非病毒载体介导脑源性神经营养因子基因的体外转染小鼠耳蜗螺旋神经节细胞实验研究  

Experimental study of non-viral vector-mediated transfection of BDNF gene into mouse cochlea SGCs

作　　者：吴雄辉[1] 赵斯君[1] 李赟[1] 谢江 WU Xionghui;ZHAO Sijun;LI Yun;XIE Jiang(Department of Otolaryngology Head and Neck Surgery,Hunan Provincial Children's Hospital,Changsha,Hunan,410007,China)

机构地区：[1]湖南省儿童医院耳鼻咽喉头颈外科,湖南长沙410007

出　　处：《中国耳鼻咽喉头颈外科》2020年第10期576-579,共4页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：湖南省卫生健康委员会资助项目(B2015-126)。

摘　　要：目的探究经聚醚酰亚胺(polyetherimide,PEI)表面修饰后的纳米羟基磷灰石载体介导脑源性神经营养因子(derived neurotrophic factor,BDNF)体外转染效率,表达产物对庆大霉素所致小鼠耳蜗螺旋神经节细胞(spiral ganglion cells,SGCs)细胞损伤的保护作用。方法构建经PEI表面修饰的纳米羟基磷灰石载体和BDNF基因真核表达质粒,体外培养并转染小鼠耳蜗SGCs细胞;分别采用实时定量PCR(real-time quantitative PCR,qRT-PCR)、蛋白质免疫印迹和细胞计数试剂盒kit-8(cell counting kit-8,CCK-8)细胞毒实验,检测体外培养小鼠耳蜗SGCs细胞及纳米羟基磷灰石载体介导BDNF基因的体外转染及表达效率,对耳毒性药物庆大霉素所致SGCs细胞损伤的保护作用。结果 DNA核酸电泳结果显示成功构建带有巨细胞病毒(cytomegalovirus,CMV)启动子的BDNF真核表达质粒;qRT-PCR和蛋白质免疫印迹结果显示与转染空质粒对照组相比,转染BDNF表达载体构建后SGCs原代细胞中BDNF的mRNA水平显著上调4.19倍(t=5.744,P<0.05),蛋白水平显著上调3.03倍(t=6.627,P<0.05),差异有统计学意义。此外,蛋白质免疫印迹和CCK8细胞毒实验结果表明,庆大霉素处理可显著抑制小鼠耳蜗SGCs细胞内BDNF基因表达,加剧细胞毒性,转染纳米羟基磷灰石载体介导qRT-PCR BDNF基因可明显逆转庆大霉素对SGCs的细胞毒作用,差异有显著统计学意义(t=10.015,P<0.01)。结论经PEI表面修饰的纳米羟基磷灰石载体可显著增强BDNF基因的跨膜能力,显著上调SGC细胞内BDNF表达产物的表达及对庆大霉素所致SGCs细胞损伤的保护作用,为内耳基因治疗的临床开展及应用提供实验和理论依据。OBJECTIVE To explore the protective effect of BDNF gene mediated by polyetherimide(PEI) surface-modified nano-hydroxyapatite vector in vitro and the protective effect of BDNF gene on gentamicin-induced SGCs cell damage in mice.METHODS A nano-hydroxyapatite vector modified with PEI surface and a BDNF gene eukaryotic expression plasmid were constructed,cultured and transfected into mouse cochlear SGCs cells in vitro.Real-time quantitative PCR(qRT-PCR),western blot and CCK8(cell counting kit-8) cytotoxicity experiments were used to detect the mouse cochlear SGCs cells cultured in vitro and nano HA vector-mediated transfection and expression efficiency of the BDNF gene,as well as protective effects oncell damage of SGCs caused by the ototoxic drug gentamicin.RESULTS DNA nucleic acid electrophoresis results showed that BDNF eukaryotic expression plasmid with CMV promoter was successfully constructed.The results of qRT-PCR and Western Blot showed that the mRNA level of transfection efficiency of mouse cochlear SGCs cells transfected with BDNF eukaryotic expression plasmid in vitro was increased by 4.19 times(t=5.744,P<0.05) and the protein level was increased by 3.03 times(t=6.627,P<0.05) compared with the control group transfected with empty plasmid,the difference was statistically significant.In addition,the results of Western Blot and CCK8 cytotoxicity experiments showed that gentamicin treatment can significantly inhibit the expression of BDNF gene in mouse cochlear SGCs cells,thereby aggravating cytotoxicity.Transfection of BDNF gene mediated by nano HA vector can significantly reverse the cytotoxic effect of gentamicin on SGCs.The difference was statistically significant(t=10.015,P<0.01).CONCLUSIONPEI surface modified nano-hydroxyapatite carrier can significantly enhance the transmembrane ability of BDNF gene,and then significantly up-regulate the expression of BDNF expression products in SGC cells and its protective effect on SGCs cell damage induced by gentamicin.The clinical development and application of gene

关 键 词：动物实验 小鼠 耳蜗 转染 纳米载体 脑源性神经营养因子基因 

分 类 号：R764[医药卫生—耳鼻咽喉科]