中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (20): 3709-3713.doi: 10.3969/j.issn.1673-8225.2010.20.023

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

应用Cyclin D1和bcl-2为靶标的siRNA慢病毒载体构建和鉴定

吴小三1，张春林1，赵 曜1，高田田2，孙 佼1，曾炳芳1   

  1. 1上海交通大学附属第六人民医院骨科，上海市   200233；
    2上海市第八人民医院骨科，上海市200235
  • 出版日期:2010-05-14 发布日期:2010-05-14
  • 通讯作者: 张春林，男，副主任医师，上海交通大学附属第六人民医院骨科，上海市 200233
  • 作者简介:吴小三，男，1984年生，安徽省安庆市人，苏州大学在读硕士，医师，主要从事骨肿瘤的基础与临床研究。 Xiaosan96@live.cn
  • 基金资助:

    国家自然科学基金资助项目（30700850）

Construction and identification of lentiviral vector targeting human bcl-2 and cyclin D1 gene

Wu Xiao-san1, Zhang Chun-lin1, Zhao Yao1, Gao Tian-tian2, Sun Jiao1, Zeng Bing-fang1   

  1. 1Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai  200233, China;
    2Department of Orthopaedics, Eighth People’s Hospital of Shanghai, Shanghai  200235, China
  • Online:2010-05-14 Published:2010-05-14
  • Contact: Zhang Chun-lin, Associate chief physician, Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
  • About author:Wu Xiao-san, Studying for master’s degree, Physician, Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China Xiaosan96@live.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30700850*

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摘要：

背景：近年来，与骨肉瘤耐药相关的基因研究大多局限于单个基因或单个通路。而进行细胞凋亡和细胞周期调控双通道同时阻断有可能逆转药物耐受机制。

目的：构建bcl-2和cyclin D1特异性siRNA慢病毒载体，拟将其转入骨肉瘤耐药细胞株，探讨对骨肉瘤耐药性的逆转作用。

方法：采用限制性内切酶酶切、T4DNA连接酶连接等方法，将bcl-2和cyclin D1基因分别插入慢病毒载体pSIH1-H1-copGFP shRNA Vector中，构建bcl-2和cyclin D1与pSIH1-H1-copGFP共表达的慢病毒载体(pSIH1-H1-copGFP- bcl-2-siRNA和pSIH1-H1-copGFP-cyclinD1-siRNA)。构建成功后的慢病毒质粒系统和pPACK包装质粒共转染293T细胞，过滤，浓缩病毒，利用荧光蛋白作为报告基因，对病毒滴度和感染效率进行检测。

结果与结论：4对bcl-2和cyclin D1特异性siRNA与双酶切慢病毒载体pSIH1- H1-copGFP shRNA Vector连接成功。共转染293T细胞包装病毒并浓缩后滴度达1.14×104 ifu/μL，适合感染目的细胞。实时荧光定量PCR检测结果显示对bcl-2和cyclinD1基因的干扰效率最高分别达88%和87%。证实将siRNA技术应用于bcl-2和cyclin D1，能够构建出有效的bcl-2和cyclin D1特异性siRNA慢病毒载体。

关键词: 骨肉瘤, bcl-2基因, cyclin D1基因, RNA干扰, 慢病毒, 组织工程

Abstract:

BACKGROUND: Studies concerning osteosarcoma drug resistance are limited for single gene or passway. However, dual-channel blocker of apoptosis and cell cycle regulation may reverse drug resistance in osteosarcoma.

OBJECTIVE: To construct an effective bcl-2, cyclin D1 specific siRNA lentiviral vector and transfect it into drug-resistant osteosarcoma cell lines of osteosarcoma, and to discuss the reversal of drug resistance.

METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. Bcl-2 and cyclin D1 genes were cloned into the site of pSIH1-H1-copGFP shRNA vector to construct the pSIH1-H1-copGFP-bcl-2-siRNA and pSIH1-H1-copGFP-cyclinD1-siRNA. The cloned lentiviral plasmids and the packaging plasmids system were transfected into 293T cells. The supernatant was collected and the titer and infection efficiency of the recombinant lentivirus were determined.

RESULTS AND CONCLUSION: The ligation of four pairs of bcl-2 and cyclin D1 specific siRNAs to the double digested lentiviral pSIH1-H1-copGFP shRNA vector were successful. The titer of concentrated virus was 1.14×104 ifu/μL in the supernatant of the infected cells. The highest interference efficiency of bcl-2, cyclin D1 genes was respectively 88% and 87% detected by real-time fluorescence quantitative PCR. The results demonstrated an effective bcl-2, cyclin D1 specific siRNA lentiviral vector has been constructed by the application of siRNA technique.

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