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GPER通过cAMP/PKA途径调节衰老血管纹周细胞上BKCa通道的研究
GPER Regulates BKCa Channel on Senile Vascular Pericytes by cAMP/PKA Pathway
【作者】 周颖；
【导师】 李丽；
【作者基本信息】 石河子大学 ， 医学生理学， 2019， 硕士
【摘要】 目的:以豚鼠耳蜗血管纹PCs为研究对象,探讨雌激素受体GPER对耳蜗PCs上BKCa的表达和功能的调节及其与年龄相关性听力损伤的关系。方法:动物实验:选取8周龄健康豚鼠随机分为Control组、D-半乳糖衰老模型组（D-galactose,D-gal）和D-gal+G1组。采用Morris水迷宫检测各组豚鼠行为的变化;用紫外分光光度法检测各组豚鼠不同组织中SOD活性和MDA含量;用听性脑干反应（auditory brainstem response,ABR）检测不同组豚鼠听力的变化;用免疫荧光技术检测血管纹PCs上BKCa和EPER表达的改变;用膜片钳技术检测GPER对PCs上BKCa电流的影响。细胞实验:在无菌条件下提取豚鼠血管纹周细胞进行培养,实验分为Control、D-gal、D-gal+G1、D-gal+G1+SQ22586和D-gal+G1+H89组。采用免疫荧光技术和Western技术检测GPER激动剂G-1对PCs上BKCa和cAMP/PKA表达的改变;用膜片钳技术检测GPER对PCs上BKCa电流的影响。结果:动物实验:（1）Morris水迷宫行为测试显示D-gal组豚鼠的空间学习能力和记忆有损伤;SOD活性和MDA含量检测结果显示D-gal组存在氧化应激损伤;（2）与Control组相比,D-gal组ABR阈值升高且Ⅰ波振幅明显降低（P<0.01）,D-gal+G1组较D-gal组相比,ABR阈值减低且Ⅰ波振幅升高（P<0.01）;（3）与Control组相比,D-gal组血管纹PCs上GPER表达明显减少（P<0.01）,D-gal+G1组较D-gal组相比没有差异;（4）与Control组相比,D-gal组血管纹PCs上BKCa表达明显减少（P<0.01）,G1干预后,血管纹PCs上BKCa表达升高;（5）与Control组相比,D-gal组血管纹PCs的电流密度和BKCa净电流均明显减小（P<0.01）,G1干预后,血管纹PCs的电流密度和BKCa净电流均增大（P<0.01）;细胞实验:（6）与Control组相比,D-gal组PCs上BKCa表达明显减少（P<0.01）,G1干预后,PCs上BKCa表达明显升高,但给予cAMP阻断剂（SQ22586）和PKA阻断剂（H89）后可以部分抑制G-1对PCs上BKCa表达的上调;（7）与Control组相比,D-gal组PCs的电流密度和BKCa净电流均明显减小（P<0.01）,D-gal+G1组较D-gal组相比,PCs的电流密度和BKCa净电流均增大（P<0.01）,但给予cAMP阻断剂（SQ22586）和PKA阻断剂（H89）后可以部分抑制了G-1对PCs上BKCa电流密度的上调。结论:GPER可能激活cAMP/PKA信号传导途径上调血管纹周细胞上BKCa表达和功能,从而发挥对年龄相关性听力损伤的保护作用。
【Abstract】 Objective:The guinea pig cochlear vascular striate PCs were used as the research object to investigate the relationship between regulation of expression and function of BKCa on PCs by estrogen receptor GPER and age-related hearing impairment.Methods:Animal experiments:8 week old healthy guinea pigs were randomly divided into Control group,D-galactose model group（D-galactose,D-gal）and D-gal+G1 group.Morris water maze was used to detect the changes of behavior in different groups of guinea pigs.UVD spectrophotometry was used to detect the activity of SOD and MDA in different tissues of guinea pigs.The auditory brainstem response（ABR）was used to detect the changes of hearing in different groups of guinea pigs.The expression of BKCa and EPER on vascular striate PCs was detected by immunofluorescence technique.The effect of GPER on BKCa current on PCs was detected by patch clamp technique.Cell experiments:primary cultured guinea pig cochlear vascular pericytes,the experiment was divided into Control,D-gal,D-gal+G1,D-gal+G1+SQ22586 and D-gal+G1+H89 groups.The expression of BKCaa and cAMP/PKA on PCs was detected by immunofluorescence technique and Western technique.The effect of GPER on BKCa current on PCs was detected by patch clamp technique.Results:（1）Morris water maze behavior test showed that the spatial learning ability and memory of D-gal group were damaged.The results of SOD activity and MDA content showed oxidative stress damage in D-gal group;（2）compared with Control group The ABR threshold was increased and the amplitude of I wave was significantly decreased in the D-gal group（P<0.01）.The ABR threshold was decreased and the amplitude of the I wave was increased in the D-gal+G1 group compared with the D-gal group（P<0.01）.3)Compared with the Control group,the GPER expression in the D-gal group was significantly decreased（P<0.01）,and the D-gal+G1 group was not different from the D-gal group;（4）Compared with the Control group,the expression of BKCa was significantly decreased in the D-gal group（P<0.01）.After G1 intervention,the expression of BKCa in the vascular striate PCs was increased.（5）Compared with the Control group,The net current of BKCa in the vascular striate PCs was significantly reduced in the D-gal group（P<0.01）.After G1intervention,the net current of BKCa in vascular striate PCs increased（P<0.01）.（6）Compared with the Control group,the expression of BKCa in PCs of D-gal group was significantly decreased（P<0.01）.After G1 intervention,the expression of BKCa in PCs was significantly increased,but cAMP blocker was given.（SQ22586）and PKA blocker（H89）can partially reverse the up-regulation of BKCa on PCs by G-1;（7）Compared with Control group,the net current of BKCa in PCs of D-gal group is significantly reduced（P<0.01）,compared with the D-gal group,the net current of BKCa in PCs increased in the D-gal+G1 group（P<0.01）.But after cAMP blocker（SQ22586）and PKA blocker（H89）were administered,the up-regulation of BKCa current density on PCs can be partially inhibited by G-1.Conclusions:GPER may activate the cAMP/PKA signaling pathway to up-regulate the expression and function of BKCa on vascular pericytes,thereby exerting a protective effect on age-related hearing impairment.