中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2013年
10期
732-736
,共5页
康小红%王立芳%曹飞%范方田%徐振晔
康小紅%王立芳%曹飛%範方田%徐振曄
강소홍%왕립방%조비%범방전%서진엽
肺肿瘤%受体,表皮生长因子%肝细胞生长因子%基因,erbB-1%Afatinib
肺腫瘤%受體,錶皮生長因子%肝細胞生長因子%基因,erbB-1%Afatinib
폐종류%수체,표피생장인자%간세포생장인자%기인,erbB-1%Afatinib
Lung neoplasms%Receptor,epidermal growth factor%Hepatocyte growth factor%Genes,erbB-1%Afatinib
目的 观察肿瘤微环境中肝细胞生长因子(HGF)和afatinib对H1975肺癌细胞增殖的影响,探讨肿瘤微环境中HGF介导afatinib耐药的机制.方法 采用四甲基偶氮唑蓝(MTT)法检测肿瘤微环境中HGF、转化生长因子α和afatinib对H1975细胞增殖的作用,采用酶联免疫吸附试验(ELISA)检测MRC-5和H1975细胞中HGF的表达水平；采用Western blot检测HGF和(或)afatinib作用后H1975细胞中表皮生长因子受体(EGFR)、Met信号通路相关蛋白及EMT标志蛋白的表达.结果 MTT检测结果显示,在HGF存在的情况下,H1975细胞对afatinib的敏感性降低.ELISA检测结果显示,细胞常规培养48 h,2.0 ×106个H1975细胞分泌的HGF＜0.1 ng,而2.0×106个MRC-5细胞分泌HGF的水平为(151.37±2.07)ng.H1975细胞与MRC-5细胞共培养72 h后,H1975细胞上清液中HGF水平为(61.13±16.21) ng/ml.Western blot检测结果显示,在HGF存在的情况下,p-Met、p-Akt和p-ERK等蛋白明显上调,afatinib能抑制p-EGFR,但对p-Met、p-Akt和p-ERK蛋白表达无影响；在afatinib存在的情况下,HGF可上调波形蛋白的表达,下调E-钙黏蛋白的表达.结论 肿瘤微环境中的HGF可能通过激活Met/PI3K/Akt、Met/MAPK/ERK信号通路以及参与EMT进程介导了afatinib原发耐药.
目的 觀察腫瘤微環境中肝細胞生長因子(HGF)和afatinib對H1975肺癌細胞增殖的影響,探討腫瘤微環境中HGF介導afatinib耐藥的機製.方法 採用四甲基偶氮唑藍(MTT)法檢測腫瘤微環境中HGF、轉化生長因子α和afatinib對H1975細胞增殖的作用,採用酶聯免疫吸附試驗(ELISA)檢測MRC-5和H1975細胞中HGF的錶達水平；採用Western blot檢測HGF和(或)afatinib作用後H1975細胞中錶皮生長因子受體(EGFR)、Met信號通路相關蛋白及EMT標誌蛋白的錶達.結果 MTT檢測結果顯示,在HGF存在的情況下,H1975細胞對afatinib的敏感性降低.ELISA檢測結果顯示,細胞常規培養48 h,2.0 ×106箇H1975細胞分泌的HGF＜0.1 ng,而2.0×106箇MRC-5細胞分泌HGF的水平為(151.37±2.07)ng.H1975細胞與MRC-5細胞共培養72 h後,H1975細胞上清液中HGF水平為(61.13±16.21) ng/ml.Western blot檢測結果顯示,在HGF存在的情況下,p-Met、p-Akt和p-ERK等蛋白明顯上調,afatinib能抑製p-EGFR,但對p-Met、p-Akt和p-ERK蛋白錶達無影響；在afatinib存在的情況下,HGF可上調波形蛋白的錶達,下調E-鈣黏蛋白的錶達.結論 腫瘤微環境中的HGF可能通過激活Met/PI3K/Akt、Met/MAPK/ERK信號通路以及參與EMT進程介導瞭afatinib原髮耐藥.
목적 관찰종류미배경중간세포생장인자(HGF)화afatinib대H1975폐암세포증식적영향,탐토종류미배경중HGF개도afatinib내약적궤제.방법 채용사갑기우담서람(MTT)법검측종류미배경중HGF、전화생장인자α화afatinib대H1975세포증식적작용,채용매련면역흡부시험(ELISA)검측MRC-5화H1975세포중HGF적표체수평；채용Western blot검측HGF화(혹)afatinib작용후H1975세포중표피생장인자수체(EGFR)、Met신호통로상관단백급EMT표지단백적표체.결과 MTT검측결과현시,재HGF존재적정황하,H1975세포대afatinib적민감성강저.ELISA검측결과현시,세포상규배양48 h,2.0 ×106개H1975세포분비적HGF＜0.1 ng,이2.0×106개MRC-5세포분비HGF적수평위(151.37±2.07)ng.H1975세포여MRC-5세포공배양72 h후,H1975세포상청액중HGF수평위(61.13±16.21) ng/ml.Western blot검측결과현시,재HGF존재적정황하,p-Met、p-Akt화p-ERK등단백명현상조,afatinib능억제p-EGFR,단대p-Met、p-Akt화p-ERK단백표체무영향；재afatinib존재적정황하,HGF가상조파형단백적표체,하조E-개점단백적표체.결론 종류미배경중적HGF가능통과격활Met/PI3K/Akt、Met/MAPK/ERK신호통로이급삼여EMT진정개도료afatinib원발내약.
Objective To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.Methods The effects of HGF,TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay.The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay.Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.Results The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF.The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0 × 106 cells,but HGF production by MRC-5 cells was (151.37 ± 2.07) ng/2.0 × 106 cells incubated for 48 h.When H1975 cells and MRC-5 cells were co-cultured for 72 h,the concentration of HGF in the culture supematant was (61.13 ± 16.21) ng/ml.In the presence of HGF,the expression of p-Met,p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated.afatinib inhibited p-EGFR,but did not affect the expression of p-Met,p-Akt and p-ERK proteins.In the presence of afatinib,HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.Conclusions HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways,and is involved in the epithelial-mesenchymal transition process.
