摘要
目的观察脑血管痉挛中缝隙连接蛋白Cx43的磷酸化位点及其$368位点磷酸化水平的变化．探讨其与脑血管痉挛的关系。方法新西兰大白兔78只按随机数字表法分为4组：正常对照组（n=6）、单纯脑池注血组（n=24）、甘珀酸（cBx）脑池处理组（n=24）和溶媒脑池处理组（n=24），后3组应用枕大池二次注血法建立兔蛛网膜下腔出血后脑血管痉挛模型及相应给药，并按1d、3d、7d、14d分成4亚组，每亚组6只。采用磷酸化蛋白富集试剂盒富集各组基底动脉中Cx43磷酸化总蛋白．再利用质谱技术鉴定出其磷酸化位点：应用Westernblotting方法分析各组Cx43$368位点磷酸化水平的变化；通过数字减影血管造影技术（DSA）观察各组基底动脉直径变化情况。结果（1）质谱技术成功鉴定出Cx43的4个磷酸化位点，分别为Y265、$364、$365、$368。（2）Westernblotting结果显示：正常对照组基底动脉Cx43$368位点磷酸化水平较低（17．0％±2-3％1；单纯脑池注血组及溶媒脑池处理组与正常对照组相比，Cx43S368位点磷酸化水平在ld、3d、7d、14d各时间点均显著升高，差异有统计学意义（尸〈0．05），且以7d表达最高，14d开始下降；CBX脑池处理组各时间点基底动脉Cx43$368位点磷酸化水平显著低于单纯脑池注血组及溶媒脑池处理组，差异有统计学意义（P〈0．05）。（3）DSA显示正常对照组第二次与首次造影基底动脉直径的百分比值平均为99．1％±1_3％，单纯脑池注血组、CBX脑池处理组、溶媒脑池处理组分别为66．1％±7．2％、91_3％±5.3％、63．7％±6．6％，CBX脑池处理组基底动脉直径显著狭窄于单纯脑池注血组．差异有统计学意义（P〈0．05）。结论缝隙连接蛋白Cx43$368位点磷酸化可能与脑血管痉挛密切相关，且其可能是CBX缓解脑血管痉挛的机制之一。
Objective To investigate the phosphorylation site ofconnexin Cx43 and the changes of $368 site phosphorylation levels in rabbits after cerebral vasospasm （CVS）. Methods Seventy-eight New Zealand rabbits were randomly assigned into four groups： healthy control group （n=6）, vehicle group （n=24）, earbenoxolone （CBX） treatment group （n=24） and menstruum treatment group （n=24）. Models of CVS after subara chnoid hemorrhage in the later three groups were established via double blood injection into the cisterna; medications were given, respectively, to each group, and according to the different observation times （1, 3, 7 and 14 d after the success of model making）, they were each divided into 4 subgroups （t~=6）. The phosphorylated Cx43 protein in the basilar arteries was gathered with PhosphoProtein Purification Kit, and mass spectrometric technique was employed to screen out its phosphorylated sites; Western blotting was used to analyze the changes of Cx43 phosphorylation levels with its $368 antibody. And then, digital subtraction angiography （DSA） was performed to observe the diameter changes of the basilar arteries. Results Four phosphorylation sites （Y265, S364.S365 and $368） in Cx43 were detected by mass spectrometry. As compared with that in the healthy control group, the $368 site phosphorylation level in the vehicle group and menstruum treatment group was significantly increased at each observation time point （P〈0.05）, reaching their peak level on the 7~ d of observation, and decreasing from the 14~ d of observation; the $368 site phosphorylation level in the vehicle group and menstruum treatment group was significantly higher than that in the CBX treatment group （P〈0.05）. Meanwhile, DSA showed that the CBX treatment group had significantly reduced CVS areas as compared with the vehicle group （P〈0.05）. Conclusion The phosphorylation of Cx43 $368 site may be associated with CVS, which might be one of the mechanisms of CBX releasing CVS.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2013年第4期330-334,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（81060099）
