【作者】 熊宇泉；

【导师】 汤钊猷；

【作者基本信息】 复旦大学 ， 疾病蛋白组学， 2009， 博士

【摘要】 原发性肝癌是常见的恶性肿瘤,手术切除仍是最有效的治疗方法,但根治术后5年转移复发率高达60-70%,是影响肝癌远期疗效的主要原因。抑制肿瘤血管生成,可减少肿瘤赖以生存的营养供应,从而抑制肿瘤生长。对不能切除的肝癌和肝癌术后转移复发的预防,抗血管生成是一条有希望的途径。血管生成抑制剂治疗肿瘤的理论基础在于新生与重塑的肿瘤血管不同于静止的正常血管。血管异质性是近年肿瘤血管生成研究的热点,从分子水平研究肿瘤血管的异质性,有助于监测肿瘤的演进,估计肿瘤的恶性程度,并可能为肿瘤治疗提供新靶点。证据表明,肿瘤血管内皮细胞的生长因子受体,信号转导分子及黏附分子等与细胞活性相关的分子表达都与正常血管内皮细胞有所差异,这些分子的异常表达可能促进了肿瘤血管内皮细胞在细胞功能上与正常血管内皮细胞有所差异。迄今已有报道从乳腺癌、结肠癌及神经胶质瘤等肿瘤中分选培养的内皮细胞比正常内皮细胞具有更强的血管生成能力及化疗抵抗能力,但尚未见检测肿瘤血管内皮细胞对抗血管生成药物敏感性的研究报道,也未见分离培养肝癌血管内皮细胞并研究其血管生成能力等特性的研究报道。目前,sorafenib等抗血管生成药物在肝癌的治疗上取得了进展,可延长患者生存期,但其疗效并不持久,通常几个月后即出现药物抵抗,肿瘤又重新生长,而其耐药机制尚不明了。本课题拟通过分离人肝癌及癌周组织的血管内皮细胞进行传代培养,并检测肿瘤血管内皮细胞及正常血管内皮细胞间的功能差异,同时检测肿瘤血管内皮细胞及正常血管内皮细胞对抗血管生成药物反应的差异,并探讨其发生差异的分子机制,以期为提高抗血管生成药物疗效找到新的途径。1.肿瘤血管内皮研究的平台建设一人肝癌及癌周组织血管内皮细胞的分离、鉴定与传代培养血管内皮细胞的数量极为有限,其分离与纯化受多种因素影响,如何从肿瘤组织的多种混杂细胞中分离纯化内皮细胞成为问题的关键。我们采用CD105抗体交联免疫磁珠分选法,并加以优化,对人肝癌及癌周组织进行血管内皮细胞分选,并进行传代培养。从肝癌癌周组织中分离的CD105阳性细胞呈典型的“铺路石样”内皮细胞形态特征;从肝癌组织中分离的CD105阳性细胞则无此特征,而呈纤维状的梭长表型。RT-PCR检测AFP及流式检测CD68的结果显示阳性分选细胞的CD105表达高达99%,但不表达AFP及CD68,从而排除了肿瘤细胞及巨噬细胞的污染;细胞免疫化学检测显示阳性分选细胞中Ⅷ因子表达为阳性,超过95%的阳性分选细胞表达Ⅷ因子及呈乙酰化低密度脂蛋白摄取实验阳性;分选细胞在Matrigel胶中可形成毛细管结构;流式结果显示CD105、CD31、CD34、CD144、VEGFR1及VEGFR2等血管内皮细胞标记在分选所得CD105阳性细胞上都有所表达。同时可将阳性分选细胞进行传代培养至20代以上。上述结果表明,通过优化的免疫磁分选法,我们成功分选获得高纯度的CD105阳性肝癌血管内皮细胞及CD105阳性正常血管内皮细胞,并能够成功传代培养,分离培养的内皮细胞可用于肝癌血管生成的相关研究。2.肿瘤血管内皮细胞及正常血管内皮细胞的功能比较为比较CD105~+TEC(肿瘤血管内皮细胞)与CD105~+NEC(正常血管内皮细胞)及HUVEC(脐静脉内皮细胞)的功能差异,我们在完全血清培养液中连续培养细胞,结果CD105~+TEC的增殖显著高于CD105~+NEC及HUVEC;在无血清培养液中培养细胞显示CD105~+TEC的抗凋亡能力显著高于CD105~+NEC及HUVEC;划痕实验显示CD105~+TEC的迁移能力高于CD105~+NEC及HUVEC;3D Magtrigel胶无血清培养液中形成毛细网状结构功能的结果显示CD105~+TEC的血管生成能力高于CD105~+NEC及HUVEC;细胞黏附结果显示CD105~+TEC与肿瘤细胞相互作用的能力高于CD105~+NEC及HUVEC;药物试验显示CD105~+TEC对化疗药物阿霉素及5-氟尿嘧啶的抵抗能力显著高于CD105~+NEC及HUVEC。以上说明CD105~+TEC与CD105~+NEC及HUVEC具有功能差异,表现出更强的增殖、抗凋亡、迁移、血管生成能力、与肿瘤细胞相互作用能力以及更强的化疗药物抵抗能力,应该更适用于肿瘤血管生成机制的体外研究。3.肿瘤血管内皮细胞及正常血管内皮细胞的对sorafenib处理的反应差异及其机制研究目前虽已有分选培养肿瘤血管内皮细胞及探索其功能的研究,但还没有关于肿瘤血管内皮细胞对抗血管生成药物反应的研究报道。我们比较了肿瘤血管内皮细胞及正常血管内皮细胞对sorafenib的反应差异:细胞增殖实验显示sorafenib在低浓度时对各细胞系的影响无显著差异,当sorafenib的浓度大于5μM时,CD105~+TEC比CD105~+NEC及HUVEC显示出更高的抵抗能力;5μM sorafenib的浓度时,小管形成实验及spheroid assay实验显示,HuVEC及CD105~+NEC不能形成毛细网状结构或发生出芽结构,而CD105~+TEC仍能形成毛细网状结构及发生出芽结构,说明肿瘤血管内皮细胞对sorafenib具有更强的抵抗作用。接着我们检测了各细胞系在sorafenib处理前后其p-STAT3、STAT3、p-Akt、Akt、p-MAPK及MAPK信号分子的变化,结果显示相对于HUVE及CD105~+NEC,CD105~+TEC在sorafenib作用24小时前后都表达更高水平的p-AKT及p-STAT3,其P-MAPK的基本表达水平更低,且P-MAPK的表达在sorafenib处理后发生反弹性上升。提示缩短给药间隔时间或联合其他药物治疗可能有助于提高sorafenib的疗效。结论1.采用优化的免疫磁分选法可有效分离纯化肝癌及癌周组织中的血管内皮细胞,并可进行传代培养。2.CD105~+TEC较CD105~+NEC及HUVEC在内皮细胞特异性分子的表达方面有差异。3.CD105~+TEC较CD105~+NEC及HUVEC具有功能差异,表现出更强的生存能力及化疗抵抗性。4.CD105~+TEC较CD105~+NEC及HUVEC对soraafenib具有更强的抵抗作用,其机制与CD105~+TEC表达更高水平的p-AKT及p-STAT3,但表达更低水平的P-MAPK,且P-MAPK的表达在sorafenib处理后发生反弹性上升相关。应用价值1.分选获得纯化的肝癌及癌周血管内皮细胞,并能进行传代培养,可用于肝癌血管生成机制及抗血管生成治疗的相关研究。2.探索了CD105~+TEC对sorafenib更具抵抗能力的机制,为提高sorafenib对肝癌的疗效提供了新线索。创新点1.首次成功传代培养从肝癌组织分选的CD105~+血管内皮细胞。2.首次验证了肿瘤血管内皮细胞对抗血管生成药物sorafenib更具抵抗性,并探索其可能的分子机制。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in Asia and Africa.Despite endeavors have been made to improve its prognosis,the overall survival,however,is still unsatisfied.The high rate of metastasis and recurrence after curative resection was responsible for the poor prognosis of resectable HCC.Anti-angiogenesis is an attractive treatment for HCC,because HCC is a typical hypervascular cancer,its growth,metastasis or even hepatocarcinogenesis depend on angiogenesis.As a major part of tumor vessels,tumor-drived endothelial cells(TEC) are heterogeneous in different organs,tissues and tumors.Alterations in growth factor receptors,cellular survival pathways,surface adhesion molecules,and passage-related changes in EC markers are all consistent with molecular perturbations in TEC,which may contribute to the function distinction between TEC and normal endothelial cells (NEC).Moreover,recent studies indicate that TEC from breast tumor,renal tumor and nasopharyngeal carcinoma,compared with NEC,were less sensitive to chemotherapeutic agents such as vincristine and doxorubicin in vitro.While whether acquired drug resistance to anti-angiogenic therapies might be a feature of the TEC has not yet been reported.The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma1.Isolation and culture TECs or NECs from HCC or adjacent normal liver tissueAs a main target of anti-angiogenesis therapy,TECs represent only a minor fraction of the total cell population within tumor tissues.Along with the fact that endothelial cells are enmeshed in a complex tissue consisting of a variety of cells,analysis of TEC has long been considered a tough task due to the difficulty of isolation and purification.In this study,a modified immunomagnetic methods using magnetic beads conjugated with anti-CD 105 antibody were used to isolate vascular endothelial cells from human HCC or adjacent normal liver tissue.The isolated NEC presented typical“cobblestone”appearance while TEC not.Negative expression of alpha fetoprotein (AFP)and CD68 in isolated ECs excludes the contamination of tumor cells and macrophages.The expression of CD105 and von Willebrand factor(vWF)was positive in more than 99%of isolated cells.Internalization of acetylated low-density lipoprotein(DiI-Ac-LDL)was positive in more than 95%of isolated cells.The isolated cells could form capillary-like tubes on BD Matrigel Matrix.Also,the isolated cells could express a panel of endothelial markers including CD105,CD31, CD34,VE-cadherin(CD144),vascular endothelial growth factor receptor-1 (VEGFR1),VEGFR2 with different ratio.Thus,TEC and NEC can be isolated from HCC and adjacent normal liver tissue by using modified immunomagnetic methods, and these cells can be serially subcultured and used for angiogenesis related researches in HCC.2.Functional characteristics of CD105~+ TECTo compare the function distinction between CD105~+ TEC and CD105~+ NEC or human umbilical vein endothelial cells(HUVEC),a series experiments have been done.Cell proliferation was measured over a period of 72 hrs in full medium. Proliferation of CD105+ TEC was significantly more active than CD105+ NEC and HUVEC when cultured in the serum supplemented medium for 24,48,and 72 hrs(P＜0.05).CD105+ TEC was more resistant to apoptosis in the serum-free medium compared with CD105+ NEC and HUVEC.Wound healing assay revealed increased migration ability of CD105+ TEC and endothelial cell-tumor cell interaction assay revealed increased ability to adhere more HCC cells of CD105+ TEC,with respect to CD105+ NEC and HUVEC.CD105+ TEC also could form capillary-like structures on Matrigel in the absence of serum while HUVEC not.Moreover,CD105+ TEC was presented increased drug resistance to cytotoxic therapy including adriamycin and 5-fluorouracil(5-Fu)than CD105+ NEC and HUVEC.Those data confirmed CD105+ TECs did not undergo cell senescence in serum-free medium and showed enhanced proliferation,motility,pro-angiogenesis properties,and resistance to cytotoxic drug treatment.3.The increased resistance of CD105+ TECs to sorafenib and its mechanism A handful of laboratories have reported successful isolation and subsequent culture of TEC and explored some characteristics of TEC,while whether acquired drug resistance to anti-angiogenic therapies might be a feature of the TEC has not yet been reported.In this study,we have compared the response of CD105+ TEC,CD105+ NEC,and HUVEC to the anti-angiogenic drug sorafenib.Cell proliferation revealed that HUVECs and CD105+ NECs were sensitive to sorafenib in a dose-dependent manner when the concentration was over 2μM,while CD105+ TEC were resistant to sorafenib at the higher dosage of 10μM.The tube formation experiment and EC-spheroid assay showed CD105+ TEC could form capillary-like tubes and produce sprouts at 5μM sorafenib while CD105+ NEC,and HUVEC not.These results demonstrated CD105+ TECs acquire more resistance to anti-angiogenic therapies than normal EC.Then we investigated the expression levels of p-STAT3,STAT3, p-Akt,Akt,p-MAPK and MAPK of different types of EC after sorafenib treatment, and found that the expression of p-STAT3 and p-Akt in CD105+ TECs before and after sorafenib treatment were higher than NECs.We also found that the baseline level of p-MAPK in CD105+ TEC was lower than NEC,and after sorafenib treatment for 24 hrs,in CD105+ TECs p-MAPK expression was significantly higher than untreated cells.These changes may contribute to the resistance of CD105+ TEC to sorafenib,and suggest a shorter dosing interval of sorafenib may of clinical benefit.Conclusions1.The modified immunomagnetic affinity purification can successfully isolate CD105+ endothelial cells from HCC and adjacent normal liver tissue,and these cells can be serially subcultured.2.Differential endothelial markers expression does exist between CD105+ TEC, CD105+ NEC,and HUVEC.3.Differential functional characteristics do exist between CD105+ TEC,CD105+ NEC,and HUVEC.CD105+ TEC acquire more pro-angiogenesis properties and resistance to cytotoxic drug treatment.4.CD105+ TEC acquire more resistance to sorafenib than normal ECs.The underlying mechanism may be related to the differential expression of p-STAT3, p-Akt and P-MAPK between CD105+ TEC and normal EC.The novelties of this work:1.Isolated and serially subcultured endothelial cells from HCC and adjacent normal liver tissue,and these cells can be used for angiogenesis related researches in HCC.2.For the first time,identified tumor endothelial cells acquired drug resistance to anti-angiogenic therapies,and explored the underlying mechanism.更多还原