电针刺激干预脊髓损伤大鼠c-fos基因和脑源性神经营养因子mRNA的表达

Effects of electro-acupuncture on expressions of c-fos gene and brain-derived neurotrophic factor mRNA in the injured spinal cord in rats

目的:通过对参与脊髓损伤过程的c-fos基因及脑源性神经营养因子不同时间点mRNA阳性细胞数的结果观察,探讨电针对脊髓损伤的影响,并以地塞米松做标准对照。方法:实验于2003-12/2004-06在哈尔滨医科大学实验中心完成。以72只Wistar大鼠为观察对象,将其分为4组,模型对照组(12只)、电针组(24只)、地塞米松组(24只)及假手术组(12只)。假手术组仅切除椎板,不损伤脊髓。其余3组用改良的Allen’s撞击法致大鼠T10脊髓损伤。电针组将大鼠置于特制治疗台上,建立四肢固定状态下的大鼠电针模型。在距损伤处上下端两个椎体的棘突间隙距中线旁开3.0～4.0mm处取穴,用30号1寸毫针垂直刺入4.0～5.0mm,使针尖触及椎板,正、负极导线分别夹在头、尾两侧的毫针针柄上(正极在上,负极在下)。电针组于造模成功后30min进行夹脊电针连续脉冲电流,频率1Hz,电压0.5V,输出强度是以背部肌肉出现轻微抽动为度。6h后进行第2次治疗,以后1次/d,20min/次。地塞米松组于造模成功后5min开始皮下给予地塞米松5mg/kg治疗。模型对照组不给予治疗。应用原位杂交方法及MoticImagesAdvanced3.0图像定量分析法观察脊髓损伤后1,3,7,14d时各组c-fos基因mRNA和脑源性神经营养因子mRNA的表达变化。结果:72只Wistar大鼠均进入结果分析。①c-fos基因mRNA的表达:在脊髓损伤后1d时地塞米松组和电针组均明显低于模型对照组(57.8±6.4,65.4±5.9,81.6±4.2,P<0.05);14d时地塞米松组和电针组均明显高于模型对照组(68.2±4.5,73.3±7.0,61.4±5.4,P<0.05),而电针组的表达又明显高于地塞米松组(P<0.05)。②脑源性神经营养因子mRNA的表达:有逐渐增高趋势,电针组、地塞米松组明显高于模型对照组(P<0.05),且在7d和14d时电针组表达明显高于地塞米松组(141.1±10.3,107.4±8.3;103.0±9.3,78.2±7.9,P<0.05)。结论:大鼠脊髓损伤后早期c-fos基因mRNA表达增强,后期表达减弱。脑源性神经营养因子mRNA的表达不断增加。说明电针组干预早期能抑制c-fos基因mRNA表达,后期可升高其表达,减少脊髓的继发性损伤,并能上调脑源性神经营养因子mRNA表达,促进脊髓神经的再生及修复,电针组干预后1周和2周时表现出的效果优于地塞米松组。

AIM：To prohe into the effect of electro-acupuncture in treatment for spinal cord injury through observing the number of mRNA positive cells of cfos gene which participants the process of spinal injury and brain-derived neurotrophic factor at different time point~ and compare with the effect of dexamethasone. METHODS：This experiment was conducted in the Experimental Center of Harbin Medical University from Decemher 2003 to June 2004. Totally 72 Wistar rats were set as the observational subjects and divided into for 4 groups ： model control group （n=12）, electro-acupuncture group （n=24）, dexamethasone group（n=24） and sham operation group （ n=24 ）. Lamina of vertebra was cut without spinal cord injury in the sham-operation group. Injury of T,o spinal cord was induced by modified Alien＇ s method in the other 3 groups. Rats in the electro-acupuncture group were put on the treatment table made specially to establish the electro-acupuncture rat models with the four limbs fixed. Point selection was performed on the 3.0-4.0 mm away from mideline . No. 30 1-inch needle was stieked into the rats for 4.0-5.0 mm, with the tip of needle touching the lamina of vertebra. Positive and negative pole wire was conneeted to the needle handle of the head and tail respectively （positive pole above and negative pole below）. Jiaji point was performed eleetro-aeunpuneture with continuous pulsed current 30 minutes ＇after successful model preparation in the eleetrn-aeupuncture group. The frequency of continuous pulsed current was 1 Hz and the pressure was 0.5 V. Output intensity was set as the slight twitch of dorsal muscle. The second treatment begun 6 hours later , then once a day, 20 minutes per time . 5 mg/kg of dexamethasone was injected subcutaneously 5 minutes after the models were prepared successfully in the dexamethasone group. Anv treatment was not given in the model control group. The expression of mRNA of c-fos gene and brainderived neurotrophic factor in each group was observed 1,3,7,14 days after spinal injury by in situ hybridization method and image quantitative analysis of Motie Images Advanced 3.0 RESULTS： Totally 72 Wistar rats entered result analysis. ① Expression of c-los gene mRNA： During the 1 day after injury, expression of c-los gene mRNA in the dexamethasone group and electro-acupuncture group was significantly lower than that in the model control group （57.8±6.4, 65.4±5.9,81.6±4.2,P 〈 0.05）;At day 14, expression of c-los gene mRNA in the dexamethasnne group and electro-acupuncture group was significantly higher than that in the model control group （68.2±4.5,73.3±7.0,61.4±5.4,P 〈 0.05）, while that in the electro-acupuncture group was significantly higher than that in the dexamethasone group （P 〈 0.05）, ② Expression of brainderived neurotrophie factor mRNA had the gradual increasing tendency,and that in the electro-acupuncture group and dexamethasone group wassignificantly higher than that in the control group （P 〈 0.05）. Expression of brain-derived neurotrophie faetor mRNA in the eleetro-aeupuneture group was significantly higher than that in the dexamethasone group at days 7 and 14 （141.1±10.3,107.4±8.3 ; 103.0±9.3,78.2±7.9,P 〈 0.05）. CONCLUSION：mRNA expression of e-fos gene is increased in the early stage of spinal cord injury and is decreased in the later stage of spinal eord injury. Expression of brain-derived neurotrophic faetor mRNA is eontinuously increased, suggesting that early intervention in the eleetro-aeupuncture group ean inhibit mRNA expression of e-fos gene and ean inerease its expression at the later stage , reduce the secondary injury of spinal eord, up regulate the expression of brain-derived neurotrophie faetor mRNA, promote the regeneration and repair of spinal nerves . Interventional effect in the eleetro-aeupuneture group is superior to that in the dexamethasone group at weeks 1 and 2 after intervention.