大鼠Olig2基因慢病毒表达载体的构建及其转染后对少突胶质前体细胞分化的影响

Construction of lentivector encoding rat Olig2 gene and its effect on differentiation of oligodendrocyte progenitor cells into oligodendrocytes

目的构建大鼠少突胶质细胞转录因子2(Olig2)的慢病毒表达载体,并转染少突胶质前体细胞(OPCs),观察Olig2过表达对OPCs向少突胶质细胞(OLs)分化的影响。方法设计合成PCR引物,从大鼠pEGFP-N3-Olig2表达质粒中扩增并克隆大鼠Olig2-EGFP片段,将其插入到pLenti6.3慢病毒表达载体中,形成pLenti6.3-Olig2-EGFP慢病毒表达载体。将其与包装质粒混合后共转染293T细胞,包装产生慢病毒后感染培养的OPCs,观察Olig2在感染细胞中的表达并鉴定其表达对OPCs向OLs分化的影响。结果成功将大鼠Olig2-EGFP片段克隆入pLenti6.3慢病毒表达载体,构建的pLenti6.3-Olig2-EGFP慢病毒表达载体能够高效表达。与空质粒转染的OPCs相比,重组质粒转染的OPCs体外诱导分化后产生更多的OLs(n=5,P<0.01)。结论成功建立大鼠pLenti6.3-Olig2-EGFP慢病毒表达系统,慢病毒介导的Olig2过表达促进大鼠OPCs向OLs分化。

Objective To construct lentivector encoding rat Olig2 gene and identify its effect on differentiation of oligodendrocyte progenitor cells （OPCs） into oligodendrocytes after transfection. Methods Polymcrase chain reaction （PCR） method was used to obtain the Olig2-EGFP gene from expression vector pEGFP-N3-Olig2. The gene fragment was then inserted into pLenti6. 3-IRES-EGFP lentivector. The lentiviral expression vector pLenti6.3-Olig2-EGFP was obtained after screening followed by sequencing. The lentiviral vector and package virus were co-transfected into 293T cells, and the virus titers were determined. The pLenti6.3-Olig2-EGFP vector was transfected into OPCs and the expression of Olig2 in OPCs was detected by observing EGFP fluorescence. The differentiation of rat OPCs into oligodendrocytes was detected by Rip immunofluorescence staining. Results The pLenti6. 3-Olig2-EGFP vector was constructed successfully and was transfected into OPCs efficiently. The pLenti6. 3-Olig2-EGFP-transfected OPCs differentiate into more receptor interaction protein （RIP）-positive OLs than the pLenti6.3-IRES-EGFP-transfectedd OPCs （P 〈 0. 01 ）. Conclusion The construction of rat Olig2-overexpressed lentivector is successful and Olig2 overexpression may significantly promote the differentiation of rat OPCs into OLs.