卵巢癌细胞株SKOV3中诱导产生的多倍体瘤巨细胞生物学特性
Biological characteristics of induction-produced polyploid tumor giant cells in ovarian cancer cell line SKOV3
乔爱琪 1闫晓燕 2梁钢 1郗彦凤 3李灵敏4
作者信息
- 1. 山西医科大学第一医院病理科,太原 030001
- 2. 山西医科大学第一医院妇科,太原 030001
- 3. 山西省肿瘤医院 中国医学科学院肿瘤医院附属山西医院 山西医科大学附属肿瘤医院病理科,太原 030013
- 4. 山西医科大学病理教研室,太原 030001
- 折叠
摘要
目的 探讨CoCl2诱导卵巢癌细胞株SKOV3产生的多倍体瘤巨细胞(PGCC)的形态及生物学特性.方法 采用300μmol/LCoCl2模拟缺氧环境诱导培养人卵巢癌细胞株SKOV3 36 h,活细胞继续常规培养、传代,20 d后收集细胞,即为PGCC组细胞;以常规培养的SKOV3细胞为对照组细胞.采用倒置显微镜观察PGCC的形成过程及形态学特点;采用免疫细胞化学法检测两组细胞肿瘤干细胞标志物O CT4、CD117的表达;采用人骨髓间质干细胞成脂诱导分化试剂盒、人骨髓间质干细胞成骨诱导分化试剂盒检测PGCC的脂肪分化及骨分化潜能;划痕实验检测PGCC的细胞迁移能力.采用1 μmol/L紫杉醇分别处理PGCC组和对照组SKOV3细胞,分别在0、24、48 h用显微镜观察两组细胞形态,检测PGCC对化疗药物的抗性.结果 显微镜下观察,常规培养液培养的SKOV3细胞中有极少量PGCC;CoCl2可以诱导SKOV3细胞形成PGCC,其形态近似圆形、缺乏分枝,体积为SKOV3细胞的3倍及以上,细胞核多为巨核或多核,PGCC可以出芽方式产生子代细胞.免疫细胞化学染色结果显示,部分PGCC中OCT4阳性表达,无CD117阳性表达;SKOV3细胞中OCT4和CD117均未表达.使用人骨髓间质干细胞成脂诱导分化培养液培养,在第3个周期时可观察到PGCC胞质内有较大的空泡形成,油红O染色显示为橙红色、类圆形的脂滴;使用人骨髓间质干细胞成骨诱导分化培养液培养20 d,茜素红染色可观察到PGCC组细胞较对照组细胞有明显的钙结节形成.细胞划痕实验结果显示,划痕后24、48 h,使用无血清培养液培养PGCC的迁移率[(59±1)％、(66±3)％]均高于对照组细胞的迁移率[(1l±3)％、(14±5)％](t值分别为32.20、19.55,均P＜0.001);使用10％血清培养液培养PGCC的迁移率[(92±3)％、(100±0)％]均高于对照组细胞的迁移率[(20±6)％、(59±9)％](t值分别为16.19、8.00,均P＜0.001).1 μmol/L紫杉醇处理48 h后,PGCC组细胞仍大部分存活,对照组SKOV3细胞大部分死亡.结论 PGCC以出芽方式产生子代细胞;PGCC具有肿瘤干细胞的特性:表达肿瘤干细胞标志物,具有多向分化潜能和较强的化疗药物抗性.
Abstract
Objective To investigate the morphological and biological characteristics of polyploid tumor giant cells(PGCC)produced by ovarian cancer cell line SKOV3 induced by CoCl2.Methods Human ovarian cancer cell line SKOV3 was induced-cultured with 300 μmol/L CoCl2 in the simulated hypoxic environment for 36 h,the live cells continued to be conventionally cultured and passaged,and the cells collected 20 days later were PGCC group;SKOV3 cell line cultured conventionally was the control group.The formation process and morphological characteristics of PGCC were observed by inverted microscope.The expression of tumor stem cell markers OCT4 and CD117 were detected by immunocytochemistry.The adipogenic differentiation and osteogenic differentiation potential of PGCC were detected by using human bone marrow mesenchymal stem cell adipogenic differentiation assay kit and human bone marrow mesenchymal stem cell osteogenic differentiation assay kit.The cell migration ability of PGCC was detected by scratch assay.PGCC group and control group SKOV3 cells were treated with 1 μmol/L paclitaxel,and the cell morphology of the two groups was observed by microscope at 0,24 and 48 h to detect the resistance of PGCC to chemotherapy drugs.Results A small amount of PGCC was observed in SKOV3 cell line cultured in conventional medium under the microscope.CoCl2 can induce SKOV3 cells to form PGCC,which was nearly round in shape and lacked branching.Its volume was 3 times or more than that of SKOV3 cells,and the nuclei were usually megakaryons or multinucleates,PGCC can produce daughter cells by budding.Immunocytochemical staining showed that OCT4 was positive in some PGCC,but no CD117 was positive.Neither OCT4 nor CD117 was expressed in SKOV3 cells.When cultured with lipid-induced differentiation medium of human bone marrow mesenchymal stem cells,the formation of large vacuoles in the cytoplasm of PGCC was observed at the 3rd cycle,and orange-red,round-like lipid droplets were shown by oil red O staining.Human bone marrow mesenchymal stem cells were cultured in osteogenic induction culture medium for 20 days,and alizarin red staining showed that calcium nodules formed significantly in cells of PGCC group compared with the control group.The cell scratch assay results showed that the migration rates of PGCC cultured in serum-free medium[(59± 1)％,(66±3)％]were higher than those of the control group[(11±3)％,(14±5)％]at 24 and 48 h after scratch(t values were 32.20 and 19.55,both P＜0.001).The migration rates of PGCC cultured in 10％serum medium[(92±3)％,(100±0)％]were higher than those of the control group[(20±6)％,(59± 9)％](t values were 16.19 and 8.00,both P＜0.001).After 1 μmol/L paclitaxel treatment for 48 h,most of the cells in the PGCC group still survived,while most of the SKOV3 cells in the control group died.Conclusions PGCC produces daughter cells by budding.PGCC has the characteristics of tumor stem cells:it expresses tumor stem cell markers and has the potential for multidirectional differentiation and strong resistance to chemotherapy drugs.
关键词
卵巢肿瘤/多倍性/肿瘤干细胞/细胞分化/抗药性Key words
Ovarian neoplasms/Polyploidy/Neoplastic stem cells/Cell differentiation/Drug resistance引用本文复制引用
出版年
2024
NETL
NSTL
万方数据