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  • 胡晓1,王建怡2,袁军3,万兴4,黄伟琨5,秦新月1*.小鼠雌激素受体α重组慢病毒的构建及其感染神经细胞的鉴定[J].第二军医大学学报,2011,32(2):160-166    [点击复制]
  • HU Xiao1, WANG Jian-yi2, YUAN Jun3, WAN Xing4, HUANG Wei-kun5, QIN Xin-yue1*.Construction of recombinant lentivirus carrying mouse estrogen receptor α and identification in infected neurons[J].Acad J Sec Mil Med Univ,2011,32(2):160-166   [点击复制]
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小鼠雌激素受体α重组慢病毒的构建及其感染神经细胞的鉴定
胡晓1,王建怡2,袁军3,万兴4,黄伟琨5,秦新月1*
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(1.重庆医科大学附属第一医院神经内科,重庆 400016; 2.贵州省人民医院小儿内科,贵阳 550002; 3.贵州省人民医院中心实验室,贵阳 550002; 4.贵州省人民医院肿瘤科,贵阳 550002; 5.贵州省人民医院口腔科,贵阳 550002)
摘要:
\[摘要\]目的构建携带C57BL/6小鼠雌激素受体α(estrogen receptor α,Erα)的重组慢病毒,感染原代培养的小鼠神经细胞,观察是否能提高Erα的表达,为进一步研究Erα与神经系统疾病的关系奠定基础。方法将Erα基因片段插入慢病毒主体载体LV-GFP-flag,构建重组慢病毒载体LV-Erα-flag。通过琼脂糖凝胶电泳(agarose gel electrophoresis,AGE)、基因测序验证后,将LV-Erα-flag与包装质粒pHelper1.0、pHelper2.0共同转染293T细胞,测定病毒滴度,获得携带Erα基因的重组慢病毒V-Erα-flag。无血清培养C57BL/6小鼠的神经细胞,对照病毒V-GFP-flag感染神经细胞,在荧光显微镜下每天观察荧光表达情况,用流式细胞仪检测感染效率和凋亡率以获得最佳感染复数(multiplicity of infection,MOI)。然后用最佳MOI值的重组慢病毒V-Erα-flag感染神经细胞,采用实时定量PCR、蛋白质印迹方法检测感染后细胞中Erα的转录和蛋白表达水平。结果AGE及测序鉴定均表明重组慢病毒载体构建成功,包装、扩增后得到V-Erα-flag,感染滴度为2×108 TU/ml;MOI=5的对照病毒V-GFP-flag能感染神经细胞,感染效率达(89.8±4.03)%,而凋亡率仅为(3.6±0.29)%。神经细胞至少能存活8周,在此期间GFP能持续表达,说明慢病毒能有效而稳定感染神经细胞。用MOI=5的V-Erα-flag感染神经细胞后,能显著增强神经细胞中的Erα mRNA和蛋白表达水平。结论成功构建了携带Erα基因的重组慢病毒,其能成功感染神经细胞,使Erα mRNA和蛋白表达水平增高。
关键词:  重组慢病毒  神经元  雌激素受体α
DOI:10.3724/SP.J.1008.2011.0160
投稿时间:2010-09-17修订日期:2011-01-04
基金项目:贵州省科技厅攻关项目\[黔科合sy(2009)3054\],贵州省优秀科技教育人才省长专项资金\[黔省专合字(2010)86号\].
Construction of recombinant lentivirus carrying mouse estrogen receptor α and identification in infected neurons
HU Xiao1, WANG Jian-yi2, YUAN Jun3, WAN Xing4, HUANG Wei-kun5, QIN Xin-yue1*
(1. Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 2. Department of Pediatrics, the People’s Hospital of Guizhou, Guiyang 550002, Guizhou, China; 3. Central Laboratory, the People’s Hospital of Guizhou, Guiyang 550002, Guizhou, China; 4. Department of Oncology, the People’s Hospital of Guizhou, Guiyang 550002, Guizhou, China; 5. Department of Stomatology, the People’s Hospital of Guizhou, Guiyang 550002, Guizhou, China)
Abstract:
\[Abstract\]ObjectiveTo construct a recombinant lentivirus carrying C57BL/6 mouse estrogen receptor α (Erα) and to infect mouse neurons, so as to pave a way for further studying the relationship of Erα with some nervous system diseases. MethodsErα gene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag, which was confirmed by agarose gel electrophoresis (AGE) and DNA sequencing. Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0, and the virus titer was examined. The neurons of C57BL/6 mouse were cultured using a serum-free culture medium, and then control lentivirus V-GFP-flag was used to infect the neurons. The infection efficiency and apoptosis rate were examined by flow cytometry to obtain optimal multiplicity of infection (MOI). GFP expression was detected daily under an inverted fluorescent microscope. After that, V-Erα-flag with the optimal MOI was used to infect neurons; the expression of Erα mRNA and protein in the neurons was detected by quantitative real-time PCR and Western blotting analysis. ResultsAGE and DNA sequencing confirmed that LV-Erα-flag was successfully constructed, and packaged into 293T cells with a titer of 2×108 TU/ml. Control lentivirus V-GFP-flag could infect neurons, with the infection efficiency being (89.8±4.03)% and the cell apoptosis rate being only (3.6±0.29)% when MOI=5. Neurons could survive in the culture for at least 8 weeks, during which the GFP was persistently expressed, indicating the lentivirus could efficiently and stably infect the neurons. The expression of Erα mRNA and protein was greatly increased in neurons infected with V-Erα-flag (MOI=5). ConclusionThe recombinant lentivirus carrying Erα has been constructed successfully, which can infect neurons and lead to increase the expression of Erα mRNA and protein.
Key words:  recombinant lentivirus  neurons  estrogen receptor α alpha