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唐彬秩, 屈艺, 赵凤艳等. 以慢病毒为载体的整合素β8 RNAi在新生鼠脑内干扰效率的研究[J]. 四川大学学报(医学版), 2018, 49(3): 325-330.
引用本文: 唐彬秩, 屈艺, 赵凤艳等. 以慢病毒为载体的整合素β8 RNAi在新生鼠脑内干扰效率的研究[J]. 四川大学学报(医学版), 2018, 49(3): 325-330.
TANG Bin-zhi, QU Yi, ZHAO Feng-yan. et al. Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats’ Brain[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(3): 325-330.
Citation: TANG Bin-zhi, QU Yi, ZHAO Feng-yan. et al. Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats’ Brain[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(3): 325-330.

以慢病毒为载体的整合素β8 RNAi在新生鼠脑内干扰效率的研究

Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats’ Brain

  • 摘要: 目的 制备稳定表达pSicoR-β8 shRNA的慢病毒载体系统,并评估其在新生鼠脑内RNA干扰(RNAi)效率。方法 将慢病毒表达载体pSicoR-β8 shRNA、pSicoR-对照序列与慢病毒包装质粒系统pDM2Gg/p RREpRSV Rev分别进行扩增及质粒酶切鉴定,然后共转染293T包装细胞株,通过293T细胞的包装和分泌以收集携带目的干扰片段的慢病毒颗粒。利用PEG-it病毒浓缩试剂进行浓缩,50%组织培养感染剂量法(TCID50)测定病毒滴度。将浓缩后的慢病毒颗粒通过侧脑室注射对新生SD乳鼠中枢神经系统野生表达的整合素β8进行干扰,荧光显微镜下观察慢病毒侧脑室注射后脑组织中绿色荧光蛋白(GFP)的表达,结合RT-PCR和Western blot方法检测β8 mRNA和蛋白在干预后表达水平的变化,以评估RNAi效率及选择最佳干预时间。结果 质粒扩增及酶切结果显示各质粒片段大小分别与质粒图谱大小一致,浓缩后测得的病毒滴度为LV-对照序列:1.0×108PFU/mL,LV-β8 shRNA:5.0×108PFU/mL。慢病毒侧脑室注射后1 d,侧脑室周围即可观察到携带GFP的慢病毒整合到宿主细胞基因组并发出绿色荧光,注射后2 d、3 d和5 d可在脑组织切片中观察到较强的绿色荧光。β8 mRNA表达水平在RNAi后1~3 d出现降低(P<0.05),第3天达到最大抑制作用,其β8 mRNA抑制率约为56%。β8蛋白表达变化趋势与β8 mRNA表达变化趋势基本一致,RNAi后第3天达到最大抑制作用,其β8蛋白抑制率约为51%。结论 成功获得滴度达到体内实验要求的含有β8 shRNA的慢病毒颗粒,并具有较好的体内干扰活性,为进一步研究整合素β8在新生鼠脑组织中的生理功能奠定了基础。

     

    Abstract: ObjectiveTo construct lentiviral vectors expressing pSicoR-β8 shRNA and evaluate its efficiency of RNA interference in neonatal rats’ brain. Methods Plasmid vectors pSicoR-β8 shRNA and pSicoR-control, as well as lentiviral packaging system pDM2G, g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-β8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing β8-shRNA or control sequence packaged and secreted by 293T were collected, concentrated by PEG-it, and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin β8 in neonatal rats’ brain was performed by intraventricular injection of lentivirus expressing β8-shRNA and rats received lentivirus expressing β8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope, β8 mRNA and β8 protein expression were detected by RT-PCR and Western blot respectively, all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention. Results Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×108 PFU/mL, and for LV-β8 shRNA 5.0×108 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence, lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d, 3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV-β8 shRNA, the inhibitory rate for β8 mRNA and β8 protein were 56% and 51%, respectively. Conclusion Lentiviral vectors expressing β8-shRNA are successfully constructed and lentiviral mediated β8-RNAi is successfully applied for in vivo use.

     

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