摘要
目的观察大鼠脐带华通胶来源MSCs(umbilical cord MSCs,UCMSCs)体外培养特性及其对大鼠脊髓损伤(spinal cord injury,SCI)后神经再生的作用。方法取足天Wistar孕鼠脐带分离、培养UCMSCs,体外扩增并采用流式细胞仪鉴定。细胞移植前1d取第5代细胞以Hoechst33258染液标记,调整终浓度为1×105个/μL备用。另取成年雌性Wistar大鼠60只,体重(300±10)g,随机分为3组,每组20只。A、B组以Impactor model-Ⅱ型打击器制备大鼠T10SCI模型,并分别于损伤区注射1μL标记后的细胞悬液和等量DMEM培养液;C组行单纯椎板减压,不予脊髓打击及治疗措施。于术后1d,1、2、3、4、5、6周,采用BBB评分法行肢体功能评分;术后6周行神经丝蛋白200(neurofi lament200,NF-200)免疫组织化学染色观察细胞分化情况;术后10周行生物素葡聚糖胺(biotinylated dextran amine,BDA)顺行示踪观察皮质脊髓束(corticospinal tract,CST)再生情况。结果培养的UCMSCs为梭形细胞,呈漩涡状或平行贴壁生长。流式细胞仪检测显示,UCMSCs表达CD29,不表达CD31、CD45及HLA-DR。术后A、B组BBB评分均明显上升,3周后A组BBB评分明显高于B组,差异有统计学意义(P<0.05);各时间点A、B组评分均低于C组(P<0.05)。术后6周,荧光显微镜观察可见Hoechst33258染液标记的UCMSCs存活并向损伤区聚集;A、C组NF-200免疫荧光染色阳性面积分别为(11943±856)像素及(13117±945)像素,明显高于B组的(7986±627)像素,差异有统计学意义(P<0.05);A、C组差异无统计学意义(P>0.05)。BDA顺行神经示踪观察显示,术后10周A组部分CST再生神经纤维可延伸至损伤区远端;B组神经纤维极少通过损伤空洞;C组CST纤维在对侧脊髓白质后索中行走。A、B组各点再生轴突数与T5横断面阳性染色轴突百分比与C组比较,差异均有统计学意义(P<0.05)。结论UCMSCs能在体外快速增殖,移植后在大鼠SCI部位存活并向神经细胞分化,可有效促进SCI后运动功能恢复和神经轴突再生。
Objective To study the growth characteristics of umbilical cord MSCs （UCMSCs） in vitro and its effect on the nerve regeneration after spinal cord injury （SCI）. Methods UCMSCs isolated from pregnant rats umbilical cord were cultured and purified in vitro. Sixty female Wistar rats weighing （300 ± 10） g were randomized into three groups （n=20 per group）. UCMSCs group （group A） in which UCMSCs suspension injection was conducted; DMEM control group （group B） in which 10% DMEM injection was conducted; sham group （group C） in which the animal received laminectomy only. Establish acute SCI model （T10） by Impactor model-II device in group A and group B. The recovery of the lower extremity was observed using BBB locomotor scoring system,neurofilament 200 （NF-200） immunofluorescence staining was performed to detect the neural regeneration,and then the corticospinal tract （CST） was observed using the biotinylated dextran amine （BDA） tracing. Results Cultured UCMSCs were spindle-shaped fibrocyte-like adherent growth,swirling or parallelly. The USMSCs expressed CD29,but not CD31,CD45,and HLA-DR. The BBB score was higher in group A than group B 4,5,and 6 weeks after operation,and there was a significant difference between two groups （P 〈 0.05）. The BBB scores at different time points were significantly lower in groups A and B than that in group C （P 〈 0.05）. UCMSCs was proved to survive and assemble around the injured place by frozen section of the cords 6 weeks after injury. NF-200 positive response area in groups A,B,and C was （11 943 ± 856）,（7 986 ± 627）,and （13 117 ± 945） pixels,respectively,suggesting there was a significant difference between groups A,C and group B （P 〈 0.05）,and no significant difference was evident between group A and group C （P 〉 0.05）. BDA anterograde tracing 10 weeks after operation demonstrated that more regenerated nerve fibers went through injured area in group A,but just quite few nerve fibers in group B went through the injuried cavity. The ratios of regenerative axons amount to T5 axons in group A and group B were smaller than that of group C （P 〈 0.05）. Conclusion UCMSCs can proliferate rapidly in vitro,survive and differentiate to neurons after being grafted into injured spinal cord. The transplantation of UCMSCs is effective in promoting functional recovery and axonal regeneration after SCI.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第12期1491-1496,共6页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30872603)
教育部新世纪优秀人才支持计划(NCET-06-0251)
天津市应用基础和前沿技术支持计划(07JCYBJC10200)~~
关键词
脊髓损伤
脐带MSCs
细胞移植
神经再生
大鼠
Spinal cord injury Umbilical cord MSCs Cell transplantation Neural regeneration Rat
