摘要
目的通过腺病毒（adenovirus，Ad）载体介导bcl-2基因转染体外培养的新生大鼠耳蜗螺旋神经节细胞（spiral ganglion cells，SGC），探讨bcl-2蛋白过度表达对顺铂所致SGC损伤的拮抗作用。方法体外培养新生大鼠SGC，携带绿色荧光蛋白（green fluorescent protein，GFP）基因的腺病毒载体Ad—GFP转染SGC，并行神经丝蛋白（NF200）免疫细胞化学染色鉴定，激光共聚焦扫描荧光显微镜下观察。携带bcl-2基因的腺病毒载体Ad—bcl-2转染SGC，蛋白质印迹法（WesternBlot）检测bcl-2蛋白表达。设立Ad—bcl-2转染加顺铂组（A组），Ad．GFP转染加顺铂组（B组），顺铂组（C组）和正常对照组（D组），顺铂作用浓度为2ug/ml；NSt3作用48h后，行各组SGC计数，并通过ImageJ软件测量各组SGC轴突长度。结果成功分离并培养新生大鼠SGC。激光共聚焦扫描荧光显微镜下观察到腺病毒载体可安全高效转染体外培养的SGC。Ad—bcl-2转染3d后，Western Blot检测有外源性人bcl-2基因的高效表达，而Ad—GFP转染组和顺铂组未检测到表达。顺铂作用后。A、B、C组部分SGC细胞突起变短、萎缩，胞体缩小、变圆，甚至浮起。A组SGC数目明显多于B组和C组（P值均〈0．01），但少于D组（P〈0．05）；A组SGC轴突长度明显长于B组和C组，但短于D组（P值均〈0．01）。结论腺病毒能够安全高效地转染体外培养的新生大鼠SGC。bcl-2蛋白过表达对顺铂所致SGC损伤有一定的拮抗作用。
Objective To assess the protection against cisplatin-induced ototoxicity by adenovirusmediated overexpression of the bcl-2 gene in cultured spiral ganglion cells（SGC）. Methods SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene （Ad- GFP） , followed by immunocytochemieal analysis for expression of the neuron-specific marker Neurofilament 200（NF200） and detection under laser scanning confoeal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups： the group of Ad-bcl-2 transfeetion followed by cisplatin treatment, the group of Ad-GFP transfectian followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 ug/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software. Results SGC were cultured successfully in vitro and transfected by adenovims vector safely and efficiently. By Western Blot, human bel-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad- GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration. Conclusions Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.
出处
《中华耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2009年第11期930-934,共5页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
国家自然科学基金（30471879）
北京市自然科学基金（7082024）
北京市教育委员会科技发展计划项目（KM200810025004）
