膀胱癌干（起始）细胞分离、鉴定实验研究
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摘要
目的:膀胱癌是常见恶性肿瘤,预后较差容易复发。目前手术治疗、放疗和化疗的疗效并不令人十分满意。“肿瘤干细胞学说”认为肿瘤组织中的肿瘤干细胞是肿瘤生长、转移和复发的主要原因。肿瘤干细胞(Cancer stem cells,CSCs)这一概念的提出为肿瘤真正意义上的靶向治疗找到了靶细胞。在靶向CSCs治疗策略开发之前,研究者首先应该分离出所有未确定的CSCs。几种人类CSCs即较新概念的肿瘤起始细胞(Tumor initiating cells,TICs)已被成功分离鉴定,但大部分CSCs(TICs)还未被分离鉴定,包括膀胱癌干(起始)细胞。在分离未确定的CSCs(TICs)时,最困难的工作在于可供分选的标记的匮乏。本研究在天津医科大学2005级博士创新基金(200604)和天津市2007年自然科学基金重点项目(07JCZDJC03400)资助下延续畅继武教授1985年首次提出的“两类癌”理念及其近30年的相关研究,进行肯定膀胱癌干(起始)细胞存在并分离、鉴定不同膀胱癌细胞亚群起始和维持肿瘤生长能力,为最终分离、鉴定膀胱癌干(起始)细胞打下实验基础;总结分离未确定肿瘤干细胞的实验研究,为最终分离、鉴定膀胱癌干(起始)细胞打下方法学基础。方法:1.肯定膀胱癌干(起始)细胞存在:采用Agilent人全基因组表达谱芯片G4110B,结合激光捕获显微切割、RNA线性扩增等技术构建低恶性膀胱移行上皮细胞癌全基因组表达图谱,进而观察干细胞密切相关基因(Bmi-1和EZH2)在膀胱癌表达情况;结合形态学方法确定膀胱癌细胞中存在侧群细胞(Sider population cells,SP cells);免疫组化方法观察胚胎干细胞标志OCT4在胱癌细胞中表达情况。2.确定可能的膀胱癌干(起始)细胞表面标记:检索人类白细胞分化抗原工作组网站得到所有人类细胞表面标记。PUBMED检索上述所有CD分子中符合干细胞或CSCs(TICs)表面标记或多年以来一直被报道的在多种肿瘤中与肿瘤发生、进展、转移和复发密切相关的表面标记。进而结合免疫组化实验筛选、确定可能的膀胱癌干(起始)细胞表面标记。3.应用可能的膀胱癌干(起始)细胞表面标记局限具有CSCs(TICs)特性的膀胱癌细胞于特定亚群:通过克隆形成实验、裸鼠成瘤实验和裸鼠成瘤连续传代实验确定不同亚群膀胱癌细胞起始和维持肿瘤生长能力。最后,总结分离未确定CSCs(TICs)方法。结果:1.干细胞密切相关基因Bmi-1和EZH2在膀胱癌细胞中高表达;膀胱癌细胞中存在SP细胞;胚胎干细胞标志OCT4在胱癌细胞细胞表达。2.共得到227个人类细胞表面标记。其中,CK34BE12,EMA,CD9,CD10,CD14,CD19,CD20,CD24,CD33,CD34,CD38,CD44v6,CD45RO,CD49d,CD56,CD61,CD62L,CD71,CD72,CD81,CD82,CD90,CD95,CD117,CD133,CD178,MUC-1(CD227)和CD243等28个表面标志可能是CSCs(TICs)表面标志。进而结合免疫组化结果筛选、确定其中EMA和CD44v6可能是膀胱癌干细胞(起始细胞)表面标志。3.膀胱癌中,EMA~-CD44v6~+亚群细胞具有起始和维持肿瘤生长能力,接近已分离的CSCs(TICs)的界定标准;免疫组化方法有助于缩小可能的表面标记范围,易化分离未确定CSCs(TICs)过程。结论:1.膀胱癌组织中存在膀胱癌干(起始)细胞;本研究首次提出形态学观察SP细胞方法,有助于判断其他未确定肿瘤干(起始)细胞的存在。2.本研究首次确定膀胱癌干(起始)细胞存在于EMA~-CD44v6~+亚群。3.本研究首次解决了分离未确定肿瘤干细胞中最困难、最耗时的问题——快速获得可能的未确定CSCs(TICs)表面标记,有助于最终分离膀胱癌干(起始)细胞,也有助于分离其他未确定上皮肿瘤CSCs(TICs)。
Objective: Bladder transitional cell carcinomas (BTCC) is the most common urologic tumor in China and it have unfavourable prognosis. Cancer stem cells hypothesis indicate that the reason for tumorigenesis, metastasis and recurrence of tumors is the existence of cancer stem cells in tumors. Cancer stem cell(CSCs) is the targets for tumor targeting therapy. Before therapy targeting to CSCs is developped,all undetermined CSCs should be isolated. CSCs have been positively identified and successfully isolated from some but not all cancers,including bladder cancer stem cells. However, the isolation and identification of undetermined CSCs has proven difficult because of the paucity of specific cell surface markers for cell sorting. This work was supported by the Doctor Foundation for Innovation, Tianjin Medical University grant yjs200604;Tian Jin Natural Science Foundation, Tianjin Municipal Science and Technology Commission grant 07JCZDJC03400.This study for isolating bladder cancer stem cells is on the basis of the previous work of Prof. Chang JiWu for bladder cancer research. Methods: 1. To confirm the existence of bladder cancer stem cells: Identifying the differentially expressed stem cells related genes, Bmi-1 and EZH2,between laser-microdissected low malignant bladder-cancer tissues and corresponding noncancerous mucosae by the combination of laser capture microdissection, oligo microarray analysis and mRNA linear amplification. Side population cells in bladder cancer were detected under fluorescence microscope. Expression of Marker of embryonic stem cells,OCT4, in bladder cancer was detected by immunohistochemistry. 2. To confirm the markers of bladder cancer stem cells : Human CD molecules were retrieved on the web. Either markers of homologous or heterologous somatic stem cells or progenitor cells or markers of tumorigenesis, progression, metastasis and recurrence in many malignant tumors, or both were retrieved on the PUBMED web.Then, confirming the potential markers of bladder cancer stem cells by immunohistochemistry. Confirming the markers of bladder cancer stem cells by colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. In the end, summarizing the methods for isolating undetermined cancer stem cells .Results: 1. Highly expressed stem cells related genes, Bmi-1 and EZH2, were found in bladder cancer cells. Side population cells in bladder cancer were confirmed under fluorescence microscope. Expression of marker of embryonic stem cells,OCT4, in bladder cancer was found 2. 227 human CD molecules were found on the web. Among the 227 human CD molecules,28 CD molecules might be the markers of cancer stem cells. They were CK34BE12,EMA ,CD9,CD10,CD14,CD19,CD20,CD24,CD33, CD34,CD38,CD44v6,CD45RO,CD49d,CD56,CD61, CD62L,CD71 ,CD72,CD81 ,CD8 2,CD90,CD95,CD117 ,CD133,CD178,MUC-1(CD227) and CD243. EMA and CD44v6 might be the markers of bladder cancer stem cells by immunohistochemistry, colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers. Condusion: We conclude the persistence of BCICs. BCICs might be among EMA- CD44v6+ subset. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers.
Objective: Bladder transitional cell carcinomas (BTCC) is the most common urologic tumor in China and it have unfavourable prognosis. Cancer stem cells hypothesis indicate that the reason for tumorigenesis, metastasis and recurrence of tumors is the existence of cancer stem cells in tumors. Cancer stem cell(CSCs) is the targets for tumor targeting therapy. Before therapy targeting to CSCs is developped,all undetermined CSCs should be isolated. CSCs have been positively identified and successfully isolated from some but not all cancers,including bladder cancer stem cells. However, the isolation and identification of undetermined CSCs has proven difficult because of the paucity of specific cell surface markers for cell sorting. This work was supported by the Doctor Foundation for Innovation, Tianjin Medical University grant yjs200604;Tian Jin Natural Science Foundation, Tianjin Municipal Science and Technology Commission grant 07JCZDJC03400.This study for isolating bladder cancer stem cells is on the basis of the previous work of Prof. Chang JiWu for bladder cancer research. Methods: 1. To confirm the existence of bladder cancer stem cells: Identifying the differentially expressed stem cells related genes, Bmi-1 and EZH2,between laser-microdissected low malignant bladder-cancer tissues and corresponding noncancerous mucosae by the combination of laser capture microdissection, oligo microarray analysis and mRNA linear amplification. Side population cells in bladder cancer were detected under fluorescence microscope. Expression of Marker of embryonic stem cells,OCT4, in bladder cancer was detected by immunohistochemistry. 2. To confirm the markers of bladder cancer stem cells : Human CD molecules were retrieved on the web. Either markers of homologous or heterologous somatic stem cells or progenitor cells or markers of tumorigenesis, progression, metastasis and recurrence in many malignant tumors, or both were retrieved on the PUBMED web.Then, confirming the potential markers of bladder cancer stem cells by immunohistochemistry. Confirming the markers of bladder cancer stem cells by colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. In the end, summarizing the methods for isolating undetermined cancer stem cells .Results: 1. Highly expressed stem cells related genes, Bmi-1 and EZH2, were found in bladder cancer cells. Side population cells in bladder cancer were confirmed under fluorescence microscope. Expression of marker of embryonic stem cells,OCT4, in bladder cancer was found 2. 227 human CD molecules were found on the web. Among the 227 human CD molecules,28 CD molecules might be the markers of cancer stem cells. They were CK34BE12,EMA ,CD9,CD10,CD14,CD19,CD20,CD24,CD33, CD34,CD38,CD44v6,CD45RO,CD49d,CD56,CD61, CD62L,CD71 ,CD72,CD81 ,CD8 2,CD90,CD95,CD117 ,CD133,CD178,MUC-1(CD227) and CD243. EMA and CD44v6 might be the markers of bladder cancer stem cells by immunohistochemistry, colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers. Condusion: We conclude the persistence of BCICs. BCICs might be among EMA- CD44v6+ subset. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers.
引文
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