PKH67示踪骨髓间充质干细胞迁移至损伤脊髓的实验研究  

Study on the utilization of PKH67 in the transplantation of bone marrow mesenchymal stem cells to in-jured spinal cord

作　　者：马娇[1] 张雪梅[1] 孙杨[1] 俞冰倩 Ma J iao Zhang Xuemei Sun Yang et al.(Department of Neurology, Second Affiliated Hospi- tal of Harbin Medical University,Harbin 150086)

机构地区：[1]哈尔滨医科大学附属第二医院,150086

出　　处：《卒中与神经疾病》2016年第6期432-437,共6页Stroke and Nervous Diseases

基　　金：于维汉院士杰出青年培养基金项目

摘　　要：目的 探讨SCI后体外移植PKH67标记的BMSCs迁移至脊髓损伤处并进行增值和分化的动员情况。方法 用梯度离心法分离和培养出SD大鼠第3代BMSCs,用绿色荧光染料PKH67标记;采用钳夹法制备脊髓损伤（SCI）模型,分为实验组（n=15）、对照组（n=16）、假手术组（n=16）;SCI术后对脊髓损伤组织进行HE染色,实验组和假手术组于术后尾静脉移植含有1×10^7个BMSCs的0.5 mL生理盐水,对照组注射等量生理盐水;分别于术后1、7、14、21 d观察大鼠后肢运动功能恢复情况,并做BBB分;术后21 d后取脊髓组织,行免疫荧光染色,观察BMSCs的迁移,增值和分化情况。结果 （1）镜下可见损伤脊髓形成的空洞、坏死及炎性细胞的增多;（2）共聚焦荧光显微镜观察显示术后21 d实验组脊髓损伤部位可见移植的BMSCs,部分BMSCs呈GFAP和Nestin阳性表达;假手术组无PKH67标记的BMSCs;实验组GFAP和Nestin阳性细胞数较对照组和假手术组明显增加（P〈0.05）,对照组较假手术组增加不明显（P〉0.05）;（3）实验组和对照组BBB评分均有增加,但实验组BBB评分显著高于对照组（P〈0.05）。结论 PKH67示踪的BMSCs可迁移至损伤脊髓部位,进行增值并分化为神经元样细胞,促进损伤脊髓的神经功能恢复。Objective To investigate the utilization of PKH67 in the transplantation of bone marrow mesenchymal stem cells to injured spinal cord and the migration, proliferation and differentiation of the BM- SCs. Methods Density gradient centrifugation method was used for isolating the rat BMSCs and the 3rd passage of BMSCs were labeled with green fluorescent dye PKH67. SCI model was generated by using aneurysm clip to clamp the the spinal cord of the rat. There were three groups ： BMSCs group, sham operation group andcontrol group. After SCI, the lesion of spinal cord was taken for HE, 1.0×10^7ceils labeled with PKH67 in 0. 5 mL 0. 9% sterile saline solution were transfused intravenously in BMSCs group and sham-operation group, the same volume of sterile saline solution were transfused in control group. The BBB scale was performed at 1 day, 7 days, 14 days, 21 days after surgery for evaluating hind limbs movement. The migration and differentiation of BMSCs were determined by using triple immunofluorescent staining. Results （1） Syringomyelia after injury, necrosis and increased inflammatory cells could be seen under microscopy. （2）Immunofluoresence analysis were performed at 21 days post-transplantation and the transplanted BMSCs were centralized in the epicenter of in- jured spinal-cord, part of BMSCs expressed by GFAP and Nestin. There were no PKH67＋ cells in sham opera- tion group. The GFAP＋ and Nestin＋ cells in BMSCs group were significantly higher than Other two groups （P〈0. 05）, while there was no obviously difference between sham operation group and control group （P〈0. 05）. （3）The BBB scores of BMSCs group and control group both improved, but the rats in BMSCs group improved rapidly than control group （P〈0. 05）. Conclusion The bone marrow mesenchymal stem cells labeled with PKH67 could migrate to the lesion of spinal cord, proliferate and differentiate into neurocyte-like cells, which could promote the recovery of spinal cord＇s function.

关 键 词：PKH67 骨髓间充质干细胞 脊髓损伤 增值 

分 类 号：R744[医药卫生—神经病学与精神病学]