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

发布时间：2019-04-17 17:56

【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,P0.001。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,P0.001,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,P0.001;C组小于A组,F=3.278×103,P0.001。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,P0.001;C组也降低,F=562.385,P0.001。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。
[Abstract]:Aim to construct a co-culture system between human nasopharyngeal carcinoma cell line CNE-1 and human umbilical vein endothelial cell (human umbilical vein endothelial cells,HUVEC), and to observe the effect of HUVEC microenvironment on the biological behavior of nasopharyngeal carcinoma cells and the inhibitory effect of chemotherapeutic drugs on the growth of nasopharyngeal carcinoma cells. Methods Nasopharyngeal carcinoma cells were divided into A, B and C groups. 100%CNE-1 supernatant was added to group A, 50%HUVEC supernatant and 50%CNE-1 supernatant were added to group B, and 100%HUVEC supernatant was added to group C. Cell adhesion rate, growth curve and inhibitory effect of cisplatin (DDP), 5-fluorouracil (5-FU) on nasopharyngeal carcinoma (NPC) cells were detected by thiazolyl blue (MTT) assay. Laser scanning confocal microscopy (laser scanning confocal microscopy,) was used to detect the inhibitory effect of cisplatin on the growth of nasopharyngeal carcinoma (NPC) cells. LSCM) was used to detect the mitochondrial membrane potential and calcium ion concentration of living cells in each group. Results the co-culture system of CNE-1 cells and HUVEC cells was successfully constructed. The adherent rate of CNE-1 cells in group B and C was higher than that in group A at different time points. At 2 h, the adhesion rate of cells in group B and C was the most significantly higher than that in group A (F = 37.303 and 20.299, respectively). The cell doubling time was (42.26 卤0.21) h in group A, (41.24 卤0.30) h in group B and (45.90 卤0.53) h in group C, respectively. The cell proliferation rate in group B was significantly faster than that in group A (P < 0.05). F = 33. 506, P = 0. 004; In group C, the inhibition concentration (IC50) was (81.65 卤0.07) 渭 g / mL and (118.38 卤0.11) 渭 g / L, respectively, compared with that in group A (109.353) and P0.001.5 渭 g / U (P < 0.05), and that in group B was (118.38 卤0.11) 渭 g / L, while that in group C was (81.65 卤0.07) 渭 g / mL. Group C was (82.95 卤0.06) 渭 g / L, compared with group A, group B increased significantly (F = 2.381 脳 105, P0.001), and there was no significant difference between group C (P < 0.05) and group C (P < 0.01). After treated with cisplatin, the number of cells in group B was significantly higher than that in group A (P < 0.01). The IC 50 was (12.44 卤0.04) 渭 g / L in group A, (16.70 卤0.01) 渭 g / L in group B, (8.13 卤0.01) 渭 g / L in group C, higher than that in group A (F = 3.203 脳 103, P0.001). Group C smaller than Group A, F = 3.278 脳 103, P0.001. The results suggest that the sensitivity of group B cells to chemotherapeutic drugs is lower than that of group B cells. Compared with group A, the transmembrane potential of mitochondria in group B was significantly higher than that in group A (P < 0.05). The mitochondrial transmembrane potential in group B was significantly higher than that in group A (P < 0.05), while that in group B was significantly higher than that in group B (P < 0.05). Compared with group A, the concentration of calcium in group B was significantly lower than that in group A, the concentration of calcium in group B was significantly lower than that in group A, and the concentration of calcium in group B was significantly lower than that in group A (P < 0.01). Conclusion changing the microenvironment of tumor cells will change the biological characteristics of the cells. Vascular endothelial cells are beneficial to maintain the normal function of nasopharyngeal carcinoma cells and enhance the viability of the cells. Furthermore, the chemosensitivity of nasopharyngeal carcinoma cells to chemotherapeutic drugs was decreased in the interactive culture system.
【作者单位】：广西医科大学药学院;
【基金】：国家自然科学基金(81460561) 广西自然基金(2014GXNSFAA118225)
【分类号】：R739.63

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