质膜钙ATP酶异构体1～3在新生大鼠耳蜗基底膜的表达  

The Expression of Plasma Membrane Ca2＋-ATPase Isoforms 1~3in the Basilar Membrane of Neonatal Rat Cochlea

作　　者：陈请国[1] 褚汉启[1] 周良强[1] 陈金[1] 刘云[1] 罗蜜[2] 陶雁玲[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院耳鼻咽喉-头颈外科,武汉430030 [2]湖北文理学院附属襄阳市中心医院耳鼻咽喉-头颈外科

出　　处：《听力学及言语疾病杂志》2016年第4期366-370,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金青年基金(81300827)资助

摘　　要：目的通过Western blot方法检测质膜钙ATP酶异构体1～3（plasma membrane Ca2＋-ATPase isoforms 1～3,PMCA1～3）在新生大鼠耳蜗基底膜（basilar membrane,BM）的表达,并检测PMCA2在单个新生大鼠耳蜗毛细胞纤毛的表达量。方法 1取出生后2天（P2）和8天（P8）健康SD大鼠各4只,断头后分离出BM,提取总蛋白,分别取20μg,通过Western blot法检测BM的PMCA1～3表达。2取P2和P8大鼠BM总蛋白3μg,通过Western blot方法检测BM的PMCA2的表达。3取4只P8大鼠,分离出BM,提取总蛋白,分别取5、10、20μg总蛋白并取100、400和800ng牛血清白蛋白（BSA）作为对照组,跑完电泳后将胶分为2部分,部分泳道用于SYPRO染胶,其余泳道用于Western blot检测PMCA2的表达。结果 1在20μg的P2和P8大鼠BM总蛋白中,PMCA1仅有微弱的表达（分别为0.126±0.024、0.131±0.012）,PMCA2表达水平较高（分别为4.16±0.528、4.25±0.319）,PMCA3几乎没有表达（均为0）,三者间差异有统计学意义（P〈0.05）。2在3μg的P2和P8大鼠BM总蛋白中,P8大鼠PMCA2的表达水平（4.571±0.336）高于P2大鼠（3.622±0.285）,差异有统计学意义（P〈0.05）。3PMCA2在单个P8大鼠BM的含量约为2.5ng。结论 PMCA1～3在新生大鼠耳蜗BM的表达水平不同,PMCA2表达量最大,这可能是由于耳蜗毛细胞对这几种PMCA亚型有不同的Ca2＋调节需求。Objective To study the expression of plasma membrane Ca2＋-ATPase isoforms 1~3（PMCA1~3）in the basilar membrane（BM）of the neonatal rat cochlea by Western blot.The PMCA2 content in single BM of the neonatal rat was also examined.Methods Four rats at postnatal 2days（P2）and 8days（P8）were respectively decapitated and their BMs were isolated.The total proteins of BMs were extracted.The 20μg total proteins were respectively loaded to the gel.The expression of PMCA1-3was detected by Western blot.Likewise,3μg total proteins from P2 and P8rat BM were loaded.The expression of PMCA2 was detected by Western blot.Four rats at P8 were decapitated and their BM was isolated.The 5μg,10μg and 20μg total proteins of P8 rat BM were added to the gel and 100 ng,400ng and 800 ng bovine serum albumin（BSA）were also loaded as reference.After electrophoresis,the gel was separated into two parts.One part was used for SYPRO staining and the other part was used for PMCA2 detection by Western blot.Results In the 20μg BM total proteins of P2 and P8rats,the expression of PMCA1 was weak（0.126±0.024,0.131±0.012,respectively）,PMCA2 was strong（4.16±0.528,4.25±0.319,respectively）,and PMCA3 was barely expressed（0）.There was a statistical difference among PMCA1,PMCA2 and PMCA3（P〈0.05）.In the 3μg BM total proteins of P2 and P8rats,the expression of PMCA2 in P8（4.571±0.336）was higher than P2（3.622±0.285）.There was a statistical difference（P〈0.05）.The PMCA2 content in the BM of a P8 rat was about 2.5ng.Conclusion There was a different-level expression of PMCA1~3in the neonatal rat BM with highest expression of PMCA2,which could be explained that cochlear hair cells had different requirements of Ca2＋turning for these PMCA isoforms.

关 键 词：质膜钙ATP酶异构体1 质膜钙ATP酶异构体2 质膜钙ATP酶异构体3 基底膜 耳蜗 

分 类 号：R339.16[医药卫生—人体生理学]