【作者】 朱峰；

【导师】 秦仁义；

【作者基本信息】 华中科技大学 ， 普通外科学， 2010， 博士

【摘要】 目的：探讨肿瘤干细胞标记物CD24、CD44、EpCAM和CD34在胆管癌中的表达,初步明确其能否用于胆管癌干细胞的分选。方法：免疫细胞化学、RT-PCR、Western Blotting和流式细胞术检测CD24、CD44、EpCAM和CD34在胆管癌细胞系QBC939和胆囊癌细胞系GBC-SD中的表达水平；免疫组织化学检测CD24、CD44、EpCAM和CD34在50例胆管组织(10例正常胆管组织、20例肝门部胆管癌、20例胆总管下段肿瘤)中的表达,流式细胞术检测其中8例胆管癌组织CD24、CD44、EpCAM和CD34的表达率。结果：在QBC939细胞系中,流式细胞术检测CD24+、CD44+和EpCAMhigh的表达率分别为57.57%、89.41%和97.74%,CD24+CD44+EpCAMhigh的表达率为51.01%；在GBC-SD细胞系中,流式细胞术检测CD24+、CD44+和EpCAMhigh的表达率分别为82.61%、93.56%和98.36%,CD24+CD44+EpCAMhigh的表达率为80.59%；在两株细胞系中,RT-PCR检测CD24、CD44和EpCAM mRNA高表达,CD34mRNA呈弱表达；免疫细胞化学、Western Blotting检测CD24、CD44和EpCAM蛋白均表达,CD34蛋白呈无表达；在人胆管癌组织中,免疫组织化学检测CD24、CD44和EpCAM蛋白均表达,而CD34蛋白无表达；流式细胞术检测CD24、CD44、EpCAMhigh CD24+EpCAMhigh/CD44+EpCAMhigh、CD44+CD24+的表达率分别为5.19-21.39%、3.79-42.32%、70.83-94.48%、4.27-19.73%、1.88-21.61%、0.43-6.60%,CD24+CD44+EpCAMhigh表达率为0.39-2.27%(均值：0.94%)。结论：CD24、CD44和EpCAM是胰腺癌干细胞和乳腺癌干细胞表面标记物,其在胆管癌中的表达率和以上两者肿瘤干细胞的分选比例十分相似,可作为胆管癌干细胞表面标记物。目的：检测及分选人胆管癌中的CD24+CD44+EpCAMhigh细胞亚群,探讨其是否具有肿瘤干细胞样生物学特性。方法：将新鲜人胆管癌标本种植到NOD/SCID小鼠皮下,建立荷人胆管癌小鼠模型。流式细胞术分选CD24+CD44+EpCAMhigh亚群细胞,NOD/SCID小鼠移植瘤试验鉴定其成瘤和分化能力。结果：建立稳定的NOD/SCID小鼠移植瘤模型,分选得到CD24+CD44+EpCAMhigh亚群细胞。NOD/SCID小鼠移植瘤试验,103个CD24+CD44+EpCAMhigh细胞能成瘤(3／8),而CD24-CD44-EpCAMlow/-细胞需5x104才能成瘤(1／8)。CD24+CD44+EpCAMhigh胆管癌细胞NOD/SCID小鼠移植瘤的组织类型及标记物的表达率和原代肿瘤相似。结论：人胆管癌中含有CD24+CD44+EpCAMhigh细胞亚群,具有强成瘤能力、自我更新和分化的能力,可能是胆管癌干细胞。更多还原

【Abstract】 Objective:To investigate the expression of CD24, CD44, EpCAM and CD34 in cholangiocarcinoma and determine whether they may be candidate marker isolated cancer stem cells.Methods:Immunocytochemistry staining, RT-PCR, Western blotting and Flow cytometry were employed to examine the expression and rate of CD24, CD44, EpCAM and CD34 at protein and mRNA levels both in Human cholangiocarcinoma cells (QBC-939) and Human gallbladder carcinoma cells (GBC-SD), respectively. The samples resected from patients undergoing surgical resection in our hospital, used for immunohistochemistry included 10 normal bile duct tissues,20 perihilar cholangiocarcinomas, and 20 distal biliary cancers. Of the 40 Extrahepatic cholangiocarcinoma,8 fresh cancer samples were used to perform the expression and rate of CD24, CD44, EpCAM and CD34 by Flow cytometry.Results:In the QBC939 cell line, the mean frequencies of CD24+, CD44+, and EpCAMhigh cells were 57.57%,89.41% and 97.74%, respectively; 51.01% of QBC939 cells were CD24+CD44+EpCAMhigh. In the GBC-SD cell line, the mean frequencies of CD24, CD44 and EpCAMhigh cells were 82.61%,93.56% and 98.36%, respectively; 80.59% of GBC-SD cells were CD24+CD44+EpCAMhigh. In both cell lines, CD34 was weakly detected only by RT-PCR, but not by immunohistochemistry examination、western blot and flow cytometry analysis. On the contrary, CD24, CD44, EpCAM were highly expressed at mRNA and protein level by RT-PCR, Immunocytochemistry staining and western blot. The results were analogue to achieve in cholangiocarcinoma by immunohistochemistry examination, and the frequency of CD24, CD44, EpCAMhigh,CD24+EpCAMhigh, CD44+EpCAMhigh, CD44+CD24+were 5.19-21.39%,3.79-42.32%,70.83-94.48%,4.27-19.73%, 1.88-21.61%,0.43-6.60%. Furthermore, the expression rate of CD24+CD44+EpCAMhigh subpopulation was 0.39-2.27%(mean:0.94%). Conclusion:CD24 combined with CD44 and EpCAM may be a candidate marker isolated cancer stem cells from cholangiocarcinoma.Objective:To isolate CD24+CD44+EpCAMhigh subset cells of human cholangiocarcinoma and identify their cancer stem cell-like properties.Methods:The procedure of subcutaneous implant of NOD/SCID mice was performed to establish the xenografts model with cholangiocarcinoma specimens. CD24+CD44+EpCAMhigh subpopulation of cholangiocarcinoma cells were sorted from xenografts by flow cytometry, and their tumorigenic potential, self-renewal ability and differentiated ability were assessed.Results:CD24+CD44+EpCAMhigh subset cells isolated from 2 xenografts were found to be highly tumorigenic in NOD/SCID mice. CD24+CD44+EpCAMhigh cells consistently formed new tumors at 1×103 cells in three of eight mice tested, In contrast, CD24"CD44"EpCAMlow/- tumor cells were less tumorigenic and gived rise to tumors when 5×104 cells were s.c. inoculated in only one of eight mice animals. Tumors originated from CD24+CD44+EpCAMhigh cells in NOD/SCID mice recapitulated the histological features and heterogeneity of the original patient tumor.Conclusion:CD24+CD44+EpCAMhigh subset cells were discriminated in human cholangiocarcinoma, which had highly tumorigenic, self-renewal ability and differentiated ability. It was first confirmed that CD24+CD44+EpCAMhigh cells may be human cholangiocarcinoma cancer stem cells.更多还原