慢病毒介导兔脑红蛋白基因感染骨髓间充质干细胞的实验研究

Lentivirus-mediated rabbit neuroglobin gene transfer to bone marrow mesenchymal stem cells

[目的]构建携带兔脑红蛋白(neuroglobin,Ngb)基因的重组慢病毒,感染兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs),建立稳定表达兔Ngb的BMSCs/Ngb细胞。[方法]构建携带Ngb基因的重组慢病毒载体,在脂质体Lipofectamine 2000作用下转染293 T细胞,Real-time PCR检测慢病毒滴度;以Ngb重组慢病毒感染BMSCs,通过荧光表达法判定感染复数(multiplicities of infection,MOI),实时荧光定量PCR(real-time PCR)、蛋白质印迹(Western blot)和酶联免疫吸附试验(ELISA)判定感染后的BMSCs中Ngb的表达情况。[结果]构建Ngb重组慢病毒载体,经酶切及测序鉴定完全正确,能够转染293 T细胞并表达,滴度为2×108 TU/ml,Ngb重组慢病毒感染BMSCs最佳MOI值为100,且Ngb重组慢病毒感染BMSCs能持续稳定高水平表达Ngb mRNA和蛋白。[结论]成功构建Ngb重组慢病毒,并将Ngb基因稳定感染至BMSCs中,实现Ngb的持续稳定高水平表达,为Ngb基因修饰的BMSCs移植治疗脊髓损伤(spinal cord injury,SCI)的研究提供基础。

[Objective] To construct lentiviral vector carrying the rabbit neuroglobin（Ngb） gene,and then to get Ngb gene-modified bone marrow mesenchymal stem cells（BMSCs） by infecting the BMSCs.[Method]The recombinant lentiviral vector carrying Ngb gene was constructed and transfected to the 293T cells through liposome Lipofectamine 2000,and the lentiviral titer was detected by Real-time PCR,BMSCs were infected with Ngb recombined lentivirus,then the multiplicities of infection（MOI） was determined by detecting the intensity of fluorescence expression.The expressions of Ngb in the infected BMSCs were determined by real-time polymerase chain reaction（PCR）,western blot and enzyme-linked immunosorbent assay（ELISA）. [Result]The Ngb recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis.It could efficiently transfect and express in 293T cells.The lentiviral titer was 2×108 TU/ml,the best MOI that Ngb recombined lentivirus infected BMSCs was 100,and the mRNA and protein expressions of Ngb in infected BMSCs were stable and high.[Conclusion]Ngb recombinant lentvirus were successfully structured and the Ngb gene was stably infected to BMSCs with stable and high expressions of Ngb in infected cells.This study lay a foundation of investigation of neuroglobin genetically modified BMSCs transplantation on spinal cord injury（SCI）.