顺铂对离体培养小鼠耳蜗毛细胞凋亡及calpain表达的影响

发布时间：2018-04-21 04:17

本文选题：顺铂 + 耳蜗　；参考：《辽宁医学院》2011年硕士论文

【摘要】：目的建立新生昆明小鼠离体顺铂耳毒性模型,研究顺铂对离体培养小鼠耳蜗毛细胞凋亡和钙蛋白酶(calpain)表达的影响,为进一步探讨顺铂的耳毒性机制及其防护办法提供实验依据。方法分离生后3～5 d的昆明小鼠耳蜗基底膜,在含有1%牛血清白蛋白的Dulbecco改良Eagle培养基/ F12(Dulbecco's Modified Eagle Media: Nutrient Mixture F-12,DMEM / F12)中培养24h,然后弃去原培养基。对照组加入2ml新鲜培养基,顺铂组分别加入含有不同浓度顺铂(4μg/ml、8μg/ml、16μg/ml和64μg/ml )的新鲜培养基,继续培养24h。采用异硫氰酸四甲基罗丹明(tetramethylrhodamine isothiocyanate,TRITC)标记的鬼笔环肽荧光染色方法观察小鼠耳蜗毛细胞的形态改变,耳蜗毛细胞计数检测毛细胞缺失情况;同时应用Hoechst 33258荧光染色方法观察耳蜗毛细胞的凋亡,耳蜗凋亡毛细胞计数检测毛细胞凋亡率;并采用免疫荧光染色和免疫印迹技术检测calpain 1(μ-calpain)和calpain 2(m-calpain)在小鼠耳蜗毛细胞的表达。结果 1. TRITC-鬼笔环肽荧光染色结果显示,对照组耳蜗内毛细胞(inner hair cell,IHC)和外毛细胞(outer hair cell,OHC)均结构完好,纤毛排列有序。顺铂组小鼠耳蜗毛细胞出现纤毛倒伏、排列紊乱等形态改变。耳蜗毛细胞计数结果显示,当顺铂浓度为4μg/ml时,IHC和OHC缺失率均明显增大,与对照组比较有显著性差异(P0.01);并且随着顺铂浓度的增高,IHC和OHC缺失率亦显著增大,呈现明显的量效关系(P0.01)。 2. Hoechst 33258荧光染色结果显示,对照组小鼠耳蜗毛细胞的细胞核呈圆形,淡蓝色,核内染色质分布均匀。顺铂组小鼠耳蜗毛细胞的细胞核则出现固缩,核内染色质由于浓集而呈亮蓝色,核呈分叶、碎片状;并且随着顺铂浓度的增高,致密浓染和破碎的细胞核亦显著增多。耳蜗凋亡毛细胞计数结果显示,当顺铂浓度为4μg/ml时,毛细胞凋亡率明显增大,与对照组比较有显著性差异(P0.01);并且随着顺铂浓度的增高,毛细胞凋亡率亦显著增大,呈现明显的量效关系(P0.01)。 3. Calpain免疫荧光染色结果显示,μ-calpain和m-calpain在对照组耳蜗毛细胞均呈微弱红色荧光;不同浓度顺铂组耳蜗毛细胞μ-calpain和m-calpain红色荧光均较对照组有所增强,但顺铂组间μ-calpain荧光强度基本相同,而m-calpain荧光则随顺铂浓度的增高而明显增强。 4. Calpain免疫印迹检测结果显示,对照组有μ-calpain和m-calpain的微弱表达;不同浓度顺铂组μ-calpain和m-calpain的表达均较对照组明显增强(P0.01),但顺铂组间μ-calpain的表达无显著性差异,而m-calpain的表达则随顺铂浓度的增高而明显增强(P0.01)。结论应用耳蜗螺旋器离体培养技术可以建立新生昆明小鼠离体顺铂耳毒性模型;顺铂可通过凋亡机制对小鼠耳蜗毛细胞造成损伤,并且顺铂可影响calpain在离体培养小鼠耳蜗毛细胞的表达,随着顺铂浓度的增高,m-calpain的表达亦逐渐增强,提示顺铂可通过calpain通路诱导小鼠耳蜗毛细胞的凋亡,进一步证实了凋亡可能是顺铂的耳毒性机制之一。
[Abstract]:Purpose

To study the effect of cisplatin on apoptosis and calpain expression of cochlear hair cells in vitro and to provide experimental basis for further exploring the mechanism of ototoxicity of cisplatin .

method

The cochlear basement membrane was cultured in Dulbecco ' s Modified Eagle Medium / F12 ( Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media : Dulbecco ' s Modified Eagle Media : Fluorescence Mixture F - 12 , DMEM / F12 ) for 24 h .

Results

1 . The results showed that both the inner hair cell ( IHC ) and outer hair cell ( OHC ) of the control group were intact and the ciliary arrangement was orderly . When the concentration of cisplatin was 4 渭g / ml , the level of IHC and OHC deletion increased significantly ( P0.01 ) , and the IHC and OHC deletion rate increased significantly with the increase of cisplatin concentration ( P0.01 ) .

2 . The results showed that the nuclei of the cochlear hair cells in the control group were round , pale blue and homogeneous in the nucleus . The nuclei of the cochlear hair cells in the cisplatin group were significantly increased . The apoptosis rate of the hair cells increased significantly with the increase of cisplatin concentration ( P0.01 ) .

3 . Calpain immunofluorescence staining showed that 渭 - calpain and m - calpain were in weak red fluorescence in the control group . The fluorescence intensity of 渭 - calpain and m - calpain in the cochlear hair cells in the cisplatin group was significantly higher than that in the control group , but the fluorescence intensity of 渭 - calpain in the cisplatin group was almost the same , while the m - calpain fluorescence increased obviously with the increase of the platinum concentration .

4 . Calpain Western blot showed that the expression of 渭 - calpain and m - calpain in the control group was significantly higher than that in the control group ( P0.01 ) , but the expression of 渭 - calpain and m - calpain in the cisplatin group was significantly higher than that in the control group ( P0.01 ) .

Conclusion

It is suggested that cisplatin can induce apoptosis of cochlear hair cells in mice by calpain pathway , and it is suggested that cisplatin can induce apoptosis of cochlear hair cells in mice by calpain pathway , and it is further confirmed that apoptosis may be one of the ototoxicity mechanisms of cisplatin .

【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.43

【参考文献】