三维细胞反应器及其在中药活性成分筛选中的应用

发布时间：2018-06-09 12:07

本文选题：多孔支架 + 三维细胞培养　；参考：《陕西师范大学》2012年博士论文

【摘要】：药物发现已经产生了许多成功的药物处理和治疗方法,然而,也遇到了高失败率、高成本等问题,也受到许多科学技术挑战的限制,例如需要用更快的速度和更准确的方式分析药物候选物。利用活细胞作为生物识别元素来进行化合物功能验证和毒性测试的基于细胞的分析是解决这些问题的一个有效方法。已有文献报道荧光蛋白、微管蛋白、G-蛋白偶联受体、细胞膜和活细胞等与生物活性成分之间的作用。基于活细胞的分析对于药物筛选而言可以产生与生化分析相比更接近体内生理的结果。在药物发现的各个阶段,已有微型和纳米级的基于活细胞的分析技术。然而,传统的体外细胞试验是利用二维培养的细胞,缺乏细胞原有组织的三维微环境,缺乏细胞生长的基质,也缺乏细胞生长的支架,细胞在二维条件下丧失了许多组织学相关的功能,不能表达其在体内条件下的许多特性。因此,二维细胞培养不能模拟细胞在体内生长的微环境,在药物功效预测方面准确度不高。不同于传统的二维细胞培养,三维细胞培养是将具有三维结构的载体与细胞在体外共培养,最大程度地模拟体内微环境,使细胞能够在载体的三维空间结构中生长、迁移、分化,产生一定的三维组织特异性结构,具有细胞培养直观性和条件可控性的优势,填补了二维细胞培养和动物实验的鸿沟,有非常大的发展潜力。由于传质的局限性,三维培养可得到不同表型的细胞,如增殖、非增殖和坏死细胞。在三维培养时癌细胞的异质性与完整实体瘤的多重表型相似,而这比二维培养时癌细胞的同质性更真实。三维培养的细胞在形状和环境上与体内条件更为相似,而细胞的形状和环境决定细胞的行为和基因表达。因此,三维细胞培养可以更真实地模拟体内细胞生长的三维微环境,细胞在三维条件下的响应更能代表其在体内条件下的响应,基于三维细胞的药物筛选方法将会是更有效的筛选方法。支架具有制备过程简单、快速,对细胞所需营养物和细胞的代谢物扩散的阻力小,可以满足多细胞的聚集等优点。已经有很多支架被用于细胞的三维培养,其中复合支架由于整合了各单一材料的优点、克服了各单一材料的缺点而引起了广泛的关注。细胞在支架上的接种方法和培养方法影响细胞的粘附、增殖和分化等活性,影响细胞与药物作用。与传统的静态接种和培养方法相比,动态接种和灌流培养中细胞悬液直接通过支架的孔,对于不同组成、孔结构和孔隙率的支架都可以更方便的使营养物质和气体通过。因此,动态接种和灌流培养在细胞生长和存活方面更有优势,利用这种方式三维培养的细胞与药物之间的作用更能反映药物在体内作用的真实情况。目前,已经有许多种类的填充床细胞反应器被用于细胞的动态接种和灌流培养,特别是被用于肝细胞高密度培养。这些反应器与静态细胞接种和培养方法相比,在细胞粘附、分布、长时间存活和各种类型的细胞功能等方面已经显示出无可比拟的优势。然而,目前所报道的大多数填充床细胞反应器由于不能提供细胞生长的必须环境,而依赖于二氧化碳培养箱,这就限制了它们的使用。并且,目前还没有利用填充床细胞反应器进行药物筛选的报道。因此,本论文旨在制备适合细胞生长的多孔支架,利用多孔支架建立不依赖于二氧化碳培养箱的三维细胞反应器,并利用这个三维细胞反应器筛选分析传统中药中能够与三维培养的癌细胞特异性结合的活性成分。本论文由六章组成。第一章为引言。引言部分介绍了药物筛选模型、高通量药物筛选和基于细胞模型的高通量药物筛选；指出了三维细胞培养的优点以及常用方法；总结了三维细胞反应器的设计原则与类型；介绍了以多孔支架为填充物的三维细胞反应器,以及几种常见的用于制备支架的生物材料和方法；简要指出了本论文的研究目的意义以及研究内容与关键技术。第二章为PLGA/羟基磷灰石/胶原蛋白复合多孔支架的制备、表征及生物相容性评价。研究了超临界二氧化碳压力、温度、作用时间对PLGA/纳米羟基磷灰石/胶原蛋白复合多孔支架孔结构的影响,结果表明通过调节超临界二氧化碳压力、温度、作用时间可得到不同孔径和孔隙率的PLGA/纳米羟基磷灰石/胶原蛋白复合多孔支架。PLGA/羟基磷灰石/胶原蛋白复合多孔支架的化学表征结果表明,羟基磷灰石、胶原蛋白均匀的分布于PLGA/羟基磷灰石/胶原蛋白复合多孔支架中。体外生物相容性实验表明,PLGA/羟基磷灰石/胶原蛋白复合多孔支架适合人骨肉瘤细胞MG-63粘附和增殖。体内生物相容性实验表明,支架的植入会引起一些炎症反应,术后7、14和28d后与支架相连的组织中的TNF-α、IL-1β、IL-6的含量逐渐减小,而IL-10的含量逐渐增加,且TNF-α、IL-1β、IL-6和IL-10的含量与空白对照组在28d时基本相似,表明由PLGA/羟基磷灰石/胶原蛋白复合多孔支架的植入引起的炎症反应在28d后消除。因此,PLGA/羟基磷灰石/胶原蛋白复合多孔支架具有良好的体内、体外生物相容性,在组织工程中有很大的应用潜力。第三章为PLGA-PEG-PLGA/纳米羟基磷灰石/胶原蛋白复合多孔支架的制备、表征及生物相容性评价。研究了超临界二氧化碳压力、温度、作用时间对PLGA-PEG-PLGA/纳米羟基磷灰石/胶原蛋白复合多孔支架孔结构的影响,结果表明通过调节超临界二氧化碳压力、温度、作用时间可以得到不同孔径和孔隙率的PLGA-PEG-PLGA/纳米羟基磷灰石/胶原蛋白复合多孔支架。体外、体内生物相容性实验表明,该支架具有良好的体外、体内生物相容性。第四章为丝素蛋白/胶原蛋白/纳米羟基磷灰石复合多孔支架的制备、表征及生物相容性评价。研究了氯化钠粒径、氯化钠和纳米羟基磷灰石的重量比及丝素蛋白和胶原蛋白的体积比对丝素蛋白/胶原蛋白/纳米羟基磷灰石复合多孔支架孔结构的影响,结果表明采用该方法可以制备孔径和孔隙率可调的生物相容性好的丝素蛋白/胶原蛋白/纳米羟基磷灰石复合多孔支架,操作简单,耗时短。体外、体内生物相容性实验表明,该支架具有良好的体外、体内生物相容性。第五章为填充床细胞反应器耦合HPLC/MS药物筛选系统的建立及其在朱砂七活性成分筛选中的应用。建立了一个通过填充床细胞反应器耦合HPLC/MS药物筛选系统,比较药物与固定的癌细胞和活的癌细胞作用之后生物指纹图谱的峰面积来评价药物与癌细胞的结合度。两种已知抗癌药物(紫杉醇和白藜芦醇)和两种已知非抗癌药物(酮洛芬和青霉素G)与模型癌细胞(Lovo细胞)之间的相互作用证明,该分析系统用于药物筛选是可行的,并将该分析系统应用于朱砂七提取物中活性成分的筛选分析,筛选出两种生物活性成分,马兜磷酸A和马兜磷酸B。第六章为三维细胞反应器及其在桃儿七活性成分筛选中的应用。建立了一个新型的三维细胞反应器,通过比较药物与固定的癌细胞和活的癌细胞作用之后生物指纹图谱的峰面积之间有无显著性差异来判断该药物是否能与癌细胞特异性结合。两种已知抗癌药物(紫杉醇和白藜芦醇)和两种已知非抗癌药物(酮洛芬和青霉素G)与癌细胞的作用证明,该三维细胞反应器在筛选与细胞特异性结合的生物活性成分方面是可行的。利用该三维细胞反应器对桃儿七提取物中的活性成分进行筛选分析。对桃儿七提取物中已鉴定的10个成分的筛选表明,槲皮素-3-O-β-D-葡萄糖苷,山奈酚-3-O-β-D-葡萄糖苷和山荷叶素这3种组分与与Lovo细胞之间没有特异性结合。而L-鬼臼毒素-4-0-β-D-葡萄糖苷、槲皮素、二十六烷酸、山奈酚、鬼臼毒素、鬼臼毒酮或异鬼臼苦酮和去氧鬼臼毒素或4’-去甲鬼臼毒酮可以与Lovo细胞特异性结合,它们的结合度分别为：32.6%±3.1%、29.3%±3.0%、15.2%±1.7%、57.1%±4.8%、68.9%±9.2%、63.6%±8.7%、46.3%±4.8%。本论文采用不同的方法制备了三种多孔支架,并对各种支架进行了表征和生物相容性评价,为三维细胞反应器的建立奠定了基础。并对现有的三维细胞反应器进行突破,提出了两种不依赖于二氧化碳培养箱的三维细胞反应器,分别将这两种三维细胞反应器用于中药提取物中与癌细胞特异性结合的活性成分的筛选分析,得到了一些能与癌细胞特异性结合的活性成分。本论文提供了一个从多组分混合物中特异性的筛选和分析生物活性成分新方法。同时,也可以为从天然产物和植物药物中筛选抗癌候选药物提供一个新的思路。
[Abstract]:however , conventional in vitro cell assay is an effective method to address these problems .

The three - dimensional cell culture is different from the traditional two - dimensional cell culture , and the three - dimensional cell culture is characterized in that the carrier and the cell with the three - dimensional structure are cocultured in vitro to maximally simulate the in vivo microenvironment , so that the cell can grow , migrate and differentiate in the three - dimensional space structure of the carrier , and the three - dimensional culture can obtain cells of different phenotypes , such as proliferation , non - proliferation and necrotic cells .

The stent has the advantages of simple preparation process , fast speed , small resistance to the diffusion of metabolites of nutrients and cells required by cells , and the like , and can meet the advantages of multi - cell aggregation and the like .

Compared with the traditional static inoculation and culture method , the cell suspension in the dynamic inoculation and perfusion culture can be more convenient for the nutrient substance and the gas to pass through the hole of the stent . Therefore , the dynamic inoculation and perfusion culture are more advantageous in cell growth and survival , and the effect between the three - dimensional cultured cells and the drug can reflect the real situation of the action of the drug in vivo .

Currently , many kinds of packed bed cell reactors have been used for the dynamic inoculation and perfusion culture of cells , in particular for high density culture of hepatocytes . These reactors have shown an unparalleled advantage in terms of cell adhesion , distribution , long - term survival and various types of cellular functions , as compared to static cell inoculation and culture methods . However , most of the packed bed cell reactors currently reported have limited their use because they do not provide the necessary environment for cell growth , and this limits their use .

Therefore , the purpose of this paper is to prepare a porous scaffold suitable for cell growth , to establish a three - dimensional cell reactor independent of the carbon dioxide incubator by using a porous scaffold , and to use the three - dimensional cell reactor to screen and analyze the active ingredients capable of specifically binding to the three - dimensional cultured cancer cells in the traditional Chinese medicine .

The first chapter introduces drug screening model , high flux drug screening and high flux drug screening based on cell model .
The advantages and common methods of three - dimensional cell culture are pointed out .
The design principle and type of three - dimensional cell reactor are summarized .
Three - dimensional cellular reactor with porous scaffold as filler and several common biological materials and methods for preparing stents were introduced .
This paper briefly points out the research objectives and the research contents and key technologies of this paper .

In chapter 2 , the preparation , characterization and biocompatibility evaluation of PLGA / hydroxyapatite / collagen composite porous scaffolds were studied . The results showed that PLGA / hydroxyapatite / collagen composite porous scaffolds were suitable for the adhesion and proliferation of human osteosarcoma cells MG - 63 . The results showed that PLGA / hydroxyapatite / collagen composite porous scaffolds were suitable for human osteosarcoma cell MG - 63 adhesion and proliferation .

In chapter 3 , the preparation , characterization and biocompatibility evaluation of PLGA - PEG - PLGA / nano hydroxyapatite / collagen composite porous scaffold were studied . The effects of supercritical carbon dioxide pressure , temperature and action time on the pore structure of PLGA - PEG - PLGA / nano hydroxyapatite / collagen composite porous scaffold were studied .

In chapter 4 , the preparation , characterization and biocompatibility of silk fibroin / collagen / nano hydroxyapatite composite porous scaffolds were evaluated . The effects of the weight ratio of sodium chloride , sodium chloride and hydroxyapatite and the volume ratio of silk fibroin and collagen on the pore structure of silk fibroin / collagen / nano hydroxyapatite composite porous scaffolds were studied .

In the fifth chapter , an HPLC / MS drug screening system for packed bed cell reactor was established and its application in the selection of active ingredients in Zhucheng 7 was established . An HPLC / MS drug screening system was established by a packed bed cell reactor . The peak area of the fingerprint was compared between the drugs and fixed cancer cells and living cancer cells .

A new type of three - dimensional cell reactor was established . The three - dimensional cellular reactor was used to screen the active components of the cell - specific cell . The results showed that the three components were : 36.6 % 卤 3.1 % , 29.3 % 卤 3.0 % , 15.2 % 卤 1.7 % , 57.1 % 卤 4.8 % , 68.9 % 卤 9.2 % , 63.6 % 卤 8.7 % , 46.3 % 卤 4.8 % respectively .

Three kinds of porous scaffolds were prepared by different methods , and the characterization and biocompatibility evaluation of various scaffolds were carried out . Two three - dimensional cell reactors which were not dependent on carbon dioxide incubator were established . Two kinds of three - dimensional cell reactors , which were not dependent on the specific binding of the cancer cells , were screened and analyzed in the traditional three - dimensional cell reactor .
【学位授予单位】：陕西师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

【参考文献】