【摘要】：目的利用透明質(zhì)酸(HA)修飾分支型聚乙烯亞胺(b PEI)納米顆粒制備新型非病毒基因載體,包載Atoh1-EGFP質(zhì)粒,檢測(cè)其在活體豚鼠內(nèi)耳的轉(zhuǎn)染效率。方法質(zhì)粒提取后按照COOH/N/P=4:10:1合成納米載體基因復(fù)合物并進(jìn)行表征。利用圓窗膜滲透的方法,導(dǎo)入實(shí)驗(yàn)動(dòng)物耳蝸。術(shù)后7天,通過激光共聚焦掃描觀察基底膜鋪片和切片了解轉(zhuǎn)染情況,并利用Western Blot和RT-PCR技術(shù)分別從蛋白和核酸水平驗(yàn)證轉(zhuǎn)染結(jié)果。結(jié)果按照本研究實(shí)驗(yàn)方法可成功合成表面帶有負(fù)電荷的納米級(jí)別的基因載體復(fù)合物顆粒,導(dǎo)入實(shí)驗(yàn)動(dòng)物耳蝸后7天取材,基底膜鋪片的共聚焦顯微鏡觀察結(jié)果顯示:基底膜內(nèi)外毛細(xì)胞可檢測(cè)到綠色熒光蛋白顯色,基底膜底轉(zhuǎn)的轉(zhuǎn)染效率達(dá)81.7±4.71%,中轉(zhuǎn)可達(dá)33.8±9.02%。結(jié)合冰凍切片結(jié)果發(fā)現(xiàn),表達(dá)的綠色熒光蛋白主要位于耳蝸底轉(zhuǎn)和部分中轉(zhuǎn),頂轉(zhuǎn)及內(nèi)外毛細(xì)胞以外區(qū)域未見綠色熒光蛋白表達(dá)�；啄ぜ�(xì)胞未見明顯變形損傷。Western Blot和RT-PCR結(jié)果也驗(yàn)證了Atoh1基因在基底膜上的成功轉(zhuǎn)染。結(jié)論 HA修飾b PEI納米顆粒制備基因載體可成功實(shí)現(xiàn)耳蝸的基因轉(zhuǎn)染,且未見對(duì)基底膜細(xì)胞產(chǎn)生明顯的毒性。合成簡(jiǎn)單、成本較低,是理想的內(nèi)耳基因轉(zhuǎn)染載體。
[Abstract]:Objective To prepare a novel non-viral gene vector by using hyaluronic acid (HA) modified branched polyethyleneimine (b-PEI) nanoparticles, and to coat the Ath1-EGFP plasmid to detect the transfection efficiency of the inner ear of the in-vivo guinea pig. Methods After extraction, the nano-carrier gene complex was synthesized and characterized by COOH/ N/ P = 4:10:1. The experimental animal cochlea was introduced by the method of the penetration of the circular window membrane. After 7 days of operation, the transfection of basement membrane was observed by laser confocal scanning, and the transfection results were verified by Western Blot and RT-PCR. Results According to the experimental method of this study, the nano-scale gene-carrier complex particles with negative charge on the surface can be successfully synthesized, and the results of the observation of the confocal microscope of the basement membrane patch on 7 days after the introduction of the experimental animal's cochlea showed that: The color of the green fluorescent protein can be detected by the inner and outer hair cells of the basement membrane, and the transfection efficiency of the basement membrane is 81.7% to 4.71%, and the transfer rate can reach 33.8% to 9.02%. Combined with the results of the frozen section, the expressed green fluorescent protein was mainly located in the basal and partial transfer of the cochlea, and no green fluorescent protein expression was found in the region other than the top and outer hair cells. No significant deformation damage was observed in the basement membrane cells. Western Blot and RT-PCR also demonstrated the successful transfection of the Atho1 gene on the basement membrane. Conclusion The gene vector of HA-modified b-PEI nanoparticles can successfully realize the gene transfer of the cochlea and has no obvious toxicity to the basement membrane cells. The invention has the advantages of simple synthesis and low cost, and is an ideal inner ear gene transfection carrier.
【作者單位】： 解放軍總醫(yī)院耳鼻咽喉研究所聾病教育部重點(diǎn)實(shí)驗(yàn)室;首都醫(yī)科大學(xué)附屬北京安貞醫(yī)院耳鼻咽喉頭頸外科;國家納米科學(xué)中心;
【基金】：國家863青年科學(xué)家項(xiàng)目(2014AA020510) 國家973計(jì)劃重大科學(xué)研究計(jì)劃干細(xì)胞項(xiàng)目(2012CB967900) 國家科技部新藥創(chuàng)制重大專項(xiàng)(2014ZX09J14101-06C) 國家自然科學(xué)基金項(xiàng)目(NSFC 81470700) 中國科協(xié)創(chuàng)新驅(qū)動(dòng)助力工程(2016CXQD01)
【分類號(hào)】：R764

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1 陳嘉寶;劉俊華;;Atoh1基因在癌癥中的研究進(jìn)展[J];醫(yī)學(xué)綜述;2011年06期

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