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

发布时间：2019-06-07 15:22

【摘要】：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(P0.05,0.05,0.01);联合组神经功能恢复情况明显优于细胞移植组、电针组(P0.05);而细胞移植组、电针组之间比较无明显差异(P0.05)。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。
[Abstract]:Background: it is considered that directional nerve differentiation of bone marrow stroma cells in injured and ischemic cerebral and spinal cord tissues is related to the changes of local microenvironment, especially the induction of neurotrophic factors. Previous experiments have confirmed that acupuncture can promote nerve regeneration and repair by increasing the expression of various cytokines and nutritional factors. Objective: to observe the effect of electro-acupuncture combined with bone marrow stroma cell transplantation on the recovery of nerve function in rats with spinal cord injury. Design, time and place: a randomized controlled animal experiment was conducted in the Cell Biology Laboratory of Harbin Medical University in 2005 / 03 / 2006 / 07. Materials: 80 healthy pure SD rats were used for the isolation and culture of bone marrow stroma cells. The remaining 72 rats were randomly divided into 4 groups: blank control group, cell transplantation group, acupuncture group, combined group. KWD- 808II pulse needle instrument is produced by Jiangsu Wujin No. 3 Radio Factory. Methods: the third passage bone marrow stroma cells isolated and cultured in vitro were labeled with BrdU 72 hours before transplantation, and the cell concentration was adjusted to 1 脳 1011L-1. Spinal cord injury models were established in all four groups. In the cell transplantation group, the bone marrow stroma suspension was slowly injected into the gray and white matter junction of the adjacent area of spinal cord injury, and the total number of cells was 6 脳 105. The blank control group was injected with the same amount of phosphate buffer. In the EA group, 24 hours after the successful establishment of the model, the patients in the EA group were treated with pulse electro-acupuncture instrument. The acupoints were taken at the distance between the spinous process space of the two vertebrae at the upper and lower ends of the injury and the 3.0~4.0mm of the midline, and the acupuncture was done once a day for 20 minutes. The combined group was treated with bone marrow stroma cell transplantation with spinal acupuncture. Main outcome measures: the improvement of nerve function after spinal cord injury was evaluated by combined behavior score at 3, 7 and 14 days after transplantation. The expression of glial fibrillar acidic protein and neuron enolase in bone marrow stroma cells labeled with BrdU was detected by double immunostaining. Results: compared with the blank control group at 3, 7 and 14 days after injury, the combined behavior scores of cell transplantation group, acupuncture group and combined group were significantly different (P0.05, 0.05, 0.01). The recovery of nerve function in the combined group was significantly better than that in the cell transplantation group and the electro-acupuncture group (P 0.05), while there was no significant difference between the cell transplantation group and the acupuncture group (P 0.05). Compared with the blank control group, the spinal cord structure of the injured area in the cell transplantation group and the combined group was relatively complete, and the spinal cord structure in the combined group was more complete. In the tissue of bone marrow stroma cell transplantation, BrdU labeled cells were significantly aggregated and survived in the injured area and its surrounding area. On the 14th day after transplantation, the positive cell rates of neuron enolase and glial fibrillar acidic protein in the cell transplantation group were 7.2% and 1.5%, respectively, and those in the combined group were 7.9% and 2.1%, respectively. Conclusion: bone marrow Stromal cells can survive in the host body after transplantation, and EA can promote the differentiation of Bone Marrow Stromal cells into nerve cells. The combined application of electro-acupuncture and bone marrow stroma cell transplantation can significantly improve the motor and sensory function of spinal cord injury rats.
【作者单位】：深圳市第六人民医院疼痛科;
【基金】：黑龙江省研究生创新基金资助项目~~
【分类号】：R651.2;R245

【参考文献】