【摘要】： 目的:利用RNA干擾(RNAi)技術,以AE3為靶基因,設計構建siRNA表達質粒,進行測序鑒定,并檢測該表達質粒對大鼠心肌樣細胞系H9c2 AE3基因表達的影響,為進一步研究AE3基因在心肌細胞損傷及保護中的作用奠定基礎。 方法: 1.設計具有短發(fā)夾結構的三條DNA序列,經(jīng)退火成互補雙鏈,再克隆至載體pSilencer3.1-H1-hygro中構建重組表達載體,轉化DH5α菌株,提取質粒并進行序列測定。 2.轉染攜帶綠色熒光蛋白的pEGFP-N2質粒,熒光顯微鏡觀察檢測瞬時轉染不同時相的轉染效率,選擇最佳時相用于后續(xù)瞬時轉染靶基因表達抑制率的分析。 3.利用構建成功的siRNA表達質粒,通過脂質體介導轉染H9c2心肌樣細胞,并通過RT-PCR、Western blot檢測細胞中AE3 mRNA和蛋白的表達。 結果: 1.重組質粒轉化DH5α菌株經(jīng)氨芐青霉素抗性篩選可見有細菌克隆長出。 2.測序鑒定表明重組質粒中含有針對AE3基因的目的序列。 3.將pEGFP-N2通過脂質體介導,轉染H9c2心肌樣細胞,分別于轉染24 h、48 h、72 h后在熒光顯微鏡下觀察轉染效果,可見H9c2心肌樣細胞胞漿內有綠色熒光,其中轉染48 h的轉染效率82±6%顯著高于轉染24 h的轉染效率43±6% (P0.01)和轉染72 h的轉染效率60±7% (P0.05)。因此,按48 h最佳轉染時間轉染構建好的siRNA表達質粒。 4. RT-PCR檢測細胞中AE3 mRNA的表達水平,顯示pSilencer-AE3-A可明顯降低H9c2心肌樣細胞中AE3 mRNA的表達。 5. Western blot檢測細胞中AE3蛋白的表達量,顯示pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C轉染的H9c2心肌樣細胞AE3蛋白表達分別降低61%,48%,25%。 結論: 1.成功構建AE3基因siRNA表達質粒pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C。 2.重組質粒pSilencer-AE3-A可明顯降低H9c2心肌樣細胞AE3基因在mRNA和蛋白水平的表達。
[Abstract]:Objective: to design and construct siRNA expression plasmid using AE3 as target gene by RNA interference (RNAi) technique, and to detect the effect of siRNA expression plasmid on the expression of H9c2 AE3 gene in rat cardiomyoid cell line. It provides a basis for further study of the role of AE3 gene in myocardial injury and protection. Methods: 1. Three DNA sequences with short hairpin structure were designed and annealed into complementary double strands, then cloned into vector pSilencer3.1-H1-hygro to construct recombinant expression vector. The recombinant expression vector was transformed into DH5 偽 strain. The plasmid was extracted and sequenced. 2. After transfection of pEGFP-N2 plasmid carrying green fluorescent protein, the transfection efficiency of different phases of transient transfection was observed by fluorescence microscope, and the optimal phase was selected to analyze the inhibition rate of target gene expression in subsequent transient transfection. 3. The siRNA expression plasmid was constructed and transfected into H9c2 cardiomyoid cells by liposome. The expression of AE3 mRNA and protein was detected by RT-PCR,Western blot. Results: 1. The recombinant plasmid transformed DH5 偽 strain was screened for ampicillin resistance. 2. Sequencing analysis showed that the recombinant plasmid contained the target sequence for AE3 gene. 3. PEGFP-N2 was transfected into H9c2 cardiomyocytes via liposome, and the transfection effect was observed under fluorescence microscope at 24 h, 48 h or 72 h after transfection. Green fluorescence was observed in the cytoplasm of H9c2 cardiomyoid cells. The transfection efficiency of 48 h was significantly higher than that of 24 h (P 0.01) and 72 h (P 0.05). Therefore, the siRNA expression plasmid was constructed at the best transfection time of 48 h. 4. The expression of AE3 mRNA was detected by RT-PCR, which showed that pSilencer-AE3-A could significantly decrease the expression of AE3 mRNA in H9c2 cardiomyocytes. 5. The expression of AE3 protein was detected by Western blot. The results showed that the expression of AE3 protein in H9c2 cardiomyocytes transfected with pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C decreased by 61% and 25% respectively. Conclusion: 1. Construction of siRNA expression plasmid pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C. of AE3 gene 2. Recombinant plasmid pSilencer-AE3-A could significantly reduce the expression of AE3 gene in H9c2 cardiomyocytes at the level of mRNA and protein.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R541;R346

【共引文獻】

相關期刊論文 前10條

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本文編號：2310108