构建和比较不同方法介导Gα（i2）C-terminal peptide转染乳鼠心肌细胞去迷走神经治疗心房颤动的研究
发布时间：2018-07-15 16:25
【摘要】：目的：构建携带Gαi2ctp的相关质粒和病毒,通过比较电穿孔法、腺病毒转染法和9型腺相关病毒转染法转染Giα2ctp基因至乳鼠心肌细胞,比较不同方法的转染效率、心肌毒性以及对胆碱类药物迷走效应的拮抗作用,探寻Gαi2ctp基因功能表达的最适转导途径。方法：构建重组质粒pDC316-Gαi2ctp-mCMV-EGFP、重组腺病毒rAd-Gαi2ctp-mCMV-EGFP和重组9型腺相关病毒rAW9-Gαi2ctp-IRES-EGFP以及相对应未携带Gαi2ctp的对照组。三种方法转染携带免疫荧光蛋白(EGFP)的Gαi2ctp目的基因至乳鼠心肌细胞,分别找寻最佳电转参数和病毒最佳感染复数(MOI)；比较最佳参数下不同方法转染情况和最大效率；AlamarBlue法测各时间点还原比率评估心肌毒性,western-blot法检测Gαi2ctp蛋白表达情况。最后分别将Gαi2ctp基因组、无基因组和空白对照组,以最适浓度的卡巴胆碱干预各组细胞,通过计数细胞整体搏动频率比较三种方法下Gαi2ctp基因的抗迷走效应差异。结果：成功构建携带Gαi2ctp的真核转染质粒、rAd和rAW9。电穿孔最佳设置为：电压：200V,脉冲时程：2ms,脉冲次数：4次/分,脉冲间隔：60s,细胞浓度：1×106/ml,质粒浓度：5ug/ml,电穿孔12h后细胞平均存活率为57.3%; rAd和rAW9最佳MOI分别为250pfu/cell和1×107vg/cell。三种方法最佳参数下转染效率分别为：质粒组：(54.2±4.8)%于2d、rAd组：100%于1.5d和rAW9组(59.2±4.4)%于4d；第11d转染效率分别为(30.1±6.6)%(12±3.4)%和(36±6.1)%; Western-blot法验证三组Gαi2ctp-EGFP蛋白成功表达；rAW9组在全观察期11d内细胞活性接近空白对照组(P0.05),3d后电穿孔组和rAd组细胞活性开始明显下降且后者较显著(P0.05)。卡巴胆碱以最佳起效浓度1μmol·L-1干预培养3d的各组细胞,发现Gαi2ctp基因组较无基因组具有明显的抗卡巴胆碱迷走效应(P0.05),但Gαi2ctp基因组间比较未见明显差异(P0.05)。结论：三种转染方法皆可有效转染乳鼠心肌细胞,但各自具有不同特点：电穿孔法转染迅速、转染效率尚可,但对细胞具有较大的破坏力；rAd法转染效率最高可达100%、但表达时间较短、心肌毒性较大；AVV9法转染起效较慢但能够稳定长时间表达且生物安全性最好。三种方法皆可有效表达Gαi2ctp并发挥拮抗迷走变时效应且各具特点,其中AVV9法更为稳定和高效。综合三种方法的有效性和毒副作用评价,AVV9法优于电穿孔法和rAd法。该实验为Gαi2ctp基因转染治疗房颤提供了可靠的理论支持和方法学依据。
[Abstract]:Aim: to construct G 伪 i2ctp carrying plasmids and viruses, and to compare the transfection efficiency of GI 伪 2ctp gene into neonatal cardiomyocytes by electroporation, adenoviral transfection and adeno-associated virus 9 transfection. Myocardial toxicity and antagonism to the aberrant effect of choline drugs were used to explore the optimal transduction pathway for the functional expression of G 伪 i2ctp gene. Methods: the recombinant plasmid pDC316-G 伪 i2ctp-mCMV-EGFP, the recombinant adenovirus rAd-G 伪 i2ctp-mCMV-EGFP, the recombinant adeno-associated virus type 9, rAW9-G 伪 i2ctp-IRES-EGFP, and the control group without G 伪 i2ctp were constructed. Three methods were used to transfect G 伪 i2ctp target gene carrying immunofluorescence protein into neonatal rat cardiomyocytes to find out the best electroporation parameters and the optimal infection complex number (moi), to compare the transfection conditions and the maximum efficiency of different methods under the best parameters. The expression of G 伪 i2ctp protein was evaluated by AlamarBlue method and Western blot method. Finally, G 伪 i2ctp genome, no genome and blank control group were treated with the optimal concentration of carbachol. The anti-vagal effects of G 伪 i2ctp gene were compared by counting the overall pulsatile frequency of the cells. Results: the eukaryotic transfection plasmids containing G 伪 i2ctp were constructed successfully. The optimum conditions of electroporation were as follows: voltage: 200V, pulse duration: 2ms, pulse number: 4 / min, pulse interval: 60s, cell concentration: 1 脳 10 6 / ml, plasmid concentration: 5 ugrml. the average survival rate of cells was 57.3% after 12 h electroporation, and the optimal moi of rAd and rAW9 were 250pfu/cell and 1 脳 107 VG / cell, respectively. The transfection efficiency of the three methods was (54.2 卤4.8)% vs (54.2 卤4.8)% at 1.5 d and (59.2 卤4.4)% at 4 days in the rAW9 group and (30.1 卤6.6)% (12 卤3.4)% and (36 卤6.1)% on the 11th day respectively, and the expression of G 伪 -i2ctp-EGFP protein was confirmed by Western-blot. The cell activity in rAW9 group was similar to that in the blank control group (P0.05) within 11 days of the whole observation period. After 3 days, the cell activity of the electroporation group and rAd group began to decrease significantly and the latter group was more significant (P0.05). The G 伪 i2ctp genome had a significant anti-carbachol aberrant effect (P0.05), but there was no significant difference between G 伪 i2ctp genome and G 伪 i2ctp genome (P0.05). Conclusion: all three transfection methods can effectively transfect neonatal rat cardiomyocytes, but each has different characteristics: electroporation is rapid, transfection efficiency is good, but it is destructive to the cells. The transfection efficiency of rAd was up to 100%, but the time of expression was shorter. The transfection of rAd was more toxic, but it could stabilize the expression for a long time and had the best biosafety. All of the three methods can express G 伪 i2ctp effectively and play a role in antagonizing the vagal time effect. AVV9 method is more stable and efficient. To evaluate the effectiveness and toxicity of the three methods, AVV9 is superior to electroporation and rAd. This experiment provides reliable theoretical support and methodological basis for G 伪 i2ctp gene transfection in the treatment of atrial fibrillation.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.75
，
本文编号：2124679
[Abstract]:Aim: to construct G 伪 i2ctp carrying plasmids and viruses, and to compare the transfection efficiency of GI 伪 2ctp gene into neonatal cardiomyocytes by electroporation, adenoviral transfection and adeno-associated virus 9 transfection. Myocardial toxicity and antagonism to the aberrant effect of choline drugs were used to explore the optimal transduction pathway for the functional expression of G 伪 i2ctp gene. Methods: the recombinant plasmid pDC316-G 伪 i2ctp-mCMV-EGFP, the recombinant adenovirus rAd-G 伪 i2ctp-mCMV-EGFP, the recombinant adeno-associated virus type 9, rAW9-G 伪 i2ctp-IRES-EGFP, and the control group without G 伪 i2ctp were constructed. Three methods were used to transfect G 伪 i2ctp target gene carrying immunofluorescence protein into neonatal rat cardiomyocytes to find out the best electroporation parameters and the optimal infection complex number (moi), to compare the transfection conditions and the maximum efficiency of different methods under the best parameters. The expression of G 伪 i2ctp protein was evaluated by AlamarBlue method and Western blot method. Finally, G 伪 i2ctp genome, no genome and blank control group were treated with the optimal concentration of carbachol. The anti-vagal effects of G 伪 i2ctp gene were compared by counting the overall pulsatile frequency of the cells. Results: the eukaryotic transfection plasmids containing G 伪 i2ctp were constructed successfully. The optimum conditions of electroporation were as follows: voltage: 200V, pulse duration: 2ms, pulse number: 4 / min, pulse interval: 60s, cell concentration: 1 脳 10 6 / ml, plasmid concentration: 5 ugrml. the average survival rate of cells was 57.3% after 12 h electroporation, and the optimal moi of rAd and rAW9 were 250pfu/cell and 1 脳 107 VG / cell, respectively. The transfection efficiency of the three methods was (54.2 卤4.8)% vs (54.2 卤4.8)% at 1.5 d and (59.2 卤4.4)% at 4 days in the rAW9 group and (30.1 卤6.6)% (12 卤3.4)% and (36 卤6.1)% on the 11th day respectively, and the expression of G 伪 -i2ctp-EGFP protein was confirmed by Western-blot. The cell activity in rAW9 group was similar to that in the blank control group (P0.05) within 11 days of the whole observation period. After 3 days, the cell activity of the electroporation group and rAd group began to decrease significantly and the latter group was more significant (P0.05). The G 伪 i2ctp genome had a significant anti-carbachol aberrant effect (P0.05), but there was no significant difference between G 伪 i2ctp genome and G 伪 i2ctp genome (P0.05). Conclusion: all three transfection methods can effectively transfect neonatal rat cardiomyocytes, but each has different characteristics: electroporation is rapid, transfection efficiency is good, but it is destructive to the cells. The transfection efficiency of rAd was up to 100%, but the time of expression was shorter. The transfection of rAd was more toxic, but it could stabilize the expression for a long time and had the best biosafety. All of the three methods can express G 伪 i2ctp effectively and play a role in antagonizing the vagal time effect. AVV9 method is more stable and efficient. To evaluate the effectiveness and toxicity of the three methods, AVV9 is superior to electroporation and rAd. This experiment provides reliable theoretical support and methodological basis for G 伪 i2ctp gene transfection in the treatment of atrial fibrillation.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.75
，
本文编号：2124679
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