一种尺寸可控三维肿瘤阵列芯片及其抗肿瘤药物分析应用

发布时间：2018-02-22 01:13

本文关键词： 微流控芯片三维肿瘤制备抗肿瘤药物测试　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：在全世界,癌症是人类死亡的主要原因之一。利用单层细胞培养进行体外高通量的抗肿瘤药物测试,用于评估药物功效。但是,单层培养对于一种药物是否最终产生临床效果预测能力差。普遍认为三维肿瘤能够增强预测能力,减少动物评估实验,降低鉴定新药的时间和费用。传统的三维肿瘤培养操作方法包括,非粘附表面培养法,回旋法,悬滴法。悬滴法的优点是有效的形成尺寸均一的三维肿瘤,同时与高通量的测试仪器兼容。回旋法是一种形成三维肿瘤的简单方法。但这些方法的缺点是耗时、费力及试剂消耗大。与传统的细胞操作方法不同,微流控芯片具有试剂消耗少,通量高的优势。芯片材料聚二甲基硅氧烷具有良好透气性,适合活细胞操作;同时其具有优良的透光及生物相容性,在开展芯片内细胞操作分析方面具有显著优势。然而,目前建立的肿瘤芯片系统往往针对于特定尺寸的肿瘤操作,不具备三维肿瘤尺寸可控性,在系统性开展肿瘤生物学分析方面仍存在不足。基于此,本研究设计制备了一种可实现不同尺寸三维肿瘤制备、操作与在线分析的微流控芯片。通过开展微流控芯片设计与制备条件优化、芯片内细胞捕获定位操作、不同尺寸人肝癌HepG2和人胶质瘤U251三维肿瘤制备及其抗肿瘤药物分析应用四个部分研究,具体结果如下:1、采用软光刻技术,成功制备出本研究使用的双层微流控芯片,用于三维肿瘤操作分析研究。结果显示,用于制备芯片上下层的PDMS胶与固化剂比例分别为10:1和20:1,固化时间分别为13和23min时,芯片键合效果最好,有效避免了漏液现象。2、通过对细胞接种密度、流速和灌注时间条件优化,实现了芯片内HepG2和U251细胞的精确捕获定位,为进一步开展三维肿瘤培养研究奠定基础。3、完成了芯片内不同尺寸HepG2和U251三维肿瘤的培养。结果显示,所制备的特定尺寸三维肿瘤均一性较好,肿瘤细胞活性高,适于开展后期的抗肿瘤药物分析应用。4、实现了基于三维肿瘤芯片的两种抗肿瘤药物分析研究。结果显示随着药物浓度的增大,细胞活性降低。博来霉素和替拉扎明两种抗肿瘤药物处理人肝癌HepG2三维肿瘤,在相同的浓度条件下,替拉扎明的处理人肝癌细胞活性较低。该研究结果表明设计的微流控芯片能够实现不同尺寸的三维肿瘤制备及其抗肿瘤药物分析应用。
[Abstract]:Cancer is one of the leading causes of death around the world. Monolayer cell culture is used to test high-throughput antitumor drugs in vitro to assess drug efficacy. Monolayer culture is a poor predictor of whether a drug will eventually produce clinical effects. It is widely believed that three-dimensional tumors enhance predictive power and reduce animal evaluation experiments. Reducing the time and cost of identifying new drugs. Traditional three-dimensional tumor culture methods include non-adherent surface culture, cyclotron, and droplet. The advantage of the droplet method is the effective formation of three-dimensional tumors of uniform size. Also compatible with high-throughput testing instruments. Cyclotron is a simple way to form three-dimensional tumors. But the disadvantages of these methods are time-consuming, laborious, and reagent consuming. The microfluidic chip has the advantages of less reagent consumption and high flux. The chip material polydimethylsiloxane has good permeability and is suitable for the operation of living cells, at the same time, it has excellent light transmission and biocompatibility. It has significant advantages in the analysis of intrachip cell operations. However, currently established tumor chip systems are often targeted at tumor operations of specific size, and do not have three-dimensional tumor size controllability. There are still some shortcomings in the systematic analysis of tumor biology. Based on this, we designed and prepared a kind of three-dimensional tumor preparation with different sizes. Operation and on-line analysis of microfluidic chips. By optimizing the design and preparation conditions of microfluidic chips, the in-chip cell capture and localization operations are carried out. Three dimensional tumor preparation of human hepatocellular carcinoma (HepG2) and human glioma (U251) with different sizes and the application of anti-tumor drugs were studied in four parts. The results are as follows: 1. The double layer microfluidic chip used in this study was successfully fabricated by soft lithography. The results show that when the ratio of PDMS glue to curing agent is 10: 1 and 20: 1, and the curing time is 13 and 23 minutes, respectively, the chip bonding effect is the best. By optimizing the cell inoculation density, flow rate and perfusion time, the accurate capture and localization of HepG2 and U251 cells in the chip can be achieved. In order to lay a foundation for the further study of three-dimensional tumor culture, the three-dimensional tumor culture of different sizes of HepG2 and U251 was completed. The results showed that the specific size of the three-dimensional tumor was uniform and the tumor cell activity was high. The results show that two kinds of antitumor drugs based on 3D tumor microarray can be analyzed with the increase of the concentration of antitumor drugs, which is suitable for the later application of antineoplastic drug analysis. Two antitumor drugs, bleomycin and telazamine, were used to treat three-dimensional HepG2 tumors of human liver cancer at the same concentration. The results showed that the microfluidic chip could be used to prepare three dimensional tumors of different sizes and to analyze antitumor drugs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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