【作者】 陈伟；

【导师】 吴浩荣；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 肝细胞肝癌(hepatocellular carcinoma, HCC)是世界范围内死亡率最高的恶性肿瘤之一,也是我国常见的恶性肿瘤之一,恶性程度高,发展迅速,治疗困难,总体疗效不理想。肿瘤干细胞(Tumor stem cell, TSC;亦称癌干细胞,cancer stem cell, CSC)的概念是在长期的研究中逐步发展形成的。肿瘤干细胞是指癌瘤组织中少量存在的具有自我更新能力、分化潜能的细胞,该群细胞属于成体干细胞的范畴且自我更新失控。研究认为肿瘤干细胞是肿瘤形成的起始细胞,可能是肿瘤发生、发展、转移、复发的根源。目前研究表明,肿瘤干细胞存在于包括白血病、乳腺癌、脑肿瘤、结直肠癌及胰腺癌等恶性肿瘤中。而对肝细胞肝癌的研究显示肝细胞癌的发生发展过程中可能也有肿瘤干细胞的参与。在不同类型的肿瘤中研究者们发现了不同的肿瘤干细胞表面标记分子。而CD133分子则是其中的重要成员之一。人CD133基因位于4号染色体,基因全长约160Kb,含有27个外显子,开放性读框(ORF)为2598bp,编码865个氨基酸(从),分子量约为120KD,基因的编码产物——CD133抗原为5跨膜糖蛋白。大量研究表明,通过细胞表面标记CD133分子,可有效地识别和鉴定多种肿瘤的肿瘤干细胞,从而对肿瘤干细胞的特性进行分析。并且CD133与肿瘤的转移、肿瘤的血管生成都有关系。另外研究者报道,可通过检测CD133 mRNA水平预测肿瘤的复发,并且认为CD133可以作为某些肿瘤的预后指标。而对肝癌干细胞的研究表明CD133抗原可能是肝癌干细胞的表面标记物。鉴此,本研究以普外科常见病肝癌为研究对象,以肿瘤干细胞表面标记分子CD133为切入点,初步探讨CD133在肝癌临床标本的表达及其临床意义。本研究利用高质量的cDNA基因文库提供的模板,为克隆CD133全长基因提供了可靠的物质基础,采用PCR分段克隆重叠片段,从技术层面解决了此类长基因克隆的瓶颈。进而,本研究将CD133基因克隆入真核表达载体,转染小鼠成纤维细胞L929细胞,获得了基因工程细胞株L-CD133,这为进一步研究CD133的生物学功能提供了物质基础,同时,基因工程细胞株的构建也使CD133抗原胞外段的天然空间结构充分展示,获得了后续的抗人CD133单抗得研制过程关键的筛选抗原。鉴于与CD133相互作用的生物因子尚未被发现,利用特异性抗体刺激细胞膜表面分子是目前能用于激发CD133分子生物学功能的有效手段。然而目前国际上已有的抗CD133单抗中,尚无功能性CD133的报道。因此,本部分研究在克隆人CD133全长基因和建立其基因工程细胞株的基础上,进而研制抗人CD133单克隆抗体,具有重要的理论意义和应用价值。为了进一步深入探讨CD133分子在肿瘤干细胞的的表达和临床意义,获得探索诊断和治疗肿瘤的新手段,本研究以单核细胞性白血病细胞株U937细胞株免疫Balb/c小鼠,L-CD133基因工程细胞株为筛选抗原,采用B淋巴瘤杂交瘤技术,成功地建立了稳定分泌抗人CD133单克隆抗体(686)的杂交瘤细胞株,并分离纯化单克隆抗体和进行了单抗特性的初步鉴定。单抗686的识别谱与商品化抗人CD133单抗AC141类似,该抗体还可用于肠癌组织的免疫组织化学分析,在肠癌组织标本上检测到CD133抗原的表达。进而发现,单抗686可有效抑制CD133+肠癌细胞Caco-2的增殖。继而,本研究用自制鼠抗人CD133单克隆抗体686作为工具,以免疫组织化学为主要方法对CD133抗原在人肝癌组织中的表达情况进行系统的研究,并对其在肝癌组织中的表达同患者的主要临床病理特征之间的关系进行探讨。研究发现：单抗6B6可用于肝癌组织的免疫组织化学分析,可在肝癌组织标本上检测到CD133抗原的表达。且大部分标本阳性细胞比率<1%,一般不超过5%。这说明CD133+肝癌干细胞在整个肝癌组织中所占比例是比较低的。本研究发现CD133抗原的表达同患者的血清AFP水平、肝癌病理分化等级有显著相关性P<0.05。血清AFP水平高、肿瘤分化程度低的病例更多见CD133抗原表达。而CD133抗原表达同患者的年龄、性别、肿瘤的大小没有相关性。综上,我们获得了一株特异性抗人CD133单克隆抗体,在肿瘤临床诊断和免疫治疗中具有潜在应用价值。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide, one of the most common malignant tumors in China as well. It is difficult to cure because of its high-grade malignancy and rapid progress.The theory of tumor stem cell (TSC), or cancer stem cell (CSC) is well established based on numerous data that accumulated in dozens of decades.TSC is a small population within tumors, and belongs to adult stem cells with characteristic of self-renewing, differentiation and malignant proliferation. More and more reports demonstrated that CD133+ cell subpopulation from tumor was identified as tumour initiating cells. In a way, the CD133+ cancer stem cells are the origin of chemoresistance, apoptosis resistance, metastasis and recurrence of various kinds of cancers. TSC was found in many types of tumors, including leukemia, breast cancer, brain cancer, colorectal cancer, pancreatic cancer. It is reported that TSC is involved in the pathologic process of hepatocarcinoma as well.Many kinds of biomarkers of TSC were found on different types of tumors.CD133 is one of the most important biomarkers to identify TSC.CD133 gene is located in chromosome 4. The full length CD133 gene is 160Kb, and it is composed of 27 exons with 1589bp of ORF(open reading frame), edcoding 5-transmembrane glycoprotein with 865 aminoacide residues, and its expected molecule weight is 120KD.Many reports demonstrated that CD133 is a perfect surface marker to identify TSC from many types of tumor. CD133+ cells are associated with tumor metastasis and tumor microvessels formation. Tumor relapse could be detected by quantitative CD133 expression in mRNA level. CD133 is also the surface biomarker of TSC of hepatocarcinoma. Hence, our study is focused on the expression of CD133 of hepatocacinoma and its clinical significance.First, we break through the bottle neck of long gene (>2Kb) cloning by using high quality fetal liver cDNA library as PCR template and cloning 3 overlaped fragments. And then, all the fragments were linked together to get the full length CD 133 gene by PCR.Then, the full lentgh CD 133 gene was subcloned into eukaryotic expression vector pIRES/EGFP. And the mouse fibroblast cell L929 was infected with the pIRES/EGFP/CD133 expression vector. CD 133 antigen expressed on the L929 cell surface and it is critical for preparation of monoclonal antibody against human CD 133.However, the counterpart of CD133 is unknown. As our knowledge, the specific monoclonal antibody may ligate and convey the signal to a cell surface receptor. But there is no report of functional monoclonal antibodies against CD133. In this study, Balb/c mice were immunized with CD133+ monocytic cell line U937. Hybridoma was obtained with B-lymphocyte hybridoma technology. Briefly, splenocytes from immunized mice and myeloma cells SP2/0 were fused and screened with CD133 transfectant L-CD133 and its negative control cells L-mock. And then, the monoclonal antibody against human CD133 (6B6) was purified and its characteristic was identified. Similar to commercial antibody AC141, mAb6B6 could recognize CD133 molecules on different type of cell lines by immunostaining and flow cytometric analysis. MAb 6B6 could detect CD133+ cancer cells in colorectal samples by immunohistochemical staining. And then, we stimulated colorectal cell line Caco-2 that expressed CD133 with mAb 6B6 in vitro. Interestingly, we found that the proliferation of Caco-2cells was obviously inhibited in presence of MAb 6B6.Furthermore, we analyzed CD133 expression on hepatocellular carcinoma samples by immunohistochemical staining and discussed its clinical significance. MAb 6B6 could detect CD133+ cells on hepatocellular carcinoma tissue. The ratios of CD133+ cells were less than 5% and were lower than 1% on most of the samples. It is demonstrated that CD133+ hepatocellular carcinoma TSC is exist but it is a small population. Our data showed that the expression of CD133 is correlated with serum AFP level and pathological differentiation of hepatocellular carcinoma patients.(P<0.05) The higher lever of serum AFP and more poorly differentiation of hepatocellular carcinoma, the higher ratio of CD133+ cells. And there is no correlation of age, sex and tumor volumn with CD133+ TSC ratio.In conclusion, we have obtained a specific anti-human CD133 MAb with the potential for research and clinical applications in tumor diagnosis and immunotherapy.更多还原