摘要
目的探究大鼠垂体柄损毁后精氨酸加压素(AVP)神经元通过双皮质素(DCX)促进神经元骨架修复的机制。方法取32只SD大鼠,随机分为实验组(损毁后1d组,n=4;损毁进神经元骨架恢后3d组,n=7;损毁后7d组,n=7;损毁后14d组,n=7)和对照组(假手术组,n=7),经颅顶损毁垂体柄,记录尿量、饮水量和尿比重,通过免疫荧光染色比较各组视上核(SON)AVP神经元中Ⅲ型β微管蛋白(Tuj1)、DCX和caspase3表达情况,并通过蛋白质印迹法探讨其调控机制。结果垂体柄损毁后,饮水量、尿量和尿比重出现了典型的三相性尿崩症(损毁后1~2d为第一相,术后3~5d为第二相,6d以后为第三相)。免疫荧光的结果提示了AVP神经元损伤后修复现象,主要表现为DCX表达在损毁后不同程度的升高。AVP神经元中DCX阳性率分别为:假手术组,(37.93±1.83)%;损毁后1d,(44.67±1.79)%;损毁后3d,(48.09±2.96)%;损毁后7d,(71.34±2.18)%;损毁后14d,(61.08±2.15)%,(P<0.01)。同时,神经元骨架蛋白Tuj1也呈现出相同时间规律,即垂体柄损毁后7d最高[假手术组,(42.82±2.02)%;损毁后7d,(60.70±1.28)%,P<0.01]。另一方面,神经元凋亡现象与骨架修复呈现相反的时间规律,主要表现为caspase3+DCX+双阳性细胞占DCX阳性细胞的比例逐渐下降:损毁后3d,(66.03±3.58)%,损毁后7d,(43.42±4.45)%,损毁后14d,(35.11±1.73)%。通过蛋白质印迹技术,进一步研究发现,神经元损伤相关通路(JNK/c-Jun)在损毁后不同时间点出现上调,表现为:p-JNK仅在损毁后3d时表达上调(P<0.05),c-Jun,p-c-Jun表达于损毁后3d时表达上调(P<0.05),并于损毁后7d时达到高峰(P<0.01),这分别与神经元凋亡与修复的时间规律相符合。结论垂体柄损毁后,JNK/c-Jun信号通路激活,一方面诱发早期AVP神经元凋亡,另一方面可能通过诱导DCX表达,促进Tuj1合成从而修复细胞骨架,保护神经元免受凋亡以及促进神经内分泌功能重建。
Objective To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL).Methods Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (n=25) and sham operation group (n=7),and the daily water consumption (DWC),daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded.Four rats on day 1 and 7 rats on each of days 3,7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1),doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting.Results After PEL,the rats exhibited a typical triphasic pattern of diabetes insipidus,with the postoperative days 1-2 as the phase one,days 3-5 as the phase two,and days 6-14 as the phase three.Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL;similarly,the expression of Tuj1 also increased progressively after PEL,reaching the peak level on day 7 after PEL.The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation,peaking on postoperative day 3 followed by progressive reduction till day 14.Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 (P<0.05) and 7 (P<0.01) after PEL,while an upregulated p-JNK expression was detected only on day 3 (P<0.05),as was consistent with the time-courses of neuronal recovery and apoptosis after PEL.Conclusion JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.
作者
李凯
冯展鹏
欧毅超
周明锋
彭君洁
龚浩东
武广森
刘亚伟
漆松涛
LI Kai;FENG Zhanpeng;OU Yichao;ZHOU Mingfeng;PENG Junjie;GONG Haodong;WU Guangsen;LIU Yawei;QI Songtao(Department of Neurosurgery,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;First Clinical Medical College,Southern Medical University,Guangzhou 510515,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2019年第9期1099-1106,共8页
Journal of Southern Medical University
基金
国家自然科学基金青年基金(81900709)
广州市科技计划项目(201902020017)
广东省科技计划项目(2016A020213006,2016A040403053)
南方医科大学科研启蒙计划(B219339033)~~
关键词
垂体柄损毁
视上核
精氨酸加压素
双皮质素
大鼠
pituitary stalk electrical lesions
supraoptic nucleus
arginine vasopressin
doublecortin
Rats