摘要
目的:检测重症肌无力脾气虚证患者酸负荷前后唾液淀粉酶(s AA)活性改变情况,并从s AA N-糖基化角度探讨重症肌无力脾气虚证s AA活性改变的机制。方法:收集36名健康(健康组)志愿者和30例重症肌无力脾气虚证患者(脾气虚组)酸负荷前后的唾液,Bernfeld法检测唾液s AA活性;Western blot法检测酸负荷前后唾液s AA蛋白表达,从糖基化蛋白表达条带的"有"或"无"判断s AA N-糖基化情况;凝集素亲和沉淀法检测s AA与刀豆凝集素(con A)结合率,以观察s AA高甘露糖型及杂合型糖链含量;凝集素亲和层析法检测s AA三种糖链(糖基化程度从高到低分别为复合型、杂合型、高甘露糖型)所占比例,以判断s AA糖基化程度。结果:与健康组比较,脾气虚组酸负荷前后s AA活性比值(后/前)降低(P<0.05);43.3%(13/30)的脾气虚患者在酸负荷前后均出现s AA糖基化蛋白条带缺失,高于健康组的13.3%(4/36,P<0.05);脾气虚组酸负荷前s AA与con A结合率高于健康组(P<0.05),提示其高甘露糖型及杂合型糖链所占比例增高;健康组弱结合峰与未结合峰(含复合型糖链)峰面积大于脾气虚组(P<0.05);脾气虚组强结合峰(以高甘露糖型及杂合型糖链为主)峰面积大于健康者组(P<0.05);提示脾气虚组s AA高甘露糖型及杂合型糖链构成比高于健康组。结论:N-糖基化不完全可能是重症肌无力脾气虚症患者s AA活性改变的主要影响因素之一。
Objective：The saliva amylase activity（sAA） ratio of before to after acid load is an important index for spleen dei ciency syndrome,and N-glycosylation of sAA is an important sign for sAA function.The purpose of this paper is to explore the mechanism underlying the activity changes of sAA in myasthenia gravis（MG） patients with syndrome of dei ciency of spleen qi based on N-glycosylation.Methods：1Saliva were sampled from 36 healthy volunteers and 30 MG patients with spleen dei ciency syndrome before and after acid load,and sAA activity were detected by Bernfeld method;2The expression of sAA N-glycosylation protein before and after acid load were detected by western blotting;3To observe the content of highmannose glycan chains and hybrid glycan chains in sAA,the combination rate of sAA and concanavalin A（con A） was detected via affinity precipitation.4The three main types of glycans（High-mannose,Hybrid,Complex） were detected by affinity chromatography with con A to assess the N-glycosylation level of sAA.Results：Compared with healthy group,the sAA ratio of before to after acid load significantly decreased in the spleen dei ciency syndrome group（P〈0.05）.There were 43.3%（13/30） in the spleen dei ciency syndrome group with sAA glycosylation bands dei cient,which was higher than the healthy group（13.3%,4/36,P〈0.05）.The combined ratio of sA A and conA in spleen def iciency syndrome group was higher than those of healthy group（P〈0.05）,suggesting the percentage of sA A High-mannose and Hybrid in spleen def iciency syndrome group increased.The peak area combined weakly and peak area unconjugated in healthy group were higher than those of spleen def iciency syndrome group（P〈0.05）;peak area combined strongly in spleen deficiency syndrome group was higher than those of healthy group（P〈0.05）,suggesting the percentage of High-mannose and Hybrid was higher than healthy group.Conclusion：Incomplete N-glycosylation may be one of main factors af fecting sAA activity.
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2016年第9期3516-3521,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.81403297)
广州中医药大学"青年英才培养工程"资助项目(No.QNYC20140114)
广州中医药大学优秀青年学者科研基金(No.2013-07)
财政部特色重点学科项目[No.财教(2013)339号]
华南中医药协同创新中心"中医药防治脾胃病脑病创新研究团队项目"(No.粤财教[2014]448号)~~
关键词
脾气虚证
重症肌无力
唾液淀粉酶
N-糖基化
Syndrome of deficiency of spleen qi
Myasthenia gravis
Ptyalin
N-glycosylation
