SIRT3通過抑制氧化應激保護糖尿病大鼠心肌缺血再灌注損傷的研究
發(fā)布時間：2018-10-05 21:48
【摘要】：目的:1.在離體心臟灌流模型上研究心肌缺血/再灌注后SIRT3的表達變化以及心肌乙�；阶兓�2.構建糖尿病大鼠模型,研究SIRT3在糖尿病大鼠心肌缺血/再灌注模型中對蛋白乙�；把趸瘧さ挠绊�,同時驗證SIRT3對糖尿病大鼠心肌缺血/再灌注損傷的保護作用。方法:第一部分:10只雄性SD大鼠,2~3月齡,體重(220-250)g,隨機分為對照組(A組,n=5)、實驗組(B組,n=5)。兩組均采用Langendorff心臟離體灌流模型。A組平行灌注20min心臟功能穩(wěn)定后再接著平行灌注60min;B組平行灌注20min心臟功能穩(wěn)定后,全心停止灌注40min再復灌60min制成心肌缺血/再灌注模型。灌注結束后分別用熒光定量PCR(RT-q PCR)和冷凍切片免疫熒光法檢測心肌Sirt3的表達情況及心肌整體乙�；健５诙糠�:24只雄性SD大鼠,2~3月齡,體重(250-300)g,通過腹腔注射鏈脲佐菌素(Streptozotocin,STZ)誘導建立1型糖尿病(Diabetes mellitus,DM)模型,在注射后的72h、7、14、28天分別取大鼠尾靜脈血監(jiān)測血糖,血糖16.70mmol/L,尿糖+++以上者表示造模成功。造模成功后將DM大鼠隨機分為對照組(A組,n=8)、SIRT3抑制劑組(B組,n=8)和SIRT3激活劑組(C組,n=8)。飼養(yǎng)5周后,A組行生理鹽水灌胃4周;B組行尼克酰胺灌胃4周;C組行白藜蘆醇灌胃4周。三組DM大鼠均采用Langendorff心臟離體灌流建模方法,平行灌注20min、全心停止灌注40min、復灌60min,制成心肌缺血/再灌注模型。灌注過程中,心肌缺血前后記錄不同實驗組的心功能變化包括心率(HR)、左心室發(fā)展壓(LVDP)、左室收縮壓最大上升/下降速率(±dp/dtmax)等。分別在平行灌注20 min時和再灌注1h時收集冠脈流出液,運用ELISA方法檢測肌鈣蛋白T(Tn-T)含量。灌注結束后,通過TTC染色測定灌注后心肌梗死面積,分別用冰凍切片免疫熒光和western bolt測定caspase3的表達水平以反映心房細胞凋亡,并測定心肌組織乙�；�,以及SOD活性及丙二醛(MDA)的含量。結果:第一部分:各組灌注結束后心肌Sirt3 m RNA表達量比較,A組是B組的1.7倍,差異有統(tǒng)計學意義(p0.05)。各組免疫熒光檢測心肌組織乙�；较啾容^,B組比A組明顯升高。第二部分:各組心肌缺血/再灌注后的心率、左心室發(fā)展壓、左心室收縮壓最大上升速率及左心室收縮壓最大下降速率比較,差異均有統(tǒng)計學意義(p0.05),且結果顯示C組上述各指標高于A組、B組,B組各指標最低,差異有統(tǒng)計學意義(p0.05)。各組缺血/再灌注后的心肌梗死面積比較,差異有統(tǒng)計學意義,B組明顯大于A組、C組而C組最低,差異有統(tǒng)計學意義(p0.05)。各組心肌缺血/再灌注后用冰凍切片免疫熒光檢測心肌caspase 3蛋白表達量,B組明顯高于A、C組,C組最低,差異有統(tǒng)計學意義(p0.05)。各組心肌缺血/再灌注后用western blot檢測心肌乙�；�,結果表明B組明顯高于A、C組,C組最低,差異有統(tǒng)計學意義(p0.05)。各組缺血/再灌注后心肌組織SOD活性相比較,C組高于A組、B組而B組最低,差異有統(tǒng)計學意義(p0.05)。各組缺血/再灌注后心肌MDA水平相比較,B組明顯高于A、C組,C組最低,差異有統(tǒng)計學意義(p0.05)。結論:1.正常成年大鼠心臟經(jīng)離體缺血/再灌注后SIRT3的表達量有明顯下調(diào),而心肌整體乙�；斤@著升高。2.SIRT3活性增強可以降低糖尿病大鼠心肌缺血/再灌注損傷,其保護糖尿病心肌的機制可能與抑制心肌氧化應激有關。
[Abstract]:Purpose: 1. The changes of the expression of SIRT3 after myocardial ischemia/ reperfusion and the changes of myocardial calcium level were studied in the model of heart perfusion. To study the effect of SIRT3 in myocardial ischemia/ reperfusion model of diabetic rats and to verify the protective effect of SIRT3 on myocardial ischemia/ reperfusion injury in diabetic rats. Methods: The first part: 10 male SD rats, 2 ~ 3 months old and 220-250 g, randomly divided into control group (group A, n = 5), experimental group (group B, n = 5). Langendorff perfusion model was used in both groups. In group A, the heart function was stable after 20min, and then perfused with 60min in parallel. After the cardiac function of 20min in group B was stable, the whole heart stopped perfusion for 40min and then poured for 60min to prepare the model of myocardial ischemia/ reperfusion. At the end of perfusion, the expression of Srt3 and the level of myocardial ischemia were detected by fluorescence quantitative PCR (RT-q PCR) and frozen section immunofluorescence assay, respectively. Part 2: 24 male SD rats, 2-3 months old and 250-300 g body weight (250-300) g were induced by Streptozoocin (STZ), and blood glucose was monitored at 72h, 7, 14 and 28 days after injection. More than + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + DM rats were randomly divided into control group (group A, n = 8), SIRT3 inhibitor group (group B, n = 8) and SIRT3 activator group (group C, n = 8). After 5 weeks of feeding, group A was given normal saline for 4 weeks, and the group B was given intragastric intragastric administration for 4 weeks, and the group C was given intragastric intragastric intragastric administration for 4 weeks. Three groups of DM rats were perfused with Langendorff's heart-to-body perfusion model, perfused in parallel for 20min, the whole heart was stopped for 40min, and the reperfusion was 60min to prepare the model of myocardial ischemia/ reperfusion. The cardiac function changes of different experimental groups were recorded before and after myocardial ischemia, including heart rate (HR), left ventricular development pressure (LVDP), maximum systolic blood pressure rising/ descending rate (Vdp/ dtmax), etc. The levels of troponin T (Tn-T) were measured by ELISA in 20 min parallel perfusion and 1h reperfusion respectively. At the end of perfusion, the area of myocardial infarction was determined by TCTC staining, and the expression level of caspase3 was measured by using frozen section immunofluorescence and western boldt, respectively, to reflect the apoptosis of atrial cells, and to determine the level of myocardial infarction and the levels of SOD and MDA. Results: In the first part, the expression of Sirt3 mRNA in myocardium was compared with that of group B, and group A was 1. 7 times of group B. The difference was statistically significant (P0.05). Compared with group A, group B was significantly higher than group A in group B. In the second part, the heart rate, left ventricular development pressure, maximum left ventricular systolic blood pressure and the maximum descending rate of left ventricular systolic blood pressure in each group were statistically significant (P0.05), and the results showed that the above indexes of group C were higher than group A and group B. The indexes of group B were the lowest and the difference was statistically significant (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). After myocardial ischemia/ reperfusion, the expression of caspase-3 protein was detected by immunofluorescence using frozen section. The group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). The level of myocardial infarction was detected by western blot after myocardial ischemia/ reperfusion. The results showed that group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). Compared with group A, group B and group B, there was significant difference between group B and group B (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). Conclusion: 1. In normal adult rats, the expression level of SIRT3 was down-regulated after ischemia/ reperfusion, and the level of myocardial ischemia/ reperfusion increased significantly. Mechanisms for protecting diabetic myocardium may be associated with inhibition of myocardial oxidative stress.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R542.2
本文編號：2254988
[Abstract]:Purpose: 1. The changes of the expression of SIRT3 after myocardial ischemia/ reperfusion and the changes of myocardial calcium level were studied in the model of heart perfusion. To study the effect of SIRT3 in myocardial ischemia/ reperfusion model of diabetic rats and to verify the protective effect of SIRT3 on myocardial ischemia/ reperfusion injury in diabetic rats. Methods: The first part: 10 male SD rats, 2 ~ 3 months old and 220-250 g, randomly divided into control group (group A, n = 5), experimental group (group B, n = 5). Langendorff perfusion model was used in both groups. In group A, the heart function was stable after 20min, and then perfused with 60min in parallel. After the cardiac function of 20min in group B was stable, the whole heart stopped perfusion for 40min and then poured for 60min to prepare the model of myocardial ischemia/ reperfusion. At the end of perfusion, the expression of Srt3 and the level of myocardial ischemia were detected by fluorescence quantitative PCR (RT-q PCR) and frozen section immunofluorescence assay, respectively. Part 2: 24 male SD rats, 2-3 months old and 250-300 g body weight (250-300) g were induced by Streptozoocin (STZ), and blood glucose was monitored at 72h, 7, 14 and 28 days after injection. More than + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + DM rats were randomly divided into control group (group A, n = 8), SIRT3 inhibitor group (group B, n = 8) and SIRT3 activator group (group C, n = 8). After 5 weeks of feeding, group A was given normal saline for 4 weeks, and the group B was given intragastric intragastric administration for 4 weeks, and the group C was given intragastric intragastric intragastric administration for 4 weeks. Three groups of DM rats were perfused with Langendorff's heart-to-body perfusion model, perfused in parallel for 20min, the whole heart was stopped for 40min, and the reperfusion was 60min to prepare the model of myocardial ischemia/ reperfusion. The cardiac function changes of different experimental groups were recorded before and after myocardial ischemia, including heart rate (HR), left ventricular development pressure (LVDP), maximum systolic blood pressure rising/ descending rate (Vdp/ dtmax), etc. The levels of troponin T (Tn-T) were measured by ELISA in 20 min parallel perfusion and 1h reperfusion respectively. At the end of perfusion, the area of myocardial infarction was determined by TCTC staining, and the expression level of caspase3 was measured by using frozen section immunofluorescence and western boldt, respectively, to reflect the apoptosis of atrial cells, and to determine the level of myocardial infarction and the levels of SOD and MDA. Results: In the first part, the expression of Sirt3 mRNA in myocardium was compared with that of group B, and group A was 1. 7 times of group B. The difference was statistically significant (P0.05). Compared with group A, group B was significantly higher than group A in group B. In the second part, the heart rate, left ventricular development pressure, maximum left ventricular systolic blood pressure and the maximum descending rate of left ventricular systolic blood pressure in each group were statistically significant (P0.05), and the results showed that the above indexes of group C were higher than group A and group B. The indexes of group B were the lowest and the difference was statistically significant (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). After myocardial ischemia/ reperfusion, the expression of caspase-3 protein was detected by immunofluorescence using frozen section. The group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). The level of myocardial infarction was detected by western blot after myocardial ischemia/ reperfusion. The results showed that group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). Compared with group A, group B and group B, there was significant difference between group B and group B (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). Conclusion: 1. In normal adult rats, the expression level of SIRT3 was down-regulated after ischemia/ reperfusion, and the level of myocardial ischemia/ reperfusion increased significantly. Mechanisms for protecting diabetic myocardium may be associated with inhibition of myocardial oxidative stress.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R542.2
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