中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (9): 1340-1345.doi: 10.3969/j.issn.2095-4344.2017.09.006

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

基因重组腺病毒载体与慢病毒载体转染兔骨髓间充质干细胞的对比

李诗鹏,李  强,石正松,蔡伟良,宁寅宽,陶  旋   

  1. 桂林医学院附属医院急诊创伤外科,广西壮族自治区桂林市  541001
  • 出版日期:2017-03-28 发布日期:2017-03-31
  • 通讯作者: 李强,主任医师,教授,硕士生导师,桂林医学院附属医院急诊创伤外科,广西壮族自治区桂林市 541001
  • 作者简介:李诗鹏,男,1990年生,汉族,甘肃省兰州市人,桂林医学院在读硕士,主要从事骨组织工程研究。
  • 基金资助:

    国家自然科学基金资助项目(31160199);广西区自然科学基金资助项目(2014GXNSFAA118263)

Comparison of lentiviral vector and adenoviral vector mediated gene transfer into rabbit bone marrow mesenchymal stem cells

Li Shi-peng, Li Qiang, Shi Zheng-song, Cai Wei-liang, Ding Yin-kuan, Tao Xuan   

  1. Department of Emergency Traumatic Surgery, the Affiliated Hospital of Guilin Medical University, Guilin 541001, Guangxi Zhuang Autonomous Region, China
  • Online:2017-03-28 Published:2017-03-31
  • Contact: Li Qiang, Chief physician, Professor, Master’s supervisor, Department of Emergency Traumatic Surgery, the Affiliated Hospital of Guilin Medical University, Guilin 541001, Guangxi Zhuang Autonomous Region, China
  • About author:Li Shi-peng, Studying for master’s degree, Department of Emergency Traumatic Surgery, the Affiliated Hospital of Guilin Medical University, Guilin 541001, Guangxi Zhuang Autonomous Region, China
  • Supported by:

    the National Natural Science Foundation of China, No. 31160199; Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 2014GXNSFAA118263

摘要:

文章快速阅读:

 

文题释义:
腺病毒载体:
是无包膜的双链DNA病毒,基因组DNA片段在感染和触发时是瞬时表达,但仍是游离体,其宿主范围广,能够容纳较大的外源基因片断,然而载体基因不整合到宿主基因组中,且体内转染后对细胞产生很强的细胞免疫反应,因此腺病毒治疗潜力仅限于病毒蛋白的免疫反应,临床上已大幅度减少腺病毒载体应用。
慢病毒:是单链RNA病毒,基因组的基因传递系统可以随机将外源基因整合到宿主细胞基因组中,并能灭活关键的管家基因或肿瘤抑制基因,他们能够高效率的感染分裂细胞,如骨髓间充质干细胞,有效的把目的基因转入细胞中,进行基因治疗。

 

摘要
背景:
腺病毒载体和慢病毒载体均是较好的组织工程基因载体,两者在介导骨形态发生蛋白2转染兔骨髓间充质干细胞中的差异尚待探究。
目的:比较腺病毒和慢病毒载体介导EGFP/骨形态发生蛋白2转染体外培养兔骨髓间充质干细胞的转导效率、持续时间及外源基因表达差异。
方法:将第5代兔骨髓间充质干细胞分3组培养,A组以腺病毒载体介导EGFP/骨形态发生蛋白2(Ad-EGFP/BMP-2)转染细胞,B组以慢病毒载体介导EGFP/骨形态发生蛋白2(Lenti-EGFP/BMP-2)转染细胞,C组为未进行转染。转染24 h、48 h、72 h、1周、3周,检测EGFP基因表达;转染72 h后,免疫组织化学观察细胞骨形态发生蛋白2的表达;转染后72 h、1周、3周,Western blot检测骨形态发生蛋白2蛋白表达。
结果与结论:①转染24 h后,A、B组可见EGFP表达,A组明显强于B组;转染48 h后,A、B组荧光进一步增强;转染72 h后,A组荧光强度近高峰,B组荧光持续增强。转染1周后,A组荧光强度开始下降,B组荧光强度仍然增强。转染3周后,A组荧光强度明显下降甚至消失,B组荧光强度有所下降,但仍然保持一定的表达。C组在各时间点均无EGFP表达;②A、B组胞浆均呈骨形态发生蛋白2阳性表达,C组呈阴性表达;③转染72 h后,A组骨形态发生蛋白2蛋白表达量强于B组;转染1周后,A组表达下降,B组表达增强并强于A组;转染3周后,A组表达微弱,B组持续表达且明显强于A组;④结果表明,相对腺病毒载体,慢病毒载体介导EGFP/骨形态发生蛋白2基因转染兔骨髓间充质干细胞在表达持续时间上具有一定的优势。

 

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程
ORCID: 0000-0001-9327-3574(李强)

关键词: 干细胞, 骨髓干细胞, 慢病毒载体, 腺病毒载体, 骨髓间充质干细胞, 基因转染, 骨形态发生蛋白类, 国家自然科学基金

Abstract:

BACKGROUND: Both lentiviral vector and adenoviral vector are considered as good vectors for gene mediation, and their differences in transferring bone morphogenetic protein 2 (BMP-2) into rabbit bone marrow mesenchymal stem cells (BMSCs) are unclear.

OBJECTIVE: To compare the duration, efficiency and the deviation of exogenous gene expression after rabbit BMSCs transfection using lentiviral vector and adenoviral vector which are used to mediate enhanced green fluorescent proteins (EGFP) and BMP-2.
METHODS: Rabbit BMSCs at passage 5 were exposed to Ad-EGFP-BMP-2 (group A) or Lenti-EGFP-BMP-2 (group B) with multiplicity of infection of 100, as transfection groups. And in control group (group C), the same quality of culture medium was required equivalent to the groups A and B. The expression of EGFP was observed by inverted fluorescence microscope at various time intervals. And the expression of exogenous gene BMP 2 in cells was detected and analyzed by immunohistochemical staining at 72 hours after transfection as well as by western blot at 72 hours, 1, 3 weeks after transfection.

RESULTS AND CONCLUSION: The intense green fluorescence emerged under the microscope at 24-48 hours after transfection in group A, which was stronger than group B, reached the peak at 72 hours, and then decreased at 1 week until disappearance at 3 weeks. No EGFP expression was detected in group C. High expression of BMP-2 was found in group A but was dramatically downregulated after 1 week. Group B showed the high expression of EGFP/BMP-2 persisted for a longer period after transfected that even lasted for 3 weeks. Overall, the lentiviral vector and adenoviral vector can efficiently transfect rabbit BMSCs and stably express the target gene of EGFP/BMP-2. Under the same MOI, compared to the adenoviral vector, transfection of lentiviral vector to rabbit BMSCs is more effectively and expression of EGFP/BMP-2 can be persistent in a longer term.

 

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

Key words: Stem Cells, Adenoviridae, Lentivirus, Mesenchymal Stem Cells, Tissue Engineering

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