慢病毒介导NEP1-40及NT-3双基因转染神经干细胞的实验研究  

Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells

作　　者：王林楠[1] 汪雷[1] 宋跃明[1] 刘立岷[1] 杨曦[1] 丰干均[1] 周春光[1] WANG Linnan, WANG Lei, SONG Yueming, LIU Limin, YANG Xi, FENG Ganjun, ZHOU Chunguang(Department of Orthopedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.Chin)

机构地区：[1]四川大学华西医院骨科,成都610041

出　　处：《中国修复重建外科杂志》2018年第4期420-427,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：四川省科技厅科技支撑项目(2015SZ0170)~~

摘　　要：目的通过慢病毒载体将NEP1-40(Nogo extracellular peptide residues 1-40)及神经营养因子_3(neurotrophin 3,NT-3)双基因转染入神经干细胞(neural stem cells,NSC_s)内进行表达,探讨NEP1-40及NT-3双基因转染NSC_s的可行性,为NSC_s体内实验奠定基础。方法将SD大鼠胚胎室管膜区NSC_s采用空载慢病毒载体(A组)、NEP1-40慢病毒载体(B组)、NT-3慢病毒载体(C组)及NEP1-40和NT-3慢病毒载体(D组)进行转染,以未转染病毒的细胞作为对照组(E组)。用感染复数(multiplicity of infection,MOI)为5、10、15的慢病毒载体分别转染24、48、72 h,荧光显微镜观察转染后细胞内荧光表达情况,确定慢病毒载体的最佳MOI和收样时间。再分别通过实时荧光定量PCR及Western blot,检测转染后细胞中NEP1-40及NT-3基因的表达,以及细胞和培养基中NEP1-40及NT-3蛋白的表达。结果荧光显微镜观察示,MOI为10时NEP1-40和NT-3基因慢病毒载体在NSC_s内转染率最高,最佳时间为转染48 h时。实时荧光定量PCR及Western blot检测示,B、D组NEP1-40 m RNA相对表达量和蛋白相对表达量均显著高于A、C组,差异有统计学意义(P<0.05);A、C组间及B、D组间比较差异无统计学意义(P>0.05)。C、D组NT-3 m RNA相对表达量和蛋白相对表达量均显著高于A、B组,差异有统计学意义(P<0.05);A、B组间和C、D组间比较差异无统计学意义(P>0.05)。结论通过慢病毒载体可将NEP1-40及NT-3双基因成功转染入NSC_s内,在NSC_s内稳定表达,且两种目的基因在表达过程中无相互拮抗或促进作用。ObjectiveTo explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 （NEP1-40） gene and neurotrophin 3 （NT-3） gene into neural stem cells （NSCs）.MethodsNSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups： no-load lentiviral vector transducted NSCs （group A）, NEP1-40 transducted NSCs （group B）, NT-3 transducted NSCs （group C）, NEP1-40 and NT-3 corporately transducted NSCs （group D）, and blank control （group E）. Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection （MOI; 5, 10, 15） for different time （24, 48, 72 hours）. Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium.ResultsThe optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C （P〈0.05）, but no significant difference was found between groups B and D, and between groups A and C （P〉0.05）. The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B （P〈0.05）, but no significant difference was found between groups A and B, and between groups C and D （P〉0.05）.ConclusionNEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.

关 键 词：神经干细胞 NEP1-40 神经营养因子3 慢病毒载体 转染 

分 类 号：Q78[生物学—分子生物学]