天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

當(dāng)前位置:主頁(yè) > 科技論文 > 基因論文 >

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

發(fā)布時(shí)間:2018-03-07 13:26

  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

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 石麗萍 ,顏光濤 ,張凱 ,李英麗;酸敏脂質(zhì)體的制備及其生物學(xué)活性[J];中國(guó)藥物與臨床;2003年04期

2 任浩洋;常紅霞;蔡雯雯;;一種具有肝細(xì)胞選擇性的脂質(zhì)體的導(dǎo)向性評(píng)價(jià)[J];解放軍藥學(xué)學(xué)報(bào);2007年01期

3 平其能,郭健新;脂質(zhì)體在基因治療中的應(yīng)用[J];藥學(xué)進(jìn)展;1998年02期

4 徐昊,王明榮,顏光濤;脂質(zhì)體轉(zhuǎn)染技術(shù)進(jìn)展及其應(yīng)用[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);2000年06期

5 毛曉春;張虹;李貴剛;;脂質(zhì)體介導(dǎo)兔角膜內(nèi)皮細(xì)胞基因轉(zhuǎn)染的觀察[J];眼科新進(jìn)展;2007年07期

6 徐蓓;王昕榮;朱桂金;;宮腔內(nèi)脂質(zhì)體轉(zhuǎn)染技術(shù)在圍著床期小鼠子宮內(nèi)膜表達(dá)的研究[J];生殖醫(yī)學(xué)雜志;2008年03期

7 嚴(yán)文偉,齊憲榮,魏來(lái),費(fèi)然,叢旭,王宇;陽(yáng)離子脂質(zhì)材料和聚乙二醇對(duì)脂質(zhì)體細(xì)胞轉(zhuǎn)染率及膜流動(dòng)性的影響[J];藥學(xué)學(xué)報(bào);2003年09期

8 王全興,曹雪濤,章衛(wèi)平,王建莉,葉天星;脂質(zhì)體介導(dǎo)的體內(nèi)外細(xì)胞因子基因轉(zhuǎn)移效果的初步研究[J];中國(guó)腫瘤生物治療雜志;1995年03期

9 王澤平,張景迎,龐呈義;內(nèi)含子增強(qiáng)脂質(zhì)體介導(dǎo)的轉(zhuǎn)基因表達(dá)[J];泰山醫(yī)學(xué)院學(xué)報(bào);2000年02期

10 程序;宋必衛(wèi);章春麗;馮海;;含氣脂質(zhì)體的超聲波介導(dǎo)體外基因輸送的實(shí)驗(yàn)研究[J];浙江工業(yè)大學(xué)學(xué)報(bào);2010年05期

相關(guān)會(huì)議論文 前4條

1 王海蛟;劉艷紅;張?bào)K;余孝其;;基于Cyclen的雙尾脂質(zhì)體作為高效基因轉(zhuǎn)染試劑的研究[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第35分會(huì):納米生物醫(yī)學(xué)中的化學(xué)問(wèn)題[C];2014年

2 田景振;張玉娟;;脂質(zhì)體的研究進(jìn)展[A];脂質(zhì)體及紫杉醇脂質(zhì)體學(xué)術(shù)論文集[C];2005年

3 王永權(quán);彭毅志;王強(qiáng);王逸濤;游波;;脂質(zhì)體載體介導(dǎo)的基因轉(zhuǎn)染對(duì)人未成熟樹突狀細(xì)胞成熟特性的影響[A];第四屆全國(guó)燒傷救治專題研討會(huì)燒傷感染救治新進(jìn)展論文匯編[C];2006年

4 毛曉春;張虹;李貴剛;;脂質(zhì)體介導(dǎo)兔角膜內(nèi)皮細(xì)胞基因轉(zhuǎn)染的觀察[A];中華醫(yī)學(xué)會(huì)第十二屆全國(guó)眼科學(xué)術(shù)大會(huì)論文匯編[C];2007年

相關(guān)重要報(bào)紙文章 前1條

1 楊曉;基因工程與人類健康長(zhǎng)壽[N];保健時(shí)報(bào);2014年

相關(guān)博士學(xué)位論文 前2條

1 孔敏堅(jiān);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[D];浙江大學(xué);2016年

2 趙浩;轉(zhuǎn)鐵蛋白靶向脂質(zhì)體跨血腦屏障轉(zhuǎn)導(dǎo)VEGF基因治療大鼠缺血性腦卒中[D];中國(guó)協(xié)和醫(yī)科大學(xué);2010年

相關(guān)碩士學(xué)位論文 前10條

1 蔣莉;聚乙烯亞胺對(duì)兩性離子型脂質(zhì)體及陰離子型脂質(zhì)體結(jié)構(gòu)和性質(zhì)的影響[D];廣西大學(xué);2016年

2 石麗萍;酸敏脂質(zhì)體的制備及其性質(zhì)的研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2002年

3 吳昊;靶向脂質(zhì)體運(yùn)載基因藥物通過(guò)血腦屏障能力的初步研究[D];吉林大學(xué);2010年

4 陳琴;環(huán)氧烷胺衍生物修飾siRNA負(fù)載脂質(zhì)體的肺部給藥研究[D];蘇州大學(xué);2015年

5 唐劭年;制備和鑒定腦靶向性脂質(zhì)體P-MMA-DOSPER[D];第一軍醫(yī)大學(xué);2006年

6 李丹;脂質(zhì)體介導(dǎo)Survivin基因過(guò)表達(dá)對(duì)人臍靜脈內(nèi)皮細(xì)胞生物學(xué)活性的影響[D];蚌埠醫(yī)學(xué)院;2012年

7 林瑩;EGFR受體介導(dǎo)肺腫瘤靶向siRNA脂多胺修飾脂質(zhì)體的制備與評(píng)價(jià)[D];蘇州大學(xué);2014年

8 蘭晶;人核心蛋白聚糖真核載體構(gòu)建及其體內(nèi)外抑瘤作用研究[D];山西醫(yī)科大學(xué);2007年

9 徐肖;脂質(zhì)體介導(dǎo)BCSG1反義寡核苷酸對(duì)人食管癌細(xì)胞株TE-13生物學(xué)行為的影響[D];河北醫(yī)科大學(xué);2008年

10 薛同春;單抗Au_(14-1)-脂質(zhì)體-p53基因轉(zhuǎn)染載體的制備及其在宮頸癌U_(14)細(xì)胞中的表達(dá)[D];江西醫(yī)學(xué)院;2004年



本文編號(hào):1579478

資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/kejilunwen/jiyingongcheng/1579478.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權(quán)申明:資料由用戶6417a***提供,本站僅收錄摘要或目錄,作者需要?jiǎng)h除請(qǐng)E-mail郵箱bigeng88@qq.com
青青操视频在线播放免费| 欧美在线观看视频三区| 国产女优视频一区二区| 青青操日老女人的穴穴| 国产精品一区二区三区日韩av | 亚洲国产成人久久99精品| 日韩女优视频国产一区| 欧美激情一区=区三区| 国产欧美一区二区三区精品视| 中文字幕有码视频熟女| 亚洲天堂精品在线视频| 99热九九热这里只有精品| 精品精品国产自在久久高清| 亚洲黄色在线观看免费高清| 风间中文字幕亚洲一区| 日本人妻精品中文字幕不卡乱码| 欧美小黄片在线一级观看| 日韩国产亚洲欧美激情| 欧美黑人在线一区二区| 亚洲一区二区精品福利| 国产精品一区二区丝袜| 国产永久免费高清在线精品| 毛片在线观看免费日韩| 国产成人精品在线一区二区三区| 日本加勒比系列在线播放| 国产福利一区二区三区四区| 欧美中文字幕日韩精品| 九九热这里有精品20| 国产中文字幕一区二区| 色播五月激情五月婷婷| 久久少妇诱惑免费视频| 色综合久久六月婷婷中文字幕| 欧美丰满大屁股一区二区三区| 亚洲精品成人午夜久久| 国产精品欧美一区二区三区不卡 | 情一色一区二区三区四| 日韩精品综合免费视频| 中国日韩一级黄色大片| 青青草草免费在线视频| 亚洲中文字幕综合网在线| 成人午夜视频在线播放|