shRNA介導(dǎo)IGF-IR基因沉默抑制鼠心肌肥厚的體內(nèi)外實(shí)驗(yàn)研究
本文選題:心肌肥厚 切入點(diǎn):基因治療 出處:《浙江大學(xué)》2016年博士論文 論文類型:學(xué)位論文
【摘要】:目的:評(píng)價(jià)磁性納米顆粒作為基因載體介導(dǎo)小發(fā)夾結(jié)構(gòu)RNA (shRNA)靶向沉默IGF1R對(duì)去甲腎上腺素誘導(dǎo)的鼠心肌肥厚的抑制作用及其可能的機(jī)制。方法:檢索IGF-1R的基因序列,依照此序列構(gòu)建同時(shí)表達(dá)綠色熒光蛋白基因(GFP)和特異性針對(duì)小鼠心肌細(xì)胞胰島素樣生長(zhǎng)因子-1受體(IGF-1R)基因的shRNA的質(zhì)粒pGFPshIGF-1R,共設(shè)計(jì)了兩條序列,用聚合物作為載體,將質(zhì)粒pGFPshlGF-1R轉(zhuǎn)染進(jìn)鼠心肌細(xì)胞,篩選轉(zhuǎn)染效率較高的基因序列;根據(jù)上述篩選的質(zhì)粒,轉(zhuǎn)染體外培養(yǎng)的鼠心肌細(xì)胞,同時(shí)與單純的脂質(zhì)體轉(zhuǎn)染方法相比較,通過(guò)western blot檢測(cè)兩種轉(zhuǎn)染方法對(duì)平滑肌細(xì)胞中IGF-1R蛋白表達(dá)下調(diào)情況;外加磁場(chǎng)作用,篩選轉(zhuǎn)染效率最高的磁場(chǎng)參數(shù)。在去甲腎上腺素誘導(dǎo)的鼠心肌肥厚細(xì)胞及動(dòng)物模型中,通過(guò)心重/體重比、心肌橫截面積、心超檢測(cè)左室大小及心功能等評(píng)價(jià)心肌肥厚模型建立情況。轉(zhuǎn)染質(zhì)粒pGFPshlGF-1R與聚合物的復(fù)合物,分子水平用westernblot檢測(cè)聚合物載體體內(nèi)轉(zhuǎn)染24h、48h、72h后,組織中IGF-1R蛋白表達(dá)受抑情況;注射結(jié)束后1周,再次通過(guò)心重/體重比、心肌橫截面積、心超檢測(cè)左室大小及心功能等評(píng)價(jià)聚合物質(zhì)粒pGFPshlGF-1R體內(nèi)抑制心肌肥厚程度,并探索IGF1R下游信號(hào)通路蛋白活化情況。結(jié)果:首先通過(guò)測(cè)序驗(yàn)證了RNA干擾序列成功插入質(zhì)粒內(nèi),經(jīng)過(guò)western blot和RT-PCR檢測(cè)IGF1R表達(dá),篩選出序列2為抑制效率較高的序列,對(duì)心肌肌細(xì)胞IGF-1R在mRNA水平和蛋白水平的抑制率分別達(dá)到47-%-1±1.8%,和41%±1.2%。在轉(zhuǎn)染載體篩選方面,不論脂質(zhì)體轉(zhuǎn)染還是應(yīng)用聚合物轉(zhuǎn)染方式,都可以將質(zhì)粒pGFPshlGF-lR轉(zhuǎn)染進(jìn)心肌細(xì)胞內(nèi)。相對(duì)于空白對(duì)照組和脂質(zhì)體轉(zhuǎn)染,聚合物作為載體展現(xiàn)了較高的效率。流式細(xì)胞結(jié)果表明,綠色熒光蛋白基因(GFP)陽(yáng)性的細(xì)胞數(shù)占35.1±3.1%,而在脂質(zhì)體轉(zhuǎn)染組綠色熒光蛋白基因(GFP)陽(yáng)性的細(xì)胞數(shù)只有13.0±5.1%;協(xié)同磁場(chǎng)轉(zhuǎn)染,在250mT輻照15min的條件下轉(zhuǎn)染效率最高,可以達(dá)到58%左右。在心肌肥厚模型的構(gòu)建上,通過(guò)去甲腎上腺素成功誘導(dǎo)心肌細(xì)胞和小鼠心臟肥厚的模型,并檢測(cè)出IGF1R明顯增高、ANP及J3-MHC的niRNA表達(dá)也明顯增高。在體內(nèi)轉(zhuǎn)染條件的實(shí)驗(yàn)中,經(jīng)尾靜脈注射表達(dá)IGFlRshRNA的聚合物,能有效到達(dá)心肌組織,轉(zhuǎn)染24h、48h、72h均能明顯抑制IGF1R表達(dá),顯著減少去甲腎上腺素誘導(dǎo)的心肌肥厚,改善心功能,表明抑制IGF1R可以作為一種有效可行的用于預(yù)防或逆轉(zhuǎn)去甲腎上腺素誘導(dǎo)的心肌肥厚,并且與IGF1R下游通路信號(hào)蛋白ERK1/1,AKT的磷酸化有關(guān)。結(jié)論:沉默IGF1R表達(dá)能預(yù)防或逆轉(zhuǎn)去甲腎上腺素誘導(dǎo)的心肌肥厚,有望為探尋高血壓心肌肥厚發(fā)病的機(jī)制和防治的策略提供新的靶點(diǎn)和思路。
[Abstract]:Aim: to evaluate the inhibitory effect of magnetic nanoparticles on myocardial hypertrophy induced by norepinephrine and its possible mechanism in IGF1R silencing with small hairpin IGF1R mediated by gene vector. Methods: the gene sequence of IGF-1R was searched. According to this sequence, the plasmid pGFPshIGF-1R was constructed, which simultaneously expressed the green fluorescent protein (GFP) gene and the shRNA specific to the insulin-like growth factor-1 receptor (IGF-1R) gene of mouse cardiomyocytes. The plasmid pGFPshlGF-1R was transfected into rat cardiomyocytes to screen the gene sequence with higher transfection efficiency. According to the selected plasmids, the cultured rat cardiomyocytes were transfected in vitro, and the transfection methods were compared with the simple liposome transfection method. The expression of IGF-1R protein in smooth muscle cells was down-regulated by two transfection methods by western blot, and the magnetic field parameters with the highest transfection efficiency were screened under the action of external magnetic field. In the myocardial hypertrophy cells induced by norepinephrine and animal models, the expression of IGF-1R protein was down-regulated by western blot. The establishment of myocardial hypertrophy model was evaluated by heart weight / body weight ratio, myocardial cross-sectional area, left ventricular size by echocardiography and cardiac function. The complex of plasmid pGFPshlGF-1R and polymer was transfected, and the molecular level was detected by westernblot for 24 h, 48 h and 72 h after transfection. The inhibition of IGF-1R protein expression in the tissue, the degree of inhibition of myocardial hypertrophy in polymer plasmid pGFPshlGF-1R was evaluated by cardiac weight / body weight ratio, myocardial cross-sectional area, left ventricular size and cardiac function 1 week after injection. Results: firstly, the RNA interference sequence was successfully inserted into the plasmid by sequencing. IGF1R expression was detected by western blot and RT-PCR. Sequence 2 was selected as the sequence with higher inhibition efficiency. The inhibition rates of IGF-1R at mRNA level and protein level were 47--1 鹵1.8 and 41% 鹵1.2, respectively. In the selection of transfection vectors, both liposome transfection and polymer transfection were used. Compared with blank control group and liposome transfection, polymer showed high efficiency. Flow cytometry showed that, The number of GFP positive cells was 35.1 鹵3.1, while in liposome transfection group, the number of GFP-positive cells was 13.0 鹵5.1.The efficiency of co-transfection was the highest at 250mT irradiation for 15min, while the GFP-positive cells were only 13.0 鹵5.1cells in liposome transfection group. It can reach about 58%. In the construction of myocardial hypertrophy model, the model of cardiac hypertrophy induced by norepinephrine was successfully used to induce cardiac hypertrophy in mice. The niRNA expression of IGFlRshRNA and J3-MHC were also significantly increased by IGF1R. In the experiment of transfection condition in vivo, the expression of IGF1R could be effectively reached by injection of polymer expressing IGFlRshRNA through tail vein, and the expression of IGF1R could be inhibited significantly at 24 h, 48 h and 72 h after transfection. Myocardial hypertrophy induced by norepinephrine was significantly reduced and cardiac function was improved. It was suggested that inhibition of IGF1R could be used to prevent or reverse norepinephrine induced myocardial hypertrophy. Conclusion: silent expression of IGF1R can prevent or reverse noradrenaline induced myocardial hypertrophy. It is expected to provide new targets and ideas for exploring the pathogenesis and prevention of hypertensive myocardial hypertrophy.
【學(xué)位授予單位】:浙江大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2016
【分類號(hào)】:R542.2
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