NGF过表达质粒修饰BMSCs移植促进大鼠急性脊髓损伤修复  

Transplantation of BMSCs Modified with NGF Overexpression Plasmid Promotes Repair of Acute Spinal Cord Injury in Rats

作　　者：石勇[1] 霞晓燕[1] Shi Yong;Xia Xiaoyan(Department of Neurosurgery,Mianyang People's Hospital,Mianyang 621000,China)

机构地区：[1]绵阳市人民医院神经外科,四川绵阳621000

出　　处：《实用骨科杂志》2020年第8期707-711,715,共6页Journal of Practical Orthopaedics

摘　　要：目的探究神经生长因子(nerve growth factor,NGF)基因过表达载体转染骨髓间充质干细胞移植促进大鼠急性脊髓损伤的修复。方法抽取人源骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),采用流式细胞仪检测BMSCs的表面蛋白,利用CRISPR/Cas 9技术构建NGF过表达载体质粒,通过慢病毒技术转染BMSCs,利用钳夹法构建(sprague dawley,SD)大鼠急性脊髓损伤(spinal cord injury,SCI)的动物模型,根据处理方案的不同,将SCI动物模型分成三组,即模型组、BMSCs组和过表达质粒组,每组5只。利用反转录聚合酶链反应(reverse transcriotion-polymerase chain reaction,RT-PCR)、Western blot、免疫荧光等方法检测NGF的表达和神经的修复。结果流式细胞仪检测第2代BMSCs的表面蛋白,CD 44和CD 29的表达为60.2%和58.3%;CD 34和CD 45的表达为3.4%和2.6%。慢病毒转染效率为90%以上。模型组术后1 d的运动功能(basso-beattie-bresnahan,BBB)评分降至最低,过表达质粒组高于模型组和BMSCs组(P<0.05)。后肢踏空率的比较中,过表达质粒组要明显高于模型组和BMSCs组(P<0.05)。RT-PCR结果显示,过表达质粒组NGF mRNA的相对表达水平要明显高于模型组和BMSCs组(P<0.05)。Western blot的结果与RT-PCR结果相同。免疫荧光结果显示,过表达质粒组NGF可与神经元细胞核(neuronal nuclei,NeuN)共染,神经元细胞量增多。过表达质粒组脊髓修复程度要优于BMSCs组和模型组,可见断裂间距明显缩小,断端修复较好。结论NGF过表达质粒转染后可以促进BMSCs向神经元细胞分化,促进SCI动物模型的功能恢复。Objective To investigate the effect of bone marrow mesenchymal stem cell transplantation transfected with nerve growth factor(NGF)gene overexpression vector on the repair of acute spinal cord injury in rats.Methods Human bone marrow mesenchymal stem cells(BMSCs)were extracted and the surface proteins of BMSCs were detected by flow cytometry.NGF overexpression vector plasmid was constructed by Cas-9 technology.BMSCs were transfected by lentivirus technology.The animal models of acute spinal cord injury in SD rats wereestablished by clamp method.SCI was constructed according to different treatment schemes.The models were divided into three groups:model group,BMSCs group and overexpression plasmid group.There were5models in each group.The NGF expression and nerve repair were detected by RT-PCR,Western blot and the immunofluorescence.Results The expression of CD44 and CD29 was 60.2%and 58.3%in the second generation BMSCs,while the expression of CD34 and CD45 was 3.4%and 2.6%in the second generation BMSCs by flow cytometry.The transfection efficiency of lentivirus was over 90%.The BBB score of the model group was the lowest one day after operation,and that of the overexpression plasmid group was higher than that of the model group and BMSCs group(P<0.05).Compared with model group and BMSCs group,the vacancy rate of hind limbs in overexpression plasmid group was significantly higher(P<0.05).RT-PCR results showed that the relative expression level of NGF in overexpression plasmid group was significantly higher than that in model group and BMSCs group(P<0.05).The result of Western blot was the same as that of RT-PCR.Immunofluorescence results showed that NGF could co-stain with NeuN in the overexpressed plasmid group,and the number of neurons increased.The degree of spinal cord repair in overexpression plasmid group was better than that in BMSCs group and model group.It was found that the fracture spacing was obviously reduced and the repair of the broken end was better.Conclusion NGF overexpression plasmid can promote B

关 键 词：神经生长因子 骨髓间充质干细胞 脊髓损伤 慢病毒转染 

分 类 号：R651.2[医药卫生—外科学]