GDNF慢病毒载体感染食蟹猴骨髓间充质干细胞

GDNF Lentivirus Infection of Cynomolgus Monkey Bone Marrow Mesenchymal Stem Cells

骨髓间充质干细胞(mesenchymal stem cells,MSCs)是基因工程和细胞治疗的种子细胞之一,本研究利用含胶质源性神经营养因子(glial cell derived neurotrophic factor,GDNF)基因的慢病毒载体感染成年食蟹猴MSCs,探讨转染后GDNF在MSCs中的体外表达水平及其影响因素。首先,通过密度梯度离心法分离食蟹猴骨髓单核细胞(marrow mononuclear cells,MNCs),体外培养食蟹猴MSCs。同时构建表达GDNF的慢病毒载体,并感染食蟹猴MSCs,分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法,测定感染不同拷贝数病毒和不同转染组细胞GDNF的蛋白分泌水平和基因表达水平。实验结果显示,表达GDNF基因的慢病毒载体成功转染成年食蟹MSCs,体外培养的MSCs持续表达分泌GDNF。感染慢病毒的拷贝数可以影响GDNF分泌水平,相同条件下感染拷贝数越高,GDNF分泌量越多,其基因表达水平越高。

Mesenchymal stem cells（MSCs） have received considerable attention for various therapeutic approaches in recent years.MSCs are also easy to genetically modify to express therapeutic genes using lentiviral vectors.In this study,we transfected cynomolgus monkey mesenchymal stem cells（MSCs） using lentiviral vectors encoding glial cell line derived neurotrophic factor（GDNF） to study its expression level in vitro.First,Lentiviral vectors encoding GDNF were packaged by 293T cells through three plasmids transient co-transfection method using standard lipofectamine reagent.The viral titers were tested by the transfection efficiency of 293T cells.At the same time,MSCs were transfected under different multiplicity of infection.GDNF gene expression level and protein secretion level of MSCs were tested by real-time PCR and ELISA methods after transfection.The results indicated lentiviral vectors encoding GDNF successfully transfected cynomolgus monkey MSCs,and over-expressed GDNF in vitro for over 1 month.Furthermore,GDNF expression was influenced by the multiplicity of infection.It might provide technical assistance for cynomolgus monkey MSCs and GDNF as gene therapy applied to the treatment of neurodegenerative diseases.