人浆液性卵巢癌细胞体外培养体系的建立及其在化疗药物敏感性检测中的应用  

Establishment of human serous ovarian cancer cell culture system in vitro and its application in chemotherapeutic drug sensitivity detection

作　　者：葛京京 徐红霞 谢丽霞 杜凯丽 桑明 孙晓东[1,2,3] GE Jingjing;XU Hongxia;XIE Lixia;DU Kaili;SANG Ming;SUN Xiaodong(Center for Translational Medicine,Xiangyang No.1 People’s Hospital,Hubei University of Medicine,Xiangyang 441000,China;Hubei Clinical Research Center of Parkinson’s Disease,Xiangyang No.1 People’s Hospital,Hubei University of Medicine,Xiangyang 441000,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]湖北医药学院附属襄阳市第一人民医院转化医学中心,湖北襄阳441000 [2]湖北医药学院附属襄阳市第一人民医院湖北省帕金森病临床研究中心,湖北襄阳441000 [3]湖北医药学院武当特色中药研究湖北省重点实验室,湖北十堰442000

出　　处：《吉林大学学报（医学版）》2021年第4期999-1007,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81703015);湖北省科技厅技术创新专项(对外科技合作类)项目(2019AHB068);湖北省科技厅实验动物资源开发及利用项目(2020DFE025)。

摘　　要：目的:建立人浆液性卵巢癌细胞体外培养体系,为浆液性卵巢癌患者的临床用药提供实验模型和理论依据。方法:采用Ⅱ型胶原酶消化和差速离心法分离培养人浆液性卵巢癌细胞,培养至第3代的浆液性卵巢癌细胞用于实验。倒置相差显微镜下观察卵巢癌细胞形态表现,绘制并分析细胞生长曲线。免疫荧光法检测细胞中人癌抗原125(CA125)、配对盒基因8(PAX8)和人附睾蛋白4(HE4)表达情况,对所培养原代细胞进行鉴定,同时检测胚胎干细胞同源转录因子Nanog和八聚体结合转录因子4(Oct-4)的表达情况。药物敏感性检测分为不同浓度5-氟尿嘧啶(5-FU)(0、0.625、1.250、2.500、5.000 mg·L^(-1))组、紫杉醇(PTX)(0、0.0625、0.1250、0.2500、0.5000 mg·L^(-1))组、替莫唑胺(TMZ)(0、0.125、0.250、0.500、1.000 mg·L^(-1))组、卡铂(CBP)(0、0.625、1.250、2.500、5.000 mg·L^(-1))组、阿霉素(DOX)(0、0.125、0.250、0.500、1.000 mg·L^(-1))组和褪黑素(MLT)(0、0.625、1.250、2.500、5.000 mg·L^(-1))组,其中0 mg·L^(-1)组为对照组,CCK-8法检测各组细胞存活率。结果:原代浆液性卵巢癌细胞接种4 h后开始贴壁,培养8~10 d生长融合成片;连续传代后细胞形态均一,生长迅速,细胞接种后2~7 d为线性增长期。免疫荧光法检测细胞中CA125、PAX8、HE4、Nanog和Oct-4均呈高表达。药物敏感性检测,卵巢癌细胞对5-FU、TMZ、CBP和MLT均有较高的敏感性,与相对应对照组比较,不同浓度5-FU组、TMZ组、CBP组、MLT组及0.0625和0.1250 mg·L^(-1) PTX组细胞存活率明显降低(P<0.05或P<0.01),且细胞存活率随药物浓度升高逐渐降低。结论:成功建立了人浆液性卵巢癌细胞体外培养体系,该细胞对5-FU、TMZ、CBP和MLT具有较高的敏感性,该结果对浆液性卵巢癌患者术后化疗药物的选择有指导意义。Objective:To establish a culture system in vitro of human serous ovarian cancer cells and to theoretical basis to provide the experiment model for the clinical adminstration of the patients with serous ovarian cancer.Methods:The human serous ovarian cancer cells were isolated and cultured from tumor tissue by typeⅡcollagenase digestion and differential centrifugation method,and the third of generation serous ovarian cancer cells were used in subsequent experiments.The morphology of ovarian cancer cells was observed under inverted phase contrast microscope,and the cell growth curve was plotted and analyzed.The expression amounts of the human cancer antigen 125(CA125),paired box gene 8(PAX8)and epididymis protein 4(HE4)in the cells,and the expression amounts of embryonic stem cell homologous transcription factor(Nanog)and octamer binding transcription factor 4(Oct-4)in the cancer stem cells were detected by immunofluorescence.The drug sensitivity test was divided into 5-fluorouracil(5-FU)groups(0,0.625,1.250,2.500,5.000 mg·L^(-1)),paclitaxel(PTX)groups(0,0.0625,0.1250,0.2500,0.5000 mg·L^(-1)),temozolomide(TMZ)groups(0,0.125,0.250,0.500,1.000 mg·L^(-1)),carboplatin(CBP)groups(0,0.625,1.250,2.500,5.000 mg·L^(-1)),adriamycin(DOX)groups(0,0.125,0.250,0.500,1.000 mg·L^(-1))and melatonin(MLT)groups(0,0.625,1.250,2.500,5.000 mg·L^(-1));among them,0 mg·L^(-1) groups were used as control group;CCK-8 method was used to evaluate the survival rates of the cells.Results:The primary serous ovarian cancer cells began to adhere to bottom of culture bottles 4 h after enzymatic digestion.The cells grew rapidly and had uniform morphology during continuous passages.The cells grew in a linearly phase during 2-7 d after inoculation,and grow and fuse into pieces after 8-10 days of culture.The results of immunofluorescence assay showed that CA125,PAX8,HE4,Nanog and Oct-4 were highly expressed in the cells.The drug sensitivity test showed that the ovarian cancer cells had high sensitivity to 5-FU,TMZ,CBP and MLT;compared with corr

关 键 词：卵巢肿瘤 原代细胞培养 化疗药物敏感性 肿瘤标志物 肿瘤干细胞 

分 类 号：R737.31[医药卫生—肿瘤] R979.1[医药卫生—临床医学]