發(fā)布時間：2019-08-08 11:01

【摘要】：目的利用透明質酸(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質粒,檢測其在活體豚鼠內耳的轉染效率。方法質粒提取后按照COOH/N/P=4:10:1合成納米載體基因復合物并進行表征。利用圓窗膜滲透的方法,導入實驗動物耳蝸。術后7天,通過激光共聚焦掃描觀察基底膜鋪片和切片了解轉染情況,并利用Western Blot和RT-PCR技術分別從蛋白和核酸水平驗證轉染結果。結果按照本研究實驗方法可成功合成表面帶有負電荷的納米級別的基因載體復合物顆粒,導入實驗動物耳蝸后7天取材,基底膜鋪片的共聚焦顯微鏡觀察結果顯示:基底膜內外毛細胞可檢測到綠色熒光蛋白顯色,基底膜底轉的轉染效率達81.7±4.71%,中轉可達33.8±9.02%。結合冰凍切片結果發(fā)現(xiàn),表達的綠色熒光蛋白主要位于耳蝸底轉和部分中轉,頂轉及內外毛細胞以外區(qū)域未見綠色熒光蛋白表達。基底膜細胞未見明顯變形損傷。Western Blot和RT-PCR結果也驗證了Atoh1基因在基底膜上的成功轉染。結論 HA修飾b PEI納米顆粒制備基因載體可成功實現(xiàn)耳蝸的基因轉染,且未見對基底膜細胞產生明顯的毒性。合成簡單、成本較低,是理想的內耳基因轉染載體。
[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實驗室;首都醫(yī)科大學附屬北京安貞醫(yī)院耳鼻咽喉頭頸外科;國家納米科學中心;
【基金】：國家863青年科學家項目(2014AA020510) 國家973計劃重大科學研究計劃干細胞項目(2012CB967900) 國家科技部新藥創(chuàng)制重大專項(2014ZX09J14101-06C) 國家自然科學基金項目(NSFC 81470700) 中國科協(xié)創(chuàng)新驅動助力工程(2016CXQD01)
【分類號】：R764

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

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