HA-bPEI纳米颗粒携带Atoh1质粒豚鼠耳蜗转染观察

In vivo transduction of Atoh1 gene into cochlear cells mediated by HA-PEI nanoparticle vectors

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。

Objective To test in vivo transfection efficiency of Atoh1-EGFP plasmids using hyaluronic acid modified branched polyethylenimine nanoparticles as a novel non-viral gene vector. Methods Plasmids were extracted following kit instructions. The gene-vector nanoparticles were synthesized（COOH/N/P=4：10：1） and then characterized before being transduced into the inner ear through intact round window membrane. At 7 days after transduction operation, transfection efficiency was evaluated by examination of both basilar membrane surface preparation and frozen sections under a confocal microscope. Transfection results were finally verified by Western blot and RT-PCR at protein and nucleic acid levels respectively. Results We successfully synthesized a non-viral gene vector which loaded Atoh1 gene well and had a negatively-charged surface to reduce possible cytotoxicity. At 7 days after transfection, confocal microscope observation of basilar membrane showed EGFP in OHCs and IHCs. OHC transfection efficiency was 81.7 ± 4.71% in the basal turn and 33.8 ±9.02% in the middle turn. Frozen section results revealed expression of EGFP mainly in the basal turn and only part of the middle turn of the cochlea. No signs of EGFP transduction were seen in either the top of the cochlea or in cells other than hair cells. Furthermore, Basilar membrane cells showed no obvious damage after operation. Both Western Blot and RT-PCR results verified successful transfection of the Atoh1 gene on the basilar membrane. Conclusion The Atoh1 gene can be successfully transduced into the inner ear by the HA modified b PEI nanoparticles and there is no obvious cytotoxicity to cochlear cells. The new nanoparticles are easy to produce at a low cost, which makes it an ideal non-viral vector for inner ear gene transfection.