AAV介导NGF过表达载体转染BMSCs对高强度聚焦超声致活体SD大鼠坐骨神经损伤模型保护作用  

Protective effect of AAV-mediated NGF overexpression vector transfection on BMSCs on sciatic nerve injury model of living SD rats induced by high-intensity focused ultrasound

作　　者：程光[1] 王梅[2] 刘爱华[3] CHENG Guang;WANG Mei;LIU Ai-hua(Department of Ultrasound,Fourth People's Hospital of Tai'an City,Tai'an 271000,P.R.China;Tai'an Central Hospital,Tai'an 271000,P.R.China)

机构地区：[1]泰安市第四人民医院超声科,山东泰安271000 [2]泰安市中心医院超声科,山东泰安271000 [3]泰安市中心医院中心实验室,山东泰安271000

出　　处：《社区医学杂志》2020年第10期721-727,731,共8页Journal Of Community Medicine

摘　　要：目的干细胞是当前研究的热门领域,利用干细胞定向分化作用可以促进神经损伤修复,但是干细胞具有分化方向不定和易老化的特点。本研究探究腺相关病毒(adeno associated virus,AAV)介导神经生长因子(nerve growth factor,NGF)过表达载体转染骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)对高强度聚焦超声致活体SD大鼠坐骨神经损伤模型的保护作用。方法分离和培养人源BMSCs,采用流式细胞仪检测BMSCs的CD29,CD45和CD90等表面蛋白表达。采用免疫组织化学染色鉴定BMSCs表面CD34和CD44表达。AAV腺病毒构建NGF过表达质粒,通过高强度聚焦超声制备SD大鼠坐骨神经损伤模型,根据处理方式不同,将SD大鼠分成空白对照组,模型组,BMSCs组和NGF过表达组。利用HE染色,电镜方法观察坐骨神经形态,示踪金染色观察神经接头的聚集,TUNEL染色检测细胞凋亡,RT-qPCR检测NGF和caspase-3mRNA表达。结果流式细胞仪检测第2代BMSCs表面蛋白,CD29阳性率为98.03%,CD90阳性率为96.92%,CD45阴性率为77.2%,共表达CD29、CD90且CD45表达阴性的细胞数占总细胞数的96.89%。BMSCs细胞表面CD34和CD44免疫组织化学染色呈阳性。慢病毒转染效率>90%,转染效率较高。HE染色结果中模型组大鼠坐骨神经有明显断裂,BMSCs组大鼠坐骨神经较模型组裂痕缩小,而NGF过表达组断裂处几乎不可见。电镜结果显示,模型组大鼠坐骨神经密度少,且形态不规则,BMSCs组大鼠坐骨神经较模型组密度大,且周边为纤维组织填充,NGF过表达组大鼠坐骨神经密度大,排列较为规则。示踪金染色显示NGF过表达组神经接头处可见神经聚集。RT-qPCR结果显示空白对照组、模型组、BMSCs组和NGF过表达组NGF mRNA相对表达量分别为1.18±0.05、0.77±0.06、1.75±0.07和3.09±0.08,F=9.082,P=0.019;空白对照组、模型组、BMSCs组和NGF过表达组caspase-3mRNA表达量分别为2.03±0.09、5.05±0.23、3.12±0.09和1.2OBJECTIVE Stem cells are a hot field of current research.Stem cells can be used to promote the repair of nerve damage through the differentiation of stem cells,but stem cells have the characteristics of uncertain differentiation direction and easy aging.This study explores the effect of adeno associated virus(AAV)-mediated nerve growth factor(NGF)overexpression vector transfection on bone marrow mesenchymal stem cells(BMSCs)on high intensity focused ultrasound Protective effect of sciatic nerve injury model in living SD rats.METHODS Isolate and culture human BMSCs,and use flow cytometry to detect the expression of CD29,CD45 and CD90 on BMSCs.Immunohistochemical staining was used to identify the expression of CD34 and CD44 on the surface of BMSC.AAV adenovirus constructs NGF overexpression plasmid and prepares SD rat sciatic nerve injury model by high-intensity focused ultrasound.According to different treatment methods,SD rats were divided into blank control group,model group,BMSCs group and NGF overexpression group.HE staining and electron microscopy were used to observe the sciatic nerve morphology,tracer gold staining was used to observe the aggregation of nerve joints,TUNEL staining was used to detect apoptosis,and RT-qPCR was used to detect the expression of NGF and caspase-3.RESULTS Flow cytometry was used to detect the surface protein of the second generation BMSCs.The positive rate of CD29 was 98.03%,the positive rate of CD90 was 96.92%,and the negative rate of CD45 was 77.2%.The number of cells that co-expressed CD29 and CD90 and negative for CD45 expression accounted for 96.89%.The immunohistochemical staining of CD34 and CD44 on the surface of BMSCs was positive.The lentivirus transfection efficiency was>90%.Transfection efficiency was high.In the HE staining results,the sciatic nerve of the model group was obviously broken,the sciatic nerve of the BMSCs group was smaller than the model group,and the fracture of the NGF overexpression group was almost invisible.Electron microscopy results showed that t

关 键 词：坐骨神经损伤 骨髓间充质干细胞 腺相关病毒 神经生长因子 过表达质粒 

分 类 号：R745.42[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]