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目的肿瘤干细胞(cancer stem cells,CSCs)在恶性肿瘤的发生发展中起着重要作用,且是肿瘤耐药及复发的根源。本研究探讨CSCs表面标记蛋白CD44在分离及鉴定胃癌干细胞中的重要作用。方法收集2015-11-06-2015-12-01华中科技大学同济医学院附属同济医院3例胃癌患者手术标本。首先采用胶原酶消化法获取胃癌细胞,部分细胞分别贴壁及悬浮培养,并采用免疫荧光及流式分析的方法检测胃癌球形体中CD44的表达情况。部分细胞采用流式细胞仪分选得到CD44~+及CD44~-胃癌细胞,并通过无血清悬浮培养法比较两群细胞的球形体形成能力。结果免疫荧光显示,CD44蛋白在球形体中高表达;流式分析发现,贴壁的原代胃癌细胞中CD44~+细胞的比例为(30.73±7.6)%,而球形体中CD44~+细胞的比例高达(56.4±5.58)%,差异有统计学意义,P=0.009。球形体形成实验结果表明,与CD44~-细胞相比,CD44~+胃癌细胞具有更强的球形体形成能力,1 000个CD44~+及CD44~-胃癌细胞分别形成(14.33±2.52)和(1.67±0.58)个球形体,差异有统计学意义,P=0.001。通过比较发现,球形体培养对胃癌干细胞的富集效率为0.3%,而CD44对胃癌干细胞的富集效率为1.4%。结论 CD44是胃癌干细胞的表面标记蛋白,其可能成为胃癌靶向治疗的可靠靶标。
OBJECTIVE: Cancer stem cells (CSCs) play an important role in the development of malignant tumors and are the root cause of tumor drug resistance and relapse. This study was to investigate the important role of CSCs surface marker protein CD44 in the isolation and identification of gastric cancer stem cells. Methods Collecting 2015-11-06-2015-12-01 3 cases of gastric cancer patients with Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. First, gastric cancer cells were obtained by collagenase digestion, and some cells were attached to the wall and suspension culture respectively. The expression of CD44 in spheroids was detected by immunofluorescence and flow cytometry. Some of the cells were sorted by flow cytometry to obtain CD44 ~ + and CD44 ~ - gastric cancer cells, and the spheroid formation ability of the two groups of cells was compared by serum-free suspension culture. Results Immunofluorescence showed that CD44 protein was highly expressed in spheroids. Flow cytometry analysis showed that the percentage of CD44 + cells in adherent primary gastric cancer cells was (30.73 ± 7.6)%, while the proportion of CD44 + cells in spheroids Up to (56.4 ± 5.58)%, the difference was statistically significant, P = 0.009. The results of spheroid formation experiment showed that CD44 ~ + cells had stronger ability of spheroid formation than CD44 ~ - cells, and the number of 1 000 CD44 ~ + and CD44 ~ - gastric cancer cells was (14.33 ± 2.52) and 1.67 ± 0.58) spheroids, the difference was statistically significant, P = 0.001. By comparison, the enrichment efficiency of spheroid culture on gastric cancer stem cells was 0.3%, while the enrichment efficiency of CD44 on gastric cancer stem cells was 1.4%. Conclusion CD44 is a surface marker protein of gastric cancer stem cells, which may be a reliable target for targeted therapy of gastric cancer.