人宫颈癌干细胞分离、鉴定及其特性  

Isolation,identification and characterization of human cervical cancer stem cells

作　　者：吴琳琳 张茹 李英 熊川 WU Linlin;ZHANG Ru;LI Ying;XIONG Chuan(Department of Physical Examination,Sichuan Armed Police Corps Hospital,Leshan,Sichuan 614000,China;Department of Gynecology,Sichuan Armed Police Corps Hospital,Leshan,Sichuan 614000,China;Department of Military Medicine and Special Disciplines,Sichuan Armed Police Corps Hospital,Leshan,Sichuan 614000,China)

机构地区：[1]武警四川省总队医院体检科,四川乐山614000 [2]武警四川省总队医院妇科,四川乐山614000 [3]武警四川省总队医院军事医学与特种学科,四川乐山614000

出　　处：《新疆医科大学学报》2022年第4期365-370,共6页Journal of Xinjiang Medical University

基　　金：四川省基层卫生事业发展研究中心科研项目(SWFZ20-Y-021)。

摘　　要：目的探讨人宫颈癌干细胞(CCSCs)的分离、培养、鉴定方法以及CD44、CD24对其生物学特性的作用。方法选择2017年1月—2018年2月武警四川省总队医院妇科收治的32例宫颈癌患者为研究对象,收集其宫颈癌组织作为实验组,另选32例子宫全切术患者,留取其正常宫颈黏膜组织作为空白组。蛋白酶消化联合体外细胞培养,在流式细胞术下分选细胞,分为CD44^(+)CD24^(+)组、CD44^(-)CD24^(-)组与对照组。通过四甲基偶氮唑盐(MTT)法和平板集落形成实验检测细胞增殖能力。取4~6周龄,雄性Balb/c裸小鼠24只,将CD44^(+)CD24^(+)、CD44^(-)CD24^(-)细胞在裸小鼠腋下进行体内移植瘤实验观察细胞成瘤率。通过端粒重复序列扩增法(TRAP)联合电泳法测定端粒酶活性。结果实验组中的CD44^(+)、CD24^(+)阳性细胞百分比均高于空白组(P<0.05)。CD44^(+)CD24^(+)组细胞的体外增殖活性高于对照组和CD44^(-)CD24^(-)组细胞(P<0.05)。CD44^(+)CD24^(+)组细胞端粒酶相对表达强度和细胞集落形成率高于对照组,CD44^(-)CD24^(-)组细胞端粒酶相对表达强度和细胞集落形成率低于对照组,差异均有统计学意义(P<0.05)。CD44^(+)CD24^(+)组体内成瘤时间早于对照组,且成瘤率更高(P<0.05)。结论CD44、CD24是分选人CCSCs可行的表面标记分子,获取的CD44^(+)CD24^(+)细胞可显现出肿瘤干细胞生物学特性。Objective To investigate the isolation,culture and identification of stem cells of human cervical cancer(CC⁃SCs)and the maintenance of biological characteristics of CD44 and CD24.Methods Thirty^(-)two patients with cervical cancer treated in the hospital from January 2017 to February 2018 were collected as the experimental group.Another 32 patients underwent total hysterectomy were selected to retain their normal cervical mucosa as the blank group.By protease digestion combined with in vitro cell culture,the mixed cell divided into CD44^(+)CD24^(+)cell group,CD44^(-)CD24^(-)cell group and the control group were separated and purified by flow cytometry.MTT assay and plate col⁃ony formation assay were used to detect the ability of cell proliferation,and tumor formation was confirmed by tumor transplantation in nude mice.CD44^(+)CD24^(+)and CD44^(-)CD24^(-)cells were subcutaneously inoculated into the axilla of 4^(-)6^(-)week^(-)old 24 male Balb/c nude mice to confirm the tumor^(-)forming ability of the cells.Telomerase activity was deter⁃mined by telomere repeat amplification protocol(TRAP)combined with electrophoresis.Results The positive percent⁃age of CD44^(+)and CD24^(+)in the experimental group were higher than the blank group.The proliferation activity of cells in CD44^(+)CD24^(+)group was higher than that in control group and CD44^(-)CD24^(-)group(P<0.05).The relative expression intensity of telomerase and colony formation rate in CD44^(+)CD24^(+)group were higher than those in the control group,and the relative expression intensity of telomerase and colony formation rate in CD44^(-)CD24^(-)group were lower than those in the control group(P<0.05).The tumor formation time of CD44^(+)CD24^(+)cells in vivo was earlier than that of the control group,and the tumor formation rate was higher(P<0.05).Conclusion CD44 and CD24 are feasible surface markers for human CCSCs,and the obtained CD44^(+)CD24^(+)cells can show biological characteristics of tumor stem cells.

关 键 词：宫颈癌 干细胞 流式细胞术 CD44 CD24 

分 类 号：R737.4[医药卫生—肿瘤]