针刺血清对无镁培养的新生大鼠海马神经元微管相关蛋白2和生长相关蛋白43表达的影响  

Effect of acupuncture serum on expression of microtubule associated protein-2 and nerve growth associated protein-43 in Mg;-free-cultured hippocampal neurons of neonatal rats

作　　者：张慧[1] 杨帆[1] 龙学平 吴旭[2] 刘振邦 陈浩[1] 吴生兵 昂文平[1] ZHANG Hui;YANG Fan;LONG Xue-ping;WU Xu;LIU Zhen-bang;CHEN Hao;WU Sheng-bing;ANG Wen-ping(Clinical College of Acupuncture,Anhui University of Chinese Medicine,Hefei 230038,China;College of Life Science,University of Science and Technology of China,Hefei 230012;Research Laboratory Center,Anhui University of Chinese Medicine,Hefei 230038,China)

机构地区：[1]安徽中医药大学针灸推拿学院,合肥230038 [2]中国科技大学生命科学学院,合肥230012 [3]安徽中医药大学科研实验中心,合肥230038

出　　处：《针刺研究》2021年第12期1029-1035,1066,共8页Acupuncture Research

基　　金：国家自然科学基金项目(No.81072872);安徽高校自然科学研究项目(No.KJ2016A858)。

摘　　要：目的:观察针刺血清对无镁培养的新生大鼠海马神经元微管相关蛋白2(MAP-2)和生长相关蛋白43(GAP-43)表达的影响,探讨针刺血清改善神经元损伤的可能作用机制。方法:SD大鼠腹腔注射戊四唑诱发急性惊厥后分为针刺组及对照组。针刺组取"百会""大椎"针刺,每日1次,共7 d。末次针刺后腹主动脉取血,提取针刺组大鼠血清作为针刺血清,对照组大鼠血清作为非针刺血清。将原代培养新生大鼠海马神经元分为正常组、无镁组、针刺血清组和非针刺血清组。正常组用细胞外液培养3 h后换无血清培养液培养,无镁组用无镁细胞外液培养3 h后换无血清培养液培养,针刺血清组用无镁细胞外液培养3 h后换成无血清培养液和针刺血清混合培养液培养,非针刺血清组用无镁细胞外液培养3 h后换成无血清培养液和非针刺血清混合培养液培养。4组分别于换液培养2、12、48 h 3个时间点用免疫荧光法和Western blot法检测海马神经元MAP-2和GAP-43蛋白表达情况。结果:与正常组比较,无镁组各时点海马神经元MAP-2蛋白的表达均明显降低(P<0.05,P<0.01),GAP-43蛋白的表达在12、48 h明显降低(P<0.05);与无镁组比较,针刺血清组各时点海马神经元MAP-2和GAP-43蛋白的表达均明显升高(P<0.05,P<0.01),非针刺血清组无明显变化(P>0.05);与非针刺血清组比较,针刺血清组各时点海马神经元MAP-2和GAP-43蛋白的表达均明显升高(P<0.05,P<0.01)。结论:针刺血清能明显上调海马神经元MAP-2和GAP-43蛋白的表达,可能在提高突触可塑性,改善神经元损伤方面发挥重要的作用。Objective To observe the effect of acupuncture serum on the expression of microtubule associated protein-2(MAP-2)and nerve growth associated protein-43(GAP-43)in cultured hippocampal neurons of convulsive rats.Methods The acute convulsion model was induced by intraperitoneal injection of pentylenetetrazol in SD rats who were then randomized into model group and acupuncture group.Rats of the acupuncture group received manual acupuncture stimulation of“Baihui”(GV20)and“Dazhui”(GV14)for 30 min,once daily for 7 days.Then,the blood samples taken from the abdominal aorta of rats in the convulsion model and acupuncture groups were processed into serum samples,i.e.non-acupuncture serum and acupuncture se-rum.The primary-cultured hippocampal neurons of fetal rats were cultured for 10 days and then divided into normal extracellular fluid(normal)group,magnesium(Mg;)free extracellular fluid group,acupuncture serum group and non-acupuncture serum group.At the 10 th day,the neurons in the normal group were cultured continuously in extracellular fluid for 3 h,and then cultured in DMEM/F12(1∶1)medium(planting fluid);neurons in the Mg;free group were cultured in magnesium-free fluid medium to induce epileptic-like discharge;neurons in the acupuncture serum group were cultured in the mixed medium of planting fluid and 10%acupuncture serum;and neurons in the non-acupuncture serum were cultured in the mixed culture medium of planting fluid and non-acupuncture serum(10%).At last,these neurons in the above-mentioned groups were cultured in the magnesium-free extracellular fluid continuously for 2,12 and 48 h,respectively,followed by detecting the expression levels of MAP-2 and GAP-43 proteins at the 3 time points by using immunofluorescence and Western blot,separately.Results The rate of MAP-2 positive cells and protein expression at 2,12 and 48 h,and the rate of GAP-43 positive cells and protein expression at 12 and 48 h in the hippocampal neurons were significantly down-regulated in the Mg;free group in contrast to the nor

关 键 词：针刺血清 惊厥 微管相关蛋白2 生长相关蛋白43 

分 类 号：R245.97[医药卫生—针灸推拿学]