CAJ | 학술논문

目的动态观察Nav1.2、Nav1.6在氯化锂-匹罗卡品癫痫模型大鼠海马CA3区的表达变化,探讨两者在癫痫发病机制中的可能作用.方法采用随机数字表法,将90只健康雄性SD大鼠随机分为实验组(n=45)和对照组(n=45),实验组予以腹腔注射氯化锂-匹罗卡品致痫,对照组予生理盐水假处理.根据时间点又分为3个亚组:24 h(n=15)组(n=15)、7d组(n=15)和60 d组(n=15),采用随机数字表法将每亚组再随机分为3个小组(n=5)分别进行免疫组化、Western印迹及RT-PCR方法检测Nav1.2、Nav1.6在大鼠海马CA3区的动态表达.结果 (1)免疫组化检测示实验24 h组海马CA3区Nav1.2蛋白表达相对对照组差异无统计学意义(P=0.492),而实验组Nav1.6蛋白表达(0.398±0.019)较对照组(0.313 ±0.017)显著升高,差异具有统计学意义(P =0.034);实验7d组海马CA3区Nav1.2和Nav1.6蛋白相对对照组差异无统计学意义(P=0.157,0.109);实验60 d组Nav1.2蛋白表达(0.117±0.009)较对照组(0.155±0.010)显著降低,差异具有统计学意义(P =0.002),而实验组Nav1.6蛋白表达(0.400±0.009)较对照组(0.318±0.010)显著升高,差异具有统计学意义(P=0.018).(2) Western印迹检测示实验24 h组海马CA3区Nav1.2蛋白表达较对照组差异无统计学意义(P=0.472),实验组Nav1.6蛋白表达(0.419±0.027)较对照组(0.290±0.007)显著升高,差异具有统计学意义(P=0.001);7 d组Nav1.2和Nav1.6蛋白表达较对照组差异无统计学意义(P=0.517,0.514);60 d组实验组Nav1.2蛋白表达(0.209 ±0.077)较对照组(0.339±0.080)明显降低,差异具有统计学意义(P=0.024),实验组Nav1.6蛋白表达(0.772±0.029)较对照组(0.489±0.014)显著升高,差异具有统计学意义(P=0.00I).(3)RT-PCR检测示实验24 h组海马CA3区Nav1.2 mRNA表达较对照组差异无统计学意义(P =0.453),实验组Nav1.6mRNA表达(2.250±0.117)较对照组(0.998±0.139)显著升高,差异具有统计学意义(P=0.001);7d组Nav1.2和Nav1.6 mRNA表达较对照组差异无统计学意义(P=0.493,0.624);60 d组实验组Nav1.2 mRNA表达(0.718±0.056)较对照组(1.000±0.026)明显降低,差异具有统计学意义(P=0.027),实验组Nav1.6 mRNA表达(2.445±0.167)较对照组(1.003±0.060)显著升高,差异具有统计学意义(P =0.001).结论 Nav1.2和Nav1.6均参与氯化锂-匹罗卡品癫痫大鼠慢性自发性癫痫形成,且Nav1.6在大鼠急性期痫性发作中发挥了作用. 目的動態觀察Nav1.2、Nav1.6在氯化鋰-匹囉卡品癲癇模型大鼠海馬CA3區的錶達變化,探討兩者在癲癇髮病機製中的可能作用.方法採用隨機數字錶法,將90隻健康雄性SD大鼠隨機分為實驗組(n=45)和對照組(n=45),實驗組予以腹腔註射氯化鋰-匹囉卡品緻癇,對照組予生理鹽水假處理.根據時間點又分為3箇亞組:24 h(n=15)組(n=15)、7d組(n=15)和60 d組(n=15),採用隨機數字錶法將每亞組再隨機分為3箇小組(n=5)分彆進行免疫組化、Western印跡及RT-PCR方法檢測Nav1.2、Nav1.6在大鼠海馬CA3區的動態錶達.結果 (1)免疫組化檢測示實驗24 h組海馬CA3區Nav1.2蛋白錶達相對對照組差異無統計學意義(P=0.492),而實驗組Nav1.6蛋白錶達(0.398±0.019)較對照組(0.313 ±0.017)顯著升高,差異具有統計學意義(P =0.034);實驗7d組海馬CA3區Nav1.2和Nav1.6蛋白相對對照組差異無統計學意義(P=0.157,0.109);實驗60 d組Nav1.2蛋白錶達(0.117±0.009)較對照組(0.155±0.010)顯著降低,差異具有統計學意義(P =0.002),而實驗組Nav1.6蛋白錶達(0.400±0.009)較對照組(0.318±0.010)顯著升高,差異具有統計學意義(P=0.018).(2) Western印跡檢測示實驗24 h組海馬CA3區Nav1.2蛋白錶達較對照組差異無統計學意義(P=0.472),實驗組Nav1.6蛋白錶達(0.419±0.027)較對照組(0.290±0.007)顯著升高,差異具有統計學意義(P=0.001);7 d組Nav1.2和Nav1.6蛋白錶達較對照組差異無統計學意義(P=0.517,0.514);60 d組實驗組Nav1.2蛋白錶達(0.209 ±0.077)較對照組(0.339±0.080)明顯降低,差異具有統計學意義(P=0.024),實驗組Nav1.6蛋白錶達(0.772±0.029)較對照組(0.489±0.014)顯著升高,差異具有統計學意義(P=0.00I).(3)RT-PCR檢測示實驗24 h組海馬CA3區Nav1.2 mRNA錶達較對照組差異無統計學意義(P =0.453),實驗組Nav1.6mRNA錶達(2.250±0.117)較對照組(0.998±0.139)顯著升高,差異具有統計學意義(P=0.001);7d組Nav1.2和Nav1.6 mRNA錶達較對照組差異無統計學意義(P=0.493,0.624);60 d組實驗組Nav1.2 mRNA錶達(0.718±0.056)較對照組(1.000±0.026)明顯降低,差異具有統計學意義(P=0.027),實驗組Nav1.6 mRNA錶達(2.445±0.167)較對照組(1.003±0.060)顯著升高,差異具有統計學意義(P =0.001).結論 Nav1.2和Nav1.6均參與氯化鋰-匹囉卡品癲癇大鼠慢性自髮性癲癇形成,且Nav1.6在大鼠急性期癇性髮作中髮揮瞭作用.
목적 동태관찰Nav1.2、Nav1.6재록화리-필라잡품전간모형대서해마CA3구적표체변화,탐토량자재전간발병궤제중적가능작용.방법 채용수궤수자표법,장90지건강웅성SD대서수궤분위실험조(n=45)화대조조(n=45),실험조여이복강주사록화리-필라잡품치간,대조조여생리염수가처리.근거시간점우분위3개아조:24 h(n=15)조(n=15)、7d조(n=15)화60 d조(n=15),채용수궤수자표법장매아조재수궤분위3개소조(n=5)분별진행면역조화、Western인적급RT-PCR방법검측Nav1.2、Nav1.6재대서해마CA3구적동태표체.결과 (1)면역조화검측시실험24 h조해마CA3구Nav1.2단백표체상대대조조차이무통계학의의(P=0.492),이실험조Nav1.6단백표체(0.398±0.019)교대조조(0.313 ±0.017)현저승고,차이구유통계학의의(P =0.034);실험7d조해마CA3구Nav1.2화Nav1.6단백상대대조조차이무통계학의의(P=0.157,0.109);실험60 d조Nav1.2단백표체(0.117±0.009)교대조조(0.155±0.010)현저강저,차이구유통계학의의(P =0.002),이실험조Nav1.6단백표체(0.400±0.009)교대조조(0.318±0.010)현저승고,차이구유통계학의의(P=0.018).(2) Western인적검측시실험24 h조해마CA3구Nav1.2단백표체교대조조차이무통계학의의(P=0.472),실험조Nav1.6단백표체(0.419±0.027)교대조조(0.290±0.007)현저승고,차이구유통계학의의(P=0.001);7 d조Nav1.2화Nav1.6단백표체교대조조차이무통계학의의(P=0.517,0.514);60 d조실험조Nav1.2단백표체(0.209 ±0.077)교대조조(0.339±0.080)명현강저,차이구유통계학의의(P=0.024),실험조Nav1.6단백표체(0.772±0.029)교대조조(0.489±0.014)현저승고,차이구유통계학의의(P=0.00I).(3)RT-PCR검측시실험24 h조해마CA3구Nav1.2 mRNA표체교대조조차이무통계학의의(P =0.453),실험조Nav1.6mRNA표체(2.250±0.117)교대조조(0.998±0.139)현저승고,차이구유통계학의의(P=0.001);7d조Nav1.2화Nav1.6 mRNA표체교대조조차이무통계학의의(P=0.493,0.624);60 d조실험조Nav1.2 mRNA표체(0.718±0.056)교대조조(1.000±0.026)명현강저,차이구유통계학의의(P=0.027),실험조Nav1.6 mRNA표체(2.445±0.167)교대조조(1.003±0.060)현저승고,차이구유통계학의의(P =0.001).결론 Nav1.2화Nav1.6균삼여록화리-필라잡품전간대서만성자발성전간형성,차Nav1.6재대서급성기간성발작중발휘료작용.
Objective To observe the expressions of Nav1.2 and Nav1.6 in the hippocampal CA3 region of lithium chloride-pilocarpine epileptic rats to explore their potential roles in epileptogenesis.Methods A total of 90 healthy male SD rats were randomly divided into normal control group (physiological saline) and epilepstic group (lithium chloride-pilocarpine).According to different timepoints,the control and epilepstic groups were randomly divided into 3 subgroups of 24-hour,7-day and 60-day.Then immunohistochemistry,Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were employed to detect the expressions of Nav1.2 and Nav1.6 in hippocampal CA3 region of rats.Results Immunohistochemisty showed that,at 24 hours,the expression of Nav1.2 had no significant difference (P =0.492) while Nav1.6 significantly increased as compared with controls(0.398 ±0.019 vs 0.313 ±0.017,P =0.034).At Day 7,both Nav1.2 and Nav1.6 had no significant change (P =0.157,0.109).Nav1.2 significantly decreased at Day 60 (0.117 ±0.009 vs 0.155 ±0.010,P =0.002).But Nav1.6 significantly increased(0.400 ±0.009 vs 0.318 ±0.010,P =0.018).Western blot showed that Nav1.2 protein had no significant difference at 24 hours (P =0.472) while Nav1.6 significantly increased (0.419 ± 0.027 vs 0.290 ±0.007,P =0.001).At Day 7,Nav1.2 and Nav1.6 proteins had no significant change (P =0.517,0.514).At Day 60,Nav1.2 protein significantly decreased (0.209 ±0.077 vs 0.339 ±0.080,P=0.024) while Nav1.6 significantly increased (0.772 ± 0.029 vs 0.489 ± 0.014,P =0.001).RT-PCR showed the same results as Western blot and immunohistochemisty.Nav1.2 mRNA had no significantly difference at 24 hours(P =0.453) while Nav1.6 mRNA significantly increased (2.250 ± 0.117 vs 0.998 ± 0.139,P =0.001) ; at Day 7,both Nav1.2 mRNA and Nav1.6 mRNA had no significant change (P =0.493,0.624).Nav 1.2 mRNA significantly decreased at Day 60 (0.718 ± 0.056 vs 1.000 ± 0.026,P =0.027).But Nav1.6 mRNA significantly increased (2.445 ± 0.167 vs 1.003 ± 0.060,P =0.001).Conclusion Both Nav1.2 and Nav1.6 are involved in the formation of chronic spontaneous recurrent seizures.And Nav1.6 also plays an important role in acute phase of seizures.