AE3基因siRNA表達(dá)質(zhì)粒的構(gòu)建及對(duì)其轉(zhuǎn)染的心肌細(xì)胞H9c2的影響
發(fā)布時(shí)間：2018-11-04 14:34
【摘要】： 目的:利用RNA干擾(RNAi)技術(shù),以AE3為靶基因,設(shè)計(jì)構(gòu)建siRNA表達(dá)質(zhì)粒,進(jìn)行測(cè)序鑒定,并檢測(cè)該表達(dá)質(zhì)粒對(duì)大鼠心肌樣細(xì)胞系H9c2 AE3基因表達(dá)的影響,為進(jìn)一步研究AE3基因在心肌細(xì)胞損傷及保護(hù)中的作用奠定基礎(chǔ)。 方法: 1.設(shè)計(jì)具有短發(fā)夾結(jié)構(gòu)的三條DNA序列,經(jīng)退火成互補(bǔ)雙鏈,再克隆至載體pSilencer3.1-H1-hygro中構(gòu)建重組表達(dá)載體,轉(zhuǎn)化DH5α菌株,提取質(zhì)粒并進(jìn)行序列測(cè)定。 2.轉(zhuǎn)染攜帶綠色熒光蛋白的pEGFP-N2質(zhì)粒,熒光顯微鏡觀察檢測(cè)瞬時(shí)轉(zhuǎn)染不同時(shí)相的轉(zhuǎn)染效率,選擇最佳時(shí)相用于后續(xù)瞬時(shí)轉(zhuǎn)染靶基因表達(dá)抑制率的分析。 3.利用構(gòu)建成功的siRNA表達(dá)質(zhì)粒,通過(guò)脂質(zhì)體介導(dǎo)轉(zhuǎn)染H9c2心肌樣細(xì)胞,并通過(guò)RT-PCR、Western blot檢測(cè)細(xì)胞中AE3 mRNA和蛋白的表達(dá)。 結(jié)果: 1.重組質(zhì)粒轉(zhuǎn)化DH5α菌株經(jīng)氨芐青霉素抗性篩選可見(jiàn)有細(xì)菌克隆長(zhǎng)出。 2.測(cè)序鑒定表明重組質(zhì)粒中含有針對(duì)AE3基因的目的序列。 3.將pEGFP-N2通過(guò)脂質(zhì)體介導(dǎo),轉(zhuǎn)染H9c2心肌樣細(xì)胞,分別于轉(zhuǎn)染24 h、48 h、72 h后在熒光顯微鏡下觀察轉(zhuǎn)染效果,可見(jiàn)H9c2心肌樣細(xì)胞胞漿內(nèi)有綠色熒光,其中轉(zhuǎn)染48 h的轉(zhuǎn)染效率82±6%顯著高于轉(zhuǎn)染24 h的轉(zhuǎn)染效率43±6% (P0.01)和轉(zhuǎn)染72 h的轉(zhuǎn)染效率60±7% (P0.05)。因此,按48 h最佳轉(zhuǎn)染時(shí)間轉(zhuǎn)染構(gòu)建好的siRNA表達(dá)質(zhì)粒。 4. RT-PCR檢測(cè)細(xì)胞中AE3 mRNA的表達(dá)水平,顯示pSilencer-AE3-A可明顯降低H9c2心肌樣細(xì)胞中AE3 mRNA的表達(dá)。 5. Western blot檢測(cè)細(xì)胞中AE3蛋白的表達(dá)量,顯示pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C轉(zhuǎn)染的H9c2心肌樣細(xì)胞AE3蛋白表達(dá)分別降低61%,48%,25%。 結(jié)論: 1.成功構(gòu)建AE3基因siRNA表達(dá)質(zhì)粒pSilencer-AE3-A,pSilencer-AE3-B,pSilencer-AE3-C。 2.重組質(zhì)粒pSilencer-AE3-A可明顯降低H9c2心肌樣細(xì)胞AE3基因在mRNA和蛋白水平的表達(dá)。
[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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R541;R346
[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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R541;R346
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
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