本文選題：β1-腎上腺素能受體 + 心肌�。� 參考：《中南大學》2014年碩士論文

【摘要】：目的探討SHR心肌β1-AR表達下調(diào)對美托洛爾降壓效應(yīng)的影響。 方法1.病毒載體包裝及鑒定實驗：對本課題組前期成功構(gòu)建及篩選出的pAAV-ZsGreen-ADRB1-shRNA重組質(zhì)粒進行酶切及測序鑒定,采用三質(zhì)粒共轉(zhuǎn)染法進行AAV9-shRNA-ADRB1-ZsGreen重組腺相關(guān)病毒的包裝,并進行病毒純化、滴度測定及體外感染293細胞活性實驗。 2.SHR心肌轉(zhuǎn)染實驗：將36只SHR隨機分為3組：AAV9-shRNA-ADRB1-ZsGreen注射組、AAV9-CMV-ADRB1-ZsGreen注射組(陰性對照組)、假手術(shù),每組12只。病毒載體采用心包腔注射法轉(zhuǎn)染SHR心肌,4周后處死大鼠,取心肌組織。采用Real-time RT-PCR法檢測各組SHR心肌組織中β1-AR基因mRNA表達；采用免疫組化、Western-blot法檢測β1-AR蛋白的表達。 3.美托洛爾干預(yù)實驗：上述3組SHR每組同時隨機分為兩個亞組,每組6只,分別給予美托洛爾和生理鹽水灌胃。每周監(jiān)測SHR血壓水平變化,連續(xù)干預(yù)4周后比較各組SHR血壓變化情況。 結(jié)果1.病毒載體包裝及鑒定：(1)酶切及測序鑒定結(jié)果表明前期構(gòu)建的pAAV-ZsGreen-ADRB1-shRNA重組質(zhì)粒為正確克隆；(2)病毒滴度測定結(jié)果表明純化后的AAV9-shRNA-ADRB1-ZsGreen滴度達1.5x1012vg/mL;(3)病毒載體感染293細胞效率可達95%以上。 2.SHR心肌轉(zhuǎn)染的效應(yīng)：(1) Real-time RT-PCR結(jié)果顯示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR mRNA表達明顯低于陰性對照組(0.4142±0.3399vs.1.0933±0.5853,P0.05)；(2)免疫組化結(jié)果顯示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR蛋白表達明顯低于陰性對照組及假手術(shù)組(50.02±4.44vs.80.05±3.72vs.79.30±2.79, P0.05);(3) Western blot結(jié)果顯示,AAV9-shRNA-ADRB1-ZsGreen注射后β1-AR蛋白表達明顯低于陰性對照組(0.5156±0.1199vs.0.8333±0.1728, P0.05)。 3.SHR心肌β1-AR表達下調(diào)對血壓及美托洛爾療效的影響：干預(yù)4周后,(1) AAV9-shRNA-ADRB1-ZsGreen+美托洛爾組、陰性對照+美托洛爾組、假手術(shù)+美托洛爾組收縮壓明顯低于陰性對照+生理鹽水組、假手術(shù)+生理鹽水組(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+生理鹽水組收縮壓明顯低于假手術(shù)+生理鹽水組(178.1±6.3mmHg vs.198.3±4.5mmHg, P0.05);(3) AAV9-shRNA-ADRB1-ZsGreen+美托洛爾組收縮壓降低幅度明顯低于陰性對照組+美托洛爾組和假手術(shù)+美托洛爾組(22.84±1.72mmHg vs.28.78±2.72mmHg vs.30.16±5.71mmHg, P0.05);(4) AAV9-shRNA-ADRB1-ZsGreen+美托洛爾組與AAV9-shRNA-ADRB1-ZsGreen+生理鹽水組降壓幅度比較無顯著性差異(22.84±1.72mmHg vs.18.84±6.55mmHg, P0.05)。 結(jié)論1.成功包裝了高純度、高滴度的AAV9-shRNA-ADRB1-ZsGreen病毒載體。 2. AAV9-shRNA-ADRB1-ZsGreen病毒轉(zhuǎn)染SHR心肌可顯著降低心肌組織中β1-AR表達。 3.心肌β1-AR表達下調(diào)的SHR其收縮壓及對美托洛爾的敏感性均顯著降低。
[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 鹵0.3399vs.1.0933 鹵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 鹵4.44vs.80.05 鹵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 鹵0.1199vs.0.8333 鹵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 鹵3.9mmHg vs.168.3 鹵5.3mmHg vs.167.2 鹵5.1mmHg vs.199.4 鹵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 鹵6.3mmHg vs.198.3 鹵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 鹵1.72mmHg vs.28.78 鹵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 鹵1.72mmHg vs.18.84 鹵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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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