吉林大学学报(医学版) ›› 2013, Vol. 39 ›› Issue (2): 286-290.doi: 10.7694/jldxyxb20130221

• 基础研究 • 上一篇    下一篇

干细胞标志Musashi2基因RNAi腺病毒载体的构建及鉴定

张慧娟，谭诗，陈莎娜，王娟，覃凤娴，陈先春，张伶   

  1. 重庆医科大学检验医学院 临床检验诊断学教育部重点实验室,重庆市重点实验室，重庆 400016
  • 收稿日期:2012-09-12 出版日期:2013-03-28 发布日期:2013-03-26
  • 通讯作者: 张 伶(Tel:023-65714234,E-mail:lingzhang@cqmu.edu.cn) E-mail:lingzhang@cqmu.edu.cn
  • 作者简介:董肖婷（1987-），女，山西省晋中市人，在读医学硕士，主要从事口腔颌面部肿瘤发生发展机制的研究。
  • 基金资助:

    辽宁省教育厅高等学校科学研究项目资助课题（L2010315）

Construction of PTEN gene eukaryotic expressing vector and its expression in tongue squamous cell carcinoma SCC-4 cell line

ZHANG Hui-juan,TAN Shi,CHEN Sha-na,WANG Juan,QIN Feng-xian,CHEN Xian-chun,ZHANG Ling   

  1. Key Laboratory of Laboratory Medical Diagnostics,Ministry of Education,School of Laboratory Medicine, Chongqing Medical University,Chongqing 400016,China
  • Received:2012-09-12 Online:2013-03-28 Published:2013-03-26

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摘要： Abstract:Objective To construct the adenoviral vectors of RNA interference of a stem cell marker Musashi2(Msi2) gene and to  detect the interference effect and the influence on the proliferation of bladder cancer cell line BIU87.  Methods   Two interference DNA fragments named msi2-1 and msi2-2 and scramble as negative control were cloned into pSES-HUS shuttle vector and sent to sequence. After being digested and identified correctly the clones digested by PmeⅠwere recombinated with the bone plasmid pAdeasy-1 in the BJ5183 bacteria.After being digested by PacⅠ the recombinant plasmids were transfected into 293A cells to package and amplify adenovirus.Real-time PCR and Western blotting were used to test Msi2 mRNA and protein expression respectively in BIU87 cells infected by adenovirus.MTT was employed to detect whether knockdown of Msi2 affected the proliferation of BIU87 cells.Results  Two interference and the scramble fragments were cloned into pSES-HUS shuttle vectors correctly.The recombinant vectors named pAdeasy-1-pSES-HUS-msi2 and pAdeasy-1-pSES-HUS-scramble were constructed and also the adenovirus were packaged and amplified successfully.Compared with scramble group,both of the mRNA and protein expression levels of Msi2 in msi2-1 and msi2-2 groups were decreased apparently（P<0.05）；the growth of BIU87 cells was reduced after knockdown of Msi2（P<0.05）. Conclusion The adenoviral vectors of Msi2 RNA interference which could inhibit the expression of Msi2 and cellular proliferation effectively in bladder cancer cell line BIU87 is successfully constructed.

关键词: Musashi2, RNA interference, adenoviral vector

Abstract: Abstract:Objective To construct the eukaryotic expressing vector of PTEN gene and to express the gene in SCC-4 cell line and to provide theoretical foundation for gene therapy of tongue squamous cell carcinoma.Methods  Human total length of PTEN gene was obtained from HeLa cell line by RT-PCR and was cloned into pMD18-T to analyze the sequence.The correct PTEN was inserted into pEGFP-N1 vector.pEGFP-PTEN was identified by digestion with EcoRⅠ and XhoⅠ.Scc-4 cell line was transfeted by pEGFP-PTEN and the expression of PTEN was observed by fluorescence microscope and Western blotting.Results  A 1 200 bp fragment had been cloned into pEGFP-N1 vector which was further identified by sequencing and NCBI BLAST analysis.pEGFP-PTEN was expressed successfully in SCC-4 cell line.Western blotting showed the expression of PTEN protein in pEGFP-PTEN group was 1.07±0.15,there were significant differences compared with pEGFP-N1 group(0.62±0.11) and blank control group(0.57±0.08) (P<0.05).Conclusion The eukaryotic recombinant vector pEGFP-PTEN is constructed successfully and could be overexpressed in SCC-4 cell line.

Key words: PTEN gene, plasmid construction, SCC-4 cell line, gene transfection

中图分类号: 

  • R373
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