电针联合骨髓基质细胞移植对脊髓损伤大鼠神经功能恢复的影响

Effects of electro-acupuncture combined with bone marrow stromal cell transplantation on recovery of neurological function in rats with spinal cord injury

背景：研究认为骨髓基质细胞在损伤、缺血的脑脊髓组织中定向神经分化与损伤局部的微环境变化有关,特别是神经营养因子的诱导作用。课题组前期实验已证实,针刺可以通过增加各种细胞因子及营养因子的表达,促进神经的再生及修复。目的：观察电针联合骨髓基质细胞移植对脊髓损伤大鼠神经功能恢复的影响。设计、时间及地点：随机对照动物实验,于2005-03/2006-07在哈尔滨医科大学细胞生物实验室完成。材料：健康纯系SD大鼠80只,取8只用于骨髓基质细胞的分离培养,剩余72只随机分为4组：空白对照组、细胞移植组、电针组、联合组,18只/组。KWD-808II型脉冲电针仪由江苏武进第三无线电厂生产。方法：取体外分离培养的第3代骨髓基质细胞,移植前72h行BrdU标记,调整细胞浓度为1×1011L-1备用。4组大鼠均建立脊髓损伤模型,造模后细胞移植组将骨髓基质细胞悬液缓慢注入到脊髓损伤临近区域的灰白质交界处,总细胞数6×105个;空白对照组同法注射等量磷酸盐缓冲液;电针组于造模成功后24h采用脉冲电针仪进行夹脊电针治疗,在距损伤处上下端两个椎体的棘突间隙旁开距中线3.0~4.0mm处取穴,针刺20min,1次/d;联合组行骨髓基质细胞移植＋夹脊电针治疗。主要观察指标：移植后3,7,14d,应用联合行为评分评估大鼠脊髓损伤后神经功能的改善状况;免疫双标法检测BrdU标记的骨髓基质细胞胶质纤维酸性蛋白、神经元烯醇化酶的表达。结果：损伤后3,7,14d与空白对照组比较,细胞移植组、电针组、联合组联合行为评分均有显著性差异（P〈0.05,0.05,0.01）;联合组神经功能恢复情况明显优于细胞移植组、电针组（P〈0.05）;而细胞移植组、电针组之间比较无明显差异（P〉0.05）。与空白对照组相比,细胞移植组、电针组损伤区脊髓结构相对较完整,联合组脊髓结构更加完整,骨髓基质细胞移植的组织内可见BrdU标记细胞在损伤区及其周边区明显聚集并存活;移植后14d细胞移植组神经元烯醇化酶和胶质纤维酸性蛋白阳性细胞率分别分7.2%和1.5%,联合组阳性细胞率分别为7.9%和2.1%。结论：骨髓基质细胞移植后可在宿主体内存活,电针可以促进骨髓基质细胞向神经细胞的分化,电针与骨髓基质细胞移植联合应用可以明显改善脊髓损伤大鼠的运动及感觉功能。

BACKGROUND： The study indicated that directed neural differentiation of bone marrow stromal calls （BMSCs） was associated with changes in local micro-environment in injured spinal cord following cerebral ischemia, in particular neurotrephic factor induction. Group pre-test has confirmed that acupuncture can increase the expression of various cytokines and neurotrophic factors to promote nerve regeneration and repair. OBJECTIVE： To observe the effects of electro-acupuncture combined with BMSC transplantation on recovery of neurological function in rats with spinal cord injury. DESIGN, TIME AND SETTING： Randomized controlled animal experiments were performed at the Laboratory of Cell Biology, Harbin Medical University from March 2005 to July 2006. MATERIALS： A total of 80 pure healthy Sprague Dawley rats were selected. Eight were used for the isolation and culture of BMSCs, and the remaining 72 rats were equally and randomly divided into 4 groups： blank control group, cell transplantation group, electro-acupuncture group, and combination group. KWD-80811 pulse-type apparatus was produced by the third radio factory in Jiangsu Wujin production. METHODS： In vitro isolation and culture of the third passage of BMSCs were obtained and laPeled by BrdU at hour 72 before transplantation, and BMSCs at 1×10^11/L were harvested for use. Rat models of spinal cord injury were established in each group. Following model establishment, BMSC suspension was injected into the injured spinal cord near the junction of the gray matter and white matter in the cell transplantation group, totally 6 × 10^5 cells. The same volume of phosphate buffer saline was infused into rats of the blank control group by the same method. In the electro-acupuncture group, at hour 24 after the successful model establishment, Jiaji Electroacupuncture Electroacupuncture instrument treatment was performed by pulse electro-acupuncture apparatus at 3.0-4.0 mm from median line in the upper and lower end of the spinous process vertebral body adjacent to open space for 20 minutes, once a day. In the combination group, rats underwent BMSC transplantation ＋ Jiaji electro-acupuncture treatment. MAIN OUTCOME MEASURES： After transplantation for 3, 7, 14 days, neurological conditions were assessed by combined behavioral scores in rats with spinal cord injury. Glial fibrillary acidic protein and neuron-specific enolase expression was measured in BrdU-labeled BMSCs by immuno-double-labeling method. RESULTS： 3, 7, 14 days after injury, compared with the blank control group, significant differences in combined behavioral scores were determined in the call transplantation group, electro-acupuncture group, and combination group （P 〈0.05, 0.05, 0.01） The recovery of neurological functton was significantly greater in the combination group than in the cell transplantation and electro-acupuncture groups （P 〈 0.05）. No significant difference was detected between the call transplantation group and the electro-acupuncture group （P 〉 0.05）. Compared with blank control group, spinal cord structure was relatively complete in the call transplantation group and electro-acupuncture group. The structure was more integrity in the combination group. Bone marrow stromal cell transplantation presented the organization BrdU-labeled cells in the lesion and the surrounding area, with obvious aggregation and survival. At day 14 following cell transplantation, positive rates of neuron-specific enolase and glial flbrillary acidic protein were respectively 7.2% and 1.5% in the cell transplantation group, 7.9% and 2.1% in the combination group. CONCLUSION： The BMSCs after transplantation can survive in the host body. Electro-acupuncture could promote the differentiation of BMSCs into neural ceils. Combination of electro-acupuncture and BMSC transplantation can significantly improve the motor and sensory function in rats with spinal cord injury.