电针“夹脊”穴对急性脊髓损伤大鼠少突胶质前体细胞增殖分化的影响

Effect of electroacupuncture at "Jiaji"(EX-B 2) points on the proliferation and differentiation of oligodendrocyte precursor cells in rats with acute spinal cord injury

目的:观察电针"夹脊"穴对急性不完全性脊髓损伤大鼠少突胶质前体细胞增殖分化的影响,探讨电针促进脊髓损伤运动功能恢复的作用机制。方法:将72只雄性SPF级SD大鼠随机分为假手术组、模型组、电针组和药物组,每组18只,各组再分为术后1、7、14d 3个亚组,每个亚组6只。模型组、电针组和药物组采用改良Allen’s法制备T10急性不完全性脊髓损伤模型。各组分别于术后腹腔注射5-溴脱氧尿嘧啶核苷(Brd U,50mg/kg),每天1次,各亚组分别连续注射1、7、14次,用于细胞增殖标记。电针组电针刺激损伤脊髓上下节段(T9、T11)棘突旁3~4 mm的"夹脊"穴,频率2 Hz/100 Hz,强度1~2 mA,以治疗部位肌肉轻微抽动为度,每日1次,每次20min;药物组腹腔注射单唾液酸神经节苷脂(GM1)10mg/kg,每日1次。电针组与药物组各亚组分别连续干预1、7、14次。采用Basso Beattie Bresnahan(BBB)运动功能评分法评定各组大鼠的后肢运动功能,取损伤部位脊髓组织用免疫荧光法检测BrdU/NG2阳性细胞共表达,免疫蛋白印迹法检测转录因子Olig2和Sox10蛋白的表达。结果:与假手术组比较,术后第1、7、14天,模型组大鼠BBB评分较低(P<0.05),脊髓组织Olig2、Sox10表达增多(P<0.05),术后第7、14天,模型组脊髓组织BrdU/NG2共表达的阳性细胞率较高(P<0.05)。术后第7、14天,电针组和药物组BBB评分高于模型组(P<0.05),药物组脊髓组织BrdU/NG2共表达高于模型组(P<0.05);术后第14天,电针组脊髓组织BrdU/NG2共表达高于模型组(P<0.05);术后第1、7、14天,电针组、药物组脊髓组织Olig2、Sox10表达高于模型组(P<0.05)。与药物组比较,术后第14天电针组BrdU/NG2共表达的阳性细胞率较低(P<0.05),术后第1、7、14天电针组脊髓组织Olig2、Sox10表达量较低(P<0.05)。结论:电针"夹脊"穴能够促进脊髓损伤后转录因子Olig2和Sox10的表达,作用与GM1相似,可促进少突胶质前体细胞增殖分化成少突胶质细胞,从而促进大鼠运动功能的恢复。

Objective To observe the effect of electroacupuncture(EA) at "Jiaji"(EX-B 2) points on the proliferation and differentiation of oligodendrocyte precursor cells in rats with acute incomplete spinal cord injury, and to explore the mechanism of EA on improving motor function of spinal cord injury. Methods A total of 72 male SPF SD rats were randomly divided into a sham operation group, a model group, an EA group and a medication group, 18 rats in each group. Each group was further divided into 1-day subgroup, 7-day subgroup and 14-day subgroup, 6 rats in each subgroup. The T10 acute incomplete spinal cord injury model was established by modified Allen’s method in the model group, EA group and medication group. The rats in each group received intraperitoneal injection of 5-bromodeoxyuridine(BrdU, 50 mg/kg), once a day, and each subgroup received continuous injection for 1, 7, 14 times for cell proliferation labeling. The rats in the EA group were treated with EA at "Jiaji"(EX-B 2) points 3-4 mm next the spinous process of the upper and lower segments of the injured spinal cord(T9, T11) with a frequency of 2 Hz/100 Hz and intensity of 1-2 mA. The muscle twitch at the treatment site was taken as the degree. The treatment was given 20 min each time, once a day. In the medication group, monosialogangliosides(GM1) was injected intraperitoneally(10 mg/kg), once a day. The subgroups of EA group and medication group were treated for 1, 7, 14 times. The score of Basso Beattie Bresnahan(BBB) was used to evaluate the motor function of hind limbs. The co-expression of BrdU/NG2 positive cells was detected by immunofluorescence, and the expression of Olig2 and Sox10 was detected by Western blot. Results Compared with the sham operation group, the BBB score was decreased 1 day, 7 days and 14 days after operation in the model group(P<0.05), the expression of Olig2 and Sox10 was increased(P<0.05), and the co-expression of BrdU/NG2 positive cells was increased 7 days and 14 days after operation(P<0.05). Seven days and14 days after operation, the BBB score in the EA group and medication group was higher than that in the model group(P<0.05),and the co-expression of BrdU/NG2 in the medication group was higher than that in the model group(P<0.05). Fourteen days after operation, the co-expression of BrdU/NG2 in the EA group was higher than that in the model group(P<0.05);1 day, 7 days and 14 days after operation, the expression of Olig2 and Sox10 in the EA group and medication group was higher than that in the model group(P<0.05). Compared with the medication group, the co-expression of BrdU/NG2 positive cells in the EA group 14 days after operation was decreased(P<0.05);1 day, 7 days and 14 days after operation, the expression of Olig2 and Sox10 in the EA group was decreased(P<0.05). Conclusion EA at "Jiaji"(EX-B 2) points could promote the expression of Olig2 and Sox10 after spinal cord injury, which has similar effects with GM1. It could promote the proliferation and differentiation of oligodendrocyte precursor cells into oligodendrocytes, so as to promote the recovery of motor function of rats.