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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。
OBJECTIVE: The specific stem cell markers of oral squamous cell carcinoma are not clear. This study aimed to enrich the biological characteristics of oral squamous cell carcinoma stem cells by microspheres culture. METHODS: Oral squamous cell carcinoma cells were cultured in DMEM / F12 + bFGF + EGF medium to induce the growth of oral squamous cell carcinoma stem cell-like microspheres. Then the biological characteristics of the microspheres were observed, including the ability of inducing differentiation by using 10% FBS + DMEM culture method; the self-renewal ability of the microspheres cells was detected by continuous cloning method; the immunofluorescence technique and reverse transcription Enzyme-linked reaction (RT-PCR) was used to detect the changes of stem cell-related signal molecules in microspheres. The results were analyzed by SPSS15.0 software. RESULTS: Typical stem cell-like microsphere formation was seen when cultured in DMEM / F12 + bFGF + EGF for about 1 week. With the extension of incubation time, the diameter of microspheres gradually increased and reached about 1 mm in about 3 weeks, which could be continuously passaged in vitro to form new microspheres. The microspheres grew rapidly in 10% FBS + DMEM and completely adhered to the surface after 7 ~ 10 days. In vitro continuous cloning experiments show that the microspheres have a strong clonality and self-renewal capacity. The expression of stem cell related molecules Shh and Gli2 in oral squamous cell carcinoma microspheres increased significantly. CONCLUSION: Microspheres culture method successfully enriches some cells with strong self-renewal ability in oral squamous cell carcinoma and provides an important tool for further study of the biological characteristics of oral squamous cell carcinoma stem cells.