高侵袭力及稳定高表达增强型绿色荧光蛋白的膀胱癌细胞株的建立

Establishment of a highly invasive human bladder cancer cell line with enhanced expression of green fluorescent protein gene

目的:建立具有高侵袭力及稳定高表达增强型绿色荧光蛋白(EGFP)的膀胱癌细胞株。方法:通过构建侵袭小室,以穿透人工基底膜、聚碳酸脂膜能力为依据,筛选从人膀胱癌细胞系EJ中筛选具有体外高侵袭能力的膀胱癌细胞亚系(EJ-m3)。用pEGFP-C1转染人膀胱癌细胞系EJ及其高侵袭力亚系,经G418抗性筛选和96孔板有限稀释获得稳定高表达EGFP的单克降细胞株。并用流式细胞术检测EGFP表达率,通过细胞周期、生长曲线和体外侵袭实验,对转染和未转染EGFP的细胞生物学行为进行综合分析。结果:筛选出了具有高侵袭力的膀胱癌细胞EJ-m3,并获得了稳定高表达EGFP的细胞株EJ-GFP和EJ-m3-GFP。该两株细胞EGFP的表达率均为99.9%,增殖指数、生长曲线和体外侵袭力与未转染前相似(P>0.05)。结论:用改良侵袭实验筛选膀胱癌细胞系的高侵袭力亚系的方法是可靠的。转染EGFP的细胞其生物学行为并未发生改变。所获得的具有高侵袭力和稳定高表达EGFP的膀胱癌细胞为膀胱癌侵袭及转移的研究提供了良好的实验平台。

Objective： To establish a highly invasive human bladder cancer cell line with enhanced expression of the green fluorescent protein（EGFP） gene.Methods： By using a specially constructed invasive chamber,the EJ cells that had penetrated the matrigel and polycarbonate adipose membrane were selected and cultured to establish offspring cell lines with high invasive power.Human bladder cancer cell lines（EJ and EJ-m3） were transfected with pEGFP-C1 by Lipofectamine^TM2000 reagent,and screened by the culture medium containing geneticin（G418） and limited dilution in 96 we11 flat-bottomed culture plates to obtain the cells with enhanced expression of the EGFP gene.The expression rate of EGFP and the cell cycle were detected by flow cytometry,the growth curve and invasive power were observed to compare the biological behavior of the cells transfected with EGFP and those that were not.Results： A highly invasive human bladder cancer cell line with enhanced expression of EGFP was established,with an expression rate of 99.9%.The proliferation index,auxodrome and invasive power of the transfected cells showed no significant differences from the untransfected ones（P〉0.05）.Conclusion： The method of constructing a highly invasive subline of bladder neoplasm is reliable,and EGFP transfection does not change the biological behavior of the cells.The obtained sublines may provide a valuable experimental platform for further study on the molecular mechanisms of bladder neoplasm metastasis.