【作者】 张建伟；

【导师】 赵平； 赵晓航； 王成锋；

【作者基本信息】 中国协和医科大学 ， 肿瘤学， 2010， 博士

【摘要】 胰腺癌是恶性程度高,临床治疗难度最大的肿瘤之一,肿瘤治疗后出现复发和转移是目前胰腺癌治疗失败的主要原因。虽然胰腺癌的治疗已经取得很大的进展,但是预后改善并不明显,即便获得根治性手术的患者5年生存率仅20%左右。对恶性肿瘤发生远处转移和复发这些临床表现的研究认为其是由外周血内存在的循环肿瘤细胞(Circulating tumor cells, CTCs)所致。进入血液内的大部分肿瘤细胞难以生存,但小部分细胞能够继续存在于循环系统之内,并聚集成细胞簇。这部分细胞再次侵出血管,在远隔部位形成肿瘤转移灶。CTCs的分离和鉴定技术不断发展,目前已经能够高效、准确的分离和鉴定。而且CTCs在肿瘤基础研究、临床疗效评价和判断预后等方面具有重要意义。在胰腺癌开展CTCs的研究将有助于评价手术及化疗的治疗效果、早期发现远处转移和判断预后。肿瘤干细胞(Cancer stem cells, CSCs)是肿瘤内一小部分特殊细胞,对肿瘤的生长、转移起到关键作用。在多种实体瘤中均证实有肿瘤干细胞存在,包括乳腺癌、肝癌、前列腺癌、肺癌、黑色素瘤和结肠癌等。肿瘤干细胞具有在组织内寿命长,可以累积突变、能够自我更新等特点。虽然不是所有恶性肿瘤的生物学特点都符合肿瘤干细胞理论,但CSCs假说为肿瘤的复发、转移提供了细胞水平的理论基础。研究发现在胰腺癌组织内存在CD24+CD44+ESA+的肿瘤干细胞亚群,数量不超过细胞总数的1%,但其成瘤特性是其它肿瘤细胞的100倍。胰腺癌肿瘤干细胞也表达其它多种肿瘤干细胞标记物,包括CD133和AFP等。本实验通过对83例胰腺癌患者的外周血应用免疫磁珠负性富集和免疫荧光鉴定方法,即使用CD45抗体偶联的免疫磁珠去除白细胞,CK8／18／19抗体免疫荧光结合细胞核DAPI鉴定的方法确定CTCs。实验中获得治疗前及治疗后外周血标本共124份,分离并确定CTCs1807个(0-208个)。对临床资料分析后认为CTCs同患者的诊断、治疗和预后均有密切关系。CTCs和CSCs均是肿瘤复发和转移的相关因素,因此实验第三部分开展了两者相关性的研究。结果显示在胰腺癌细胞系、组织学标本和CTCs中均发现了胰腺癌肿瘤干细胞CD24、CD44、ESA和AFP等相关标记物的表达,为进一步研究奠定了基础。更多还原

【Abstract】 Pancreatic cancer is a devastating malignancy and a leading trouble to the clinical doctor. The most important reason of death is metastasis and recurrence. Although the prognosis of not only advanced and metastatic pancreatic cancer but also even that of resectable pancreatic cancer is poor, its 5 years survival less than 20%. A better understanding of tumor progression and recurrence has led to the hypothesis that distant metastasis steered by a specific subset of ’tumor-driving cells’, generically named circulating tumor cells (CTCs). Circulating tumor cells are rare malignant cells found in the peripheral blood that originate from the primary tumor or metastatic sites. CTCs in the circulating system stimulate immune response, and most of tumor cells had been killed. However a small part of tumor cells escaped and gathered into a cell cluster. These part of tumor cells contribute to the metastasis. Therefore CTCs had been the basis of tumor metastasis. New techniques have been developed to isolate and characterize those cells. CTCs enumeration has been incorporated into different fields of oncology as a prognostic marker, a tool to monitor therapy response, and a method to understand basic tumor characteristics. The study on CTCs of pancreatic cancer has important clinical significance.Cancer stem cells constitute a distinct subpopulation in the tumor and are considered to drive both tumorigenesis and metastasis. Those cells are thought to be highly resistant to standard treatment modalities. Recent progress in stem cell biology and technologies has successfully achieved the identification of CSCs in a variety of cancers, such as breast cancer, colorectal cancer and many malignant tumors. The cancer stem cells have self-renewal and multipotent capacity to generate progeny of various differentiation states that constitute the bulk of the tumor. Although not all types of cancers follow the CSC theory, it provides a possible cellular mechanism to account for the metastasis and recurrence of the tumor The expression of cell-surface markers can be used to identify and isolate cancer stem cells. These markers can serve as either positive or negative criteria for a stem cell phenotype. Pancreatic cancer stem cells’phenotype have been identified as CD44+CD24+ESA+. CD133 and AFP also had been reported as CSCs markers and more markers will be found in new researches.83 patients diagnosed as pancreatic cancer had been researched for CTCs by immunomagetic negative enrichment together with immunofluorescence. Leukocytes were removed by immunomagnetic beads coupled with CD45 antibody. The identification of CTCs can be made only both cytokeratin 8,18,19-phycoerythrin staining and DAPI staining positive.1807 (0-208) circulating tumor cells were identied from 124 blood sample collected from all patients before and/or after theropy. The relationship of CTCs and CSCs had been studied in cell line, tissue specimen and CTCs with stem cell markers. Most of markers,such as CD24, CD44, ESA and AFP were positive in CTCs, therfore, Cancer stem cell phenotype had been found in CTCs. Next reaserch may be carried out on these results.更多还原