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基于遙控機(jī)械力的重要生物分子的跨細(xì)胞膜導(dǎo)入

Transmembrane delivery of key biomolecules into the cell by remote control of mechanical force

作者: 胡明峰  王穎奇  葛琳  馮磊  李彬  楊晟  劉曉冬 
單位:清華大學(xué)醫(yī)學(xué)院生物醫(yī)學(xué)工程系(北京100084)
關(guān)鍵詞: 跨膜運輸;磁納米顆粒;機(jī)械敏感離子通道;磁控系統(tǒng);超聲微泡致孔 
分類號:R318.04
出版年·卷·期(頁碼):2016·35·3(226-230)
摘要:

目的 生物醫(yī)學(xué)基礎(chǔ)研究及其實踐應(yīng)用日益需要在分子、細(xì)胞與系統(tǒng)、活體間建立直接的關(guān)聯(lián),解決方案之一是利用非接觸方式遠(yuǎn)程控制細(xì)胞生命過程,當(dāng)前受到廣泛關(guān)注的光基因?qū)W等手段即為此方面的重要突破性進(jìn)展。研究中的核心問題是如何實現(xiàn)在細(xì)胞乃至分子水平上對關(guān)鍵生命物質(zhì)及生物學(xué)過程進(jìn)行遠(yuǎn)程調(diào)控。為此,基于磁場和超聲施加機(jī)械力,并輔助以超聲微泡、磁納米顆粒以及機(jī)械敏感通道,提出實現(xiàn)細(xì)胞遠(yuǎn)程控制的新型解決方案。方法 使用超聲輸出設(shè)備對細(xì)胞膜周邊的微泡施加機(jī)械力,以黃色熒光蛋白(yellow fluorescent protein,YFP)標(biāo)記的DNA作為轉(zhuǎn)基因標(biāo)記物,測試不同條件下DNA跨膜進(jìn)入細(xì)胞的效率。運用化學(xué)合成法合成磁納米顆粒,使用電磁鐵作為磁控系統(tǒng),利用細(xì)胞鈣成像實驗監(jiān)測機(jī)械力敏感離子通道的開放情況。結(jié)果 在機(jī)械刺激作用下,細(xì)胞外液中的YFP質(zhì)粒可以跨膜進(jìn)入細(xì)胞并有效表達(dá)。與細(xì)胞共培養(yǎng)的磁納米顆粒在電磁場的作用下,致使機(jī)械敏感離子通道開放、鈣離子進(jìn)入細(xì)胞及鈣成像熒光上升。結(jié)論 基于遙控機(jī)械力能夠?qū)崿F(xiàn)生物分子跨膜運送的遠(yuǎn)程控制。通過超聲波(強(qiáng)度>0.6W/cm2)誘發(fā)微泡爆破從而施加機(jī)械沖擊并在細(xì)胞膜上成孔,實現(xiàn)了DNA跨細(xì)胞膜導(dǎo)入及有效表達(dá)。磁納米顆粒通過可調(diào)控電磁場(18~20V,>30mT)對細(xì)胞膜施加機(jī)械刺激,通過內(nèi)源性機(jī)械敏感機(jī)制引起鈣電流及膜興奮性的增加,從而實現(xiàn)跨膜鈣信號的遠(yuǎn)程控制。

Objective Both basic and applicable research in biomedicine have growing demands in more effective approaches that build direct connections between molecules,cells,systems and bodies.One of the solutions is to establish the remote control of cellular processes via non-contact manners.Optogenetics,as one of the breakthroughs,has caught great attentions in the field.The key issue here is how to implement the remote control of crucial entities and processes at the cell and molecular levels.Therefore,based on mechanical force through magnetics and ultrasonics,assisted with ultrasound microbubble,magnetic nanoparticles and mechanosensitive ion channels,we propose a novel solution to the remotely controlled cells.Methods We used ultrasonic generator to stimulate microbubbles around cells to induce mechanical force for membrane sonoporation.Yellow fluorescent protein (YFP) expression with fluorescence as the index indicated the efficiency of sonoporation thus DNA transported under different conditions.For magnetic control of transmembrane Ca2+,chemically-synthesized magnetic nanoparticles (MNP) were driven by electromagnetic control system.Results Aided with mechanical stimuli,substantial amount of YFP plasmids successfully went across cell membrane and got expressed.The fluorescence of cells co-cultured with MNPs was enhanced upon stimulation of magnetic control system.Conclusions Our results demonstrate that based on the remote control of mechanical force,one can achieve the remote control of transmembrane transport of key biomolecules.The transmembrane transport and expression of DNA can be accomplished through sonoporation (ultrasound strength: >0.6W/cm2).Cell membrane excitability as well as Ca2+ signals could be enhanced through electromagnetics (18 to 20V,>30mT).

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