Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (20): 3780-3786.doi: 10.3969/j.issn.1673-8225.2010.20.040

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Expression of the transfected basic fibroblast growth factor gene in myoblasts and regulatory system

Zhang Li-gui1, Wang Hong-yun2, Qin Lei-lei3, Huang Xiao-hui1, Wang Chuan-fu4   

  1. 1Department of Ophthalmology, Central Hospital of Enshi, Enshi   445000, Hubei Province, China;
    2Harvard Institution of Medicine, USA;
    3Department of Ophthalmology, Qingdao Municipal Hospital, Qingdao   266401, Shandong Province, China;
    4Department of Ophthalmology, Affiliated Hospital of Qingdao University Medical College, Qingdao   266403, Shandong Province, China
  • Online:2010-05-14 Published:2010-05-14
  • Contact: Wang Chuan-fu, Master, Doctoral supervisor, Professor, Department of Ophthalmology, Affiliated Hospital of Qingdao University Medical College, Qingdao 266403, Shandong Province, China
  • About author:Zhang Li-gui, Doctor, Associate professor, Associate chief physician, Department of Ophthalmology, Central Hospital of Enshi, Enshi 445000, Hubei Province, China Liguizz@163.com

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Abstract:

BACKGROUND: Transgenosis of basic fibroblast growth factor (bFGF) gene has been successfully performed into the muscle satellite cells of rat extraocular muscles in the previous study of the research group, proving that bFGF could express in the myoblasts of extraocular muscles, also promote cell proliferation and differentiation.

OBJECTIVE: To further investigate the methods for regulating the expression of the bFGF in myoblasts following transfection.

METHODS: Target gene bFGF was connected with inducing expression vector pcDNA4/TO/myc-HisTMA, followed by masculine clone sequencing identified by colony PCR and enzyme digestion, EcoR Ⅰ and Hind Ⅲ restriction enzyme digestion, as well as Xho Ⅰ single enzyme verification. C2C12 myoblasts antibiotics sensitivity was screened and finally defined. By use of lipofection transfection technology, cell lines where C2C12 stably expressed pcDNA6/TR were estabolishd and then identified by Western blot. The pcDNA4/TO/myc-HisTMA-bFGF was transfected into pcDNA6/TR- C2C12 cells. The bFGF expression and secretion in C2C12 cells following tetracycline-induced pcDNA4/TO/myc-HisTMA-bFGF transfection were determined by immunofluorescence and Western blot, the controls were established.

RESULTS AND CONCLUSION: ① The conjunction between the bFGF and inducing expression vector pcDNA4/TO/myc-HisTMA was proved successfully by sequencing comparison, double digestion and single digestion. ②The minimal lethal concentration of blasticidin to C2C12 cells was 10 mg/L, while that of zeocin was 750 mg/L. ③ The pcDNA6/TR- C2C12 cell lines were established correctly. ④ The myoblasts treated by tetracycline and transfected with pcDNA4/TO/myc-HisTMA-bFGF were positive for gene expression, those untreated exhibited a negativity; bFGF protein could be produced in myoblasts treated by tetracycline and transfected with pcDNA4/TO/myc-HisTMA-bFGF, the production reached a peak at 24 hours, while those untreated can not produce bFGF protein. Results suggest that the bFGF expression in the myoblasts can be controlled by tetracycline inhibition and regulatory systems.

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