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

作　　者：唐彬秩 屈艺[2] 赵凤艳[2] 童煜[2] 王德健 TANG Bin-zhi;QU Yi;ZHAO Feng-yan;TONG Yu;WANG De-jian(Department of Pediatrics, Sichuan Academy of Medical Sciences ＆ Sichuan Provincial People＇s Hospital/University of Electronic Science and Technology of China, Chengdu 610072, China;Key Laboratory of Birth Defects and Related Disease of Women and Children （Sichuan University ）, Ministry of Education, Chengdu 610041, China;Department of Pharmacology, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China)

机构地区：[1]四川省医学科学院.四川省人民医院/电子科技大学临床医学院儿科,成都610072 [2]出生缺陷与相关妇儿疾病教育部重点实验室(四川大学),成都610041 [3]成都中医药大学药学院药理学教研室,成都610075

出　　处：《四川大学学报（医学版）》2018年第3期325-330,共6页Journal of Sichuan University(Medical Sciences)

基　　金：四川省人民医院博士基金资助项目(No.30305030580);四川大学出生缺陷与相关妇儿疾病教育部重点实验室开放课题资助项目(No.2016001);四川省科技厅基金(No.2014SZ0149;No.2016TD0002);成都中医药大学科技发展基金资助项目(No.030029052)资助

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pDM2G、g/pRRE和pRSV 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:1.0×10~8 PFU/mL,LV-β8 shRNA:5.0×10~8 PFU/mL。慢病毒侧脑室注射后1d,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2d、3d和5d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1~3d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。Objective To 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 systempDM2 G,g/p RREand 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 293 T.Lentiviral particles expressing β8-shRNA or control sequence packaged and secreted by 293 T 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 GFPLentivirus was observed under fluorescence microscope,β8 mRNA and β8 protein expression were detected by RTPCR 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 1.0×10^8 PFU/mL,and for LV-β8 shRNA 5.0×10^8 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 dand 5 dafter intraventricular injection.Western blot and RT-PCR demonstrated a maximum inhibition happened 3 dafter 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 lentivir

关 键 词：整合素β8 慢病毒载体 RNAI 体内干扰 

分 类 号：R450[医药卫生—治疗学]