摘要
背景:近年来国内外学者相继分离出Ep CAM^(high)CD44^+结肠癌干细胞,但大多为有关大肠癌细胞系中肿瘤干细胞的分离研究,关于结肠癌干细胞功能的报道相对较少。目的:从结肠癌组织对结肠癌干细胞进行筛选和分离培养,探讨结肠癌干细胞蛋白表达情况以及生物学特性。方法:获得结肠癌组织标本,进行原代细胞分离。分离EpC AMhighCD44^+细胞,利用流式细胞仪进行检测、分选,并利用无血清培养基对细胞进行培养和传代,观察细胞球形成情况。对小鼠进行细胞皮下回植,观察其日常表现以及肿瘤生长情况。待肿瘤生长到直径1 cm以上之后,获得肿瘤组织进行免疫组化检验,观察Ep CAM^(high)CD44^+肿瘤细胞流式细胞仪分选情况和不同时间EpC AMhighCD44^+肿瘤细胞在无血清培养基中的生长情况,以及小鼠成瘤情况和移植瘤免疫组化检查中性上皮黏蛋白和结肠特异性分化标志物CK-20表达情况。结果与结论:经检测在获得的结肠癌组织中存在Ep CAM^(high)CD44^+结肠癌干细胞,在结肠癌原代细胞中Ep CAM^(high)CD44^+细胞的比例为1.7%-38%。在无血清培养基中对Ep CAM^(high)CD44^+肿瘤细胞进行24 h的培养之后进行观察,可以发现产生一定体积较小松散的悬浮细胞球,但数量较少;培养21 d之后进行观察,可以发现形成形态一致的细胞球,且形态致密。经裸鼠皮下回植和观察,可以发现形成移植瘤。对移植瘤进行苏木精-伊红染色和观察,可以发现呈现出典型的结肠癌细胞形态特征。对移植瘤进行免疫祖化检查,所有移植瘤均能表选中性上皮黏蛋白和结肠特异性分化标志物CK-20。经检测发现,经10PPC、1PPC、0.1PPC 5-氟尿嘧啶干预,Ep CAM^(high)CD44~^+Colo205细胞的增殖均得到促进,且随着药物作用时间的延长促进作用逐渐增强。提示结肠癌组织中存在Ep CAM^(high)CD44^+结肠癌干细胞,其具有一定的增殖、更新、分化以及致瘤能力和耐化疗能力。
BACKGROUND： EpCAM^highCD44^＋ colon cancer stem cells have been isolated in recent years, and most of them are related to the study of isolating tumor stem cells in colorectal cancer cell lines. There are less reports on the function of colon cancer stem cells.OBJECTIVE： To isolate, screen and culture colon cancer stem cells from colorectal carcinoma tissues and to explore the protein expression and biological characteristics of colon cancer stem cells. METHODS： The primary cells were isolated from the colon cancer tissues. EpCAM^highCD44^＋ cells were detected and sorted by flow cytometry. Then, these cells were cultured in serum-free medium. After the cells werereplanted subcutaneously into the mice, we observed the daily performance and tumor growth in the mice. When the tumor diameter was above 1 cm, tumor tissues were taken for immunohistochemical testing. The growth of EpCAM^highCD44^＋ cells cultured in the serum-free medium was observed; expressions of neutral epithelial mucin and CK-20 in EpCAM^highCD44^＋ cells were detected. RESULTS AND CONCLUSION： EpCAM^highCD44^＋ colon cancer stem cells were detected in the colon cancer tissues, and the proportion of EpCAM^highCD44^＋ cells in primary colon cancer cells was 1.7%-38%. After 24 hours of serum-free culture, there were a few of small suspended cell spheres which were incompact. After 21 days, dense cell spheres with uniform size were found. The formation of transplanted tumor was found in the nude mice. Hematoxylin-eosin staining showed the transplanted tumor had the typical characteristics of colon cancer cells. The expression of neutral epithelial mucin and CK-20 was observed in all the transplanted tumors. Additionally, the proliferation of EpCAM^highCD44^＋ cells could be promoted by 5-fluorouracil（10, 1, and 0.1 PPC） with time. These findings indicate that EpCAM^highCD44^＋ colon cancer stem cells in the colon cancer have a certain ability of proliferation, regeneration, differentiation, tumor formation and chemotherapy resistance.
出处
《中国组织工程研究》
CAS
北大核心
2015年第45期7286-7291,共6页
Chinese Journal of Tissue Engineering Research
作者简介
张东兴，1975年生，山东省人，汉族，2010年贵阳医学院（现已更名为贵州医科大学）毕业，硕士，主治医师，主要从事结直肠外科及肛门盆底疾病研究。
