高表达asporin对L929细胞中TGF-β1及Col2α1的影响

Effect of overexpression of asporin on regulations of TGF- β1 and Col2α1 in L929 cells

目的:构建高表达asporin基因L929细胞株,研究其对子小鼠正常成纤维细胞L929 TGF-β1及Col2α1表达的影响。方法:运用RT-PCR技术从小鼠下颌骨组织中扩增asporin cDNA,连接酶连接至真核表达载体pcDNA3.1(-),所得的重组质粒pcDNA3.1(-)-ASPNⅢ用脂质体法转染小鼠成纤维细胞L929,经过G418筛选,使用RT-PCR以及Western blot技术筛选asporin基因高表达的细胞株。并使用RT-PCR以及Western blot技术检测asporin基因高表达对细胞TGF-β1及Col2α1表达的影响。结果 :成功获得asporin高表达的L929细胞株。RT-PCR以及Western blot技术检测高表达asporin对TGF-β1及Col2α1的表达有明显的抑制作用。结论:asporin基因在L929细胞中高表达能抑制细胞TGF-β1及Col2α1的表达。

Objective To establish the L929 cell line with high expression of asporin ,and to study the effect of asporin on the expressions of TGF- β1 and Col2α1 in mouse fibroblast L929. Methods Asporin cDNA was amplified from mouse mandible tissue by RT-PCR,then was inserted into the eukaryotic expression plasmid pcDNA3.1（-）. The recombinant plasmid pcDNA3.1（-）-ASPNIII was transfected into the mouse fibroblast cell line L929, followed by G418 treatment. RT-PCR and Western blot assays were used to screen the stable, asporin high expressing L929 cell line.The effect of overexpression of asporin on the regulations of TGF-β1 and Col2α1 was further studied. Results The stable, aspori high expressing n L929 cell linewas successfully established. Results of RT-PCR and Western blot assays showed that overexpression of asporin could effectively inhibit the expressions of TGF-β1 and Col2α1. Conclusion Enforced expression of asporin can inhibit the expression of TGF-β1 and Col2α1 in L929 cells.