摘要
目的探讨白细胞介素8(IL-8)在乳腺癌细胞中促血管生成作用,及其与雌激素受体之间的关系。方法收集不同的乳腺癌细胞培养上清液,通过观察这些上清液对人脐静脉内皮细胞迁移、增殖和新生毛细血管管腔形成三个体外血管生成实验以及裸鼠体内血管生成实验来研究细胞因子在乳腺癌血管生成中的作用机制;采用细胞上清液芯片实验检测MDA-MB-231细胞和雌激素受体稳定转染的MDA-MB-231细胞的IL-8表达水平;进一步将雌激素受体和IL-8启动子瞬时转染至MDA-MB-231细胞,经双荧光素酶报告基因检测雌激素受体对IL-8的调控作用。结果与IL-8低表达细胞相比较,IL-8高表达和中表达细胞上清液所培养的HUVEC更易于发生迁移(t值分别为6.94和17.75,P均<0.05),IL-8抗体可以特异性地阻断这种作用(t值分别为16.67和9.08,P均<0.05)。雌激素受体阴性MDA-MB-231细胞经过雌激素受体稳定转染后,IL-8的表达水平下降8.8倍。雌激素受体α能够下调IL-8启动子的活性(r=0.856,P<0.05),对照载体无此作用。结论IL-8具有促乳腺癌细胞血管生成作用,乳腺癌细胞中IL-8的水平与雌激素受体水平呈负相关,外源性的雌激素受体可下调乳腺癌细胞中的IL-8表达。
Objective To explore the angiogenic effect of interleukin-8 (IL-8) in breast cancer and its association with estrogen receptor (ER). Methods The supernatants of culture liquid of breast cancer cells of different lines with high expression of Il-8 (MDA-MB-231 and MDA-MB-157), moderate expression of Il-8 (SKBr-3), or low expression of Il-8 (T47D and ZR75-1) were collected. These different conditioned media and human umbilical cord vein endothelial cells (HUVECs) were used in cell migration test to calculate the number of migrating HUVECs. Neutralizing antibody of IL-8 was added into above supernatants to observe the change in HUVECs migration. Mouse-tail collagen was added into the 12-well plate and then solidified, HUVECs were added thereon, and different supernatants were used as culture fluid so as to observe the angiogenesis of HUVECs on the collagen. HUVECs were cultured in the supernatants of culture liquid of different breast cells and CyQUANT dye was added at different time points so as to observe the proliferation of HUVECs by CCD imaging system. The supernatants of the culture fluid of the MDA-MB-231, SKBr-3, or T47D cells with or without fibroblast growth factor (FGF)-2 were injected subcutaneously into mice. Five days later the mice were killed to strip the skin to observe the angiogenesis microscopically. Supernatant chip test was made to measure the concentrations of IL-8 in different supernatants. Estrogen receptor (ER)-α and pN1481 Luc containing IL-8 promoter or p Luc0 not containing IL-8 promoter were transfected into different human breast cancer cells. Dual-luciferase assay was performed to study the regulation of IL-8 level by ER. Results The numbers of migrating HUVECs cultured in the supernatants of MDA-MB-231 cells, SKB-Br-3 cells, and T47D cells were 7800±368, 6510±419, and 3470±297 respectively ( P <0.05). After the addition of IL-8 neutralizing antibody, the number of migrating HUVECs cultured in the supernatant of MDA-MB-231 cells was 4700±233 and 4900±328 respectively, both significantly lower than those before the addition (both P <0.05). In comparison with those cultured in the supernatants of the breast cancer cells expressing lower level of IL-8, the HUVECs cultured in the supernatants of the breast cancer cells expressing higher level of IL-8 tended to form more microangioid structure and proliferate more rapidly ( P <0.05). The skin of the mice injected with the supernatants of the breast cancer cells expressing higher level of IL-8 showed more blood vessel formation. Transient transfection test showed that the IL-8 level of the MDA-MB-231 cells transfected with ER was decreased by 8.8 folds compared with that of the ER negative MDA-MB-231 cells. Dual-luciferase assay showed that the activity of IL-8 promoter was significantly down-regulated by ERα ( r =0.856, P <0.05). Conclusions IL-8 is the key factor involved in angiogenesis of human breast cancer cell. The IL-8 level in human breast cancer cells is negatively correlated with ER status. Exogenous ER may down-regulate the expression of IL-8 in breast cancer cell.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第20期1419-1423,共5页
National Medical Journal of China
关键词
乳腺肿瘤
白细胞介素8
新生血管化
生理性
受体
雌激素
Breast neoplasms
Interleukin-8
Neovascularization, physiologic
Receptors, estrogen
