2.低糖培養(yǎng)H9c2心肌細(xì)胞,將其分為6組:正常對(duì)照組,脂質(zhì)體lipo2000組(lipo2000組),150pmol低濃度轉(zhuǎn)染miR-27a mimics組(miR-27a 150pmol組),300pmol高濃度轉(zhuǎn)染miR-27a mimics組(miR-27a 300pmol組)及轉(zhuǎn)染其各自陰性對(duì)照組(miR-27a N.C 150pmol組、miR-27a N.C 300pmol組)。轉(zhuǎn)染后作用48小時(shí),用TUNEL染色法檢測(cè)細(xì)胞凋亡。3.培養(yǎng)H9c2心肌細(xì)胞,分為低糖組(LG組),高糖組(HG組),高糖+miR-27a mimics組(mimics組),高糖+miR-27a inhibitor組(inhibitor組),高糖+miR-27a mimics N.C組(mimics N.C組),高糖+miR-27a inhibitor N.C組(inhibitor N.C組),高糖+脂質(zhì)體lipo2000組(lipo組),高滲組(HO組),各組轉(zhuǎn)染終濃度均為300pmol,轉(zhuǎn)染后作用48小時(shí),用熒光定量PCR方法檢測(cè)miR-27a的表達(dá)水平,Western Blotting方法檢測(cè)細(xì)胞凋亡相關(guān)蛋白Bax、Bcl-2、caspase-3、c IAP-1、Apaf-1蛋白的表達(dá)水平,并用TUNEL染色法檢測(cè)細(xì)胞凋亡。4.高糖培養(yǎng)H9c2心肌細(xì)胞,分為對(duì)照組,利拉魯肽組(LR組),miR-27a mimics組,LR+miR-27a mimics組,LR+miR-27a mimics N.C組,LR+lipo2000組,利拉魯肽的干預(yù)濃度為1000nmol/L,轉(zhuǎn)染組轉(zhuǎn)染終濃度均為300pmol。作用48小時(shí)后,用熒光定量PCR方法檢測(cè)miR-27a的表達(dá)水平,并用TUNEL染色法檢測(cè)細(xì)胞凋亡。結(jié)果:1.相較于對(duì)照組,低濃度和高濃度轉(zhuǎn)染miR-27a mimics組,其miR-27a的表達(dá)水平均升高(P0.05),且高濃度轉(zhuǎn)染組miR-27a的表達(dá)水平較低濃度轉(zhuǎn)染組明顯升高(P0.05)。與對(duì)照組相比,當(dāng)轉(zhuǎn)染150pmol的miR-27a inhibitor時(shí),miR-27a表達(dá)已被明顯抑制(P0.05),在轉(zhuǎn)染300pmol miR-27a inhibitor時(shí),miR-27a表達(dá)近乎完全被抑制(P0.05)。2.與對(duì)照組相比,轉(zhuǎn)染miR-27a mimics組,其心肌細(xì)胞凋亡指數(shù)呈濃度依賴性升高(P0.05),而lipo2000組、miR-27a N.C 150pmol組、miR-27a N.C 300pmol組的細(xì)胞凋亡指數(shù)較對(duì)照組無(wú)明顯差異(P0.05)。3.在高糖狀態(tài)下,轉(zhuǎn)染miR-27a mimics組,其miR-27a表達(dá)水平及細(xì)胞凋亡指數(shù)顯著增高(P0.05);轉(zhuǎn)染inhibitor組,其miR-27a表達(dá)明顯受到抑制且細(xì)胞凋亡指數(shù)顯著降低(P0.05),而lipo2000組、mimics N.C組和inhibitor N.C組與高糖對(duì)照組相比均未引起miR-27a表達(dá)水平的變化以及細(xì)胞凋亡指數(shù)的變化(P0.05)。而高滲組miR-27a含量與低糖對(duì)照組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4.Western Blotting實(shí)驗(yàn)結(jié)果表明,miR-27a mimics組Bax、caspase-3及Apaf-1蛋白表達(dá)水平升高,而inhibitor組Bax、caspase-3及Apaf-1蛋白表達(dá)水平降低,且兩組之間的變化有統(tǒng)計(jì)學(xué)差異(P0.05)。同時(shí),miR-27a mimics組Bcl-2、c IAP-1蛋白表達(dá)水平降低,而inhibitor組Bcl-2、c IAP-1蛋白表達(dá)水平升高,且兩組之間的變化有統(tǒng)計(jì)學(xué)差異(P0.05)。5.與對(duì)照組比較,單純給予利拉魯肽干預(yù)組,其miR-27a表達(dá)水平及心肌細(xì)胞凋亡指數(shù)明顯降低(P0.05)。而miR-27a mimics組及LR+miR-27a mimics組,其miR-27a表達(dá)水平及心肌細(xì)胞凋亡指數(shù)較對(duì)照組明顯升高(P0.05),且LR+miR-27a mimics組心肌細(xì)胞凋亡指數(shù)較miR-27a mimics組降低(P0.05)。結(jié)論:1.miR-27a可促進(jìn)心肌細(xì)胞的凋亡,且呈濃度依賴性。2.在高糖狀態(tài)下,miR-27a可通過上調(diào)促凋亡蛋白Apaf-1、caspase-3和Bax的表達(dá),下調(diào)凋亡抑制蛋白c IAP-1和Bcl-2的表達(dá)促進(jìn)心肌細(xì)胞的凋亡。3.GLP-1受體激動(dòng)劑通過抑制心肌細(xì)胞miR-27a的表達(dá),發(fā)揮抗心肌細(xì)胞凋亡的作用。
[Abstract]:Objective: diabetes (DM) is a group of metabolic diseases characterized by a variety of factors such as heredity and environment. Cardiovascular disease is one of the major complications of DM. In recent years, it has been confirmed that diabetic cardiomyopathy (DCM) is closely related to the high incidence and high mortality of cardiovascular disease in DM patients. Cardiomyocyte apoptosis is one of the main pathogenesis of DCM. LR is an artificial glucagon like peptide -1 (GLP-l) receptor agonist, which not only plays the hypoglycemic effect, but also has the protective effect of cardiovascular system. Small RNA (miRNA) is a conservative, endogenous, non coding single strand small molecule RNA, which is in the hair of cardiovascular disease. .miR-27a plays an important role in the development of miRNA., which is closely related to apoptosis. Our previous study found that GLP-1 could inhibit the expression of miR-27a in cardiac myocytes by activating AMPK. Therefore, the purpose of this study was to investigate whether the GLP-1 receptor agonist LR has passed the inhibition of the expression of miR-27a in cardiac myocytes and exerts its resistance to the expression of miR-27a. The effect of cardiomyocyte apoptosis provides a theoretical basis for its application in preventing and controlling DCM and reducing the mortality of cardiovascular disease in DM patients. Methods: 1. low sugar cultured H9c2 cardiomyocytes were transfected with 150pmol and 300pmol miR-27a mimics as well as 150pmol and 300pmol miR-27a inhibitor, respectively, for 48 hours, respectively. The normal control group was set up to detect the expression level of miR-27a in each group by fluorescence quantitative PCR, and then the transfection efficiency.2. low sugar cultured H9c2 cardiomyocytes were divided into 6 groups: normal control group, lipo2000 group of liposome (lipo2000 group), 150pmol low concentration transfected miR-27a mimics group (miR-27a 150pmol group), 300pmol high concentration transfection Group s (group miR-27a 300pmol) and its negative control group (group miR-27a N.C 150pmol, miR-27a N.C 300pmol group). After 48 hours of transfection, the apoptotic H9c2 cardiomyocytes were detected by TUNEL staining method, which were divided into low sugar group, high sugar group, high sugar group and high sugar group. Itor group (Group itor), high glucose +miR-27a mimics N.C group (Group mimics N.C), high glucose +miR-27a inhibitor N.C group (inhibitor N.C group), high sugar + liposome lipo2000 group, hypertonic group (Group), the final transfection concentration was all respectively, and the expression level was detected by fluorescein quantitative detection method after 48 hours after transfection. Apoptosis related protein Bax, Bcl-2, Caspase-3, C IAP-1, Apaf-1 protein expression level, and TUNEL staining method was used to detect the apoptotic.4. high sugar culture H9c2 myocardial cells, divided into the control group, the Li La Lu peptide group (LR group), the miR-27a mimics group, the group, the concentration of the intervention group was 1. 000nmol/L, the transfection group was transfected with the final concentration of 300pmol. for 48 hours. The expression level of miR-27a was detected by fluorescence quantitative PCR, and the apoptosis was detected by TUNEL staining. Results: 1. compared with the control group, the expression level of miR-27a in the low concentration and high concentration transfected miR-27a mimics group was all increased (P0.05), and the high concentration transfected group miR-2. The expression level of 7a was significantly higher than that in the low concentration transfection group (P0.05). Compared with the control group, the expression of miR-27a was obviously inhibited when transfected to 150pmol miR-27a inhibitor (P0.05). The miR-27a expression was almost completely suppressed in the transfection of 300pmol miR-27a inhibitor. The death index increased in a concentration dependent manner (P0.05), while the apoptotic index of group lipo2000, miR-27a N.C 150pmol group and miR-27a N.C 300pmol group was not significantly different than that of the control group (P0.05). The expression level and the cell withering index were significantly higher in the miR-27a mimics group. It was obviously inhibited and the apoptosis index decreased significantly (P0.05), while group lipo2000, mimics N.C group and inhibitor N.C group did not cause the change of miR-27a expression and the change of apoptosis index (P0.05), but there was no significant difference between the miR-27a content of the hypertonic group and the low sugar control group (P0.05).4.We. The results of stern Blotting experiment showed that the expression level of Bax, caspase-3 and Apaf-1 protein in group miR-27a mimics increased, while the expression level of Bax, caspase-3 and Apaf-1 protein in inhibitor group decreased, and there was a statistical difference between the two groups. The level of protein expression was increased and the changes between the two groups were statistically different (P0.05).5. was compared with the control group. The expression level of miR-27a and the apoptosis index of myocardial cells were significantly decreased (P0.05). The expression level of miR-27a and the apoptosis index of myocardial cells in the miR-27a mimics group and the LR+miR-27a mimics group were more than that of the control group. The apoptotic index of myocardial cells in group LR+miR-27a mimics was significantly higher than that in group miR-27a mimics (P0.05). Conclusion: 1.miR-27a can promote apoptosis of cardiac myocytes, and the concentration dependent.2. in high glucose state, miR-27a can be regulated by up regulation of apoptotic protein Apaf-1, caspase-3 and Bax, and down regulation of apoptosis inhibitory protein. The expression of Bcl-2 and.3.GLP-1 can promote the apoptosis of cardiomyocytes..3.GLP-1 receptor agonists can inhibit the apoptosis of cardiomyocytes by inhibiting the expression of myocardial cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2

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6 郁U，

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