STAT3ER慢病毒表达载体的构建及体外对神经元再生作用的研究

目的构建携带信号转导和转录激活蛋白STAT3和基因突变的雌激素受体ER的慢病毒载体,并转染受损的大鼠背根神经节神经元,研究其对神经元突起再生的作用。方法运用基因重组技术,将STAT3、ER和核糖体介导的绿色荧光蛋白(IRES-GFP)片段插入慢病毒载体pRRL-MCS+的PmeI位点构建慢病毒载体pRRL-STAT3ER-IRES-GFP,经RT-PCR、Western blotting和测序分析加以鉴定其正确性。将慢病毒载体3质粒细胞包装系统(主体质粒pRRL-STAT3ER-IRES-GFP、包装质粒pCMVdeltaR8.74和包膜质粒VSV-G)共转染293T细胞,包装慢病毒载体并测定滴度。切断大鼠左L5近神经节的神经根,摘取L5背根神经节做分离神经元培养,将pRRL-STAT3ER-IRES-GFP感染神经元细胞并观察神经元细胞核转运和突起的长度。结果构建的慢病毒载体pRRL-STAT3ER-IRES-GFP经RT-PCR和Western blotting鉴定正确,测序分析与Genbank报道的STAT3基因序列完全一致。3质粒共转染293T细胞后,测定慢病毒滴度为1010-11TU/ml。转染pRRL-STAT3ER-IRES-GFP的神经元经4-羟基三苯氧胺(4HT)激活,STAT3ER有明显的核转运,并增强突起的生长,经χ2检验与对照组有显著性差异。结论成功构建了表达小鼠STAT3ER基因的慢病毒载体并证实STAT3信号转导有利于轴突生长。

Construction of lentivirus vectors carrying STAT3ER and its effect on neurite growth

Objective To construct lentiviral vector carrying STAT3 and mutated mouse oestrogen receptor α（ER） transinfected into neurons of rat dorsal root ganglia（DRG） to investigate functions of STAT3 in adult rat primary sensory neurons.Methods Gene recombinant technology was employed to cleave and insert the PmeI fragment of STAT3ERIRES-GFP into the PmeI site of the lentivirus transfer vector pRRL-MCS＋ to construct a lentiviral vector pRRLSTAT3ER-IRES-GFP.RT-PCR,Western blotting and sequencing analysis were used for identification.Then,293T cells were transfected with main vector pRRL-STAT3ER-IRES-GFP,packaging plasmid pCMVdeltaR8.74 and coated plasmid VSV-G.Lentiviral vectors were packaged and the titer was determined.The peripheral and dorsal roots of rat left L5 DRG were cut near their ganglia,L5 DRGs were taken to separate neurons cultured in vitro.The constructed pRRL-STAT3ERIRES-GFP was used to transfect neurons and nuclear translocation of STAT3ER and neurite lengths of neurons were analyzed.Results The lentiviral vector plasmid pRRL-STAT3ER-IRES-GFP was identified correctly by RT-PCR and Western blotting,and DNA sequencing analysis confirmed that STAT3 gene sequencing was exactly the same with that reported by Genbank.The concentration of the virus titer was 10 10-11 TU /mL after transinfection of 293T cells.After transinfection with pRRL-STAT3ER-IRES-GFP,nuclear translocation of STAT3ER in transfected neurons was seen obviously after stimulation with 4HT,neurite outgrowth was enhanced by STAT3.Conclusion Lentiviral vector carrying STATSER was successfully constructed and activation of STAT3 signaling is beneficial to axonal growth.