【作者】 张妤；

【导师】 殷正丰；

【作者基本信息】 第二军医大学 ， 肿瘤学， 2013， 博士

【摘要】 研究背景和目的肿瘤远处转移是指肿瘤细胞从原发灶脱落后随血液系统或淋巴系统循环到达靶器官,继而形成转移灶。侵入血液循环中的肿瘤细胞被称为循环肿瘤细胞(circulating tumor cells, CTCs)。肝切除和肝移植是目前治疗肝癌的主要方式。然而,高达40%接受肝切除术的肝癌患者及10%以上的肝移植肝癌患者术后第一年会发生复发。人们已经认识到,肝癌细胞在疾病早期可能即发生血行转移,而循环肿瘤细胞则是肝癌转移或复发的一个主要根源。因此,循环肝癌细胞检测对转移复发预测、治疗方案选择以及预后评估都具有重要的意义。在现有众多的循环肿瘤细胞检测方法中,只有美国Veridex公司的CellSearch系统被批准用于临床检测[3]。与其他方法相类似,CellSearch System也采用基于靶向上皮细胞表面标志物EpCAM的免疫磁性分选策略分离CTCs,而EpCAM在肝癌细胞中的表达率低,不足以用来分选肝癌CTCs。去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)是一种专一表达于肝实质细胞表而的跨膜蛋白,能结合和内吞以半乳糖基和N-乙酰半乳糖胺基为端基的分子。根据肝细胞膜表面存在大量ASGPR的特性,我们创建了一种独特的基于ASGPR及其配体相互作用的循环肝癌细胞磁性分离方法。然而,该方法操作耗时长,且限制了被分选的肝癌细胞用于后续细胞培养和细胞生物学实验。近年发展起来的微流控芯片技术很适合用于CTCs分选,不仅可以高效分离非常稀少的CTCs,而且在细胞研究方面具有独特的优势。本研究论文将ASGPR及其配体之间相互作用的原理移植到微流控芯片分析系统,建立了一套用于循环肝癌细胞分选鉴定以及后续培养的微流控芯片实验平台。利用此平台从临床肝癌血样中分离、计数循环肝癌细胞,分析计数结果与肝癌病情之间的关系。另外设计微量细胞培养芯片,为循环肝癌细胞培养打下基础。研究方法1.采用L-Edit Pro8.30绘图软件设计、绘制芯片图形。2.采用紫外光刻法及注塑法制竹PDMS芯片。3.采用亲和素修饰芯片表面,与生物素化去唾液酸胎球蛋白相连,分离肝癌细胞。4.采用基于广谱抗细胞角蛋白(cytokeratin, CK)抗体CK3-6H5的免疫荧光染色方法鉴定、计数分离获得的肝癌细胞。5.通过肝癌细胞系HepG2掺入试验优化循环肝癌细胞分离芯片的设计参数及进样速度。6.通过肝癌细胞系HepG2和乳腺癌细胞系MCF-7掺入试验分析芯片分离循环肝癌细胞的回收率和特异性。7.采用所设计的微流控芯片检测6例不同分期的HCC患者及2例健康志愿者2mL血液样本中的CTCs。8.设计、制作含有流速控制、配体捕获和体积捕获的循环肝癌细胞分离芯片。9.通过HepG2肝癌细胞掺入试验分析：芯片分离循环肝癌细胞的回收率。10.设计低密度肿瘤细胞培养芯片。11.采用HepG2肝癌细胞稀释液检测芯片培养微量细胞的细胞成活率。12.采用EXCEL软件对结果进行处理。各项数据通过x±SD表示。实验结果1.L-Edit Pro8.30绘图软件设计、绘制循环肝癌细胞分离芯片,每个芯片设有8个通道,每个通道含2254个微米柱,微米柱半径设为80μm、120μm、150μm和200μm等4个参数,PDMS制成芯片通道高为50μm。2.细胞掺入实验测得最佳进样速度为0.8mL/h,微米柱最佳半径为80μm。3.HepG2肝癌细胞掺入试验显示,1mL健康志愿者外周血中分别掺入100个、300个、500个和1000个HepG2细胞,在每个掺入水平,HepG2细胞的平均回收率均≥69%。4.从6例HCC患者血液样本中均检出CTCs,2例健康人血液样本未被检出CTCs。5.循环肝癌细胞分选芯片分为三部分：第一部分为流速控制部分；第二部分为基于配体-受体结合的循环肝癌细胞捕获部分；第三部分为基于体积循环肝癌细胞捕获部分。6.以100个细胞/mL、500个细胞/mL和1000个细胞/mL稀释液进行检测,得到捕获率分别为79%、77%和84%。7.细胞密度为1×103个细胞/mL时,所取12个培养小室中的细胞在第1天、第6天、第12天和第14天的计数,平均计数分别为3个、4个、6个和9个。结论本研究在以往工作的基础上,将原有的基于ASGPR的循环肝癌细胞捕获系统移植在微流控芯片上,与原系统相比,简化了检测步骤,缩短了检测时间,减少了样本采集量,同时在检测敏感性与特异性方面也有所提高。初步检测HCC患者与健康志愿者血样的结果表明,该微流控捕获循环肝癌细胞芯片系统具有临床应用潜能。随后进行的芯片结构改进更是大大提高了循环肝癌细胞的检测速度和捕获效率。而本研究设计的网式微流控细胞培养芯片不但可以进行少量的肿瘤细胞培养,而且可以进行后续的药敏实验,为进一步开展循环肝癌细胞的生物学研究奠定了实验基础。更多还原

【Abstract】 Background and ObjectiveHepatic resection and liver transplantation are the main modalities of curative treatment for HCC at present. However, up to40%of patients undergoing hepatectomy and more than10%of liver transplant recipients develop postoperative recurrences within the first year after surgery, which leads to death in almost all patients. It has been recognized that circulating tumor cells (CTCs), which are released into the blood circulation from either primary or metastatic lesions, are an active source of HCC metastasis or recurrence. Therefore, the enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients.The CellSearch(?) system, based on EpCAM antibody-coated magnetic beads approved by the US Food and Drug Administration has been used for detection of CTCs in breast cancer, colon cancer and prostate cancer. However, in liver neoplasia, only a small percentage (0%-20%) of HCC cases express EpCAM. Obviously, all of the EpCAM-based strategies are not appropriate for detection of HCC CTCs.ASGPR is a transmembrane protein expressed exclusively on the surface of hepatocytes that can bind and internalize molecules with terminal galactose and N-acetylgalactosamine residues. Depended on the interaction of the ASGPR with its ligand, we have developed and validated of a sensitive and specific system to magnetically separate CTCs in HCC patients, combined liver cell-specific antibody Hep Par1immunofluorescence staining method. However, this method is a time-consuming, and limits the separated HCC CTC from subsequent cell culture and other biological experiments. As a new technology developed in recent years for CTCs isolation, the microfluidic chip not only can efficiently isolate CTCs, but also has advantages in cell research. In the current study, we further developed a high-throughput microfluidic device, which provides an enhanced platform for the HCC CTC isolation. Still Based on the interaction of the ASGPR with its ligand, combining this interaction with microfluidic chip, we developed a microfluidic CTC isolation system and a microfluidic cell culture system flowing up. It can be used to carry out the HCC CTC isolation, CTCs count and the HCC case analysis. Meanwhile HCC CTCs could be cultured in cell culture microfluidic chips, so that drug sensitivity test will be carried out. Methods1. Microfluidic chips were designed with L-Edit Pro8.30.2. Microfluidic chips were fabricated following the standard protocol of UV lithography and soft lithography.3. The CTC-chip surface was made functional with biotinylated asialofetuin for HCC CTC isolation.4. The CTCs isolated were identified by immunofluorescence staining with the usage of the antibodies against epithelial-specific antigen cytokeratins (CKs).5. To determine the minimum time and the most efficient micropillar radius required to achieve cell capture, we examined the cell-capture performances of the HCC CTC chip at different flow rates and different micropillar radius with HCC cell line (HepG2) spiked blood sample.6. The recovery of the HCC CTCs isolation and detection system were determined by the HepG2cells spiked blood sample experiments.7. The specificity of the HCC CTCs isolation and detection system were determined by the HepG2and MCF-7cells spiked blood sample experiments.8. The established isolation and detection system for HCC CTCs was applied to examine CTCs in blood samples from6HCC patients and2healthy volunteers.9. CTCs isolation chip contains3parts was designed with L-Edit Pro8.30.10. The recovery of the HCC CTCs isolation was determined by the HepG2cells spiked PBS.11. Low density cell culture chip was designed with L-Edit Pro8.30.12. Cell survival rate and doubling time on CTC culture microfluidic chips were determined with HepG2cells.Results1. CTC isolation microfluidic chips were designed with8isolation channels, with2254micro pillars in each channel and a set of radius (80,120,150and200μm) high in50μm. 2. For HepG2cells, the maximal cell-capture numbers were achieved at a flow rate of0.8mL/h with radius as80um.3. In recovery experiment,100,200,1000and2000HepG2cells were spiked into1mL blood samples from healthy volunteers. At each spiked level, the recovery is80%at the highest.4. In specificity experiment, a set of HepG2and MCF-7cells spiked blood sample (100,300,500and1000cells mL1) were detected by CTC isolation microfluidic chips. It turned out that the recovery of HepG2cells was69%at least, however, only4%of MCF-7cells.5. CTCs were detected in all blood samples from6HCC patients while non was detected in those from2healthy volunteers.6. CTCs isolation chip contains3parts:the first part uniformed the flow; the second part isolated CTCs depended on ligand-receptor reaction; the third part isolated CTCs on volumn.7. At the density gradient of100,500and1000cells/ml, the recovery is79%,77%and84%separately.8. At a cell density of1x103cells/ml, the average cell number of12wells is3,4,6and9on the1st,6th,12th and14th day.ConclusionsBased on the ASGPR depended magnetic-activated HCC CTC separation system, we set up a new CTC isolation system on microfluidic chips. It showed concise protocol, time saving, less sample volume and also improvement in sensitivity and specificity. Through tests with HCC patients’blood samples, it showed its’possibility in HCC CTC detection, however, more clinical trials are still needed to be took out. The changes of structure of the microfluidic isolation chips improved the recovery and shorten the detection time. Rare cells could be cultured in the "net"cell culture microfluidic chips, drug sensitivity test also can be realized on it.更多还原