【作者】 左国华；

【导师】 梁平；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 目的:肝细胞癌(hepatocellular carcinoma, HCC)是临床上最常见的恶性肿瘤之一,以其高发病率和高度恶性行为著称,占我国癌症死亡率的第二位,肿瘤切除或肝移植是治疗该病的最有效手段,但术后复发转移率高严重影响了治疗的效果。部分肝癌病人在肝癌切除或肝移植前,已有肿瘤细胞脱落进入血循环系统中,入血的循环肿瘤细胞(circulating tumor cell, CTC)是导致肝癌术后复发和转移的首要条件。因此,检测循环肿瘤细胞对判断肝癌的转移复发、指导临床治疗具有重要意义。大多数学者在肝癌患者外周血中检测AFP mRNA,作为判断循环肿瘤细胞的指标,但部分肝炎或肝硬化的患者检测结果提示该方法假阳性率较高,其特异性令人难以满意,且单标志物检测容易出现假阴性。研究显示端粒酶大多数(85-100%)的肿瘤细胞中表达,多数正常体细胞不表达,而端粒酶蛋白催化活性亚单位(human telomerase reverse transcriptase, hTERT)是合成端粒酶的限速亚基,对端粒酶活性调控起着决定性的作用。hTERT表达在AFP阴性的肝癌中也有很高的发生率,hTERT表达阳性诊断肝癌的敏感性为73.9%,特异性为100%,显示hTERT的基因表达是比端粒酶更敏感的诊断指标。因此我们推测hTERT mRNA做为循环肿瘤细胞检测的标志物,可避免合并肝炎或肝硬化的肝癌患者中假阳性的干扰,应具有良好的癌特异性。免疫磁珠(Immunomagnetic bead,IMB)技术是近年来发展起来的一项新的免疫学技术,免疫磁珠细胞分离是应用最主要的一个方面,具有简便易行、分离纯度高、特异性高、同时保留细胞活性的优点。与常规检测技术(如免疫细胞化学、RT-PCR、流式细胞仪等)相结合,能在106-7个单核细胞中分离检测出一个肿瘤细胞,可弥补这些技术的不足,并改善这些技术对循环肿瘤细胞检测的敏感性及特异性。因此,本研究以肝细胞癌为研究对象,在构建肝细胞癌免疫磁珠的基础上,联合免疫细胞荧光技术进行循环肿瘤细胞形态学分析,以及巢式RT-PCR技术联合检测AFP mRNA和hTERT mRNA,并尝试对富集分离出来的循环肿瘤细胞进行培养,为检测及防治肝癌的血液转移提供理论依据和方法。方法:(1)利用碳二亚胺偶联法,在磁珠上包被单抗HAb18,制备肝癌免疫磁珠后,将微量肝癌HepG2细胞与外周血单个核细胞悬液混匀,再通过免疫磁性细胞分离,行免疫细胞化学鉴定,检测免疫磁珠的特异性和敏感性,并计算癌细胞的回收率。(2)采集肝癌患者56例、肝炎和肝硬化患者19例、继发性肝癌患者11例血样,及18个健康志愿者的血样作为阴性对照。用Ficoll密度梯度离心与免疫磁珠技术首先对患者的外周血进行癌细胞的富集,然后运用FITC-CK标记富集癌细胞,免疫细胞荧光技术对循环肿瘤细胞行形态学分析,并用巢式RT-PCR技术联合检测AFP mRNA和hTERT mRNA。(3)对8例富集分离出来的循环肿瘤细胞进行培养,通过测定生长曲线和细胞倍增时间,锚着独立性试验了解克隆集落能力,扫描电镜观察细胞表面超微结构,以及裸鼠移植及成瘤观察等方法研究其细胞生物学特性。结果:(1)包被HAb18的免疫磁珠能与HepG2细胞敏感而特异地结合,当单个核细胞与HepG2细胞比为1×106:1时可检测到癌细胞,结合免疫细胞化学方法可检出外周血单个核细胞中57.2%的微量癌细胞,无假阳性。(2)在用肝癌HepG2细胞所做的2组预实验中,免疫磁珠联用增强了巢式RT-PCR的灵敏度、特异性,其灵敏度为5ml血中含有一个癌细胞。(3)在临床样品的实验中,免疫磁珠富集后,结合免疫细胞荧光方法观察肝癌患者外周血中循环肿瘤细胞,检出阳性率为42.9%(24/56),检出细胞的形态可分为四类:中等细胞型、大细胞型、有核细胞碎片和无核细胞碎片。(4)肝癌患者外周血中AFP mRNA及hTERT mRNA的阳性检出率分别为55.4%、48.2%。AFPmRNA及hTERT mRNA的检出率和TNM分期、远处转移临床参数密切相关。当肝癌直径大于5cm、多结节、伴门静脉癌栓,AFPmRNA及hTERT mRNA的检出率均明显提高(P<0.05)。血清AFP水平、肝癌分化程度等与AFPmRNA及hTERT mRNA的检出率无显著相关(P>0.05)。67.9%的肝癌患者至少表达一种标志物,35.7%的患者两种标志物均阳性。AFP mRNA和hTERT mRNA的检出呈正相关,相关系数为0.256。hTERT mRNA在AFP mRNA阳性和阴性的肝癌患者检出率分别为64.5%、28%(P<0.05)。AFP mRNA在肝炎和肝硬化患者中检出率为21.1%( HCC vs肝炎和肝硬化,P<0.05),继发性肝癌患者及健康成人均未检出;hTERT mRNA在继发性肝癌患者中检出率为63.6%,而肝炎和肝硬化及健康成人中均未检出。(5) 8例标本中,其中7例培养未成功,仅有1例出现肿瘤细胞增生,异型性明显,命名为HCC-27,共传代培养5代。免疫细胞化学染色证实为肝癌细胞,HCC-27细胞的增殖及独立锚着生长能力显著低于HepG2细胞(P<0.05)。两只裸鼠经脾内接种培养肿瘤的细胞,其中一只裸鼠出现肝内移植瘤生长。结论:(1)成功利用碳二亚胺耦联法制备了一种抗人肝细胞癌免疫磁性微珠,制备的肝癌免疫磁珠能从外周血单个核细胞中分离检测微量肝癌细胞,有较好特异性和敏感性。(2)通过免疫磁珠细胞分选技术富集,结合免疫细胞荧光方法可对肝癌患者外周血中的循环肿瘤细胞进行形态学检测。循环肿瘤细胞形态分为四类:中等细胞型、大细胞型、有核细胞碎片和无核细胞碎片。(3)肝癌患者外周血单核细胞经免疫磁珠富集后,联合检测肝细胞特异性AFP mRNA和癌特异性hTERT mRNA,有助于提高患者外周血循环肿瘤细胞检测的敏感性和特异性。(4)肝癌外周血中的循环肿瘤细胞经免疫磁珠细胞富集分离后培养,1例培养成活,这是肝癌血行播散的直接证据。培养成活的细胞具有肝细胞癌的组织学特征,其生长增殖及锚着独立生长能力低,但具有转移潜能。更多还原

【Abstract】 Objectives: Hepatocellular carcinoma (HCC) is the most common malignant tumor in our country with the second death rate on the cancer death list, which has a very aggressive clinical course. Surgical resection and liver transplantation are the main methods of the treatment for HCC patients, but the long-term survive rate was poor. It was reported that cancer cell disseminating from the focus into the blood circulation alreadly before or during surgery resection and liver transplantation, these circulating tumor cells (CTC) might be an important reason of its high recurrence and metastasis rate. The CTC detection of the peripheral blood is important for the judgement of the recurrence and the direction of clinic therapy in HCC patients.To date, CTC in HCC cases were evaluated mainly by detecting alpha- fetoprotein (AFP) mRNA used PT-PCR in peripheral blood that could provide useful information. However, it is controversial whether the detection of the gene transcripts truly reflects the presence of tumor cells, since AFP mRNA is not only specifically detected in HCC cases but also it was significantly found in liver innocuousness disease cases including hepatitis or liver cirrhosis. Therefore, single marker assay may lead to false negative or false positive results. Recently, investigations revealed that telomerase is reactivated in approximately 85-100% of various types of malignant tumors but that it is inactive in most nonneoplastic somatic cells. Human telomerase reverse transcriptase (hTERT) is a catalytic component of reverse transcriptase, and hTERT expression is rate limiting for telomerase, its expression significantly correlated with telomerase activity. The expression rate of hepatocarcinoma is remarkably increasing too, even in the AFP negative HCC tissue, since the sensitivity is 73.9% and the specificity is 100% in HCC, these indicates that hTERT expression is one of more sensitive diagnosis agent than telomerase in HCC. To avoided false positive in hepatitis or liver cirrhosis cases, it is then possible that hTERT mRNA would be a new molecular detection marker gene of hepatocarcinoma. Immunomagnetic bead (IMB) is a new immunologic technique developed recently decade, immmunomagnetic cell seperation is one of main application area, and it has possessed the advantages including easy performance, enhanced separating concentration, higly specificity and maintained cytoactive. Combination with immunocytochemical methods, reverse transcriptase-polymerase chain reaction and flow cytometry etc, IMB could enrich and detect one tumor cell in 106 to 107 peripheral mononuclear blood cells, and could improve the sensitivity and specificity in detection CTC using these technique. Therefore, the present study was to obtain immunomagnetic beads which can be specially and sensitively combined with human hepatocellular carcinoma cells firstly, and secondly expected to assess morphological characteristics of CTCs, and examined the expression of AFPmRNA and hTERT mRNA by nested RT-PCR after immunomagnetic cell separation of peripheral mononuclear blood cells obtained from patients with HCC. Lastly we hope to culture the CTCs from the blood samples and to finding the activities of the cells released from the primary neoplasm after immunomagnetic cell separation. It will be useful to detection and prevention hematogenous metastasis in HCC.Methods: 1. Mediated by 1-ethyl-3, 3-dimethylaminopropyl carbodiimide (EDC), immunomagnetic beads against human hepatocellular carcinoma cells were constructed by binding the monoclonal antibodies HAb18 with magnetic microbeads. Mixing small numbers of cells from the hepatocellular carcinoma line HepG2 with peripheral blood mononuclear cells performed a study. After isolated through immunomagnetic cell separation, the rare cancer cells were observed through microscope and identified by immunocytochemistry, then to evaluate the specificity and sensitivity of the immunomagnetic bead. The recovery rate of tumor cells and detecting efficiency of immunomagnetic cell seperation were caculated. 2. Ten ml of peripheral blood were collected from 56 patients with HCC, 19 with hepatitis and liver cirrhosis, and 11 with metastasis liver cancer. In addition, 18 blood samples were collected from the healthy volunteers as the control. Ficoll density gradient centrifugation and immunomagnetic beads coat HAb18 antibody coated odies were used to isolate and sort circulating carcinoma cells from peripheral blood in HCC patients, then cellar morphological characteristic was observed by immunofluorescence staining technique, and AFP mRNA and hTERT mRNA were tested by nested RT-PCR to detect the circulating tumor cells in peripheral blood and evaluate the clinic implication. 3. There were 8 cases of CTCs cultured and identificated after immunomagnetic beads sorting, cytobiologic characteristic in growth curve, time of cell doubling, anchoring independence, ultrastructre of cell surface, and metastases in nude mouse after transplantated in spleen were observed.Results: 1. The obtained immunomagnetic beads which binding the antibodies HAb18 could specially and sensitively combine HepG2 cells. We detected tumor cell when the number of tumor cell was much more than one in 1×106 peripheral blood mononuclear cells. The 57.2% rare tumor cells were detected by the above methods and no false-positive results were observed. 2. In the preliminary experiments using HepG2 cells, one HepG2 cell could be detected in 5ml blood sample with immunomagnetic beads sorting and nested RT-PCR technique. 3. In the subsequent clinic experiments, there were 42.9%(24/56) HCC patients who were detected CTCs by immunofluorescence staining technique in combination of immunomagnetic beads sorting, Four kinds of morphological characteristic CTCs were observed in peripheral mononuclear blood with HCC patients after immunomagnetic beads sorting, CTCs can be divided into 4 classes: 1) moderately cells; 2) large cells with a large nucleus; 3) nucleate cells debris; 4) enucleate cells debris. 4. The total positive detection rate of AFP mRNA and hTERT mRNA was 55.4% and 48.2% respectively in patients with HCC. The frequency of positive cases showed strong correlation with TNM stage and extrahepatic metastases. Additionally, the positive rate was increasing rapidly in patients with large (exceeding 5cm) tumors (P<0.05). The frequency of AFP mRNA and hTERT mRNA positivity was also increasing in patients with HCC with multi-nodules (P<0.05), portal cancer thrombosis (P<0.05). Other clinical parameters, such as serum AFP level, grade of differentiation, did not correlate with the presence of AFP mRNA and hTERT mRNA in peripheral blood (P>0.05). On the other hand, 67.9% HCC patients were positive for at least one marker. The presense of AFP mRNA in peripheral blood was correlated with hTERT mRNA, and the coefficient is 0.256. The presense of hTERT mRNA in peripheral blood in AFP mRNA negative and positive patients was 64.5% and 28% (P<0.05). Meanwhile, AFP mRNA expressed markedly in HCC patients compared to the hepatitis and liver cirrhosis patients with no-HCC (P<0.05), but none of samples from metastasis liver cancer patients and healthy volunteers showed AFP mRNA. The positive detection rate of hTERT mRNA in metastasis liver cancer patients was 63.6%, and respectively samples demonstrated negative hTERT expression in the hepatitis and liver cirrhosis patients or healthy volunteers. 5. We have got tumor cells in eight blood samples cultured respectively after immunomagnetic beads sorting, but seven were failed. Only one sample’s tumor cells emerged endothelial tumor cell proliferation, named HCC-27, and serial cultured for five generation. It was confirmed as hepatocarcinoma cell by morphological analysis and AFP immunocytochemistry analysis, but the growth curve, time of cell doubling, and the ablility of anchoring independence of HCC-27 was significantly lower compared with HepG2 (P>0.05). HCC-27 can survive and metastasize into liver of nude mice (1/2) after transplanted in spleen.Conclusion: 1. The obtained immunomagnetic beads can specially and sensitively separate rare HCC cells from peripheral mononuclear blood cells. 2. After using Ficoll density gradient centrifugation and immunomagnetic beads separation and sorting, there were four kinds of morphological characteristic CTCs in peripheral mononuclear blood with HCC patients such as moderately cells, large cells, nucleate and enucleate cells debris. 3. Combined with immunomagnetic beads sorting and nested RT-PCR technique may be useful to detection CTCs in HCC, detection of AFP mRNA in combination with hTERT mRNA provide useful source to improve sensitivity and specificity of detecting circulating hepatocarcinoma cells. 4. Separation and culture of CTC from peripheral blood samples taken give perfect evidence for hematogenous metastasis of HCC. The ability of growth and clony forming efficiency was low, but it had potency of metastasis.更多还原