【作者】 熊汉真；

【导师】 李学农；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 研究背景和目的结直肠癌(colorectal carcinoma，CRC)是我国常见的消化系统恶性肿瘤之一，而转移是患者的主要致死原因。肿瘤转移是一个多步骤、多阶段的复杂过程，转移的肿瘤细胞经历了细胞骨架变化、黏附特性的改变、运动能力的增强及蛋白水解酶表达增加等一系列改变。动物实验最新的研究表明，造血祖细胞与肿瘤转移有着密切联系，KaplanRN等利用β半乳糖苷酶标记骨髓细胞移植的小鼠Lewis肺癌模型、小鼠B16黑色素瘤模型和c-myc转基因小鼠自发性淋巴瘤模型发现：VEGFR-1阳性造血祖细胞(vascular endothelial growth factor receptor-1 hemopoietic progenitor cells，VEGFR-1~+HPC)首先在特定器官形成VEGFR-1~+HPC细胞簇(VEGFR-1~+HPCcellular clusters)，为瘤细胞准备了易于形成转移的微环境，这些细胞簇形成部位与肿瘤转移常见部位一致。研究表明，VEGFR-1~+HPC充当了肿瘤转移必不可少的细胞书签(cellular bookmarking)或者说特使细胞(envoy cells)。VEGFR-1~+HPC并非以前发现的血管内皮祖细胞(endothelial progenitor cells，EPC)即VEGFR-2阳性造血祖细胞，腹腔注射VEGFR-2抗体，不能阻断VEGFR-1~+HPC细胞簇形成及瘤转移灶形成，只能限制肿瘤转移灶生长。同时利用VEGFR-1抗体阻断或剔除VEGFR-1~+HPC，则能够抑制预迁移器官造血祖细胞簇的形成与肿瘤转移。但造血祖细胞在人体肿瘤的主要作用及机制仍不十分清楚，且目前尚未在人类肿瘤模型上得到证实，由于脐血来源广泛，获取较容易，成为骨髓及外周造血干祖细胞的极佳替代来源，我们选定CD133~+造血祖细胞，在减少其诱导分化的同时，观察其对低转移人大肠癌细胞体内外的增殖和侵袭作用。CD133是一个目前公认的造血祖细胞表面抗原，在正常及异常造血和非造血组织中均有表达，随着细胞的分化成熟其表达减弱、消失。在正常造血组织中，如人胚胎、肝脏和骨髓、脐血、成人骨髓和外周血CD34~+细胞中都可检测到CD133 mRNA，而在其他成熟血细胞均未检测到其表达。同时随着最新的研究表明，CD133 mRNA在外周血中的表达里，实体恶性肿瘤转移患者的表达水平明显高于非转移患者，且以骨转移患者的CD133 mRNA表达为著。但这一结果的作用机制主要是由于CD133~+造血祖细胞或者是CD133~+肿瘤干细胞的结果尚不清楚。本研究重点探讨CD133~+造血祖细胞对结直肠癌增殖、侵袭及转移的影响，阐明CD133~+造血祖细胞在结直肠癌转移中的主要生物学功能，为揭示结直肠癌转移机制及抗转移治疗奠定基础。方法1．CD133~+造血祖细胞的筛选、鉴定及培养由南方医院妇产科提供新鲜脐血标本20例，在脐血采集后6h内，利用CD133~+免疫磁珠，分离出CD133~+造血祖细胞，并分别予流式细胞仪及免疫细胞染色分析和鉴定样本量中CD133~+细胞含量，同时在减少诱导CD133~+分化的同时，培养CD133~+造血祖细胞。2．CD133~+造血祖细胞对结直肠癌细胞生物学特性的影响将CD133~+造血祖细胞与SW480共趋化培养，用MTT法检测肿瘤细胞的增殖及黏附能力的影响，用Transwell小室法检测肿瘤细胞的迁移能力的变化，用Westem blot了解VEGFR-1、MMP-2、E-Cadherin的表达情况。观察CD133~+造血祖细胞作用前后细胞增殖、体外侵袭变化。3．应用整体可视化结肠癌转移动物模型观察共移植效应利用稳定表达绿色荧光蛋白(enhanced green fluorescent protein，EGFP)的结肠癌细胞株SW480／EGFP~+；通过结肠癌尾静脉注射的方法建立结肠癌转移模型，利用肿瘤细胞表达EGFP的特点，将CD133~+细胞与SW480／EGFP~+共培养通过尾静脉注射的方法观察肿瘤细胞成瘤能力及体内转移能力的变化，应用组织病理学及分子生物学技术对结肠癌转移动物模型进行评价和鉴定。结果1、CD133~+造血祖细胞的筛选、鉴定及培养由南方医院妇产科提供，脐血标本共20份(其中18份成功提取新鲜细胞，2份失败)，50～80 ml／份，枸橼酸抗凝，经产妇和家属同意取自足月健康顺产新生儿，新鲜脐血采集后6 h内，分离出CD133~+造血祖细胞。从新鲜的脐血中得到的单个核细胞数平均为(1．48±0．19)×10~7／ml，经CD133磁珠分离系统分离得CD133~+富集细胞的平均数为(1．33±0．01)×10~5／ml，新鲜脐血单个核细胞中的CD133~+细胞所占比例为(0．8±0．01)％，经流式细胞仪鉴定CD133~+CD34~+细胞纯度达到80％以上。CD133~+细胞培养一周后，免疫细胞化学染色CD133，显微镜下观察CD133阳性细胞数>90％。2、CD133~+造血祖细胞对结直肠癌细胞生物学特性的影响CD133~+造血祖细胞能显著促进SW480细胞的生长(n=60、Z=-6．106、P=0．000)，能促进肿瘤细胞的形态学改变，即肿瘤细胞的核深染，异型明显，细胞呈多角形或不规则形，能显著促进SW480细胞的迁移能力；细胞黏附实验表明，含有CD133~+造血祖细胞的实验组其肿瘤细胞的黏附能力明显高于对照组(n=60、Z=-3．679，P=0．000)。利用丙酮沉淀法提取细胞总蛋白(即浓缩蛋白)检测MMP-2、VEGFR-1及E-Cadherin蛋白表达情况发现实验组中的3种蛋白表达水平均高于对照组。3、利用整体可视化结肠癌转移裸鼠模型观察共移植效应转染后的结肠癌细胞SW480／EGFP~+稳定、高效的表达绿色荧光蛋白，采用尾静脉注射的方法建立结肠癌可视化转移动物模型，注射2周后，实验组可见肿瘤转移，对照组未见有转移情况，注射4周后，实验组67％发生转移，对照组仅有一只发生转移，注射6周后，实验组中成瘤转移率为4／6，对照组中的成瘤转移率为1／6。经过生存分析的统计结果表明，实验组的转移率高于对照组(n=6，Breslow检验，P=0．045)。通过分子生物学技术，发现实验组中MMP-2、VEGFR-1蛋白高表达，而E-Cadherin的表达则逐渐减低。结论1、CD133~+造血祖细胞可显著促进人结直肠癌细胞的增殖及侵袭能力，CD133~+造血祖细胞与人结直肠癌细胞裸鼠共移植具有促转移作用，初步证明，人造血祖细胞可能对人结直肠癌细胞转移形成具有重要影响。2、CD133~+造血祖细胞可与结直肠癌细胞直接作用，能促进转移相关基因和血管生成因子MMP-2、VEGFR-1蛋白高表达，同时调节E-Cadherin的蛋白表达，初步表明：CD133~+造血祖细胞的促转移作用可能与转移相关因子MMP-2、VEGFR-1及E-Cadherin的表达有关。本研究的创新之处1、将人造血祖细胞与结直肠癌细胞株共趋化培养，直接在人癌实验层次揭示了造血祖细胞对结直肠癌细胞的作用。2、将人造血祖细胞与结直肠癌细胞株共趋化培养后进行人类肿瘤动物模型共移植实验研究。初步证明，CD133~+人造血祖细胞可显著促进人结直肠癌细胞的增殖及侵袭能力，CD133~+造血祖细胞与人结直肠癌细胞裸鼠共移植具有促转移作用，人造血祖细胞可能对人结直肠癌细胞转移形成具有重要影响。更多还原

【Abstract】 BACKGROUND & OBJECTIVEColorectal cancer （CRC） is the second worldwide leading cause of cancer death in the world. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, travelling through the bloodstream, and stopping at a distant site. At the new site: the cells establish a blood supply and can grow a life-threatening mass.Kaplan et al demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 （VEGFR-1; also known as Flt-1） home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR-1 function using antibodies or by the removal of VEGFR-1⁺ cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 （inhibitor of differentiation 3）-competent VEGFR-1⁺ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. They set up an ingenious experiment to track the movements of various cell populations as tumour cells metastasized in the lungs of live mice. The mice were irradiated to kill off all their bone-marrow cells, which were then replaced by bone-marrow cells tagged with green fluorescent protein; this made the cells easy to find under a microscope. Once the new bone-marrow cells were established, the mice were injected in the skin with lung carcinoma or melanoma cells, each marked with red fluorescent protein. The tumour cells were expected to form a primary tumour in the skin, and then to metastasize to the lungs. But the green bone-marrow-derived cells appeared in the lungs on days 12-14 after injection of the red cells-well before any of the tumour cells had arrived in the lung. The red tumour cells turned up only on day 18 post-injection, and by day 23 micrometastases had formed, with more than 95% of the tumour cells being found in exactly the same sites as the bone-marrow-derived cells. Mice were injected intraperitoneally with anti-VEGFR-1 antibody, it eliminated the initiating clusters and completely prevented metastasis, whereas anti-VEGFR-2 antibody did not prevent the formation of VEGFR-1⁺ clusters but limited metastatic progression. The hematopietic stem cells can now be obtained from peripheral or umbilic cord blood, as well as from bone marrow. The umbilic cord blood has more advantages than the bone marrow, with the sufficient donors and without the side effects, such as graff-versus-host disease（GVHD）.CD133 is a 120-kDa five-transmembrane-domain glycoprotein expressed on normal primitive haematopoietic, endothelial, neural and epithelial cells. Expression of CD133 mRNA is significantly increased in peripheral blood mononuclear cells in solid cancers regardless of tumour type, particularly in patients with bone metastasis; overall survival was higher in patients with low or undetectable CD133 mRNA expression, although it was not ascertained whether the mononuclear cell fraction also contained CD133⁺ tumour cells as well as CD133⁺ haematopoietic progenitor cells. In this study, we aim to clarify the possible role of CD133⁺ haematopoietic progenitor cells from cord blood in the proliferation, invasion and metastasis of CRC line SW480. It will be helpful to understand the molecular basis of CRC, and establish a new target for early metastatic diagnostic markers and novel therapeutic strategies.METHODS1、The identification and cultivation of CD133⁺ haematopoietic progenitor cells from cord bloodAll human umbilical CB samples were obtained from the Obstetrics and Gynecology Department of Southern Hospital after informed consent. CD133⁺ haematopoietic progenitor cells were isolated from human umbilical CB samples （total of 20 samples, 2 lost）. Each pooled mononuclear cells obtained from CB samples after Ficoll density gradient centrifugation separation. CD133⁺ haemato -poietic progenitor cells were positively isolated using the mini-MACS immunomagnetic separation system, according to manufacturer’s instructions. After CD133⁺ haematopoietic progenitor cells isolation, the purity of the isolated cell populations was determined by fluorescein-activated cell sorting with CD34-R-phycoerythrin-conjugated mouse anti-human monoclonal antibody, CD133-phycoerythrin anti-human monoclonal antibody （Miltenyi Biotec） and immunocytochemistry.2、The effects of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480The treatment group is the co-culture of CD133⁺ haematopoietic progenitor cells and SW480, the control group used the same culture medium without CD133⁺ cells. The effect of CD133⁺ haematopoietic progenitor cells on proliferation in SW480 cell was measured by MTT assay, the migration abilities of SW480 cell were assayed in Transwell cell culture, Cell adhesion assay was carried out in a 96 microplate well precoated with fibronection. The expression of VEGFR-1、MMP-2、E-Cadherin were analyzed by western blot.3、Using whole-body visualizing orthotopic animal model in colon cancer to demonstrate the effection of CD133⁺ haematopoietic progenitor cells in vivoThe metastatic ability evaluated by whole-body visualizing orthotopic animal model. The effection of CD133⁺ haematopoietic progenitor cells on in vivo tumor growth was assessed by tail vein injection of SW480/EGFP⁺/CD133⁺ and SW480 /EGFP⁺ cells .RESULTSThe main results and findings are as follows:1、The identification and cultivation of CD133⁺ haematopoietic progenitor cells from cord bloodIsolated CD133⁺ haematopoietic progenitor cells （purity over 80% as determined by flow cytometry） were characterized by flow cytometry and immunofluorescence staining for hematopoietic stem cells markers （CD133, CD34） and then cultured in undifferentiation conditions.2、The effects of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480 in vitroCD133⁺ haematopoietic progenitor cells could significantly improve the growth ability of SW480 cell, not only the ability of proliferation（n=60、Z=-6.106、P=0.000）, but also the morphology. The morphology of the cells was remarkably changed. Irregular nucleus, double nucleus and polymorphic nucleus appeared in the treatment group, and the cells shaped as polygon-like and leptosomatic. In the cell adhesion assay, OD₄₉₀ of the treatment group was 0.11±0.01, the control group was 0.05±0.01, （n=60、Z=-3.679、P=0.000） . The expression of VEGFR-1、MMP-2、E-Cadherin protein could be up-regulated in vitro .3、The effection of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480/EGFP⁺ in vivoSW480/EGFP⁺ cells stably expressed high-levels of enhanced green fluorescent protein （EGFP） and EGFP had no effect on cell proliferation. Visualizing orthotopic animal model developed with tail vein injection was built with metastasis rate. After injection, animals had metastasis within 2 weeks in the treatment group, and subsequently, 4 weeks after injection, 67% of animal had metastasis in the treatment group. In control group ,only one animasl had metastasis （n=6, Breslow test, P=0.045） .The whole-body visualizing orthotopic animal model was successfully validated by pathological detection. The expression of VEGFR-1, MMP-2 protein could be up-regulated in vivo, but E-cadherin protein could be down-regulated.CONCLUSION1. CD133⁺ haematopoietic progenitor cells are effective in increasing tumor cell proliferation and invasion. Hematopoietic progenitor cells may play an important role in the invasion and mesatasis of colorectal neoplasms.2. CD133⁺ haematopoietic progenitor cells may involve in direct action to mediate proliferation, invasion and metastasis of CRC. The expression of VEGFR-1、MMP-2 protein could be up-regulated in co-transplantation and two-ways regulation the expression of E-cadherin protein. These data will be helpful to elucidate the molecular mechanism of CD133⁺ haematopoietic progenitor cells and provide new clues.更多还原