NEP1-40和NT-3基因共转染施万细胞来源外泌体对神经干细胞存活与分化的影响  

Effects of co-transfection of NEP1-40 and NT-3 genes with Schwann cell-derived exosomes on the survival and differentiation of neural stem cells

作　　者：邓志鹏 王林楠[1,2] 张庄 杨曦[1,2] 宋跃明[1,2] 汪雷[1,2] DENG Zhipeng;WANG Linnan;ZHANG Zhuang;YANG Xi;SONG Yueming;WANG Lei(Department of Orthopedics,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China;Orthopedic Research Institute,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China)

机构地区：[1]四川大学华西医院骨科,成都610041 [2]四川大学华西医院骨科研究所,成都610041

出　　处：《华西医学》2022年第7期1041-1049,共9页West China Medical Journal

基　　金：四川省科学技术厅重点研发项目(2020YFS0143)。

摘　　要：目的 观察Nogo胞外肽端残基1-40(Nogo extracellular peptide residues 1-40,NEP1-40)和神经营养因子3(neurotrophin 3,NT-3)基因共转染施万细胞来源外泌体(Schwann cell-derived exosome,SCDE)对神经干细胞(neural stem cell,NSC)存活与分化的影响,为SCDE与NSC共移植的体内试验奠定基础。方法 通过慢病毒载体将NEP1-40及NT-3双基因转染入施万细胞,收集SCDE进行鉴定。选取传代3次的NSC,将其接种到接种板中,分为常规培养组、单纯外泌体培养组(加入空载质粒修饰SCDE进行培养)和双基因外泌体培养组(加入双基因修饰SCDE进行培养)。检测各组细胞的活性,并通过免疫荧光检测神经元特异核蛋白(neuronal nuclei,NeuN)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和半乳糖神经酰胺酶(galactosylceramidase,GALC)阳性的细胞来评价NSC的存活与分化情况。结果 双基因转染后荧光强度均较高且细胞状态较好。双基因组NEP1-40和NT-3的信使RNA和蛋白相对表达水平均高于空载质粒组(P<0.05)。双基因组SCDE中NEP1-40和NT-3的蛋白相对表达水平均高于空载质粒组(P<0.05),两组的SCDE中CD63的蛋白相对表达水平差异无统计学意义(P>0.05)。细胞活性方面,双基因外泌体培养组细胞活性最强,单纯外泌体培养组次之,常规培养组最弱,组间两两比较差异有统计学意义(1.28±0.04 vs. 0.72±0.09 vs. 0.41±0.04,P<0.05)。细胞存活情况方面,双基因外泌体培养组的NeuN阳性细胞(5.23±0.22 vs. 2.36±0.09 vs. 1.00±0.01)和GALC阳性细胞(2.29±0.06 vs.1.75±0.02 vs.1.00±0.04)存活情况最佳,单纯外泌体培养组次之,常规培养组最差,组间两两比较差异有统计学意义(P<0.05);常规培养组的GFAP阳性细胞存活情况最佳,单纯外泌体培养组次之,双基因外泌体培养组最差(1.00±0.04 vs. 0.52±0.04 vs. 0.30±0.01),组间两两比较差异有统计学意义(P<0.05)。细胞分化情况方面,双基因外泌体培养组的NeuN阳性细�Objective To observe the effects of co-transfection of Nogo extracellular peptide residues 1-40(NEP1-40) and neurotrophin 3(NT-3) genes with Schwann cell-derived exosomes(SCDEs) on the survival and differentiation of neural stem cells(NSCs), and lay the foundation for the in vivo experiments of SCDE and NSC co-transplantation. Methods The NEP1-40 and NT-3 genes were transfected into Schwann cells by lentiviral vector,and SCDEs were collected for identification. The NSCs that have been passaged for 3 times were selected and inoculated into the inoculation plate, and they were divided into conventional culture group, simple exosome culture group(adding empty vector plasmid to modify SCDE for culture) and two genes exosome culture group(adding two genes modified SCDE for culture). The activity of cells in each group was detected. The survival and differentiation of NSCs were evaluated by immunofluorescence detection of neuronal nuclei(NeuN), glial fibrillary acidic protein(GFAP) and galactosylceramidase(GALC) positive cells. Results After transfection of these two genes, the fluorescence intensity was higher and the cell state was better. The relative expression levels of messenger RNA and protein of NEP1-40 and NT-3 in the two gene groups were higher than those in the empty plasmid group(P<0.05). The relative expression levels of NEP1-40 and NT-3 proteins in SCDE of the two gene groups were higher than those of the empty vector group(P<0.05).There was no significant difference in the relative expression level of CD63 protein in SCDE between the two groups(P>0.05). In terms of cell activity, the cell activity of the two genes exosome culture group was the strongest, followed by the simple exosome culture group, and the conventional culture group was the weakest. The differences between any two groups were statistically significant(1.28±0.04 vs. 0.72±0.09 vs. 0.41±0.04, P<0.05). In terms of cell survival, NeuN-positive cells(5.23±0.22 vs. 2.36±0.09 vs. 1.00±0.01) and GALC-positive cells(2.29±0.06 vs. 1.75±0.0

关 键 词：外泌体 施万细胞 Nogo胞外肽端残基1-40 神经营养因子3 神经干细胞 

分 类 号：R338.1[医药卫生—人体生理学]