骨髓间充质干细胞移植大鼠脊髓损伤区脑源性神经营养因子的表达

Effects of bone marrow-derived mesenchymal stem cells transplantation on the brain derived neurotrophic factors in rats after spinal cord injury

目的:观察脊髓间充质干细胞移植对大鼠脊髓损伤后脑源性神经营养因子表达的影响。方法:实验于2002-06/2003-06在中国医科大学实验动物中心完成。选取3月龄Wistar大鼠64只,随机选4只大鼠取股骨骨髓作骨髓间充质细胞的分离与培养,经过培养、鉴定及传代。其余60只大鼠分成3组制作脊髓横断损伤模型。细胞移植24只,磷酸盐缓冲液组24只,空白对照12只。脊髓损伤后第7天,在无菌条件下,细胞移植组以微量注射器缓慢注入含骨髓间充质干细胞(106/mL)的培养液5μL,磷酸盐缓冲液组注射注入等量磷酸盐缓冲液5μL,对照组未制作脊髓损伤。分别于术后7d、14d、28d麻醉下行心脏灌流固定取T10节段脊髓,细胞移植组与磷酸盐缓冲液组取出损伤节段的脊髓(8只/时点),空白对照组于同一节段取出相应脊髓(4只/时点)。应用免疫组化法观察间充质干细胞移植后,大鼠脊髓损伤区脑源性神经营养因子的表达变化。结果:所有实验动物均全部进入结果分析,术后感染5只,均予补足。①原代间充质干细胞培养:细胞接种24h后贴壁生长,72h细胞增殖,有细胞团形成,在传代过程中,4代以前的细胞倍增时间为4～6d,至10代以后细胞增殖能力有所减弱,胞体变得扁平,若加入碱性成纤维细胞生长因子,则可维持其增殖能力和形态。②脑源性神经营养因子在正常大鼠脊髓组织中有一定表达,移植术后第7天,第14天及第28天,细胞移植组脑源性神经营养因子均高水平表达,与磷酸缓冲液组相比较差别明显。结论:骨髓间充质干细胞移植后可能通过上调脑源性神经营养因子的表达从而促进脊髓损伤区神经元轴突的再生。

AIM：To observe the effects of bone marrow-derived mesenchymal stem cells （BMMSCs） transplantation on the brain-derived neurotrophic factors （BDNF） after the spinal cord injury （SCI） of rats. METHODS：This experiment was conducted at the Experimental Animal Center, China Medical University form June 2002 to June 2003. Totally 64 3-month-old Wistar rats were used in this experiment. Four rats were randomly chosen, their bone marrow of thighbone was used for isolation and culture of BMMSCS. Culture, identification and passage were performed. The other 60 rats were divided into 3 groups to make spinal cord transection injury model： cell transplantation group （n=24）, phosphate buffer solution group （n=24） and blank control group （n=12）. Seven days after spinal cord injury, under aseptic condition, we slowly injected 5μL culture liquid containing BMMSCs（ 106/mL）into the rats with microinjector in the cell transplantation group. 5 μL phosphate buffer solution was injected into the rats in the phosphate buffer solution. Heart perfusion and fixation was performed at postoperative 7, 14 and 28 days and spinal cord at T10 segment was taken out. Spinal cord with injured segments were taken out from the rats in the cell transplantation group and phosphate buffer solution （8 rats at each time point）, and the same segment of spinal cord was taken out from the rats in blank control group （4 rats at each time point）. The expression of brain-derived neurotrophic factors （BDNF） of injured area of spinal cord was observed with immunohistochemical method after transplantation of mesenchymal stem cells. RESULTS： All the experimental animals entered the stage Of result analysis, 5 rats were infected after operation and replaced by others.① Culture of primary mesenchymal stem cell： Cells grew adhesively at 24 hours after inoculation, and proliferated at 72 hours, with cell mass formation. During the passage, proliferous time of the cells of the first four generations was 4 to 6 days. Cellular proliferative ability declined from the 10^th generation. Cell body became flat. The added basic fibroblast growth factors can maintain cellular proliferative ability and morphology. ②Expression of BDNF： BDNF had certain expression in the spinal tissue of normal rats. The expression of BDNF was higher on the 7^th, 14^th and 28^th day after transplantation in cell transplantation group as compared with phosphate buffer solution group, with significant statistical meaning. CONCLUSION：BMMSCs can promote the regeneration of neuron axon of spinal injury region after transplantation through up-regulating the expression of BDNF.