摘要
目的 构建携带脑源性神经营养因子(BDNF)、神经营养因子-3(NT-3)、融合基因(BDNF-T2A-NT-3)的慢病毒载体,通过感染人源骨髓间充质干细胞(hBMSCs)检测BDNF和NT-3的mRNA和蛋白相对表达水平。方法 采用聚合酶链反应(PCR)技术扩增制备BDNF、NT-3、BDNF-T2A-NT-3目的基因片段,离体构建携带BDNF、NT-3、BDNF-T2A-NT-3的慢病毒表达载体并感染293T细胞;采用PCR技术检测293T细胞中BDNF、NT-3和BDNF-T2A-NT-3 mRNA相对表达水平;采用流式细胞术检测hBMSCs免疫表型;将对数生长期的hBMSCs随机分为hBMSCs组、空载慢病毒组、BDNF组、NT-3组和融合基因组,分别用空载慢病毒、BDNF慢病毒载体、NT-3慢病毒载体、BDNF-T2A-NT-3慢病毒载体感染后,于倒置相差显微镜下观察细胞荧光强度;采用荧光PCR法和蛋白免疫印迹法检测BDNF、NT-3的mRNA和蛋白的相对表达水平。结果 测序分析鉴定结果显示,成功构建了携带BDNF、NT-3、BDNF-T2A-NT-3的慢病毒载体。慢病毒感染293T细胞后在倒置相差显微镜可观察到绿色荧光,感染目的基因组293T细胞的BDNF、NT-3和BDNF-T2A-NT-3 mRNA相对表达水平均高于无感染目的基因组(P值均<0.01)。流式细胞术检测结果显示,hBMSCs中分化抗原(CD)73;、CD90;、CD34;阳性表达率分别为97.77%、52.15%和0.07%。BDNF组、NT-3组hBMSCs细胞的BDNF mRNA的相对表达水平均高于hBMSCs组(P值均<0.01),融合基因组hBMSCs细胞的BDNF、NT-3 mRNA的相对表达水平均分别高于hBMSCs组、空载慢病毒组、BDNF组和NT-3组(P值均<0.01);BDNF组、NT-3组、融合基因组hBMSCs细胞的NT-3蛋白相对表达水平均分别高于hBMSCs组和空载慢病毒组(P值均<0.01)。结论 成功构建的BDNF、NT-3、BDNF-T2A-NT-3慢病毒载体可介导BDNF和NT-3在hBMSCs中稳定表达,为探索BDNF和NT-3基因在神经退行性疾病发病机制中的作用提供依据。
Objective To construct the lentiviral vector carrying brain-derived neurotrophic factor(BDNF), neurotrophin-3(NT-3) and the fusion gene(BDNF-T2 A-NT-3), and the relative expression levels of mRNA and protein of BDNF and NT-3 were detected by infecting human bone marrow mesenchymal stem cells(hBMSCs). Methods The target gene fragments of BDNF, NT-3 and BDNF-T2 A-NT-3 were amplified and prepared by polymerase chain reaction(PCR) technology, and a lentivirus expression vector carrying BDNF, NT-3 and BDNF-T2 A-NT-3 was constructed in vitro and infected with 293 T cells. The relative mRNA expression of BDNF, NT-3 and BDNF-T2 A-NT-3 in 293 T cells was detected by PCR and the immunophenotype of hBMSCs was detected by flow cytometry. The hBMSCs in logarithmic phase were randomly divided into hBMSCs, empty lentivirus, BDNF, NT-3 and fusion gene groups. The hBMSCs of last four groups were infected with empty lentivirus, BDNF lentivirus vector, NT-3 lentivirus vector and fusion gene BDNF-T2 A-NT-3 lentivirus vector respectively. The fluorescence intensity of cells was observed under an inverted phase contrast microscope. The relative expression of mRNA and protein of the target genes BDNF and NT-3 was detected by fluorescence PCR and Western blot. Results Sequencing analysis and identification results showed that the lentiviral vectors carrying BDNF, NT-3 and BDNF-T2 A-NT-3 were successfully constructed. The green fluorescence could be observed under the inverted phase contrast microscope after the 293 T cells were infected with the lentivirus. The mRNA relative expression of BDNF, NT-3 and BDNF-T2 A-NT-3 in 293 T cells infected with target genome group was higher than that in non-infected target genome group(all P<0.01). The results of flow cytometry showed that the positive expression rates of cluster of differentiation(CD)73;, CD90;and CD34;in hBMSCs were 97.77%, 52.15% and 0.07% respectively. The relative expression of BDNF mRNA in BDNF group and NT-3 group was higher than that in hBMSCs group(both P<0.01). The relative
作者
蔡瑜
彭聪
陈瑶
袁瑞
闫永建
CAI Yu;PENG Cong;CHEN Yao;YUAN Rui;YAN Yong-jian(Shandong Academy of Occupational Health and Occupational Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan,Shandong 250062,China;不详)
出处
《中国职业医学》
CAS
北大核心
2021年第6期640-647,共8页
China Occupational Medicine
基金
国家自然科学基金面上项目(81972989)。
关键词
HBMSCS
脑源性神经营养因子
神经营养因子-3
融合基因
感染
构建
慢病毒载体
Human bone marrow mesenchymal stem cells
Brain-derived neurotrophic factor
Neurotrophic factors-3
Fusion gene
Infection
Construction
Lentiviral vector
