CAJ | 학술논문

目的探讨钙调蛋白依赖性丝氨酸蛋白激酶(CASK)在氯化锂-匹罗卡品癫痫大鼠模型中不同时间点的表达及其参与癫痫发作的可能机制. 方法 30只SD大鼠按照随机数字表法分为对照组(n=5)和实验组(n=25),实验组在点燃后进一步按照随机数字表法分为1、3、7、14、30d5组(n=5).通过免疫组化、免疫荧光和Western blotting实验分别检测CASK在模型鼠海马组织中的表达变化.另将21只成年健康SD大鼠按照随机数字表法分为3组(n=7),分别为转染组、空病毒组、对照组,水合氯醛麻醉3组大鼠后分别注入等量的CASK-RNAi-LV、LV-scrRNAi和生理盐水,点燃1h内观察此3组大鼠行为学的改变,同时运用Western blotting检测转染组与对照组大鼠CASK复合物下游分子NMDAR2b在点燃后的表达. 结果 (1)免疫组化及荧光染色结果提示,CASK仅在神经元中表达,在神经胶质细胞中不表达;实验组CASK阳性细胞数明显多于对照组;且CASK在癫痫大鼠的海马组织中表达模式为1d降至最低,后逐渐增高,直到30 d.(2)转染模型相关实验结果提示,转染组(CASK表达被抑制)大鼠癫痫发作频率和发作级别(发作20 min后)均较空病毒组及对照组大鼠明显降低,差异有统计学意义(P＜0.05).对照组大鼠NMDAR2b在点燃1d后开始增高,3d达到高峰,10d下降;而转染组大鼠NMDAR2b在点燃后各个时间点的表达水平较对照组大鼠均明显降低,差异有统计学意义(P＜0.05). 结论 CASK在癫痫大鼠中的表达量明显增加,可能通过NMDAR2b途径影响癫痫的发生与发展.
목적 탐토개조단백의뢰성사안산단백격매(CASK)재록화리-필라잡품전간대서모형중불동시간점적표체급기삼여전간발작적가능궤제. 방법 30지SD대서안조수궤수자표법분위대조조(n=5)화실험조(n=25),실험조재점연후진일보안조수궤수자표법분위1、3、7、14、30d5조(n=5).통과면역조화、면역형광화Western blotting실험분별검측CASK재모형서해마조직중적표체변화.령장21지성년건강SD대서안조수궤수자표법분위3조(n=7),분별위전염조、공병독조、대조조,수합록철마취3조대서후분별주입등량적CASK-RNAi-LV、LV-scrRNAi화생리염수,점연1h내관찰차3조대서행위학적개변,동시운용Western blotting검측전염조여대조조대서CASK복합물하유분자NMDAR2b재점연후적표체. 결과 (1)면역조화급형광염색결과제시,CASK부재신경원중표체,재신경효질세포중불표체;실험조CASK양성세포수명현다우대조조;차CASK재전간대서적해마조직중표체모식위1d강지최저,후축점증고,직도30 d.(2)전염모형상관실험결과제시,전염조(CASK표체피억제)대서전간발작빈솔화발작급별(발작20 min후)균교공병독조급대조조대서명현강저,차이유통계학의의(P＜0.05).대조조대서NMDAR2b재점연1d후개시증고,3d체도고봉,10d하강;이전염조대서NMDAR2b재점연후각개시간점적표체수평교대조조대서균명현강저,차이유통계학의의(P＜0.05). 결론 CASK재전간대서중적표체량명현증가,가능통과NMDAR2b도경영향전간적발생여발전.
Objective To investigate the calcium/calmodulin-dependent serine protein kinase (CASK) expression in the hippocampus of epileptic rat models and its mechanism.Methods Thirty healthy adult male Sprague-Dawley rats were randomly divided into 6 groups (n=5):control group and experimental groups of 1,3,7,14 and 30 d after kindling by lithium chloride-pilocarpine;CASK expressions in the hippocampus tissues were measured by immunohistochemistry,immunoflurescence and Western blotting.Another 21 healthy adult male Sprague-Dawley rats were divided into 3 groups (n=7):transfected group,empty adenoviral group and control group,and after anesthesia with chloral hydrate,the same volume of CASK-RNAi-LV,LV-scrRNAi and normal saline was given,respectively;the behavior changes of rats in the three groups were observed within one h of kindling;the expression of N-methel-D-aspartate receptor subunit 2b (NMDAR2b,the downstream of CASK complex),in the transfected group and control group was further examined by Western blotting.Results CASK expressed only in neurons not gliacyte;and the number of CASK positive cells in experimental groups was larger than that in the control group.CASK expression in the hippocampus reduced to minimum level one d after kindling,and maintained at increased levels until 30 days.Both seizure frequency and seizure grade of the epilepsy in the transfected group were significantly decreased as compared with those in the empty adenoviral group and control group (P＜0.05).NMDAR2b expression in the hippocampus of control group increased at the first 24 h of kindling and maintained for 3 days,and then,it decreased 10 days after kindling;NMDAR2b expression in the hippocampus of transfected group was significantly reduced as compared with that in the control group at all time points of kindling (P＜0.05).Conclusions The CASK expression of epilepsy rat models is increased,which influences the occurrence and development of epilepsy.CASK is linked to epilepsy via its action on synaptic NMDAR2b,which may be the potential target of anti-epileptic drugs.