HA-bPEI纳米颗粒携带Atoh1质粒豚鼠耳蜗转染观察
发布时间：2019-08-08 11:01
【摘要】：目的利用透明质酸(HA)修饰分支型聚乙烯亚胺(b PEI)纳米颗粒制备新型非病毒基因载体,包载Atoh1-EGFP质粒,检测其在活体豚鼠内耳的转染效率。方法质粒提取后按照COOH/N/P=4:10:1合成纳米载体基因复合物并进行表征。利用圆窗膜渗透的方法,导入实验动物耳蜗。术后7天,通过激光共聚焦扫描观察基底膜铺片和切片了解转染情况,并利用Western Blot和RT-PCR技术分别从蛋白和核酸水平验证转染结果。结果按照本研究实验方法可成功合成表面带有负电荷的纳米级别的基因载体复合物颗粒,导入实验动物耳蜗后7天取材,基底膜铺片的共聚焦显微镜观察结果显示:基底膜内外毛细胞可检测到绿色荧光蛋白显色,基底膜底转的转染效率达81.7±4.71%,中转可达33.8±9.02%。结合冰冻切片结果发现,表达的绿色荧光蛋白主要位于耳蜗底转和部分中转,顶转及内外毛细胞以外区域未见绿色荧光蛋白表达。基底膜细胞未见明显变形损伤。Western Blot和RT-PCR结果也验证了Atoh1基因在基底膜上的成功转染。结论 HA修饰b PEI纳米颗粒制备基因载体可成功实现耳蜗的基因转染,且未见对基底膜细胞产生明显的毒性。合成简单、成本较低,是理想的内耳基因转染载体。
[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.
【作者单位】： 解放军总医院耳鼻咽喉研究所聋病教育部重点实验室;首都医科大学附属北京安贞医院耳鼻咽喉头颈外科;国家纳米科学中心;
【基金】：国家863青年科学家项目(2014AA020510) 国家973计划重大科学研究计划干细胞项目(2012CB967900) 国家科技部新药创制重大专项(2014ZX09J14101-06C) 国家自然科学基金项目(NSFC 81470700) 中国科协创新驱动助力工程(2016CXQD01)
【分类号】：R764
本文编号：2524327
[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.
【作者单位】： 解放军总医院耳鼻咽喉研究所聋病教育部重点实验室;首都医科大学附属北京安贞医院耳鼻咽喉头颈外科;国家纳米科学中心;
【基金】：国家863青年科学家项目(2014AA020510) 国家973计划重大科学研究计划干细胞项目(2012CB967900) 国家科技部新药创制重大专项(2014ZX09J14101-06C) 国家自然科学基金项目(NSFC 81470700) 中国科协创新驱动助力工程(2016CXQD01)
【分类号】：R764
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1 陈嘉宝;刘俊华;;Atoh1基因在癌症中的研究进展[J];医学综述;2011年06期
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