摘要
目的通过基因重组技术构建pcDNA3.0-RGC32重组质粒,并检测其对细胞骨架的影响。方法扩增RGC32的全长并定向克隆到真核表达载体pcDNA3.0,获得重组质粒pcDNA3.0-RGC32,转染SW480细胞,采用G418筛选单克隆,Westernblot检测转染前后SW480细胞中RGC32的表达水平,并制作转染前后SW480细胞骨架标本,并采用划痕实验观察细胞的迁移能力。结果 pcDNA3.0-RGC32真核表达载体构建成功,转染重组载体后细胞中RGC32的表达量明显增多。转染重组质粒前,SW480细胞骨架中微丝纤维束少而短、极性不明显;转染重组载体后的细胞微丝纤维束数量增多,变长,有了明显的极性,并且可见细胞膜向前伸出丝状结构的伪足或者是片状结构的伪足,细胞骨架发生了重组,并且过表达后细胞的迁移能力增强。结论通过过表达RGC32,促进细胞骨架重组,有利于细胞运动,推测细胞骨架重组是RGC32促进肿瘤转移的重要机制。
Objective To construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32（RGC32） on cell cytoskeleton in vitro.Methods The full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32.After transfection of the recombinant plasmid into SW480 cells,the expression of RGC32 in the cells was detected by Western blotting.The cytoskeleton of SW480 cells was visualized before and after the transfection,and the changes in the cell migration ability was assessed by wound-healing assay.Results The recombinant plasmid pcDNA3.0-RGC32 was successfully constructed.The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32.Before the transfection,the microfilaments of SW480 cells were few and short without obvious polarity,but after the transfection,the microfilaments were increased and elongated with also an obvious polarity,and the invasive structures of lamellae and lamellipodia occurred.The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.Conclusion Overexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration,which can be an important mechanism of RGC32 in promoting cancer metastasis.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第7期1179-1182,共4页
Journal of Southern Medical University
基金
国家自然科学基金(30770976)~~
