基于微流控芯片的体外肠道吸收模型构建及其应用进展

肠道是口服药物吸收的主要部位，肠道的上皮细胞上含有绒毛和微绒毛，它们通过增加表面积等因素促进分泌、细胞黏附和吸收。传统的二维/三维（2D/3D）细胞培养模型、动物模型在研究药物吸收方面发挥了重要作用，但是由于缺乏足够的人体药动学的可预测性或伦理问题等，其应用存在一定局限性。因此，以体外活细胞为基础，模拟人肠道的核心结构和关键功能是构建基于微流控芯片的肠道模型的研究重点。该模型是利用微加工技术制备出的模拟人体肠道的复杂微结构、微环境和生理功能的微流控芯片仿生系统。与2D细胞培养和动物实验相比，肠芯片模型能有效地模拟人体内环境，在药物筛选方面更具特异性。本综述概括了国内外肠芯片模型以及与肠道相关的多器官耦合芯片模型的研究进展，及其在疾病建模、药物吸收和转运方面的应用，并总结了当前肠芯片模拟肠道稳态和疾病面临的挑战，为进一步建立更可靠的体外肠芯片模型提供参考。     

微流控芯片技术的研究进展

微流控芯片技术是指采用微细加工技术，在一块几平方厘米的芯片上制作出微通道网络结构和其他功能单元，把生物和化学等领域所涉及的样品制备、生物与化学反应、分离和检测等基本操作单元集成或基本集成在尽可能小的操作平台上，用以完成不同的生物或化学反应过程，并对其产物进行分析的技术。它不仅使生物样品与试剂的消耗降低至纳升（nl）甚至皮升（pl）级，而且使分析速度大大提高，     

基于人肝癌细胞系SMMC-7721的三维培养条件研究

目的 基于人肝癌细胞系SMMC-7721考察合适的海藻酸钠、氯化钙浓度,形成良好的海藻酸钙凝胶,优选三维条件下肿瘤细胞的培养条件。方法 在96孔板中培养SMMC-7721细胞,基于L₉（3⁴）正交表设计实验,以MTT法检测并计算氯化钙溶液、柠檬酸钠溶液不同浓度与作用时间下的细胞存活率;结合正交优选结果,考察适宜的海藻酸钠浓度,筛选适合的成胶、溶胶条件,并在三维培养模式下进行细胞活力确证。结果 最佳凝胶应用条件为1%氯化钙溶液与1%海藻酸钠溶液作用15 min内用于成胶,10%柠檬酸钠溶液作用凝胶10 min内用于溶胶,MTT法检测细胞存活率为94.97%。该三维培养条件下的肝癌细胞72 h内生长状态良好,存活率可达92.10%,细胞堆积紧密,出现肿瘤细胞球样聚集体。结论 本实验筛选出适合肿瘤细胞三维培养的成胶、溶胶条件。在该条件下培养肝癌细胞SMMC-7721,细胞生长状态良好,且细胞生长与聚集形态与传统二维培养存在差异,更接近细胞体内生存环境。基于该方法进行肿瘤细胞三维培养,可为深入研究肿瘤细胞生长状态,为抗肿瘤药物筛选提供更有利的方式。     

微流控芯片技术研究进展

微流控芯片分析技术近年来迅速发展,在生化分析中应用广泛。本文介绍了微流控芯片技术的加工方法、分离技术及检测技术,并从DNA检测、酶联免疫分和细胞信号通路研究等方面综述了其在生化分析中的应用,对其应用中的不足进行了分析,对其应用前景做了展望。     

微流控芯片及其在肿瘤研究中的应用进展

微流控芯片具有微型化、集成化、效率高、试剂消耗量小等特点,在细胞水平药物筛选及仿生器官方面的研究取得了一系列成果。本文着重介绍了微流控芯片的加工材料和制作技术,以及在肿瘤研究领域的应用和最新研究进展,并提出了微流控芯片领域存在的问题及解决问题的方法。     

药物筛选微流控芯片的构建及其在花蕊石抗肝癌研究中的应用

目的构建一种集成有微阀结构的双层PDMS-玻璃复合芯片,考察其适用性并研究花蕊石生品、制品水煎液对肝肿瘤细胞Hep G2凋亡坏死的影响。方法制作双层微流控芯片并考察微阀的性能,同时进行了Hep G2细胞的培养,死活试剂盒检测细胞活力。将预制备的不同浓度花蕊石生品、制品水煎液通入微流控芯片,分别作用于Hep G2细胞24、48和72 h,凋亡坏死试剂盒检测Hep G2细胞凋亡坏死率。结果芯片微阀性能良好,可灵活控制液阀的开关且未表现出明显的延迟现象。细胞在芯片中生长状态良好,培养72 h期间细胞存活率≥97%。药物作用细胞后,发生凋亡坏死的细胞数量随给药浓度的升高、给药时间的延长而增加,呈现出一定的时间与浓度依赖性,且制品水煎液的效果好于生品。结论实验证明了花蕊石生品、制品水煎液对肝肿瘤细胞Hep G2具有促凋亡作用,也进一步验证了本实验室设计的芯片在细胞研究和药物筛选方面的可行性,为微流控芯片在中药抗肿瘤新药开发领域中的推广与应用提供理论依据。     

基于细胞水平药物筛选的微流控芯片系统研究进展

进入21世纪以来,药物筛选技术正朝着快速、高效的细胞水平筛选方面发展,微流控芯片技术作为现代生命科学领域的重要研究工具,以其分析微型化、高通量化、可集成化和良好的生物相容性等特点,在细胞水平药物筛选方面引起了广泛关注,并取得了一系列成果。国家“十二五”规划中启动科技重大专项“基于微流控芯片的新药研究开发关键技术”,使得微流控芯片在药物筛选领域的研究达到了一个新的高度。本文主要综述了近年来基于细胞水平药物筛选的微流控芯片系统的研究进展,并对其应用前景进行了展望。     

毛细管电泳和微流控芯片在药物筛选中的应用

药物筛选是现代药物开发流程中测试和获取特定生理活性化合物的一个步骤.毛细管电泳技术由于具有样品消耗量小、速度快、柱效高以及所用溶液体系较接近生物体液组成等特点,已经成为一种非常具有潜力的药物及先导化合物的高效筛选工具.文中就毛细管电泳技术在药物筛选中的最新应用情况进行综述,具体从测定药物解离常数pKa值、药物脂水分配系数(logP)、药物与蛋白质的结合常数的测定以及手性药物筛选等方面进行论述,同时也探讨了微流控芯片技术在高通量药物筛选方面的最新研究进展.     

血脑屏障微流控芯片模型的构建及其中药活性成分跨膜转运研究

构建血脑屏障微流控芯片模型,应用于中药活性成分跨血脑屏障渗透性研究。该芯片模型由垂直交叉的两层通道及单层聚碳酸酯膜组成,采用原代人脑微血管内皮细胞,并能模拟血管剪切应力。通过活/死细胞染色和免疫荧光染色观察芯片上细胞生长状态,细胞生长状态良好,且在动态培养下细胞间黏附连接蛋白结构完整;考察该芯片模型对荧光示踪剂和3种模型药物的渗透性和P-糖蛋白(P-gp)的表达情况,结果显示荧光示踪剂和模型药物的渗透性与文献报道一致,芯片上P-gp表达和功能正常,表明该血脑屏障芯片模型具有完整的结构和功能。将构建成功的芯片模型应用于6种中药活性成分跨血脑屏障渗透性评价,采用HPLC-MS/MS法测定跨膜转运液中的药物浓度,获得各成分的P_app结果。延胡索甲素P_app为(4.51±1.90)×10^-7cm·s^-1、延胡索乙素P_app为(9.10±6.59)×10^-7cm·s^-1、欧前胡素P_app为(9.38±2.53)×10     

浓度梯度微流控芯片平台的构建及其应用于抗白念珠菌药物快速筛选研究

本文建立了用于抗白念珠菌药物快速筛选的浓度梯度微流控芯片平台,通过荧光示踪剂荧光素钠在芯片上的分布定性考察浓度梯度生成情况,采用HPLC法对芯片内模型药物氟康唑的浓度梯度分布进行定量分析,进一步比较不同流速条件对浓度梯度形成的影响,最终确定两水相流速1∶1的比例用于后续药物筛选研究。以阿尔玛蓝为细胞活力指示剂,通过该平台分别进行了两性霉素B、氟康唑、伊曲康唑、伏立康唑、泊沙康唑、特比萘芬、5-氟胞嘧啶、卡泊芬净的药敏实验,快速高效地获得了药物的MIC范围,且与CLSI建议的白念珠菌敏感株的MIC值相一致,表明该平台可以通过一次实验快速筛选得到抗菌药物的MIC值范围。此外,该批次白念珠菌对特比萘芬呈现耐药,与96孔板法验证结果一致,表明该方法还可以用于耐药菌株的快速筛选。     

Microfluidic 3D cell culture: potential application for tissue-based bioassays

Current fundamental investigations of human biology and the development of therapeutic drugs commonly rely on 2D monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function or physiology of living tissues, nor the highly complex and dynamic 3D environments in vivo. Microfluidic technology can provide microscale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in the microfluidic technology for 3D cell culture and their biological applications.     

3D cell culture systems modeling tumor growth determinants in cancer target discovery

Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.     

B淋巴母细胞株95041的建立及生物学特性的研究

本人用微量全血 EB 病毒转化法建立了杜纳综合征患者的 B 淋巴母细胞株95041。该细胞呈悬浮生长,核型为45,XO,细胞群体倍增时间为62h/代。该细胞株表现为姐妹染色单体交换率(6.27次/细胞)及丝裂霉素 C 诱发姐妹染色单位交换率(13.56次/细胞)微核率(8‰,11.2‰)与正常细胞 JMS95031相比无显著性差异(P>0.05)。     

WT-1蛋白在卵巢颗粒细胞瘤和卵泡膜细胞瘤中的表达及意义

目的探讨WT-1蛋白在卵巢颗粒细胞瘤和卵泡膜细胞瘤中的表达及意义。方法用免疫组织化学方法研究WT-1蛋白在37例卵巢颗粒细胞瘤(GCT)和39例卵泡膜细胞瘤(TCT)及10份健康卵巢组织中的表达。结果在卵巢GCT和TCT组织中,WT-1蛋白的表达明显高于其在正常组织中的表达。且高、中分化组与低分化组之间,Ⅰ期、Ⅱ期与Ⅲ期之间的差异均有统计学意义(P<0.05),即随组织学分级与临床分期提高而降低。WT-1蛋白阳性表达的病例10年生存率高于阴性表达者,差异有统计学意义(P<0.05)。结论WT-1蛋白有望作为卵巢GCT和TCT的辅助标记物和影响预后的一个因素,能为病理诊断和判定预后提供一定的指导作用。     

Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-throughput studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long term in vitro studies are required.It has been shown that hepatocytes functionality can be improved and extended in time when cultured as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were performed. An inoculum concentration of 1.2 × 10⁵ cell/mL and a 1:2 hepatocyte:mouse embryonic fibroblast ratio allowed to improve significantly the albumin secretion rate and both ECOD (phase I) and UGT (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte monocultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration was also observed. In summary, the improved hepatocyte specific functions during long term incubation of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model for long term toxicological studies.     

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

Drug-transporter interactions could impact renal drug clearance and should ideally be detected in early stages of drug development to avoid toxicity-related withdrawals in later stages. This requires reliable and robust assays for which current high-throughput screenings have, however, poor predictability. Kidney-on-a-chip platforms have the potential to improve predictability, but often lack compatibility with high-content detection platforms. Here, we combined conditionally immortalized proximal tubule epithelial cells overexpressing organic anion transporter 1 (ciPTEC-OAT1) with the microfluidic titer plate OrganoPlate to develop a screenings assay for renal drug-transporter interactions. In this platform, apical localization of F-actin and intracellular tight-junction protein zonula occludens-1 (ZO-1) indicated appropriate cell polarization. Gene expression levels of the drug transporters organic anion transporter 1 (OAT1; SLC22A6), organic cation transporter 2 (OCT2; SLC22A2), P-glycoprotein (P-gp; ABCB1), and multidrug resistance-associated protein 2 and 4 (MRP2/4; ABCC2/4) were similar levels to 2D static cultures. Functionality of the efflux transporters P-gp and MRP2/4 was studied as proof-of-concept for 3D assays using calcein-AM and 5-chloromethylfluorescein-diacetate (CMFDA), respectively. Confocal imaging demonstrated a 4.4?±?0.2-fold increase in calcein accumulation upon P-gp inhibition using PSC833. For MRP2/4, a 3.0?±?0.2-fold increased accumulation of glutathione-methylfluorescein (GS-MF) was observed upon inhibition with a combination of PSC833, MK571, and KO143. Semi-quantitative image processing methods for P-gp and MRP2/4 was demonstrated with corresponding Z′-factors of 0.1?±?0.3 and 0.4?±?0.1, respectively. In conclusion, we demonstrate a 3D microfluidic PTEC model valuable for screening of drug-transporter interactions that further allows multiplexing of endpoint read-outs for drug-transporter interactions and toxicity.