口腔鳞状细胞癌干细胞样微球体的体外培养  

Preliminary study on the sphere-forming stem-like cell populations of oral squamous cell carcinoma

作　　者：王丽君[1] 严明[1] 叶冬霞[1] 陈万涛[1] 张萍[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院·口腔医学院口腔颌面外科,上海市口腔医学重点实验室,上海200011

出　　处：《中国口腔颌面外科杂志》2010年第2期149-153,共5页China Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(30772433);上海市科学技术委员会资助项目(08DZ2271100;08JC1414400);上海市重点学科建设项目(S30206);上海市青年科技启明星计划(07QA14038)~~

摘　　要：目的:口腔鳞状细胞癌特定的干细胞标志物并不清楚,本研究旨在通过微球体培养方式,富集口腔鳞癌干细胞,对其生物学特性进行初步研究。方法:采用DMEM/F12+bFGF+EGF培养液培养口腔鳞癌细胞,诱导口腔鳞癌干细胞样微球体生长。随后对该微球体的生物学特性进行观察,包括采用10%FBS+DMEM培养法检测其诱导分化能力;采用体外连续克隆法检测该微球体细胞的自我更新能力;采用免疫荧光技术及反转录酶链反应(RT-PCR)检测微球体内干细胞相关的信号分子的表达变化;采用SPSS15.0软件包对结果进行t检验。结果:在DMEM/F12+bFGF+EGF中培养1周左右,即见典型的干细胞样微球体形成。随着培养时间延长,微球体直径逐渐增大,3周左右可达到1mm左右,在体外可连续传代,形成新的微球体。微球体在10%FBS+DMEM中迅速贴壁生长,7～10d后完全贴壁,单层样生长。体外连续克隆实验表明,微球体有很强的克隆形成能力和自我更新能力。干细胞相关分子Shh及Gli2在口腔鳞癌微球体中的表达水平显著升高。结论:微球体培养法成功富集了口腔鳞癌中一些具有较强自我更新能力的细胞,为进一步研究口腔鳞癌干细胞的生物学特性提供了一个重要工具。PURPOSE：Cancer stem cell plays an important role in initiation,progress,recurrence and metastasis of cancer,yet till now,the specific markers for oral squamous cell carcinoma stem cell is still unavailable.In this study,we used spheric culture condition to enrichment stem like cells in oral squamous cell carcinoma,and did some pilot studies on its biological characteristics.METHODS：Oral squamous cell carcinoma cells were cultured in DMEM/F12 ＋ bFGF ＋ EGF medium to induce the sphere formation.The biological characteristics of these sphere forming cells were studied,10% FBS ＋ DMEM culture condition was used to detect the differentiation of these spheres;Successive cloning methods were used to examine the self -renewal capacity of these spheres.Immunofluorescent staining and reverse transcriptase chain reaction（RT-PCR） were used to investigate the expression of some stem cell-related molecules in these spheres.SPSS 15.0software package was used for statistical analysis of the results.RESULTS：Some tiny typical spheres were observed under the microscope 1 week after culturing in DMEM/F12 ＋ bFGF ＋ EGF.These spheres grew gradually as the culture time extended and the diameter reached to 1mm after 3 weeks of culture.These spheres could be continuously passaging in DMEM/F12 ＋ bFGF ＋ EGF.In 10% FBS ＋ DMEM culture medium,these spheres began to adherently grow and after about 7-10 days,these spheres completely formed single,adhesive growth layer.Further studies showed that these spheres had stronger continuous colony forming ability and self-renewal ability.Immunofluorescent staining and RT-PCR revealed that some stem-related molecules such as Shh,Gli2 expressed at higher levels in those sphere cells.CONCLUSIONS：The sphere culture method could enrich the stem-like cell population with higher self-renewal ability and higher expression of stem-related molecules in oral squamous cell carcinoma,which provides a useful tool for further oral squamous cell carcinoma stem cell research.

关 键 词：口腔鳞状细胞癌 干细胞 微球体 克隆形成 分化 

分 类 号：R739.8[医药卫生—肿瘤]