摘要
目的:分析骨髓间充质干细胞移植对大鼠脊髓损伤后生长相关蛋白43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,随机选取4只大鼠作骨髓间充质细胞的分离与培养。其余60只随机分成3组:细胞移植组(n=24),磷酸盐缓冲液组(n=24),空白对照组(n=12)。骨髓间充质干细胞提取自成鼠的股骨骨髓,经过培养、鉴定及传代,细胞传3代,待细胞大约长至50%汇合时,吸弃培养基,置于37℃,体积分数为0.05的CO2培养箱中培育30min,调整细胞密度以备移植。将大鼠麻醉后,无菌条件下用改良Allen打击装置,制作大鼠脊髓损伤动物模型,脊髓损伤后第7天,细胞移植组以微量注射器缓慢注入含间充质干细胞(106/mL)的培养液5μL,磷酸盐缓冲液组注入等量磷酸盐缓冲液5μL,空白对照组未制作脊髓损伤,分别于移植术后1、3、5d以及术后7、14、28d麻醉处死大鼠,细胞移植组与磷酸盐缓冲液组取出损伤节段的脊髓(4只/时点),空白对照组于同一节段取出相应脊髓(2只/时点)。应用反转录-聚合酶链反应和免疫组化法观察间充质干细胞移植后大鼠脊髓损伤区生长相关蛋白43基因表达的变化。结果:各组实验动物均全部进入结果分析,无脱落。①生长相关蛋白43mRNA的表达:生长相关蛋白43mRNA在正常大鼠脊髓组织中呈弱表达。间充质干细胞移植术后第1、3、7天时间点,细胞移植组生长相关蛋白43mRNA逐渐增高表达,与磷酸盐缓冲液组比较差别有统计学意义(P<0.05)。②生长相关蛋白43的表达:生长相关蛋白43在正常大鼠脊髓组织中有一定表达,间充质干细胞移植术后第7、14、28天,细胞移植组生长相关蛋白43持续高水平表达,与磷酸盐缓冲液组比较差别有统计学意义(P<0.05)。结论:间充质干细胞在移植后通过上调生长相关蛋白43基因的表达从而促进轴突的再生,可能是治疗脊髓损伤的重要机制。
AIM：To analyze the effects of mesenchymal stem cells （MSCs） transplantations on the expression of growth associated protein-43 （GAP- 43） after the spinal cord injury （SCI） in rats.
METHODS：The experiment was conducted at the Experimental Animal Center of Chinese Medical University between June 2002 and June 2003. Among 64 Wistar rats of 3-month old, 4 were selected randomly to perform the isolation and culture of MSCs, and other 60 rats were divided into 3 groups at random： cell transplantation group （n=24）, phosphate buffer solution （PBS） group （n-24） and control group （n=12）. MSCs were extracted from the thigh bone marrow of adult Wistar rats, then cultured, evaluated and subcultured for 3 generations. When grew to 50%, the cells were confluenced and then placed and cultured at 37℃ incubator containing 5% CO2 for 30 minutes. The densities of cells were adjusted to prepare for transplantations. The SCI models were established in the anaesthetized rats of sterile condition by the modified ALLen set. On 7^th day after SC1 operation, 5μL cultured medium containing 106/mL MSCs was injected gradually by microsyringe in the cell transplantation group, and same dose of phosphate buffer was given in PBS group. There was no SC1 model in the control group. At 1,3,5 days before transplantation and 7, 14,28 days after transplantation, all the rats were executed in anesthesia. The spinal cords were obtained from the injured segments in the cell transplantation group and PBS group （4 rats at each time point） while the corresponding spinal cord in the control group was obtained from the same segment （2 rats at each time point）. The expression changes of GAP-43 were observed with reverse transcription polymerase chain reaction and immunohistochemical method. RESULTS：All the experimental animals were entered into the result analysis without drop. ①Expressions of GAP-43 mRNA： GAP-43 mRNA expressed weakly in the spinal tissues of normal rats. Compared with PBS group, at 1, 3, 7 days after transplantation, the expressions of GAP-43 mRNA enhanced gradually in the cell transplantation group, with the significant difference （P 〈 0.05）.②Expressions of GAP-43： GAP-43 expressed certainly in the spinal tissues of normal rats. Compared with PBS group, at 7, 14, 28 days after transplantation, GAP-43 mRNA continued to express highly in the cell transplantation group, with the significant difference （P 〈 0.05）.
CONCLUSION ：The transplantations of MSCs can promote the regeneration of axons through the up-regulation of GAP-43 expressions, which is possibly an important mechanism of treating SC1.
出处
《中国临床康复》
CSCD
北大核心
2006年第13期5-7,i0001,共4页
Chinese Journal of Clinical Rehabilitation
