骨肉瘤原代癌相关成纤维细胞对骨肉瘤SOSP-9607细胞增殖的影响  

Effect of primary cancer-associated fibroblasts in osteosarcoma on the proliferation of osteosarcoma SOSP-9607 cells

作　　者：张瑞[1] 唐启胜[2] 丁瑞 杨龙飞 hang Rui;Tang Qisheng;Ding Rui;Yang Longfei(Department of Orthopedics,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China;Department of Urology,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China;Department of Medical Laboratory and Research Center,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China)

机构地区：[1]第四军医大学唐都医院骨科,西安710038 [2]第四军医大学唐都医院泌尿外科,西安710038 [3]第四军医大学唐都医院中心实验室,西安710038

出　　处：《肿瘤研究与临床》2020年第12期818-821,共4页Cancer Research and Clinic

基　　金：国家自然科学基金(81502671)。

摘　　要：目的探讨骨肉瘤微环境中的癌相关成纤维细胞(CAF)对骨肉瘤SOSP-9607细胞增殖的影响及可能机制。方法通过原代培养获得骨肉瘤来源的CAF及癌旁组织正常成纤维细胞(NF),使用流式细胞术检测CAF和NF的α平滑肌肌动蛋白(α-SMA)和成纤维细胞活化蛋白(FAP)的表达。收集CAF、NF的培养液,制备为相应条件培养液。将SOSP-9607细胞分为对照组、NF处理组、CAF处理组,分别应用新制备的未经处理的培养液及CAF、NF条件培养液处理。采用四甲基偶氮唑盐(MTT)法检测SOSP-9607细胞各处理组细胞增殖能力,实时荧光定量聚合酶链反应、蛋白质印迹法检测各SOSP-9607细胞组cyclin D1表达水平。结果CAF中α-SMA和FAP阳性率分别为(86±5)%和(74±8)%,NF中分别为(15±3)%和(10±1)%,CAF与NF间差异均有统计学意义(t=20.83,P<0.05;t=14.30,P<0.01)。MTT法检测显示,CAF处理组SOSP-9607细胞培养24 h后能够促进细胞增殖;96 h后490 nm处吸光度值达到1.83±0.26,NF处理组为1.40±0.17,对照组为1.35±0.15,差异有统计学意义(F=8.59,P<0.05)。CAF处理组SOSP-9607细胞培养48 h后,cyclin D1 mRNA相对表达量上升至1.57±0.13,NF处理组为1.05±0.15,对照组为1,三组间差异有统计学意义(F=23.09,P<0.05),同时cyclin D1蛋白的表达水平也明显增强。结论CAF能够促进骨肉瘤SOSP-9607细胞增殖,并促进cyclin D1基因的表达。Objective To investigate the effect of cancer-associated fibroblasts(CAF)in osteosarcoma microenvironment on the proliferation of osteosarcoma SOSP-9607 cells and its potential mechanism.Methods CAF and paracancerous tissue normal fibroblasts(NF)derived from osteosarcoma were obtained through primary culture.Flow cytometry was used to detect the expression ofα-smooth muscle actin(α-SMA)and fibroblast activation protein(FAP)in CAF and NF.The medium of CAF or NF was collected,and the corresponding conditioned medium was produced.The SOSP-9607 cells were divided into the control group,NF treatment group and CAF treatment group,which were treated with newly produced untreated medium and CAF or NF conditioned medium,respectively.Methylthiazolyl tetrazolium(MTT)assay was used to detect the proliferation of SOSP-9607 cells.Real-time fluorescence quantitative polymerase chain reaction and Western blot was used to determine the expression level of cyclin D1 in SOSP-9607 cells.Results The positive rates ofα-SMA and FAP in CAF were(86±5)%and(74±8)%,respectively,which were different from those in NF treated group[(15±3)%and(10±1)%],and the difference was statistically significant(t=20.83,P<0.05;t=14.30,P<0.01).MTT assay showed that CAF treatment group could promote the proliferation of SOSP-9607 cells after the treatment for 24 h;and absorbance value at 490 nm was reached to 1.83±0.26 at 96 h in CAF treatment group,1.40±0.17 in NF treatment group,and 1.35±0.15 in the control group,and the difference was statistically significant(F=8.59,P<0.05).After 48 h treatment,the relative expression level of cyclin D1 mRNA in SOSP-9607 cells was increased to 1.57±0.13 in CAF treatment group,1.05±0.15 in NF treatment group,and 1 in the control group,and there was a statistical difference among three groups(F=23.09,P<0.05).Moreover,the expression of cyclin D1 protein was also increased.Conclusion CAF can promote the proliferation of osteosarcoma SOSP-9607 cells and enhance the expression of cyclin D1 gene.

关 键 词：骨肉瘤 癌相关成纤维细胞 细胞增殖 细胞周期蛋白D 

分 类 号：R738.1[医药卫生—肿瘤]