转染碱性成纤维细胞生长因子基因在成肌细胞中表达及调控系统的建立(英文)

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

背景:作者前期研究已经成功将碱性成纤维细胞生长因子(basic fibroblast grouth factor,bFGF)基因转入鼠眼外肌的肌卫星细胞中,证明其能够在眼外肌的成肌细胞中表达并促进细胞增殖,促进其分化能力。目的:进一步探讨转染后碱性成纤维细胞生长因子基因在成肌细胞中表达的调控方法。方法:将目的基因碱性成纤维细胞生长因子与诱导表达载体pcDNA4/TO/myc-HisTMA连接,通过经菌落PCR和酶切鉴定的阳性克隆测序和EcoRⅠandHindⅢ双酶切处理和XhoⅠ单酶切处理验证。筛选并确定C2C12成肌细胞的抗生素的敏感性。通过脂质体转染技术,建立C2C12稳定表达pcDNA6/TR细胞系,经Western blot(蛋白印迹法)鉴定。将pcDNA4/TO/myc-孙HisTMA-bFGF转染到pcDNA6/TR-C2C12细胞系,免疫荧光法及Western blot法检测碱性成纤维细胞生长因子在四环素诱导的转染了pcDNA4/TO/myc-HisTMA-bFGF的C2C12细胞内的表达及其分泌情况,并设对照。结果与结论:①经测序对照,双酶及单酶切处理均证实碱性成纤维细胞生长因子与诱导表达载体pcDNA4/TO/myc-HisTMA成功连接。②blasticidin对C2C12细胞的最小致死浓度为10mg/L,zeocin对C2C12细胞的最小致死质量浓度为750mg/L。③建立的pcDNA6/TR-C2C12细胞系正确。④经四环素处理的转染了pcDNA4/TO/myc-HisTMA-bFGF的成肌细胞基因表达阳性,未经处理的则为阴性;经四环素处理的转染了pcDNA4/TO/myc-HisTMA-bFGF的成肌细胞产生碱性成纤维细胞生长因子蛋白,24h达高峰,未处理的则不能产生碱性成纤维细胞生长因子蛋白。结果提示应用四环素抑制调节系统,可以调节碱性成纤维细胞生长因子基因在成肌细胞中的表达。

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.