甲氨蝶呤对映体诱导的肺癌A549耐药细胞株中VEGF及其受体表达差异的研究  

Differential expressions of VEGF and its receptor in methotrexate enantiomer-resistant lung cancer A549 cells

作　　者：董林[1] 何晓东[2] 陶绍能[2] 孙余婕[2] 李明[2] 沈佐君[2] 

机构地区：[1]蚌埠医学院研究生院,蚌埠233000 [2]安徽医科大学附属省立医院省临床检验中心,合肥230001

出　　处：《肿瘤》2009年第5期404-408,共5页Tumor

基　　金：国家自然科学基金资助项目(编号:30672011);安徽省自然科学基金资助项目(编号:050430902);安徽省人才开发资金资助项目(编号:2005Z040)

摘　　要：目的:通过探讨甲氨蝶呤(methotrexate,MTX)对映体耐药细胞中血管内皮生长因子(vascular endothelial growth fac-tor,VEGF)及其受体表达的差异,以反映MTX对映体耐药细胞在肿瘤中血管形成能力的不同。方法:采用实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)技术分别检测D-(-)-MTX/A549组、L-(+)-MTX/A549组以及亲本细胞组中VEGF、血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)-1和VEGFR-2mRNA的表达水平;皮下接种裸鼠,成瘤后取瘤体切片,行CD34免疫组织化学检测,新生微血管密度(microvessel density,MVD)计数;采用双层软琼脂克隆形成实验检测D-(-)-MTX/A549组、L-(+)-MTX/A549组细胞的克隆形成率。结果:RFQ-PCR检测结果显示,D-(-)-MTX/A549组VEGF、VEGFR-1和VEGFR-2Ct值/β-actinCt值分别为1.668±0.127、1.872±0.133和1.919±0.107,L-(+)-MTX/A549组的比值分别为2.035±0.185、2.221±0.157和2.255±0.140,亲本细胞组的比值为2.057±0.123、2.291±0.138和2.354±0.131;3种细胞植入裸鼠成瘤后切片免疫标记CD34统计MVD显示亲本细胞为3.29±1.11,L-(+)-MTX/A549细胞为8.00±2.14,D-(-)-MTX/A549细胞为57.88±13.87;软琼脂实验检测克隆形成率发现D-(-)-MTX/A549组为(0.625±0.088)%,L-(+)-MTX/A549组为(1.050±0.095)%,亲本细胞组为(1.250±0.248)%。研究结果显示D-(-)-MTX/A549组和L-(+)-MTX/A549组以及亲本细胞组之间差异均有统计学意义(P<0.05),而L-(+)-MTX/A549组与亲本细胞组之间则差异无统计学意义(P>0.05)。结论:D-(-)-MTX诱导肺腺癌A549耐药细胞在肿瘤血管形成上的能力大于L-(+)-MTX诱导的细胞。Objective： To study the differential expressions of the vascular endothelial growth factor （VEGF） and its receptor （VEGFR） in methotrexate （MTX） enantiomer-resistant cells in order to reflect the different angiogenesis capacity of MTX enantiomer-resistant tumor cells.Methods：Real-time fluorogentic quantitative PCR（RFQ-PCR） was used to determine the level of VEGF,VEGF-R1, and VEGF-R2 mRNAs in D-（-） MTX/A549 cells, L-（＋） MTX/A549 cells, and parental cells. The nude mice were inoculated subcutaneously. The tumor body was resected after tumorigenesis and examined by CD34 immunohistochemistry. The microvessel density （MVD） was calculated. The colony formation rate was determined by double layer soft agar clony formation test in D-（-） MTX/A549 cells and L-（＋） MTX/A549 cells. Results：RFQ-PCR showed that the Ct ratios of VEGF, VEGFR-1 and VEGFR-2 and β-actin as reference were 1.668±0.127, 1.872±0.133, and 1.919±0.107 in D-（-） MTX/A549 cells, and they were 2.035±0.185, 2.221±0.157, and 2.255±0.140 in L-（＋） MTX/A549 cells. In parent cells they were 2.057±0.123, 2.291±0.138, and 2.354±0.131,respectively. The three cells were inoculated into nude mice. The tumor body was cut and examined by CD34 staining. MVD in tissue section were 3.29±1.11 in parent cells, 8.00±2.14 in L-（＋） MTX/A549 cells, and 57.88±13.87 in D-（-） MTX/A549 cells. Soft-agar clony formation test found that the colony formation rate was （0.625±0.088）% in D-（-） MTX/A549 cells, （1.050±0.095）% in L-（＋） MTX/A549 cells,and （1.250±0.248）% in parent cells. The difference was significant between D-（-）MTX/A549 cells and L-（＋） MTX/A549 and between D-（-）MTX/A549 cells and parent cells （P〈0.05）, but not significant between L-（＋）MTX/A549 and parent cells （P〉0.05）. Conclusion：The tumor angiogenesis capacity of lung denocarcinoma A549 cells induced by D-（-） MTX was greater than that induced by L-（＋）MTX.

关 键 词：肺 非小细胞肺 甲氨蝶呤 抗药性 肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]