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
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摘要
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=2.381×105,P<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=3.203×103,P<0.001).The IC50 of group C was lower than that of group A(F=3.278×103,P<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,P<0.001).The concentration of calciumion in group C also decreased(F=562.385,P<0.001).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-1cells.
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=2.381×105,P<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=3.203×103,P<0.001).The IC50 of group C was lower than that of group A(F=3.278×103,P<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,P<0.001).The concentration of calciumion in group C also decreased(F=562.385,P<0.001).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-1cells.
引文
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[2]Leonardi GC,Candido S,Cervello M.The tumor microenvironment in hepatocellular carcinoma[J].Int J Oncol,2012,40(6):1733-1747.
[3]Miki Y,Ono K,Hata S,et al.The advantages of co-culture over mono cell culture in simulating in vivo environment[J].Steroid Biochem Mol Biol,2012,131(3-5):68-75.
[4]Feliz LR,Tsimberidou AM.Anti-vascular endothelial growth factor therapy in the era of personalized medicine[J].Cancer Chemotherapy Pharmacology,2013,72(1):1-12.
[5]Asim BD,Pojul L,Biplab B.Human recombinant Cripto-1increases doubling time and reduces proliferation of HeLa cells independent of pro-proliferation pathways[J].Cancer Letters,2012,318(2):189-198.
[6]辛毅,李娜,黄益民,等.人脐带间充质干细胞的原代培养及多向分化潜能的研究[J].细胞与分子免疫学杂志,2013,29(10):390-392.
[7]陈云龙,侯华新,莫春燕,等.激光共聚焦显微镜实时动态观察不同微环境中鼻咽癌细胞摄取大黄素差异研究[J].药物分析杂志,2014,34(2):485-488.
[8]Troja W,Kil K,Klanke C,et al.Interaction between human placental microvascular endothelial cells and a model of human trophoblasts:effects on growth cycle and angiogenic profile[J].Physiol Rep,2014,2(3):2-44.
[9]马力,刘大军,李德天,等.不同分离方法及培养条件对兔骨髓间充质干细胞生长增殖及生物特性的影响[J].中国组织工程研究与临床康复,2008,12(38):7401-7406.
[10]刘江涛,王霖霖,郭雄,等.线粒体功能学研究常用实验方法[J].医学综述,2015,21(1):6-8.
[11]Mandal S,Lindgren AG,Srivastava AS,et al.Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells[J].Stem Cells,2011,29(3):486-495.
[12]Mayevsky A.Mitochondrial function and energy metabolism in cancer cells:past overview and future perspectives[J].Mitochondrion,2009,9(3):165-179.
[13]李建瑛,徐永华,张东辉,等.两种不同基质对体外培养成骨细胞促贴壁生长的影响[J].实验动物科学,2011,28(5):19-21.
[14]Cardenas C,Miller RA,Smith I,et al.Essential regulation of cell bioenergetics by constitutive InsP3receptor Ca2+transfer to mitochondria[J].Cell,2010,142(2):270-283.
[15]Rasola A,Bernardi P.Mitochondrial permeability transition in Ca2+-dependent apoptosis and necrosis[J].Cell Calcium,2011,50(3):222-233.
[16]Knizetova P,Ehrmann J,Hlobilkova A,et al.Autocrine regulation of glioblastoma cell cycle progression,viability and radioresistance through the VEGF-VEGFR2(KDR)interplay[J].Cell Cycle,2008,7(16):2553-2561.
[17]Sukmana I,Vermette P.The effects of co-culture with fibroblasts and angiogenic growth factors on microvascular maturation and multi-cellular lumen formation in HUVEC-oriented polymer fibre constructs[J].Biomaterials,2010,31(19):5091-5099.
[18]Chen Z,Htay A,Dos Santos W,et al.In vitro angiogenesis by human umbilical vein endothelial cells(HUVEC)induced by three-dimensional co-culture with glioblastoma cells[J].J Neuro Oncol,2009,92(2):121-128.
[19]Shi B,Andrukhov O,Berner S,et al.The angiogenic behaviors of human umbilical vein endothelial cells(HUVEC)in co-culture with osteoblast-like cells(MG-63)on different titanium surfaces[J].Dent Mater,2014,30(8):839-847.