Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (5): 1-7    
研究报告     
FGFR2IIIc重组慢病毒载体的构建及其在肌原细胞L6中的表达
郭淑军1, 万艳1, 李丽玲1,2, 秦丽1, 陈小佳1
1. 国家教育部基因组药物工程研究中心 广东省生物工程药物重点实验室 暨南大学生物工程研究所 广州 510632
2. 深圳南山区慢性病防治院 深圳 518000
Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors
GUO Shu-jun1, WAN Yan1, LI Li-ling1,2, QING Li1, CHEN Xiao-jia1
1. National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, Bioengineering Institute of Jinan University,Guangzhou 510632, China;
2. Shenzhen Nanshan Chronic Diseases Prevention and Cure Center, Shenzhen 518000,China
 全文: PDF(1352 KB)   HTML
摘要:

目的:构建人FGFR2IIIc重组慢病毒表达系统,感染并筛选获得大鼠肌原细胞L6表达人FGFR2IIIc的重组细胞株,初步研究FGFR2IIIc对L6细胞的作用。方法:从人胎盘组织中获得FGFR2IIIc基因,并克隆到Gateway慢病毒系统的入门载体pENTR-11,采用LR重组酶将重组的pENTR-FGFR2IIIc和表达载体pLenti6/V5-DEST进行重组反应得到pLenti6/V5-DEST-FGFR2IIIc。将该重组表达载体和Viral Packaging Mix包装质粒通过阳离子脂质体共转染293FT细胞,待细胞完全裂解后收集重组慢病毒颗粒上清液;取适量上清液感染L6细胞,杀稻瘟毒素筛选2~4周,挑单克隆细胞进一步扩大培养并建立重组细胞株。RT-PCR、间接免疫荧光和Western blot鉴定重组细胞株中目的基因FGFR2IIIc的表达,bFGF和硫酸乙酰肝素共同诱导各重组L6细胞株后流式细胞术分析细胞周期,形态观察检测成肌分化水平,并通过相关信号通路抑制剂分析与各信号通路的关系。结果:构建了含人FGFR2IIIc基因的重组慢病毒表达载体,获得的重组慢病毒颗粒能高效感染L6细胞,RT-PCR和间接免疫荧光实验说明正确表达目的基因;流式细胞术结果表明L6中高表达FGFR2IIIc的重组细胞株的G1/G0期的细胞增多;各重组细胞株分别加入Erk1/2和p38通路抑制剂抑制诱导2天,发现重组细胞株发生不同的形态变化。结论:通过慢病毒表达系统,成功构建高表达FGFR2IIIc的重组L6细胞株,FGFR2IIIc通过Erk1/2和p38通路影响了L6细胞的形态发生,该结果为下一步在大鼠L6细胞中研究FGFR2IIIc基因与成肌分化功能相关性奠定基础。

关键词: 成纤维细胞生长因子受体2IIIc慢病毒表达系统大鼠肌原细胞    
Abstract:

Objective: To establish the recombinant L6 myoblasts with exogenous FGFR2IIIc by lentivirus and to distinguish their characters. Methods: FGFR2IIIc gene was obtained by RT-PCR from human placental and was cloned into pENTA-11. Recombinant expression vector pLenti6/V5-DEST-FGFR2-IIIc was obtained by using pENTA-FGFR2IIIc and pLenti6/V5-DEST plasmids with LR recombinase. Then such vector and Viral Packaging Mix vectors were co-transfected into 293FT cells to gain recombinant lentiviral particles. After infected by Lentivirus,stable L6 cell monoclones were selected by blasticidin for 2~4 weeks. The recombinant cell lines with exogenous FGFR2IIIc were identified by RT-PCR,indirect immunofluorescence and Western blot. Co-stimulated by both bFGF and HSPG, cell cycles of recombinant L6 cells were evaluated by flow cytometry. Their myogenic differentiation was observed with or without inhibitors of ERK and p38 signal pathway. Results: The vector pLenti6/V5-DEST-FGFR2-IIIc was reconstructed to gain lentiviral particles to establish recombinant L6 myoblasts with exogenous FGFR2IIIc.The recombinant L6-FGFR2IIIc group show an increase in the percentage of cells in the G1/G0 phase compared to the L6-vector group. Phosphorylated Erk1/2 inhibition with PD98059 leads to some cells myogenesis;And phosphorylated p38 inhibition with SB203580 leads to cells apoptosis. Conclusions: FGFR2IIIc can inhibit L6 myoblasts proliferation and result in the tendency of myogenic differentiation.

Key words: FGFR2IIIc    Lentivirus expression system    L6 myoblasts
收稿日期: 2010-11-26 出版日期: 2011-05-27
ZTFLH:  Q786  
基金资助:

广东省自然科学基金资助项目(7005907)

服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  

引用本文:

郭淑军, 万艳, 李丽玲, 秦丽, 陈小佳. FGFR2IIIc重组慢病毒载体的构建及其在肌原细胞L6中的表达[J]. 中国生物工程杂志, 2011, 31(5): 1-7.

GUO Shu-jun, WAN Yan, LI Li-ling, QING Li, CHEN Xiao-jia. Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors. China Biotechnology, 2011, 31(5): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I5/1


[1] Luo Y, Lu W,Mohamedali K A,et al.The glycine box:a determinant of specificity for fibroblast growth factor.Bioehemistry,1998,37(47):16506-16515.

[2] Dionne C A,Crumley G,Bellot F,et al.Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic FGFs. EMBO J, 1990,9(9):2685-2692.

[3] Miki T,Fleming T P,Bottaro D P,et al.Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.Science, 1991,251(4989):72-75.

[4] Katoh M,Hattori Y,Tanaka M,et al.K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase.Proc Natl Acad Sci USA, 1992,89(7):2960-2964.

[5] Ornitz D M,Xu J,Colvin J S,et al.Receptor specificity of the FGF family.J Biol Chem, 1996, 271(25): 15292-15297.

[6] Zhang X,Ibrahimi O A,Olsen S K,et al.Receptor specificity of the FGF family.J Biol Chem, 2006,281(23):15694-15700.

[7] Delezoide A L,Benoist-Lasselin C,Lefeai-Mallet L,et al.Spatio-temporal expression of FGFR 1,2and 3 genes during human embryo-fetal ossification.Mech Dev,1998,77(1):19-30.

[8] Cohen M M, Kreiborg S.The central nervous system in the Apert syndrome .Am J Med Genet, 1990, 35(1):36-45.

[9] Hatano N,Mori Y,Oh-hora M,et al.Essential role for ERK2 mitogen-activated protein kinase in placental development.Genes Cells,2003,8(11):847-856.

[10] Saba-El-Leil M K,Vella F D,Vernay B,et al.An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.EMBO Rep,2003,4(10):964-968.

[11] Yao Y, Li W,Wu J,et al.Extracellular signal-regulated kinase 2 is necessary for mesoderm Differentiation.Proc Natl Acad Sci USA,2003,100(22):12759-12764.

[12] Jones N C,Fedorov Y V,Rosenthal R S,et al.ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion.J Cell Physiol,2001,186(1):104-115.

[13] Nebreda A R,Porras A. p38 MAP kinases:beyond the stress response.Trends Biochem Sci,2000,25(6):257-260.

[14] Zarubin T,Han J.Activation and signaling of the p38 MAP kinase pathway.Cell Res,2005,15(1):11-18.

[15] Natalicchio A,Laviola L,De Tullio C D,et al.Role of the p66shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of Actin Cytoskeleton in rat Myoblasts. J Biol Chem, 2004, 279(42):43900-43909.

[16] Penn B H,Bergstrom D A,Dilworth F J,et al.A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation.Genes Dev,2004,18(19):2348-2353.

[17] Li J,Johnson S E.ERK2 is required for efficient terminal differentiation of skeletal myoblasts.BBRC,2006,345(4):1425-1433.

[18] Coolican S A,Samuel D S,Ewton D Z,et al.The Mitogenic and Myogenic Actions of Insulin-like Growth Factors Utilize Distinct Signaling Pathways. J Biol Chem, 1997,272(10):6653-6662.

[19] Wu Z,Woodring P J,Bhakta K S,et al. p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps.Mol Cell Biol,2000,20(11):3951-3964.

[20] Zetser A,Gredinger E,Bengal E. p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation.Participation of the Mef2 transcription factor. J Biol Chem, 1999,274(8):5193-5200.

[21] Katoh Y, Katoh M. FGFR2-related pathogenesis and FGFR2-targeted therapeutics. International Journal of Molecular Medicine, 2009, 23(3):307-311.

[22] Arman E, Haffner-Krausz R, Gorivodsky M, et al. Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.Proc Natl Acad Sci USA,1999,96(21):11895-11899.

[23] Miraoui H, Severe N, Vaudin P, et al. Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells:implication of FGFR2 signaling. Journal of Cellular Biochemistry, 2010, 110(5):1147-1154.

[24] Walsh K, Perlman H. Cell cycle exit upon myogenic differentiation. Current Opinion in Genetics & Development, 1997, 7(5):597-602.

[25] Esko J D,Selleck S B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Ann Rev Biochem, 2002, 71:435-471.

[26] Kan M, Wang F, Xu J, et al. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science, 1993, 259(5103):1918-1921.

[27] Gospodarowicz D,Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol, 1986, 128(3):475-484.

[28] Vanden-Driessche T,Vanslembrouck V,Goovaerts I,et al.Long-term expression of human coagulation factor Ⅷ and correction of hemophilia A after in vivo retroviral gene transfer in factor Ⅷ-deficient mice.Proc Natl Acad Sci U S A, 1999, 96(18):10379-10384.

[29] Connelly S,Smith T A,Dhir G,et al.In vivo delivery and expression of physiological levels of functional human factor Ⅷ in mice.Hum Gene Ther, 1995, 6(2):185-193.

[30] Naldini L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science,1996,272(5259):263-267.

[31] Kafri T,Blomer U,Peterson D A,et al.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat Genet,1997,17(3):314-317.

[1] 郭淑军 万艳 李丽玲 秦丽 陈小佳. FGFR2IIIc重组慢病毒载体的构建及其在肌原细胞L6中的表达[J]. 中国生物工程杂志, 2011, 31(05): 0-0.