摘要
目的:探讨hepaCAM基因细胞外结构缺失突变体对膀胱癌细胞的生物学影响。方法:采用脂质体法将hepaCAM缺失突变体转染T24细胞;激光共聚焦显微镜检测突变体蛋白细胞定位;Western印迹法检测蛋白的表达;体外黏附、解离实验及伤口愈合、基质胶侵袭实验研究突变体对T24细胞黏附、活动力的影响;MTT法检测对细胞增殖的改变。结果:突变体蛋白在单个细胞中的分布与野生型hepaCAM蛋白相近,定位于细胞核周边区域,但细胞相互连接后,与野生型hepaCAM蛋白定位不同,突变体蛋白呈现非特异性分布。获得稳定表达突变体蛋白的T24细胞,在细胞黏附、活动力方面明显低于表达野生型hepaCAM蛋白组,细胞增殖与表达野生型hepaCAM蛋白组相比明显增强。结论:缺失细胞外区域对hepaCAM蛋白细胞定位有重要影响,对抑制T24细胞增殖、增强细胞黏附及活动力无明显作用。细胞外区域对hepaCAM基因发挥正常作用是必须的。
Objective：To investigate the effects of extracellular domain-trnncated mutant of hepaCAM gene on biological behaviors of bladder carcinoma cells in vitro. Methods： Human cell line T24 was transfected with pEGFP-N2-hepaCAM, pEGFP-N2-hepa CAM-mt or mock plasmid pEGFP-N2 by lipofectamine mediation. The stable colonies were obtained by G418 screening. Laser confocal microscopy was used to detect the location of hepaCAM-mt in the T24 cells, Western blotting was used to determine the expression of protein in stable cells. Cell adhesion and motility abilities were tested by cell adhesion and detachment assays and wound healing and Matrigel invasion assays. The proliferation ability was investigated by MTT assay. Results：In well-spread cells, mutant proteins were localized on the perinuclear membrane like wild-type hepaCAM protein; after the cells contacted with each other, hepaCAM-mt protein had non-specific expression in the cells, which was significantly different with the localization of the hepaCAM proteins. T24 cell clone which acquired stable expression of hepaCAM-mt protein had decreased adhesion and motility abilities and enhanced proliferation potential compared with the cells with wild-type hepaCAM gene expression. Conclusion：The deletion of extracellular domain in hepaCAM gene was important for the localization of hepaCAM protein but had no significant effect on the proliferation, adhesion, and migration of T24 cells. The extracellular domain of hepaCAM was necessary for exerting normal functions of hepaCAM gene.
出处
《肿瘤》
CAS
CSCD
北大核心
2008年第6期463-467,共5页
Tumor
