AAV9-shRNA-ADRB1-ZsGreen转染SHR心肌下调β1-AR表达对美托洛尔降压疗效的影响

发布时间：2018-04-26 16:42

本文选题：β1-肾上腺素能受体 + 心肌　；参考：《中南大学》2014年硕士论文

【摘要】：目的探讨SHR心肌β1-AR表达下调对美托洛尔降压效应的影响。方法1.病毒载体包装及鉴定实验：对本课题组前期成功构建及筛选出的pAAV-ZsGreen-ADRB1-shRNA重组质粒进行酶切及测序鉴定,采用三质粒共转染法进行AAV9-shRNA-ADRB1-ZsGreen重组腺相关病毒的包装,并进行病毒纯化、滴度测定及体外感染293细胞活性实验。 2.SHR心肌转染实验：将36只SHR随机分为3组：AAV9-shRNA-ADRB1-ZsGreen注射组、AAV9-CMV-ADRB1-ZsGreen注射组(阴性对照组)、假手术,每组12只。病毒载体采用心包腔注射法转染SHR心肌,4周后处死大鼠,取心肌组织。采用Real-time RT-PCR法检测各组SHR心肌组织中β1-AR基因mRNA表达；采用免疫组化、Western-blot法检测β1-AR蛋白的表达。 3.美托洛尔干预实验：上述3组SHR每组同时随机分为两个亚组,每组6只,分别给予美托洛尔和生理盐水灌胃。每周监测SHR血压水平变化,连续干预4周后比较各组SHR血压变化情况。结果1.病毒载体包装及鉴定：(1)酶切及测序鉴定结果表明前期构建的pAAV-ZsGreen-ADRB1-shRNA重组质粒为正确克隆；(2)病毒滴度测定结果表明纯化后的AAV9-shRNA-ADRB1-ZsGreen滴度达1.5x1012vg/mL;(3)病毒载体感染293细胞效率可达95%以上。 2.SHR心肌转染的效应：(1) Real-time RT-PCR结果显示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR mRNA表达明显低于阴性对照组(0.4142±0.3399vs.1.0933±0.5853,P0.05)；(2)免疫组化结果显示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR蛋白表达明显低于阴性对照组及假手术组(50.02±4.44vs.80.05±3.72vs.79.30±2.79, P0.05);(3) Western blot结果显示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR蛋白表达明显低于阴性对照组(0.5156±0.1199vs.0.8333±0.1728, P0.05)。 3.SHR心肌β1-AR表达下调对血压及美托洛尔疗效的影响：干预4周后,(1) AAV9-shRNA-ADRB1-ZsGreen+美托洛尔组、阴性对照+美托洛尔组、假手术+美托洛尔组收缩压明显低于阴性对照+生理盐水组、假手术+生理盐水组(174.8±3.9mmHg vs.168.3±5.3mmHg vs.167.2±5.1mmHg vs.199.4±3.1mmHg vs.198.3±4.5mmHg, P0.05);(2) AAV9-shRNA-ADRB1-ZsGreen+生理盐水组收缩压明显低于假手术+生理盐水组(178.1±6.3mmHg vs.198.3±4.5mmHg, P0.05);(3) AAV9-shRNA-ADRB1-ZsGreen+美托洛尔组收缩压降低幅度明显低于阴性对照组+美托洛尔组和假手术+美托洛尔组(22.84±1.72mmHg vs.28.78±2.72mmHg vs.30.16±5.71mmHg, P0.05);(4) AAV9-shRNA-ADRB1-ZsGreen+美托洛尔组与AAV9-shRNA-ADRB1-ZsGreen+生理盐水组降压幅度比较无显著性差异(22.84±1.72mmHg vs.18.84±6.55mmHg, P0.05)。结论1.成功包装了高纯度、高滴度的AAV9-shRNA-ADRB1-ZsGreen病毒载体。 2. AAV9-shRNA-ADRB1-ZsGreen病毒转染SHR心肌可显著降低心肌组织中β1-AR表达。 3.心肌β1-AR表达下调的SHR其收缩压及对美托洛尔的敏感性均显著降低。
[Abstract]:Objective to investigate the effect of down-regulation of myocardial 尾 1-AR expression in SHR on the hypotensive effect of metoprolol. Method 1. Virus vector packaging and identification experiment: the pAAV-ZsGreen-ADRB1-shRNA recombinant plasmid successfully constructed and screened by our team was digested and sequenced, and the AAV9-shRNA-ADRB1-ZsGreen recombinant adeno-associated virus was packaged by three plasmids cotransfection, and the virus was purified. The titer of 293 cells was measured and the activity of 293 cells was tested in vitro. 2.SHR myocardial transfection test: 36 SHR were randomly divided into 3 groups: AAV9-shRNA-ADRB1-ZsGreen injection group and AAV9-CMV-ADRB1-ZsGreen injection group (negative control group, sham-operated group, 12 rats in each group). The virus vector was injected into SHR myocardium by pericardial injection for 4 weeks, then the rats were killed and myocardial tissue was taken. The expression of 尾 1-AR gene mRNA was detected by Real-time RT-PCR method and 尾 1-AR protein expression was detected by immunohistochemistry and Western-blot. 3. Metoprolol intervention experiment: each of the three groups of SHR was randomly divided into two subgroups, 6 rats in each group were given metoprolol and normal saline respectively. The changes of blood pressure of SHR were monitored weekly and the changes of blood pressure of SHR were compared after 4 weeks of continuous intervention. Result 1. Packaging and identification of virus vector 1) digestion and sequencing analysis showed that the recombinant pAAV-ZsGreen-ADRB1-shRNA plasmid constructed in the previous period was the correct clone and the virus titer was determined. The titer of purified AAV9-shRNA-ADRB1-ZsGreen was 1.5 x 1012vg / mLmLt3) the efficiency of the virus vector infected 293 cells was more than 95%. Effect of 2.SHR on Myocardial transfection: Real-time RT-PCR results showed that the expression of 尾 1-AR mRNA in AAV9-shRNA-ADRB1-ZsGreen group was significantly lower than that in negative control group (0.4142 卤0.3399vs.1.0933 卤0.5853 P0.05P0.05). Immunohistochemical results showed that the expression of 尾 1-AR protein in AAV9-shRNA-ADRB1-ZsGreen group was significantly lower than that in negative control group and sham-operated group (50.02 卤4.44vs.80.05 卤2.79, P0.050.53) Western blot results showed that AAV9-shRNA-ADRB1-ZsGreen was significantly lower than that in negative control group and sham-operation group. The results showed that the expression of 尾 1-AR protein in AAV9-shRNA-ADRB1-ZsGreen was significantly lower than that in negative control group (0.5156 卤0.1199vs.0.8333 卤0.1728, P 0.05). Effect of down-regulation of myocardial 尾 1-AR expression of 3.SHR on blood pressure and therapeutic effect of metoprolol: after 4 weeks of intervention, the systolic blood pressure of AAV9-shRNA-ADRB1-ZsGreen metoprolol group, negative control metoprolol group, sham-operated metoprolol group was significantly lower than that of negative control group. SBP in the sham saline group (174.8 卤3.9mmHg vs.168.3 卤5.3mmHg vs.167.2 卤5.1mmHg vs.199.4 卤4.5mmHg, P0.05Hg2) SBP in the AAV9-shRNA-ADRB1-ZsGreen saline group was significantly lower than that in the sham-operation saline group (178.1 卤6.3mmHg vs.198.3 卤4.5mmHg, P0.05Hg) AAV9-shRNA-ADRB1-ZsGreen metoprolol group was significantly lower than that in the negative control group and pseudoprolol group. In the metoprolol group (22.84 卤1.72mmHg vs.28.78 卤5.71mmHg, P0.05Hg), there was no significant difference between the AAV9-shRNA-ADRB1-ZsGreen metoprolol group and the AAV9-shRNA-ADRB1-ZsGreen saline group in lowering blood pressure (22.84 卤1.72mmHg vs.18.84 卤6.55mmHg, P 0.05). Conclusion 1. The AAV9-shRNA-ADRB1-ZsGreen virus vector with high purity and high titer was successfully packaged. 2. Transfection of AAV9-shRNA-ADRB1-ZsGreen virus into SHR myocardium could significantly reduce the expression of 尾 1-AR in myocardium. 3. The systolic blood pressure and sensitivity to metoprolol decreased significantly in SHR with down-regulation of myocardial 尾 1-AR expression.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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