摘要
目的:通过无血清悬浮球状培养方法富集纯化人源前列腺癌干细胞并进行小鼠体内外实验验证。方法:采用前列腺癌DU145细胞系,通过无血清悬浮球状培养的方法分离富集、培养纯化出肿瘤干细胞微球(Tumor spheres)。通过流式细胞仪鉴定干细胞表型,并采用动物成瘤实验,将不同数量级的DU145细胞和Tumor spheres细胞接种到NOD/SCID小鼠双侧腋下,观察成瘤率和成瘤速度。对分离富集、培养纯化的Tumor spheres进行诱导分化、增殖水平、活性氧簇、细胞周期检测。为进一步明确Tumor spheres的肿瘤干细胞特性,采用Western blot法检测特异性蛋白的表达,RT-PCR法检测相关mRNA表达。结果:采用无血清悬浮球状培养的方法可使对数生长期DU145细胞分离富集形成Tumor spheres,培养3代可达到纯化的目的,流式细胞技术鉴定其表达前列腺癌干细胞表型CD44(+)CD24(-/low)比例为(63±1)%,明显高于DU145细胞[(33±4)%](P<0.05)。动物成瘤实验显示,接种Tumor spheres侧肿瘤成瘤速度和成瘤率均高于接种DU145细胞侧。Tumor spheres具有更强的自我更新能力和分化潜能,细胞周期检测发现Tumor spheres中(81.67±0.68)%处于细胞周期的G_(0)/G_(1)期(P<0.05),同时Tumor spheres具有更低水平的活性氧簇ROS水平(P<0.05)。Tumor spheres高表达肿瘤干细胞HES1蛋白(P<0.05),Tumor spheres高表达肿瘤干细胞相关Notch1、HES1、HIF-1α、Sox2、Jagged1的mRNA(P<0.05)。结论:无血清悬浮球培养分离纯化DU145肿瘤干细胞的方法简便易行、获取量大、成瘤率高,Tumor spheres具有明确的肿瘤干细胞特性,可作为前列腺癌肿瘤干细胞研究的良好实验模型。
Objective:To enrich and purify human-derived prostate cancer stem cells by a serum-free suspension spheroid culture method and to validate it in vivo and vitro.Methods:The prostate cancer DU145 cell line was used to isolate,enrich and purify tumor stem cell microspheres(tumor spheres)by serum-free suspension spheroid culture.Stem cell phenotypes were identified by flow cytometry,and animal tumorigenesis assays were used to inoculate different quantitative levels of DU145 and tumor spheres into the bilateral axils of NOD/SCID mice to observe the tumorigenic rate and tumorigenic speed.The isolated enriched and cultured purified tumor spheres were assayed for induced differentiation,proliferation level,reactive oxygen species clusters,and cell cycle.The tumor stem cell characteristics of tumor spheres were further clarified.Specific proteins were detected by Western blot and relevant mRNA expression was detected by RT-PCR.Results:Serum-free suspension spheroid culture was used to enable the isolation and enrichment of logarithmic growth stage DU145 cells to form tumor spheres,which could be cultured for 3 generations to achieve purification,and the flow cytometric identification of their expression of prostate cancer stem cell phenotype CD44(+)CD24(-/low)ratio was(63±1)%,which was significantly higher than that of DU145 cells[(33±4)%](P<0.05).Animal tumorigenesis experiments showed that the tumorigenesis rate and tumorigenicity were higher on the tumor spheres side than on the DU145 cells side.Tumor spheres had a stronger self-renewal capacity and differentiation potential,and cell cycle assays revealed that(81.67±0.68)%of tumor spheres were in the G_(0)/G_(1) phase of the cell cycle(P<0.05),while tumor spheres had lower levels of reactive oxygen species cluster ROS levels(P<0.05).Tumor spheres highly expressed tumor stem cell HES1 protein(P<0.05)and tumor stem cell-associated mRNA of Notch1,HES1,HIF-1α,Sox2,and Jagged1(P<0.05).Conclusion:The method of isolation and purification of DU145 tumor stem cells by seru
作者
毛启远
李道睿
郑琦
陈茂宽
林洪生
王学谦
MAO Qiyuan;LI Daorui;ZHENG Qi;CHEN Maokuan;LIN Hongsheng;WANG Xueqian(Department of Oncology,Guang'anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China;Department ofUrology,Surgery Department 1,Yongning District Hospital of Traditional Chinese Medicine,Guangxi Nanning 530000,China)
出处
《现代肿瘤医学》
CAS
北大核心
2023年第16期2949-2956,共8页
Journal of Modern Oncology
基金
国家自然科学基金青年项目(编号:82004179,82104656)
“优秀青年科技人才(创新类)”培养专项(编号:ZZ14-YQ-013)
北京市科学技术协会-青年人才托举工程(编号:BYESS2023357)。
关键词
无血清悬浮球状培养
前列腺癌
肿瘤干细胞
DU145细胞
肿瘤干细胞微球
实验模型
serum-free suspension spheroid culture
prostate cancer
tumor stem cells
DU145 cells
tumor stem cell microspheres(tumor spheres)
experimental model
