【作者】 刘振寰； 潘佩光； 祁岩超； 赵勇； 柴铁劬； 唐纯志； 王琴玉； 杨君军； 林锦泉；

【Author】 Liu Zhen-huan~1,Pan Pei-guang~1,Qi Yan-chao~2,Zhao Yong~1,Chai Tie-qu~3,Tang Chun-zhi~3,Wang Qin-yu~3,Yang Jun-jun~3,Lin Jin-quan~3 (1 Pediatric Department of Neurological Rehabilitation.Nanhai Obstetrics and Gynecology Hospital Affiliated to Guangzhou University of Traditional Chinese Medicine,Foshan 528200,Guangdong Province, China;2 Department of Internal Medicine,Guangzhou Municipal Cancer Hospital,Guangzhou 510095, Guangdong Province,China;3 Laboratory of Department of Acupuncture & Massage,Guangzhou University of Traditional Chinese Medicine,Guangzhou 510405,Guangdong Province,China.

【机构】 广州中医药大学附属南海妇产儿童医院小儿神经康复科； 广州市肿瘤医院内科； 广州中医药大学针灸推拿学院实验室；

【摘要】 目的:观察通督醒神针刺法对脑性瘫痪幼鼠脑神经细胞凋亡及神经生长因子蛋白表达,肢体功能的影响,分析该种影响是否与针刺时间有关。方法:①选用7d龄新生SD幼鼠109只。将大鼠结扎左侧颈总动脉后恢复2h,置于透明密闭容器中,并入37℃恒温水中以1L/min的速度通入低氧气体(体积分数0.08氧气和0.92氮气),2.5h后将动物取出,将存活者继续保温1h时后作行为测定,翻身不能、平衡异常或左旋者视为成功的模型(72只)。②将其余24只大鼠设为假手术组:只分离左颈总动脉后不结扎,亦不作低氧处理。将造模成功的大鼠72只按随机抽签法分为3组:脑性瘫痪幼鼠+针刺Ⅰ组、脑性瘫痪幼鼠+针刺Ⅱ组、模型组,每组24只。选用大鼠百会、患侧率谷透角孙、内关、曲池、足三里、涌泉。脑性瘫痪幼鼠+针刺Ⅰ组于造模后24h开始针刺,前7d仅针四肢部穴位,第2天开始加针头部穴位。脑性瘫痪幼鼠+针刺Ⅱ组造模后第8天开始针刺,头、体针同步。两组均以针灸针先在内关、涌泉速刺微出血,不留针,然后针头部及曲池、足三里。百会、率谷透角孙及曲池、足三里接G6805-Ⅱ电针仪,连续波,频率5～10Hz,持续10min,1次/d。脑性瘫痪幼鼠+针刺Ⅰ组连续针刺20d,脑性瘫痪幼鼠+针刺Ⅱ组连续针刺13d。假手术组针刺方法与脑性瘫痪幼鼠+针刺Ⅰ组相同;模型组只造模,不予以任何治疗。③于术后第7、14、21天,将胶布粘于大鼠两前爪腹侧面后,记录其撕去胶布所用时间。④术后第21天各组随机选取10只大鼠作以下检测:分别采用原位末端标记法及免疫组化方法测定大鼠额页皮质与海马区神经元凋亡及海马区神经生长因子表达,用图像分析仪在光镜下(×400)计数神经细胞凋亡数目及神经生长因子免疫阳性细胞表达数目。⑤率的比较采用x 2检验,组间多均数比较采用方差分析(F-q检查)。结果:纳入大鼠109只,因造模失败脱失1 3只,假手术组动物无死亡。造模后7d,脑性瘫痪幼鼠+针刺Ⅰ组、脑性瘫痪幼鼠+针刺Ⅱ组、模型组分别死亡2、5、3只;造模后7～14d,上述3组分别死亡2、2、4只;造模后14～21d,上述3组分别死亡0、0、2只。①大鼠海马和大脑额叶皮质神经元凋亡情况:2个针刺治疗组大鼠海马和大脑额叶皮质内原位末端标记法染色阳性细胞显著减少,与模型组相比,差异明显(P<0.05=。且脑性瘫痪幼鼠十针刺Ⅰ组神经细胞凋亡计数明显低于脑性瘫痪幼鼠十针刺Ⅱ组(P<0.01=。②大鼠海马神经元神经生长因子蛋白表达:2个针刺治疗组大鼠神经生长因子蛋白表达明显增强,在数量和强度方面均明显多于或高于假手术组和模型组(P<0.01=,脑性瘫痪幼鼠+针刺Ⅰ组明显多于或高于脑性瘫痪幼鼠+针刺Ⅱ组(P<0.01=。③术后第7天,脑性瘫痪幼鼠+针刺Ⅰ组鼠撕去胶布所用时间明显短于模型组(P<0.01=;术后第14天,模型组明显长于其他3组(P<0.05～0.01=;术后第21天,2个针刺组和模型组鼠前肢功能均显著改善,脑性瘫痪幼鼠+针刺Ⅰ组基本接近正常水平(与假手术组比较,差异不明显,P>0.05),但模型组仍明显长于脑性瘫痪幼鼠+针刺Ⅰ组和假手术组(P<0.01)。结论:针刺可抑制脑性瘫痪幼鼠脑内神经细胞的凋亡,增强脑性瘫痪幼鼠脑组织的神经生长因子的表达,改善肢体功能,对脑性瘫痪幼鼠脑组织损伤大鼠具有一定的保护作用,且越早干预效果越好。更多还原

【Abstract】 aim:To study the effect of acupuncture Anshenbunao and Tiqitongluo method on apoptosis of hypoxic-ischemic cerebral neural cells,protein expression of nerve growth factor and the function of limbs in newborn rats,and analyze whether the effect is related with the time of acupuncture.methods:The experiment was conducted in the Laboratory of Acupuncture & Massage as well as the Clean Laboratory of Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine from August to October 2005.①109 young SD rats aged 7 days were selected.Rats recovered for 2 hours after ligation of left common carotid artery and then were put into a hyaline airtight container,in which there was homeothermal water of 37℃,and hypoxia gas（including 8%of O₂ and 92%of N₂） was infused in the container at the speed 1 L/min.Animals were taken out at 2.5 hours later,and the survival rats received behavioral assessment following 1-hour incubation.Rats unable to roll over,abnormal in balance or kept on turning left were taken as successful models （n=72）.②The rest 24 rats were taken as the sham-operation group:rats were only separated of the left common carotid artery without ligation or hypoxia treatment.Rats of successful modeling were randomly divided into 3 groups:hypoxic-ischemic+acupunctureⅠgroup（group A）.hypoxic-ischemic+acupuntureⅡgroup（group B） and model group with 24 rats in each group.The acupoints of Baihui,Shuaigu to Jiaosun,Neiguan,Quchi, Zusanli,Yongquan were selected.Rats in group A were acupunctured at 24 hours after modeling,and only acupoints in four extremities were acupunctured in the first 7 days,while acupoints in head were also acupunctured from the 8^th day.Rats in group B were acupunctured at both head and body on the 8^th day.Rats of both groups were acupunctured rapidly at Neiguan and Yongquan with littlee blood and without retention,then head-acupoints,Quchi and Zusanli were acupunctured.Baihui,Shuaigu to Jiaosun,Quchi,Zusanli were connected with G6805-Ⅱelectroacupunture equipment with continuous wave at the frequency of 5-10 Hz for 10 continuous minutes once a day.Rats in group A were acupunctured for 20 continuous day.Rats in group B were continuously acupunctured for 13 days.Rats in the sham-operation group were interfered the same as group A.Rats in the model group only received no treatment but grouping.③The ventral surfaces of the upper limbs in rats were stuck with rubber cement at 7,14,21 days after operation,and the time of getting out of the rubber cement was recorded.④At 21 days after operation,10 rats were randomly selected from each group to conduct the following detection:in situ end-labeling（ISEL） and immunohistochemical method were adopted to measure the neuronal apoptosiss in cortex of temple and hippocampus as well as the expression of nerve growth factor（NGF） in hippocampus respectively,and image analyzer was used to count the number of neuronal apoptosis and number of NGF that positive in expression under light microscope（×400）.⑤The differences were compared with Chi square test,and means were compared among groups with analysis of variance（F-q test）.results:Of 109 enrolled rats,13 failed for failure modeling,and no one died in the sham-operation group. At 7 days after modeling,there were 2,5 and 3 rats died in group A,B and model group respectively.At 7-14 days after modeling,there were 2,2 and 4 respectively.At 17-21 days after modeling,they were 0,0 and 2.①Apoptosis of neurons in hippocampus frontal lobe stained with ISEL in group A and B were significantly decreased,and the differences in comparison with the model group were remarkable（P<0.05）.Moreover,the number of apoptosis in group A was obviously lower than group B（P<0.05）.②Protein expression of NGF in neurons of hippocampuss in rats:It was significantly reinforced in group A and B,while as to number and intensity,those were obviously greater and higher than sham-operation group and model group（P<0.01）,and those were obviously greater and higher in group A than group B（P<0.01）.③At the 7^th day after operation,the time of getting rid of rubber cement in group A was obviously shorter than in medel group（P<0.01）.At the 14^th day after operation,it was significantly long in model group than 3 other groups（P<0.05—0.01）.At the 21^st day after operation,the function of upper limbs in rats of group A and B as well as model group were obviously ameliorated,and that of group A approached to normal level（there was no significant differences comparing with the sham-operation group,P>0.05）,whereas that of model group was longer than group A and sham-operation group（P<0.01）.conclusion:Acupuncture can inhibit the apoptosis of hypoxic-ischemic neurocytes in brain,reinforce the expression of NGF in brain tissues of rats with hypoxic-ischemic damage and ameliorate the function of limbs,which has certain protective effects on hypoxic-ischemic damage,and the earlier the intervention is carried out,the better the effects are.更多还原