【摘要】：目的：構建攜帶Gαi2ctp的相關質粒和病毒,通過比較電穿孔法、腺病毒轉染法和9型腺相關病毒轉染法轉染Giα2ctp基因至乳鼠心肌細胞,比較不同方法的轉染效率、心肌毒性以及對膽堿類藥物迷走效應的拮抗作用,探尋Gαi2ctp基因功能表達的最適轉導途徑。方法：構建重組質粒pDC316-Gαi2ctp-mCMV-EGFP、重組腺病毒rAd-Gαi2ctp-mCMV-EGFP和重組9型腺相關病毒rAW9-Gαi2ctp-IRES-EGFP以及相對應未攜帶Gαi2ctp的對照組。三種方法轉染攜帶免疫熒光蛋白(EGFP)的Gαi2ctp目的基因至乳鼠心肌細胞,分別找尋最佳電轉參數(shù)和病毒最佳感染復數(shù)(MOI)；比較最佳參數(shù)下不同方法轉染情況和最大效率；AlamarBlue法測各時間點還原比率評估心肌毒性,western-blot法檢測Gαi2ctp蛋白表達情況。最后分別將Gαi2ctp基因組、無基因組和空白對照組,以最適濃度的卡巴膽堿干預各組細胞,通過計數(shù)細胞整體搏動頻率比較三種方法下Gαi2ctp基因的抗迷走效應差異。結果：成功構建攜帶Gαi2ctp的真核轉染質粒、rAd和rAW9。電穿孔最佳設置為：電壓：200V,脈沖時程：2ms,脈沖次數(shù)：4次/分,脈沖間隔：60s,細胞濃度：1×106/ml,質粒濃度：5ug/ml,電穿孔12h后細胞平均存活率為57.3%; rAd和rAW9最佳MOI分別為250pfu/cell和1×107vg/cell。三種方法最佳參數(shù)下轉染效率分別為：質粒組：(54.2±4.8)%于2d、rAd組：100%于1.5d和rAW9組(59.2±4.4)%于4d；第11d轉染效率分別為(30.1±6.6)%(12±3.4)%和(36±6.1)%; Western-blot法驗證三組Gαi2ctp-EGFP蛋白成功表達；rAW9組在全觀察期11d內細胞活性接近空白對照組(P0.05),3d后電穿孔組和rAd組細胞活性開始明顯下降且后者較顯著(P0.05)�？ò湍憠A以最佳起效濃度1μmol·L-1干預培養(yǎng)3d的各組細胞,發(fā)現(xiàn)Gαi2ctp基因組較無基因組具有明顯的抗卡巴膽堿迷走效應(P0.05),但Gαi2ctp基因組間比較未見明顯差異(P0.05)。結論：三種轉染方法皆可有效轉染乳鼠心肌細胞,但各自具有不同特點：電穿孔法轉染迅速、轉染效率尚可,但對細胞具有較大的破壞力；rAd法轉染效率最高可達100%、但表達時間較短、心肌毒性較大；AVV9法轉染起效較慢但能夠穩(wěn)定長時間表達且生物安全性最好。三種方法皆可有效表達Gαi2ctp并發(fā)揮拮抗迷走變時效應且各具特點,其中AVV9法更為穩(wěn)定和高效。綜合三種方法的有效性和毒副作用評價,AVV9法優(yōu)于電穿孔法和rAd法。該實驗為Gαi2ctp基因轉染治療房顫提供了可靠的理論支持和方法學依據(jù)。
[Abstract]:Aim: to construct G 偽 i2ctp carrying plasmids and viruses, and to compare the transfection efficiency of GI 偽 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 偽 i2ctp gene. Methods: the recombinant plasmid pDC316-G 偽 i2ctp-mCMV-EGFP, the recombinant adenovirus rAd-G 偽 i2ctp-mCMV-EGFP, the recombinant adeno-associated virus type 9, rAW9-G 偽 i2ctp-IRES-EGFP, and the control group without G 偽 i2ctp were constructed. Three methods were used to transfect G 偽 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 偽 i2ctp protein was evaluated by AlamarBlue method and Western blot method. Finally, G 偽 i2ctp genome, no genome and blank control group were treated with the optimal concentration of carbachol. The anti-vagal effects of G 偽 i2ctp gene were compared by counting the overall pulsatile frequency of the cells. Results: the eukaryotic transfection plasmids containing G 偽 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 鹵4.8)% vs (54.2 鹵4.8)% at 1.5 d and (59.2 鹵4.4)% at 4 days in the rAW9 group and (30.1 鹵6.6)% (12 鹵3.4)% and (36 鹵6.1)% on the 11th day respectively, and the expression of G 偽 -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 偽 i2ctp genome had a significant anti-carbachol aberrant effect (P0.05), but there was no significant difference between G 偽 i2ctp genome and G 偽 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 偽 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 偽 i2ctp gene transfection in the treatment of atrial fibrillation.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R541.75
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本文編號：2124679