【作者】 张新；

【导师】 杨广顺； 王欣；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma)是在中国位于第三位,全世界位于第六位的常见恶性肿瘤。2000年全球肝细胞癌发病数为56.4万人,其中约55%在中国。由于肝细胞癌恶性程度高,术后易复发,对放疗化疗等传统手段不敏感,因此其总体疗效仍不理想。近年来肿瘤干细胞理论的提出使人们对肿瘤的生物学行为有了新的认识,该理论认为,肿瘤组织中存在一小群具有自我更新和多向分化能力的肿瘤干细胞,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。迄今,肿瘤干细胞理论已经在包括白血病、乳腺癌、脑肿瘤、结直肠癌及胰腺癌等许多系统的恶性肿瘤中取得实验方面的支持,在这些实验中,细胞膜表面糖蛋白CD133经常被用作筛选肿瘤干细胞的表面标记物。肝癌干细胞领域的研究也表明,在肝细胞癌的发生发展过程中可能也有肿瘤干细胞的参与,这些实验结果显示,CD133阳性肝癌细胞具有更强的克隆形成能力、更高的增殖活性及体内成瘤性,表明CD133抗原可能是肝癌干细胞的表面标记物。更加深入地了解CD133抗原在肝细胞癌中的表达情况将有助于我们认识该抗原在肝细胞癌生物学行为中的作用,并为将来基于CD133阳性肝癌细胞的治疗研究提供实验参考。因为现有肝癌干细胞研究主要是以肝癌细胞系为研究对象进行的,这些细胞系在体外长期培养过程中因其培养条件的不同,其自身特性可能会发生一些未知的改变,从这些细胞系中分离得到的CD133阳性肝癌细胞及基于此所得出的结论也就可能与体内的真实情况不相吻合,也无法明确说明CD133抗原是否为肝癌干细胞的表面标记。而CD133抗原在人肝细胞癌组织中的表达情况及其临床意义,也少见报道。本研究以免疫组织化学为主要手段,系统地对CD133抗原在人正常肝脏、肝炎后肝硬化组织、肝细胞癌及癌旁肝组织、肝细胞癌门静脉癌栓、肝细胞癌肺转移瘤、肝癌细胞系移植瘤、FAH-/-小鼠诱发肝细胞癌标本中的表达情况及CD133抗原同AFP抗原及Ki-67抗原的共表达情况进行了研究,对CD133抗原表达同肝细胞癌患者主要临床病理特征的关系进行分析。我们的研究结果表明:1、人肝细胞癌组织中CD133抗原的表达同患者血清AFP水平、有无肝硬化、肿瘤分级及肝细胞癌组织中AFP抗原及Ki-67抗原的表达有显著相关性,CD133抗原多表达于血清AFP水平高、合并肝硬化、肿瘤分级差及高表达AFP抗原及Ki-67抗原的患者肝细胞癌组织内,其表达可以作为肝细胞癌诊断分级的辅助指标;2、人肝细胞癌门静脉癌栓中CD133抗原表达同原发灶无明显不同,人肝细胞癌肺转移瘤内未见CD133+肝癌细胞,显示人肝细胞癌肺转移瘤的生长可能同CD133+肝癌细胞无关;3、人及小鼠的正常肝组织汇管区胆管上皮细胞、肝炎后肝硬化组织汇管区胆管上皮细胞、肝细胞癌组织内增生的小胆管上皮细胞管腔侧细胞膜均可见普遍的CD133抗原的表达,但其表达与肝癌细胞CD133抗原的表达情况不同,其在肝癌细胞及胆管细胞内的不同表达模式可能代表两种细胞各自不同的分化或功能状态;4、在共表达CD133抗原及Ki-67抗原的肝细胞癌中,CD133+肝癌细胞多出现于高表达Ki-67抗原的区域,但多数CD133+肝癌细胞未见Ki-67抗原表达,表明CD133+细胞可能在短暂进入细胞周期,进行不对称分裂后又返回到G0期,其增殖在平时并不活跃,显示CD133+细胞具有部分肿瘤干细胞样特性。我们的研究结果显示,虽然CD133阳性肝癌细胞具有部分肿瘤干细胞样特性,并同肝细胞癌的临床病理特征间具有相关性,但其在肿瘤转移中的作用不明确,并且在胆管结构中也广泛表达。这些结果为进一步明确CD133在肝癌生物学行为中所起的作用及其同肝癌干细胞的关系提供了实验依据。更多还原

【Abstract】 Hepatocellular carcinoma is the third common in China, the sixth common maliganancies worldwide. Its global incidence was 564,000 in 2000, 55% of which was in China. Due to its high maliganancy, high incidence of recurrence after surgeries and resistance to traditional therapies, the overall surival is still unsatisfactory.Recently, the cancer stem cells theory gave us a new insight into the tumor biological behavior. According to this theory, only a small amount of tumor cells should be responsible for the initiation, progression, metastasis and recurrence of tumors, and those cells are all characteristics of self-renewal ability and multiple differentiation potential. To date, this theory has gained supports from researches in various maliganancies, include leukemia, brain tumors, breast carcinoma, colorectal carcinoma and pancreatic carcinoma. A frequently used sorting markerin these experiments is the cell surface glucosylated protein CD133. Research on CSCs of hepatocellular carcinoma also revealed that CD133-positive cells possess a greater colony-forming efficiency, higher proliferative output, and a greater ability to form tumor in vivo, all of which indicates that CD133 is a potential surface marker of HCC CSCs. Deeper understanding of its expression in HCC may help us better understand the biological behavior of HCC and provides some insight into the future anti-tumor drug research targeted at CD133-postive cells.At presence, most research on HCC CSCs are based on HCC cell lines, the characteristics of those HCC cell lines might change because of long time culture in vitro. So, it is not known to what extent can those conclusions match the real condition in human. And its little known about its expression in human HCC tissues and its correlations with clinicopathological factors.By using immunohistochemistry, we systematically investigated the expression of CD133 in normal human liver, human cirrhotic liver, HCC and its adjacent non-tumor liver tissue, portal thrombosis, metastatic HCC in lung, xenograft of HCC cell lines in NOD/SCID mice, FAH-/--induced HCC. Also, we detected co-expression of CD133 with AFP and Ki-67 antigen, and we analyzed the correlations between CD133 expression in HCC and the major clinocopathological factors.Our results shows: 1. The expression of CD133 is significantly associated with serum AFP level, the existence of cirrhosis, tumor grade, AFP and Ki-67 expressions, higher expression of CD133 can be found in high tumor grade HCC tissues expressing AFP and Ki-67 from patients with higher serum AFP level, complicating cirrhosis; 2. There is no obvious difference between the expression of CD133 in portal thrombosis and its origin, and we didn’t find the expression of CD133 in metastatic HCC in lung that we tested, which might indicates that the sustain of its growth in lung have no association with CD133; 3. We found there are universal apical staining of CD133 in bile ducts of human and mouse liver triad area, human cirrhotic liver triad area and in hyperplastic bile ducts in HCC stroma and its adjacent non-tumor liver tissues, but this pattern of its expression is different from what in HCC, indicating that different expression patterns presents different states of differentiation or function in these two types of cells; 4. In those HCC specimens expressing both CD133 and Ki-67, CD133 was highly expressed in the areas where Ki-67 was also highly expressed, but the co-expression of these two antigens showed that only a small amount of CD133-positive HCC cells expressed Ki-67 simultaneously, which indicates CD133-positive HCC cells went into cell cycle temporarily and went back to G0 soon after asymmetric cell division, and remains quiescent until next division.Our results shows, although CD133+ HCC cells have some CSCs like properties, and is correlated with clinicopathological factors, their role in tumor metastasis remains unclear, and its expression can be found in bile ductal structures universally. Our resluts gave further evidence towards the role of CD133 in the biological behavior of HCC and its relationship with the genuine hepatocarcinoma stem cells.更多还原