摘要
目的观察顺铂诱导大鼠耳蜗螺旋神经节细胞（spiral ganglion cell，SGC）死亡的模式，探讨凋亡相关基因BNIP3在顺铂诱导神经元死亡中的作用。方法体外分离培养大鼠耳蜗基底膜SGC，加入100μmol／L的顺铂建立损伤模型。通过实时定量聚合酶链反应（PCR）检测SGC凋亡相关基因BNIP3的表达情况；采用蛋白印迹杂交（Western blot）检测半胱氨酸蛋白酶蛋白-3（caspase-3）特异降解产物et血影蛋白（α-spectrin）的表达，间接确定caspase-3活性；采用免疫荧光技术，观察BNIP3蛋白的表达定位及其与顺铂耳毒性损伤的关系。结果顺铂作用后12h，细胞形态变小，神经纤维出现结节和断裂，神经丝抗体（NF-200）标记阳性的细胞数目减少。离体培养的SGC在顺铂作用3h，6h和12h，BNIP3基因转录活性明显增加，与对照组相比差异具有统计学意义（P值均〈0．05）。Westernblot结果显示，对照组中α-spectron降解带灰度值比值（X±s，下同）为0．10±0．05，顺铂作用6h、12h后该比值分别上升为0．49±0．09和0．75±0．08，与对照组相比差异具有统计学意义（q：8．63及14．61，P值均〈0．01）；同时顺铂作用12h组与顺铂作用6h组相比，差异也具有统计学意义（q=5．98，P〈0．05）。正常SGC不表达或在细胞质中微弱表达BNIP3蛋白；顺铂损伤后BNIP3蛋白表达明显增强，并且强表达的BNIP3蛋白定位于SGC细胞核，周围的支持细胞中BNIP3蛋白的表达也明显增高，但表达定位于细胞质而不是细胞核。结论顺铂诱导大鼠耳蜗SGC凋亡过程中，BNIP3在神经元及其周围的支持细胞中可能遵循不同的信号传导途径，在SGC中BNIP3可能是通过结合DNA，在转录水平调节某些凋亡相关基因的表达从而诱导细胞凋亡。
Objective To observe spiral ganglion cell（SGC） death pattern caused by cisplatin and investigate pro-apoptotic protein BNIP3 involve ment in SGC death. Methods Cochlear SGC were isolated from the neonatal rats and cultured in vitro. A cochleas insult model were induced by treatment of 100 μmol/L cisplatin. Real-time PCR were used to determine expression of apoptosis related gene in neonatal rat cochlear cultures after cisplatin treatment. Western blotting was used to detect α-spectrin and indirectly determine caspase-3 activity. Double immunohischemical staining method was performed to indicated the localization and expression of BNLP3 and NF-200. Results Morphological finding showed that SGC were smaller, and neurofiber were blebbing and broken at treatment of cisplatin for 12 h. NF-200 marker positive cell number decreased. The transcription level of BNIP3 in cisplatin treatment for 3 h,6 h and 12 h was higher than the control group （P 〈 O. 05 ）. Western blotting results showed that 120 000 of breakdown products of α-spectrin relative gray level were O. 10±0. 05 in the control group, 0. 49 ±O. 09 and 0. 75 ± 0. 08 in cisplatin treatment for 6 h and 12 h group. It increased significantly in the group of cisplatin treatment for 6 h and 12 h than the control group （q =8.63 and 14. 61 ,P 〈0. 01 ）. When compared between 6 h of cisplatin treatment and 12 h group, significant difference was detected （q =5. 98,P 〈0.05）. Therewas weak BNIP3 positive expression in cytoplasm of the control group. However, strongly BNIP3-positive labeled were seen in the nucleus of SGC and cytoplasm of some stromal ceils around SGC after cisplatin treatment. Conclusions BNIP3 played an important role in cisplatin induced SGC death and followed independent signaling transduction pathway that differ from stromal cells around SGC. It may suggest that BNIP3 enter nucleus to bind DNA and uo-reaaalate aooototic ~ene exnression to nromote cells death.
出处
《中华耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2011年第3期214-219,共6页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
国家自然科学基金（30873131）
吉林省科技厅资助项目（200805110）
