【作者】 周静；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 胰腺癌是消化系统中预后最差的肿瘤之一，即使接受根治性切除术后的患者其5年生存率也只有5～10％。因而深入阐明胰腺癌发生机制，寻找胰腺癌治疗的新靶点对于改善预后具有重要意义。最新研究认为肿瘤是一种干细胞疾病，是由少数具有成瘤能力的肿瘤干细胞增殖发育而成的异常组织。近年来国内外学者通过特异性的细胞表面标志已在急性髓细胞样白血病、乳腺癌、脑肿瘤、前列腺癌、肺癌和结肠癌等肿瘤中成功分离出相应的肿瘤干细胞，并证明它们在肿瘤的形成和生长过程中的决定性作用。亦有学者从事胰腺癌干细胞的分离鉴定，但迄今尚未确定出公认的表面标志物。Li等通过鉴定提出CD44~+CD24~+ESA~+的细胞亚群是胰腺癌干细胞，Hermann等认为胰腺癌干细胞的表型是CD133~+。另外，他们的研究主要是证明胰腺癌是由肿瘤干细胞形成的，调控其生长增殖的信号通路尚未报道。肿瘤SP细胞的分离鉴别是被一致公认的研究肿瘤干细胞生物学特性的替代方法。本研究采用流式分析证实了胰腺癌中SP细胞的存在，通过体内体外实验研究其包括干细胞特性在内的生物学特性，并初步探讨了PI3K／mTOR信号通路对其生存增殖的调控作用。第一部分胰腺癌SP细胞的分离和表型分析目的分离胰腺癌中的SP细胞亚群并确定其表型。方法应用Hoechst 33342染色，流式细胞仪检测6个胰腺癌细胞系及3个原代培养的临床胰腺癌标本中SP细胞的含量。选取典型的具SP细胞的细胞系检测SP细胞的表型。结果除了BXPC-3，其他胰腺癌细胞系及原代培养标本都存在Verapamil敏感的SP细胞。PANC-1、CAPAN-1、ASPC-1、PC-3和SW-1990中SP细胞的比例分别为7.84％，11.22％，16.88％，0.43％和2.25％。3个临床标本中SP细胞的比例分别为0.33％，2.79％和0.98％。PANC-1、CAPAN-1和ASPC-1中的绝大多数SP细胞的表型为CD133~+和CD44~+CD24~-ESA~+结论胰腺癌中的确存在SP细胞，其表型为CD133~+和CD44~+CD24~-ESA~+。第二部分胰腺癌干细胞样SP细胞生物学特性及化疗耐药机制初探目的分离鉴定胰腺癌干细胞样的SP细胞亚群并探讨其生物学特性及化疗耐药机制。方法应用Hoechst 33342染色，FACS分选胰腺癌细胞系PANC-1中的SP细胞。通过平板克隆形成试验和NOD-SCID小鼠异种移植成瘤试验比较SP细胞与non-SP细胞的克隆形成能力及成瘤能力，通过对体外培养的SP细胞和SP细胞衍生肿瘤的Hoechst 33342复染SP再分析判断其是否具有分化潜能。通过成球实验评价SP细胞的自我更新能力。采用Transwell试验和MTT试验比较分选的SP细胞与non-SP细胞的侵袭能力及其对化疗药物的耐药性。应用实时定量PCR检测SP细胞与non-SP细胞中ABCB1和ABCG2的表达。采用流式细胞术比较SP细胞与non-SP细胞的细胞周期。结果PANC-1 SP细胞具有较高的自我更新能力和成瘤能力并且能够发生不对称分裂生成non-SP细胞。SP细胞的侵袭力及其对化疗药物的耐药性明显高于non-SP细胞。SP细胞中ABCB1和ABCG2的表达水平明显高于non-SP细胞且大多数细胞处于G1期。结论胰腺癌SP细胞富集了具有高侵袭能力的干细胞样肿瘤起始细胞，其耐药机制可能与其大多数细胞处于静息期及某些ABC转运体的高表达相关。第三部分PI3K／mTOR信号通路参与胰腺肿瘤干细胞样SP细胞生存增殖的调控目的分离鉴定胰腺癌中肿瘤干细胞样的SP细胞亚群并探讨PI3K／mTOR信号通路对其生存与增殖的调控。方法应用流式分析检测胰腺癌细胞系PANC-1中SP细胞的含量。观察加入PI3K／mTOR信号通路特异性抑制剂LY294002或雷帕霉素培养后PANC-1中SP细胞的含量变化。采用MTT试验和克隆形成试验检测LY294002或雷帕霉素对分选的SP细胞和non-SP细胞的抑制作用。结果加入LY294002或雷帕霉素培养后PANC-1 SP细胞的含量明显降低(LY294002，7.60±0.27％vs 1.90±0.22％，P=0.000；雷帕霉素，7.60±0.27％vs 1.14±0.20％，P=0.000)。LY294002对SP细胞和non-SP细胞的生存抑制率(1-细胞存活率)分别是47.87±3.82％和27.64±2.09％，差异有统计学意义(P=0.001)。雷帕霉素对SP细胞的生存抑制率亦高于non-SP细胞(57.04±2.78％vs 35.99±3.11％，P=0.001)。LY294002和雷帕霉素对SP细胞的克隆形成能力的抑制率也高于non-SP细胞(LY294002，54±6.56％vs 34.67±3.06％，P=0.01；雷帕霉素，48±3.61％vs 30.67±3.21％，P=0.003，结论PI3K／mTOR信号通路参与对其生长增殖的调控，可能成为根治胰腺癌的治疗新靶点。更多还原

【Abstract】 Pancreatic cancer is one of the most lethal cancers in digestive system, as indicated by a 5year survival rate of 5％-10％even for patients received curative radical excision. It istherefore essential to develop a deeper understanding of the biological characteristics ofthis disease to develop more effective therapies. Emerging evidence showed that onlycancer stem cells, a small subset of cells within a tumor, is able to self-renew and give riseto heterogeneous populations of daughter cells that comprise the tumor. The existence ofcancer stem cells was initially proven in hematological malignancy and subsequentlyverified in breast carcinoma, brain cancers, prostate, lung and colon by specific cell surfacemakers. They were proven to play a decisive role in the pathogenesis of their correspondingtumors. Recently, the cancer stem cell hypothesis has been explored in pancreatic cancer,but the consensus marker of pancreatic cancer stem cells is undetermined. Li et al identifieda CD44+CD24+ESA+ subpopulation as putative pancreatic cancer stem cells, whileHermann et al defined human pancreatic cancer stem cells by means of the surface markerCD133. Moreover, these studies mainly indicate that pancreatic cancer is caused by cancerstem cells, the cell signaling pathways governing the maintenance and development ofpancreatic cancer stem cells, which may become targets to disrupt cancer stem cellactivities, are not well defined. Side population isolated from tumors has been proven to bea generally accepted alternative to study cancer stem cells biology since they are enriched in cancer stem cells. The present study was undertaken to investigate the prevalence of SPcells, study the biological characteristics of them and investigate the role of PI3K/mTORpathway in the survival and proliferation of them.PartⅠIsolation and phenotype analysis of side population cells inhuman pancreatic cancerObjective To identify side population in pancreatic cancer and define its phenotype.Method To detect the persistence of SP cells in 6 pancreatic cancer cell lines and 3 clinicalsamples by Hoechst 33342 dyeing and FACS analysis. To perform phenotype analysis onSP cells in three typical pancreatic cancer cell linesResults All cell lines and clinical samples were found to exhibit verapamil-sensitive SPcells except BXPC-3. The proportions of SP cells in human pancreatic cancer cell linesPANC-1、CAPAN-1、ASPC-1, PC-3 and SW-1990 were 7.84％, 11.22％, 16.88％, 0.43％,and 2.25％, respectively. The proportions of SP cells in clinical pancreatic cancer sampleswere 0.33％, 2.79％, and 0.98％, respectively. Most SP cells in PANC-1, CAPAN-1 andASPC-1 were CD133~+ and CD44~+CD24~-ESA~+Conclusion Side population cells do exist in human pancreatic cancer. The phenotype ofSP cells in pancreatic cancer were CD133~+ and CD44~+CD24~-ESA~+PartⅡThe biological characteristics and mechanism of drugresistance of cancer stem-like side population cells in pancreaticcancerObjective To identify side population cells in pancreatic cancer and investigate the biological characteristics and mechanism of drug resistance of them.Method To detect the presentation of SP cells in pancreatic cancer cell line, PANC-1, byHoechst 33342 dyeing and FACS analysis. To compare the clone formation efficiency andtumorigenicity between SP cells and non- SP cells by clone formation assay andNOD/SCID xenograft transplantation experiment. To Study the differentiation ability of SPcells by flow cytometry reanalysis of SP-derived tumors and cultured SP cells. To evaluatethe self-renew ability of SP cells by sphere formation assay. Transwell invasion assay andMTT assay were conducted to compare the invasiveness and drug resistance between SPcells and non-SP cells. Real-time PCR was used to detect the expression of ABCB1 andABCG2 in both SP cells and non-SP cells. Cell cycle analysis was also performed to thesetwo populations.Results SP cells do exist in PANC-1. SP and NSP cells were collected for subsequentexperiments, the purity was 96.5％for SP cells and 99.11％for NSP cells. PANC-1 SPwere demonstrated to possess greater self-renew ability and tumorigenicity and be capableof generating nontumorigenic non-SP cells through asymmetrical cell division. SP cellswere more invasive and more resistant to chemotherapeutic drugs than NSP cells. ABCB1and ABCG2 were found to express at higher concentrations in SP than NSP cells. And mostSP cells are in G1 cell cycle arrest.Conclusion There is a significant enrichment of stem-like invasive tumor-initiating cells inthe SP of pancreatic cancer. Their high resistance to chemotherapy was related to theincreased expression of ATP-binding cassette transporters and relative quiescence in cellcycle. It may help explain why conventional therapies often fail to improve long-termsurvival of pancreatic cancer and why even patients who respond to primary chemotherapyultimately develop to local recurrence and metastasis. PartⅢPI3K/mTOR pathway is critical for survival andproliferation of pancreatic cancer stem-like side population cellsObjective To characterize side population cells in pancreatic cancer and investigate therole of PI3K/mTOR pathway in the survival and proliferation of them.Method To detect the presentation of side population cells in pancreatic cancer cell lines,PANC-1, by FACS analysis, as well as the SP proportion change in PANC-1 treated withPI3K/mTOR-specific inhibitor LY294002 and rapamycin. MTT assay and clone formationassay were done to assess the inhibition of LY294002 and rapamycin.Results LY294002 and rapamycin decreased the SP fraction in PANC-1. Compared withcontrol PANC-1 cells, which contained 7.60±0.27％SP cells, PANC-1 cells treated withLY294042 and rapamycin had only 1.90±4.22％(P=0.400) and 1.14±0.24％(P=0.000)SP cells, respectively. Both LY294002 and rapamycin preferentially inhibited the SP ratherthan NSP cells. LY294002 and rapamycin inhibited PANC-1 SP cell proliferation by 47.87±3.82％and 57.04±2.78％(1-relative SR) respectively, while inhibited the NSP cellproliferation by 27.64±2.09％and 35.99±3.11％only. In addition to proliferation,LY294042 and rapamycin also preferentially inhibited the colony-formation ability of SPcells by 54±6.56％and 48±3.61％(1-relative CEF), respectively, compared with that ofthe NSP cells by 34.67±3.06％and 30.67±3.21％.Conclusion PI3K/mTOR pathway is critical for Pancreatic SP cells maintenance, whichcould be selectively targeted for inhibiting cancer stem-like cells for improved treatment.更多还原