【作者】 徐鸥；

【导师】 路虹；

【作者基本信息】 河北医科大学 ， 耳鼻咽喉科学， 2004， 硕士

【摘要】 目的：应用听性脑干反应（Auditory brainstem response，ABR）、血清超氧化物歧化酶（superoxide dismutase,SOD）活性和丙二醛（MDA）含量、光镜及扫描电镜技术，观察铁螯合剂—去铁胺（Deferoxamine,DFO）和银杏叶提取物—金纳多（Extract of Ginkgo Biloba leaves Injection,EGb）联合应用对抗顺铂（Cisplatin,CDDP）耳毒性的作用。 方法：选成熟健康，耳廓反应灵敏，体重 350-400 克的白毛红目豚鼠 40 只，雌雄不限，随机分为 5 组，每组 8 只。分别为 CDDP 组、EGb 组、DFO 组、EGb+DFO 组及对照组。Ⅰ组（CDDP 组）：连续 5 天腹腔注射顺铂 2 mg.kg-1.d-1；Ⅱ组（EGb 组）：给顺铂前 2 天，腹腔注射 EGb14 mg·kg-1·d-1，自第 3 天起加腹腔注射顺铂 2 mg.kg-1.d-1，共 7 天；Ⅲ组（DFO组）：股内侧皮下注射去铁胺 100 mg.kg-1.d-1，注射 1 小时后腹腔注射顺铂 2 mg.kg-1.d-1，共 5 天；Ⅳ组（EGb+DFO 组）：给顺铂前 2 天，腹腔注射金纳多 14 mg.kg-1.d-1，自第 3 天起，加股内侧皮下注射去铁胺 100 mg.kg-1.d-1，注射 1 小时后腹腔注射顺铂 2 mg.kg-1.d-1，共 7 天；Ⅴ组（对照组）：连续 5天腹腔注射生理盐水 2 ml.kg-1.d-1。给药前后分别测定各组动物听性脑干反应。停药后 1 天，采集动物血清并以断头法处死动物。测定各组动物血清 SOD 活性和 MDA 含量。光镜标本制作: 各组动物于停药后 1 天以断头法处死动物，快速取出听泡，暴露耳蜗，蜗顶钻孔，取出镫骨，挑破卵圆窗、蜗 1<WP=4>中 文 摘 要窗,用吸管以４%多聚甲醛（4℃，PH=7.4）,缓慢灌洗耳蜗。然后将其浸于固定液中，次日取出，以０.1MPBS(PH=7.4)清洗，置于 10%EDTA 中，脱钙 20 天，PBS 清洗，石蜡包埋，平行蜗轴切片，行苏木精-伊红染色及骨髓铁染色，光镜观察。扫描电镜标本制作：各组于停药后１天以断头法处死动物，快速取出听泡，暴露耳蜗，蜗顶钻孔，取出镫骨，挑破卵圆窗、蜗窗，用吸管以 2.5%戊二醛（4℃，PH=7.4）缓慢灌洗耳蜗。然后将其浸于固定液中，次日取出，以０.1MPBS(PH=7.4)清洗，在解剖显微镜下剥去耳蜗骨壳及螺旋韧带，暴露基底膜，1%四氧化锇后固定 2 小时；０.1mol/l磷酸缓冲液漂洗；2%单宁酸 30 分钟，2 次；０.1mol/l 磷酸缓冲液漂洗 1 小时；梯度乙醇脱水；醋酸异戊酯过度；临界点干燥；离子溅射镀膜；扫描电镜观察。采用统计学方法分析处理数据并观察用药前后耳蜗毛细胞形态学变化。 结果：CDDP 组动物 ABR 阈值较其它各组明显升高（P<0.01），EGb 组动物 ABR 阈值与对照组差异有显著性（P<0.05），与其余各组间差异不显著（P>.05）。DFO 组和 EGb+DFO 组与对照组及 EGb 组差异不显著（P>0.05）。血清 SOD 活性和 MDA 含量检测表明，CDDP 组血清 SOD活性较对照组明显降低（P<0.01），其它各组较对照组则差异不显著（P>.05）。血清 MDA 含量以 CDDP 组升高最显著（P<0.01），其余各组较对照组略有升高，其差异没有显著性（P>.05）。光镜观察，CDDP 组耳蜗 Corti 器严重变形,外毛细胞肿胀、变形、移位、内外隧道增宽,严重者,毛细胞溶解,核部分或全部消失, Corti 器结构破坏,病变以 2、4 回为重。EGb 组动物 Corti 器变形外毛细胞肿胀、变形、移位, 2<WP=5>中 文 摘 要间隙增宽,程度比 CDDP 组轻。DFO 组及 EGb+DFO 组动物Corti 器结构基本正常或稍变形，毛细胞结构大致正常。对照组动物 Corti 器结构正常，毛细胞完整无移位。此外在 CDDP组耳蜗螺旋韧带及蜗轴中可见较多的铁颗粒和少数小块，EGb 组耳蜗蜗轴中也有少量铁颗粒，而 DFO 组和 DFO+EGb组耳蜗螺旋韧带及蜗轴中未见含铁血黄素沉积，提示 DFO可减轻顺铂的骨髓抑制副作用。耳蜗扫描电镜显示 CDDP 组动物耳蜗各回外毛细胞均有不同程度破坏，以底回和第二回为最重，外毛细胞缺失、倒伏、粘连，内毛细胞亦有不同程度破坏。EGb 组动物耳蜗各回外毛细胞亦有散乱、倒伏、缺失，但比 CDDP 组略轻，内毛细胞轻度受损。DFO 组与EGb+DFO 组外毛细胞偶有倒伏、散乱，内毛细胞基本排列正常，与 CDDP 组相比受损明显减轻，听毛呈Ｖ字形，开口向内，整齐排列为三排，内毛细胞的听毛排列为一排。 结论：铁螯合剂（DFO）与银杏叶提取物（EGb）合用，可有效减轻 CDDP 的耳毒性作用，并可能减轻顺铂的骨髓抑制作用。其效果优于单独应用金纳多，但与单独应用去铁胺效果未见明显差异。结果提示铁离子参与的自由基反应可能是顺铂耳毒性机制之一。更多还原

【Abstract】 Objective ： The protective effect of iron chelator(Deferoxamine,DFO) and Extract of Ginkgo Biloba leavesInjection (EGb) against ototoxicity of cisplatin (CDDP) wasstudied by the test of auditory brainstem response (ABR),thevitality of superoxide dismutase (SOD) in serum,the content ofthe metabolite of lipid peroxidation (MDA) in serum, andobservesion of morphological change in hair cells with lightmicroscope,scanning electron microscope. Methods ： Forty purebred guinea pigs which weremature,healthy,sensitive to auricle reflex,300-400 gram.The sexof them was not limited.They were randomly divided into fivegroups.Each group had eight guinea pigs.They were CDDPgroup,EGb group,DFO group,EGb+DFO group and controlgroup.GroupⅠreceived 2 mg·kg-1·d-1 CDDP transperitoneallyfor five days.Group Ⅱ received 14 mg·kg-1·d-1 EGb twodays before 2 mg·kg-1·d-1 CDDP transpeiitoneally for sevendays.Group Ⅲ received 100 mg · kg-1 · d-1 DFO bysubcutaneous injection on the bosom of thigh one hour before 2mg·kg-1·d-1 CDDP transpritoneally for five days.Group Ⅳreceived 14 mg·kg-1·d-1 EGb two days before DFO andCDDP.Then it was added 100 mg·kg-1·d-1 DFO one hour before 4<WP=7>英 文 摘 要2 mg · kg-1 · d-1 CDDP for five days.Group Ⅴ recivedphysiological saline(0.9%) transpritoneally for fivedays.Auditory threshold was tested before the experiment andafter it.The next day,blood samples were obtained and all theanimals were sacrificed. The vitality of superoxide dismutase(SOD) and the content of the metabolite of lipid peroxidation(MDA) were tested in serum. Cochleas were stripped and fixedby fixing solution.Light microscope observation:killedanimals,segregated otocyst from temporal bone quickly,exposedcochlea,bored apical cochlea,took stapes out,unpacked ovoidwindow and round window,poured cochlea with 4%Polyoxymethylene (4 ℃ ,PH=7.4) and fixed in it for oneday,washed with 0.1M PBS (PH=7.4),decalcificated with 10%Calcium Disodium Versenate for 20 days,washed withPBS;embedded with petroline,used paraffin sectioning methodwhich direction was paralled with modiolus,usedhaematine-eosin stain and marrow iron stain,observed with lightmicroscope. Scanning electron microscope observation:killedanimals,segregated otocyst from temporal bone quickly,exposedcochlea,bored apical cochlea,took stapes out,unpacked ovoidwindow and round window,poured cochlea with 2.5% GlutaricDialdehyde (4℃,PH=7.4) and fixed in it for 4-6 hours.Cochleashell and spiral ganglion were stripped and basilar membranewas exposed thoroughly under anatomical microscope.Theywere postfixed with 0.1% Osmium tetraoxide for 2hours;washed with 0.1 mol/l PBS for 1 hour, dehydrated 5<WP=8>英 文 摘 要gradiently with alcohol,transited with acetate isoamyl ester,driedat critical point,sputtered with golden ion,observed withSEM.Data were analysized by SPSS. Results ： the hearing threshold of ABR was highersignifitcantly in CDDP group than in other groups (P<0.01) Thedifference of auditory threshold value between EGb group andcontrol group was significant (P<0.05).And the difference ofauditory threshold betweenⅡ，Ⅲ，Ⅳ groups and control groupwere not significant (P>0.05).The SOD in serum showed thatdifference between CDDP group and other groups wassignificant(P<0.01),while insignificant among othergroups(P>.05).The vitality of SOD was decreased obviously inCDDP group.The content of the metabolite of lipid peroxidation(MDA) in cochlea was higher significantly (P<0.01) in CDDPgroup than that in other groups.But the content of MDA in othergroups were not raised significantly (P>0.05).Light microscopeshowed that the spiral organ in CDDP group was distortedserviously.The outer hair cells swelled,modificated,displacedand the corti tunnel was broadened.Further more ,lytic necrosishappe更多还原