【作者】 马宇超；

【导师】 喻风雷；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：利用体外细胞实验、动物实验及临床实验来对膜过滤分离肿瘤细胞技术(Isolation by size of epithelial tumor cells, ISET)进行系统性研究,目的是为了建立起一整套使用ISET技术在血液循环过程中检测、筛选、分离循环肿瘤细胞(circulating tumor cells, CTC)并进行深入分析的技术方法,并为进一步的临床试验打下基础。研究方法：1.利用带EGFP报告基因的H520肺鳞癌细胞株,构建ISET技术体外细胞实验方法及技术流程,并观察ISET技术在生理盐水和外周血这两种不同介质中的肿瘤细胞回收率及敏感性、特异性。2.建立肿瘤细胞血行转移动物模型,利用ISET技术对动物外周血进行检测,寻找转移的肿瘤细胞。3.ISET外周血筛查装置的设计和建立,以及对96例肺部疾病患者外周血进行筛查,观察ISET技术应用于临床检测的敏感性和特异性,并对特殊病例进行外周血CTC的持续监视。4.ISET血液循环系统的设计、建立,应用于VX2兔肿瘤细胞血行转移模型,对比ISET外周血筛查装置和血液循环系统的敏感性,验证ISET血液循环系统的安全性。结果：1.成功将pEGFP-N3质粒转染入H520肿瘤细胞中,体外实验发现ISET技术在生理盐水和外周血这两种不同介质中的肿瘤细胞回收率分别为97.57±1.33%、79.53±2.63%,敏感性测试中基本达到了1ml外周血中检测到1个肿瘤细胞的敏感性,由于阴性对照组中未发现目标细胞,表明ISET技术有很高的特异性。2.分别建立NOD-SCID小鼠肿瘤细胞血行转移模型和VX2兔肿瘤细胞血行转移模型,利用ISET技术在3只NOD-SCID荷瘤鼠体内检测出CTC,在17只VX2荷瘤兔中有3只实验兔检测出CTC。3.设计并建立ISET外周血筛查装置并应用于96例肺部疾病患者,肿瘤组CTC的检出率为2.99%,非肿瘤组CTC的检出率为0.00%。远处转移组CTC的检出率为50.00%,显著高于未远处转移组CTC的检出率0.00%(P<0.05)。4.对结肠癌肺、骨转移患者进行外周血持续监测,治疗后期发现其外周血中开始持续存在CTC及癌栓。5.设计并建立ISET血液循环系统并应用于VX2兔肿瘤细胞血行转移模型,发现利用ISET外周血筛查装置的CTC的检出率为17.65%,显著低于利用ISET血液循环系统的检出率82.35%(P<0.05)。结论：1.ISET技术在体外实验中有较高的敏感性和特异性,且实验方法简单,不依赖任何大型设备,实验结果稳定,可重复性强。2.在国际上首次利用ISET技术在肿瘤细胞血行转移动物模型中检测到CTC,具有极高的特异性。3.ISET外周血筛查装置适用于临床筛查CTC且方便组装便于携带。4.ISET技术在临床实验中表现出极高的特异性,但是敏感性不佳。5.ISET技术在疗效监视、评估预后以及在肿瘤转移机制研究方面有巨大潜力。6.在国际上首次设计并建立ISET血液循环系统,可以显著提高ISET技术敏感性,并且直接减少循环血液中存在的CTC数量。上述技术、方法的实施对肿瘤诊断、疗效监视提供了新的思路。更多还原

【Abstract】 OBJECTIVE:Systematic study of isolation by size of epithelial tumor cells (ISET) methodology in vitro, animal experiments and clinical trials. To establish a kind of method of ISET in the process of blood circulation to detect, screen and separate circulating tumor cells. This pilot study will serve as a basis for a much larger clinical trial.METHOD:1.The method for the technical processes of ISET technology in vitro established with H520-pEGFP cells. H520-pEGFP cells will mix into the physiological saline and the blood sample from healthy volunteers respectively; detect to ascertain the recovery, the sensitivity and the specificity by ISET.2.To establish an animal model of tumor cells metastasis in the blood circulating, and detect the CTCs in the peripheral blood of animals by ISET technology.3.To design an ISET screening device, detect the CTCs in the peripheral blood of96patients with pulmonary disease. To observe the sensitivity and specificity of ISET screening device used in clinical testing. To monitor the presence of CTCs in one patient’s peripheral circulation undergoing standard chemotherapy and radiotherapy.4.To design an ISET hemoperfusion system, detect the CTCs in the blood circulating of rabbit with VX2tumor, observe and compare sensitivity and security of ISET screening device and ISET hemoperfusion system.RESULTS:1.The pEGFP-N3plasmid was transfected into H520tumor cells successfully. The recovery rate of mixed tumor cells detected by ISET technology remained about97.57±1.33in physiological saline and79.53±2.63in blood sample from healthy volunteers. We could detect1tumor cell in1ml peripheral blood while no positive cells were detected in healthy control, showing the high sensitivity and specificity of the ISET technology.2.The NOD-SCID mice model and rabbit model of tumor cells metastasis in the blood circulating have been established respectively.3samples in3NOD-SCID mice and3samples in17rabbit were detected with the CTCs by ISET technology.3.The ISET screening device was designed and built successfully to detect the CTCs in the peripheral blood of96patients with pulmonary disease.2samples in67cancer patients and0sample in29non-neoplastic patients were detected with the CTCs.2samples in4patients of stage IV and0sample in63patients of stage Ⅰ-Ⅲ were detected with the CTCs. However, CTCs were not detected in all6healthy samples by control. The detection rate of CTCs in4patients of stage IV was higher than that in63tumor patients of stage Ⅰ-Ⅲ (P<0.05).4.The persistence of CTCs in peripheral circulation in the patient of stage IV were monitored by ISET screening device.5.The ISET hemoperfusion system was designed and built successfully to detect the CTCs in the blood circulating of rabbit with VX2tumor. The detection rate of CTCs by ISET hemoperfusion system was higher than that by ISET screening device (P<0.05),82.35and17.65respectively (p<0.05).Conclusion:1.The ISET technology to detect CTCs was convenient and stable with high sensitivity and good specificity. It does not rely on any large-scale experimental equipment.2.CTCs have been detected in animal models of tumor cells metastasis in the blood circulating at the first time in the international, indicating that ISET technology has a high specificity.3.ISET screening device suitable for clinical detecting CTCs and easy to assemble and carry.4.The ISET technology exhibit high specificity in clinical trials, but the sensitivity is poor.5.We can evaluate efficacy of chemotherapy and radiotherapy and other cancer-targeting therapies by using ISET technology that has great potential for mechanistic studies of ISET-isolated CTC biology.6.A new ISET hemoperfusion system could be designed to increase sensitivity to CTCs greatly and reduce the number of CTCs directly during the blood flow at the first time in the international.The ISET hemoperfusion system gives us new insights into the diagnosis of malignant tumor and monitoring adjuvant therapy.更多还原