癌相关成纤维细胞对喉癌细胞系Hep-2生物学行为影响的体外研究

目的探讨癌相关成纤维细胞(CAFs)在体外的分泌功能及对喉癌细胞Hep-2生物学行为的影响。方法提取江西省肿瘤医院2016-09-09 1例喉鳞状细胞癌手术患者的癌组织及癌旁组织,进行喉癌CAFs及正常成纤维细胞(NFs)的原代培养。ELISA试剂盒测定CAFs和NFs培养上清液中MMP-2、MMP-9的浓度。制备CAFs及NFs条件培养基分别对Hep-2细胞进行培养,CCK-8法检测细胞的增殖情况。采用Transwell法,Hep-2细胞分别与CAFs及NFs进行共培养,设实验组(CAFs共培养组)、对照组(NFs共培养组)和空白组(DMEM培养组),流式细胞法检测Hep-2细胞凋亡率,Transwell侵袭小室法检测Hep-2细胞的侵袭能力。采用SPSS 15.0对数据进行统计学分析。结果 MMP-2于CAFs培养上清液中的浓度为(3.40±0.22)ng/mL,于NFs培养上清液中的浓度为(2.52±0.19)ng/mL,差异有统计学意义,t=-5.246,P=0.006。MMP-9于CAFs培养上清液中的浓度为(164.88±6.54)pg/mL,于NFs培养上清液中的浓度为(114.16±4.48)pg/mL,差异有统计学意义,t=-11.087,P<0.001。CCK-8检测各培养基组的A450值,各培养基组间A450值差异有统计学意义,F培养基=13.977,P培养基<0.001,显示与NFs条件培养基组相比,CAFs条件培养基可促进Hep-2细胞的增殖。流式细胞法检测实验组Hep-2细胞凋亡率为(7.17±1.24)%,而对照组和空白组Hep-2细胞凋亡率则分别为(2.45±0.73)%、(2.05±0.74)%,差异有统计学意义,F=27.664,P=0.001。Transwell法共培养后,检测各组Hep-2穿膜细胞数,实验组为(347.80±23.73)个,对照组和空白组分别为(254.00±46.11)、(191.40±23.67)个,差异有统计学差异,F=28.599,P<0.001。结论 CAFs对Hep-2细胞具有促进增殖、增强侵袭能力以及诱导凋亡的作用,且CAFs中MMP2、MMP-9分泌性表达的增加可能有助于增强Hep-2细胞的侵袭能力。

Effects of CAFs on biological behaviors of laryngeal carcinoma cell line hep-2 in vitro

Objective To investigate the secretory function of carcinoma associated fibroblasts(CAFs)and the effects of CAFs on biological behavior of laryngeal carcinoma cell line Hep-2 in vitro.Methods Tumor and adjacent histologically normal tissues of patient with laryngeal squamouos cell carcinoma from Department of Head and Neck Surgery,Jiangxi Cancer Hospital in September 9,2016 were collected for primary culture of CAFs and normal fibroblasts(NFs).The concentrations of MMP-2,MMP-9 in NFs and CAFs cell supernatant were measured by ELISA.Hep-2 cells were cultured with CAFs and NFs conditioned medium respectively,and the cell proliferation was determined by CCK-8 assays.After co-cultured with CAFs and NFs respectively,the apoptosis and invasion of Hep-2 cells were evaluated by flow cytometry and Transwell assay.The experiment was divided into 3 groups:experiment group(co-cultured with CAFs),control group(co-cultured with NFs)and empty group(cultured with DMEM).Results The levels of MMP-2 in CAFs cell supernatant and NFs cell supernatant were(3.40±0.22)ng/ml and(2.52±0.19)ng/ml respectively.The difference was statistically significant(t=-5.246,P=0.006).The difference between the levels of MMP-9 in CAFs cell supernatant and NFs cell supernatant was also statistically significant with the concentrations of(164.88±6.54)pg/ml vs(114.16±4.48)pg/ml(t=-11.087,P<0.001).CCK-8 results through statistical analysis suggested that CAFs conditioned medium could significantly promote the proliferation of Hep-2 cells compared with the NFs condioned medium(F=13.977,P<0.001).The apoptosis rate of Hep-2 cells in experiment group was(7.17±1.24)%,while in control group and empty group were(2.45±0.73)%and(2.05±0.74)%respectively,the difference was statistically significant(F=27.664,P=0.001).The number of Hep-2 cells that penetrated the upper chamber layer in experiment group was 347.80±23.73,while the control group and empty group were 254.00±46.11 and 191.40±23.67,respectively.The difference was statistically significant(F=28.599,P<0.001).Conclusions CAFs promote the proliferation,invasion and induce apoptosis of Hep-2 cells.The upregulation of MMP-2,MMP-9 secretory expression in CAFs may contribute to enhance the invasiveness of Hep-2 cells.