摘要
目的运用无血清成球培养法从人胰腺癌细胞系L3.6pl中分离/富集肿瘤干细胞,并对其"干性"特性进行鉴定。方法采用无血清与低黏附细胞培养板诱导胰腺癌L3.6pl细胞产生肿瘤细胞球。收集悬浮细胞球形成细胞,采用磁性细胞分选(magnetic activated cell sorting,MACS)技术纯化胰腺癌L3.6pl细胞株中的CD24+CD44+ESA+细胞,继续在无血清条件下培养观察细胞的球体形成能力。Western blot检测L3.6pl来源的干细胞中自我更新及上皮细胞间质化转变(epithelial-mesenchymal transition,EMT)相关癌基因表达。免疫荧光检测法检测干细胞相关标志的表达,并检测干细胞的细胞分化潜能。通过NOD-SCID小鼠皮下移植评估其致瘤能力,免疫组化检测原癌基因c-Met和RON的表达。通过MTT法检测胰腺肿瘤干细胞对吉西他滨或紫杉醇的敏感性。结果 CD24+CD44+ESA+细胞具有明显致瘤性,且在无血清条件下培养能形成细胞球体,存在干细胞所具有的自我更新、EMT和耐药性等能力,并与c-Met、RON的蛋白表达成正相关。结论成功分离获得胰腺癌干细胞,人胰腺癌L3.6pl来源的CD24+CD44+ESA+细胞具有肿瘤干细胞特性,为将来研究胰腺癌个性化治疗及疗效评价提供了很好的研究模型。
Aim To isolate cancer stem cells from human pan-creatic cancer cell line L3. 6pl and to identify their biological characteristics. Methods L3. 6pl cells were cultivated in com-mercial low adhesion plate with serum-free stem cell culture me-dium （ MEM/F12 1：1 ） supplemented with B27. The cancer stem cells reformed into floating spheres were isolated. The method of tumor sphere formation was used to isolate/enrich and characterize the cancer stem cells in pancreatic carcinoma cell line L3. 6pl. Cancer stem cell spheres were collected and sorted using magnetic cell sorting （ magnetic activated cell sorting, MACS） technology, with the cell surface markers of CD24 ＋CD44 ＋ ESA＋. Self-renewal and EMT-related oncogene expres-sion were measured with Western blot. Cancer stem cells differ-entiation potential and the expression of cancer stem cell related signs were checked with Immunofluorescence assay. To deter-mine tumorigenesis in vivo, Xenograft assay in NOD-SCID mice were performed respectively, then immunohistochemistry proto-oncogene c-Met and RON expression were also checked. West-ern blot was used to detect the changes of stemness relative tran-scriptional factors and epithelial markers expressed in spheres before and after differentiation. Drug resistance of pancreatic cancer stem cells to gemcitabine or paclitaxel was measured with MTT assay. Results CD24 ＋ CD44 ＋ ESA＋ cells were signifi-cantly tumorigenic, and cultured in serum-free conditions to form spheroids, which had the characteristics of stem cells with self-renewal, EMT and drug-resistant capabilities, and had a posi-tive correlation with the c-Met, RON protein expression. Con-clusion Human pancreatic stem cells are successfully isolated, which provides a useful model for individualized therapy and e-valuation of the therapeutic efficacy for pancreatic cancer pa-tients.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第4期583-587,共5页
Chinese Pharmacological Bulletin
基金
湖南省教育厅平台项目(No 11K052
No 09K105)
湖南省高等学校产业化培育项目(No 13cy026)
湖南省科技计划项目(No 2013FJ6090)
湖南省"十二五"植物学重点建设学科资助(201142)
民族药用植物资源研究与利用湖南省重点实验室开放基金项目(No HHUW2010-65)
