CAJ | 학술논문

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bel-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bel-2基因的腺病毒载体Ad-bel-2转染SGC,蛋白质印迹法(Western Blot)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2μg,/ml;顺铂作用48 h后,行各组SGC计数,并通过lmageJ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3 d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.bel-2蛋白过表达对顺铂所致SGC损伤有一定的拮抗作用.
목적 통과선병독(adenovirus,Ad)재체개도bcl-2기인전염체외배양적신생대서이와라선신경절세포(spiral ganglion cells,SGC),탐토bel-2단백과도표체대순박소치SGC손상적길항작용.방법 체외배양신생대서SGC,휴대록색형광단백(green fluorescent protein,GFP)기인적선병독재체Ad-GFP전염SGC,병행신경사단백(NF200)면역세포화학염색감정,격광공취초소묘형광현미경하관찰.휴대bel-2기인적선병독재체Ad-bel-2전염SGC,단백질인적법(Western Blot)검측bcl-2단백표체.설립Ad-bcl-2전염가순박조(A조),Ad-GFP전염가순박조(B조),순박조(C조)화정상대조조(D조),순박작용농도위2μg,/ml;순박작용48 h후,행각조SGC계수,병통과lmageJ연건측량각조SGC축돌장도.결과 성공분리병배양신생대서SGC.격광공취초소묘형광현미경하관찰도선병독재체가안전고효전염체외배양적SGC.Ad-bcl-2전염3 d후,Western Blot검측유외원성인bcl-2기인적고효표체,이Ad-GFP전염조화순박조미검측도표체.순박작용후,A、B、C조부분SGC세포돌기변단、위축,포체축소、변원,심지부기.A조SGC수목명현다우B조화C조(P치균＜0.01),단소우D조(P＜0.05);A조SGC축돌장도명현장우B조화C조,단단우D조(P치균＜0.01).결론 선병독능구안전고효지전염체외배양적신생대서SGC.bel-2단백과표체대순박소치SGC손상유일정적길항작용.
Objective To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated 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 immunocytochemical analysis for expression of the neuron-specific marker Neurofilament200(NF200)and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bel-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 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatmentonly and the untreated group. Cisplatin worked for 48 hours at a concentration of 2μg/ml. Outcome measures included survival humber of SGC and longest neurite length by using ImageJ software. Results SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-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 bel-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.