孙达权,石松,徐莹莹,雷霆雯,莫晓川,王筑婷,李红梅,徐国强
SUN Daquan,SHI Song,XU Yingying,LEI Tingwen,MO Xiaochuan,WANG Zhuting,LI Hongmei,XU Guoqiang

• 1:贵州医科大学基础医学院

摘要(Abstract)：

目的:构建人Spsb1基因的shRNA慢病毒干扰载体,为研究该基因在肝癌细胞生长、增殖、转移和浸润中的作用打下前期基础。方法:根据shRNA的设计原则设计一段shRNA和阴性对照shRNA,化学合成DNA双链中的两条单链,通过退火和配对后与慢病毒干扰载体LV-3(p GLVH1/GFP+Puro)连接,阳性克隆子LV3-Spsb1测序鉴定;将LV3-Spsb1和辅助质粒p CMV-delta8.91/p CMV-VSVG共转染HEK293T细胞,转染后24 h在荧光显微镜下观察细胞转染效率;用转染后收集到的病毒液感染肝癌细胞SMMC-7721,荧光显微镜下观察细胞荧光;用1 mg/L嘌呤霉素杀死未感染的细胞,Western blot检测细胞内源性Spsb1基因表达量。结果:测序鉴定证明获得的LV3-Spsb1质粒是所需要的目的慢病毒shRNA干扰载体;通过与辅助质粒共转染HEK293T细胞后,细胞在荧光显微镜下发出明亮的绿色荧光,提示细胞转染效率较高;病毒液感染SMMC-7721后,细胞内出现绿色荧光,证明共转染系统可以成功包装shRNA-Spsb1重组慢病毒颗粒;Western blot结果显示shRNA-Spsb1重组慢病毒能够抑制细胞内Spsb1基因表达,证明shRNA-Spsb1的确可以降低肝癌细胞内源性Spsb1基因表达,可用于后期的基因功能研究。结论:成功构建人Spsb1基因的慢病毒shRNA干扰载体LV3-Spsb1并包装出其慢病毒颗粒。
Objective: To construct lentivirus shRNA interference vector of human Spsb1( splA/ryanodine receptor domain and SOCS box containing 1) gene and to lay a foundation for uncovering the function of human Spsb1 gene in hepatocellular carcinoma development and metastasis. Methods:Firstly,a fragment of shRNA sequence for Spsb1 and a negative control shRNA were designed according to the shRNA design principles; Secondly,two single DNA strands of shRNAs,including shRNA of Spsb1 and the control,were synthesized by gene corporation,and then were ligated with lentivirus interference vector named LV3( p GLVH1/GFP + Puro) after their annealing and renaturation. Positive clone LV3-Spsb1 was verified by DNA sequencing; Thirdly,co-transfection was implemented to mediate expressional plasmid of LV3-Spsb1 and auxiliary plasmids of p CMV-delta8. 91 and p CMV-VSVG into HEK293 T cells. In 24 h after transfection,the transfection efficiency was confirmed with fluorescent microscopy; Fourthly,virus solution was collected by centrifugation under low temperature for 5 min at3000 g and filtrating by hydrophilic filter membrane in 48 h after transfection. The collected virus solution was then stored in-80 ℃ after subpackage; Fifthly,virus solution was added into hepatocellular carcinoma cell SMMC-7721 which was detected by fluorescent microscope in 72 h after infection; Lastly,western blot was used to detected the expression of SPSB1 protein in infected SMMC-7721 cells which survived in 1 mg/L puromycin screening culture medium for a week. Results: DNA sequencing showed that positive clone LV3-Spsb1 was just the goal lentivirus shRNA interference vector; HEK293 T cells,co-transfected by lentivirus expressional vector LV3-Spsb1 and auxiliary vectors p CMV-delta8. 91 and p CMV-VSVG,showed bright green fluorescence under fluorescent microscope which proved the transfection efficiency; SMMC-7721 cells showed green fluorescence after infection by viral particles which verified that shRNA-Spsb1. Lentivirus particle package was successful; western blot results showed the expression of SPSB1 protein decreased in infected SMMC-7721 cells which indicated that lentivirus vector LV3-Spsb1 could knock down the gene expression of Spsb1. Conclusion: Lentivirus shRNA expressional vector LV3-Spsb1 was successfully constructed and its lentivirus particle was packaged.

关键词(KeyWords)： 肝肿瘤;慢病毒干扰载体;脂质体;转染;Spsb1基因
liver neoplasms;lentiviral interference vector;liposomes;transfection;Spsb1 gene

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81560390);; 贵州省科学技术基金[黔科合J字(2010)2,黔科合J字(2014)2025号];; 贵阳医学院院基金(院基金合同字第017号)

作者(Author): 孙达权,石松,徐莹莹,雷霆雯,莫晓川,王筑婷,李红梅,徐国强
SUN Daquan,SHI Song,XU Yingying,LEI Tingwen,MO Xiaochuan,WANG Zhuting,LI Hongmei,XU Guoqiang

DOI: 10.19367/j.cnki.1000-2707.2017.06.002

参考文献(References)：

• [1]Kleiber ML,Singh SM.Divergence of the vertebrate sp1A/ryanodine receptor domain and SOCS box-containing(Spsb)gene family and its expression and regulation within the mouse brain[J].Genomics,2009(9):358-366.
• [2]Hilton DJ,Richardson RT,Alexander WS,et al.Twenty proteins containing a C-terminal SOCS box form five structural classes[J].Proc Natl Acad Sci(USA),1998(95):114-119.
• [3]Dong Z,Cheng F,Yuwen Y,et al.Identification and expression analysis of a Spsb gene in planarian Dugesia japonica[J].Gene,2015(564):168-175.
• [4]Qin Y,Mc Allister SS.SPSB1 may have MET its match during breast cancer recurrence[J].Cancer Discov,2014(4):760-761.
• [5]Feng Y,Pan TC,Pant DK,et al.SPSB1 promotes breast cancer recurrence by potentiating c-MET signaling[J].Cancer Discov,2014(4):790-803.
• [6]Treadwell JA,Singh SM.Microarray analysis of mouse brain gene expression following acute ethanol treatment[J].Neurochem Res,2004(29):357-369.
• [7]Nishiya T,Matsumoto K,Maekawa S,et al.Regulation of inducible nitric-oxide synthase by the SPRY domainand SOCS box-containing proteins[J].J Biol Chem,2011(286):9009-9019.
• [8]Lewis RS,Kolesnik TB,Kuang Z,et al.TLR regulation of SPSB1 controls inducible nitric oxide synthase induction[J].J Immunol,2011(187):3798-3805.
• [9]Sun DQ,Wang Y,Liu DG.Cancer cell growth suppression by a 62nt AU-rich RNA from C/EBPβ3'UTRthrough competitive binding with HuR[J].Biochem Biophys Res Commun,2012(426):122-128.
• [10]Singer HS,Chiu AY,Meiri KF,et al.Advances in understanding the development of the nervous system[J].Curr Opin Neurol,1994(7):153-159.
• [11]Dobbing J,Sands J.Comparative aspects of the brain growth spurt[J].Early Hum Dev,1979(3):79-83.
• [12]Gutierrez H,Dolcet X,Tolcos M,et al.HGF regulates the development of cortical pyramidal dendrites[J].Development,2004(131):3717-3726.
• [13]Nakano M,Takagi N,Takagi K,et al.Hepatocyte growth factor promotes the number of PSD-95 clusters in young hippocampal neurons,Exp Neurol,2007(207):195-202.
• [14]Liu S,Nheu T,Luwor R,et al.SPSB1,a novel negative regulator of the transforming growth factor-βsignaling pathway targeting the type II receptor.J Biol Chem,2015(29):17894-17908.

文章评论(Comment)：