摘要
目的探讨紧密连接蛋白claudin-6过表达对乳腺癌细胞MCF-7生物学行为的影响。方法用脂质体法将含有claudin-6的真核表达载体转染乳腺癌细胞系MCF-7后，采用RT—PCR、Western印迹和细胞免疫荧光的方法进行稳定克隆的筛选和鉴定；采用MTT法和克隆形成实验检测细胞生长情况；采用划痕法和Boyden小室体外侵袭实验检测细胞迁移和侵袭性。结果建立了稳定表达claudin-6基因的4株单克隆（C1，C2，C3，C4）；细胞免疫荧光结果显示，claudin-6的表达主要定位于细胞膜；与对照组和空载体组相比，稳定表达claudin-6的MCF-7细胞生长缓慢，转染6h时吸光度A值C1为1．62，C2为1．25，C3为1．30，C4为1．58与对照组A值1．78及空载组1．82比较明显降低。其克隆形成率C1为0．25，C2为0．17，C3为0．26，C4为0．21明显低于对照组（0．53）和空载组（0．45）；细胞的迁移性、侵袭性被抑制。结论claudin-6的过表达能够抑制MCF-7细胞的生长和转移等恶性表型，说明紧密连接蛋白claudin-6作为乳腺癌表型抑制基因在乳腺癌发生及转移中起负性调控作用。
Objective To explore the effect of over-expression of tight junction protein claudin-6 upon the biological characters of breast cancer cell line MCF-7. Methods The MCF-7 subline expressing a high level of clandin-6 was established by transfection with a pcDNA3.1-clandin-6 expression vector. The expression of claudin-6 in mRNA and its protein level were confirmed by RT-PCR, Western blot and immunofluorescent assays. Then the effect of claudin-6 upon cell proliferation was examined by MTT assay. Colony-forming assays were used to examine 2-D and 3-D colony-forming capacities. Invasive and migratory traits of clandin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay. The structure and function of tight junctions in both parental and claudin-6 expression MCF-7 cells were evaluated by measuring transepithelial electrical resistance. Results Immunofluorescent assays showed that transfected cells expressed claudin-6 on their membrane. Cells with a high level expression of claudin-6 grew slowly than control cells. Anchorage-independent growth, invasive and migratory traits also decreased substantially in cells with claudin-6 expression; whereas the transepithelial electrical resistance increased in the claudin-6 transfected cells. Conclusion The up-regulation of claudin-6 expression in MCF-7 breast cancer ceils suppresses their malignant phenotypes with a correlation with the restoration of tight junction integrity. Claudin-6 may function as a cancer suppressor whose down-regulation contributes to the malignant progression of certain types of breast cancers.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第6期407-412,共6页
National Medical Journal of China
基金
国家自然科学基金（30670807）
高等学科博士学科点专项科研基金（20060183071）
吉林省科技发展计划项目基金（200505141）
关键词
乳腺肿瘤
紧密连接部
侵袭
Breast neoplasms
Tight junction
Invasion
