人脐静脉血管内皮细胞微环境对鼻咽癌CNE-1细胞生长和化疗药物敏感性影响研究

Effect of the microenvironment around human umbilical vein endothelial cell on the proliferation and the chemo-sensitivity of nasopharyngeal carcinoma cell CNE-1

目的构建人鼻咽癌细胞CNE-1与人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)的交互培养体系,初步观察HUVEC微环境对鼻咽癌细胞生物学行为以及化疗药物对细胞生长抑制的影响。方法将鼻咽癌细胞分为A、B和C组,A组加入100%CNE-1上清液,B组加入50%HUVEC上清液和50%CNE-1上清液,C组加入100%HUVEC上清液。采用噻唑蓝(MTT)法检测细胞贴壁率、生长曲线和不同浓度顺铂(DDP)、5-氟尿嘧啶(5-FU)对鼻咽癌细胞的生长抑制作用,激光扫描共聚焦显微技术(laser scanning confocal microscopy,LSCM)检测各组活细胞线粒体膜电位和钙离子浓度。结果成功构建了CNE-1细胞与HUVEC细胞的交互培养体系。不同时间段CNE-1细胞贴壁率B组和C组均大于A组,2h时B组和C组与A组的贴壁率差异最为显著,F值分别为37.303和20.299,P值分别为0.004和0.001。A组细胞倍增时间为(42.26±0.21)h,B组为(41.24±0.30)h,C组为(45.90±0.53)h,交互培养B组细胞增殖速率显著快于A组,F=33.506,P=0.004;而C组慢于A组,F=109.353,P<0.001。5-FU作用3组细胞后,A组细胞的半数抑制浓度(IC50)为(81.65±0.07)μg/mL,B组为(118.38±0.11)μg/mL,C组为(82.95±0.06)μg/mL,与A组相比,B组增加,差异有统计学意义,F=2.381×105,P<0.001,C组差异无统计学意义。DDP作用3组细胞后,A组的IC50值为(12.44±0.04)μg/mL,B组为(16.70±0.01)μg/mL,C组为(8.13±0.01)μg/mL,B组大于A组,F=3.203×103,P<0.001;C组小于A组,F=3.278×103,P<0.001。提示交互培养B组细胞对化疗药物的敏感性降低。与A组细胞膜电位比较,B组细胞线粒体跨膜电位显著升高,F=25.511,P=0.007;C组显著降低,F=54.251,P=0.002。与A组钙离子浓度比较,B组钙离子浓度显著降低,F=260.972,P<0.001;C组也降低,F=562.385,P<0.001。结论改变肿瘤细胞的微环境,将改变细胞的生物学特性,血管内皮细胞有利于维持鼻咽癌细胞的正常功能,提高细胞的活力,并导致交互培养体系中鼻咽癌细胞对化疗药物敏感性降低。

OBJECTIVE To establish a co-culture system of nasopharyngeal carcinoma cell CNE-1and human umbilical vein endothelial cell（HUVEC）and observe the changes of biological behaviors and susceptibilities of Nasopharyngeal Carcinoma Cell CNE-1to chemotherapeutic in the microenvironment of HUVEC.METHODS To address the effects of HUVEC on CNE-1biological characteristics,CNE-1cells were divided into 3groups.In group A cells were cultured in the presence of CNE-1conditioned media,in group B cells were cultured in 50% CNE-1conditioned media and 50% HUVEC conditioned media,in group C cells were cultured in the presence of 100% HUVEC conditioned media.The growth curves,plating efficiency and inhibition of 5-FU and DDP on CNE-1cells were determined by MTT assay.Mitochondrial membrane potential and calcium concentration of CNE-1cells were determined by laser scanning confocal microscopy.RESULTS A co-culture system of CNE-1and HUVEC was established successfully.The plating efficiency of group B and group C were higher than that of group A at different point in time.After 2hours,there were significantly difference among group B,group C and group A（for group B,F=37.303,P=0.004;for group C,F=20.299,P=0.011）.The population doubling time was（42.26±0.21）h for group A,（41.24±0.30）h for group B and（45.90±0.53）h for group C,respectively.The proliferation of group B（F=33.506,P=0.004）and group C（F=109.353,P〈0.001）cell were higher than that of group A.After the cells treated with 5-FU,the IC50 of three groups were（81.65±0.07）μg/mL for group A,（118.38±0.11）μg/mL for group B,（82.95±0.06）μg/mL for group C,respectively.There was statistically significance between group A and group B（F=2.381×10^5P〈0.001）.There was no statistically significance between group A and group C.After the cells treated with DDP,the IC50 of three groups were（12.44±0.04）μg/mL,（16.70±0.01）μg/mL,（8.13±0.01）μg/mL respectively.The IC50 of group B was higher than that of group A（F=3.203×10^3P〈0.001）.The IC50 of group C was lower than that of group A（F=3.278×10^3P〈0.001）.The cells of group B was lower the chemo-sensitivity.Compared with group A,the MTP rose significantly in group B（F=25.511,P=0.007）.The MTP decreased significantly in group C（F=54.251,P=0.002）.The concentration of calciumion in group B decreased（F=260.972,P0.001）.The concentration of calciumion in group C also decreased（F=562.385,P0.001）.CONCLUSIONS The biological characteristics of CNE-1showed significant changes after co-culture.The microenvironment of HUVEC cells was helpful to maintain the normal function of the nasopharyngeal carcinoma cells and improved cell＇s vigor.It is concluded that co-culture with HUVEC cells can reduce the chemo-sensitivity of CNE-1 cells.