三维微图案超细纤维支架调控功能性组织形成的研究
发布时间：2018-10-15 19:27
【摘要】：体内大多数组织由多种细胞组成,且这些细胞及其分泌的细胞外基质在组织中有序排列,以发挥特定的组织功能。随着组织工程和再生医学的不断发展,研究者们一直致力于在体外模拟和重建这些复杂的组织结构,其中图案化组织工程支架在调控细胞形态、细胞分布和多细胞共培养等作用明显。静电纺纤维支架具有高的比表面积和类似于细胞外基质的结构,作为组织工程支架具有很大的优势。但目前将图案化技术与电纺技术结合起来构建图案化纤维支架,并调控不同种类细胞的空间分布、重建组织结构微环境的研究仍较少。据此,本论文设计了光刻掩模,运用光刻和磁控溅射工艺制得图案化接收板,采用普通静电纺丝设备获得了图案化纤维支架。为模拟肝小叶和心肌片层的结构,通过图案化纤维支架分别实现了肝细胞和肝组织中其他相关细胞、心肌细胞和心肌组织中其他相关细胞的共培养,较好地维持了肝细胞和心肌细胞的活力。在此基础上,将图案化共培养肝细胞体系、心肌细胞体系作为药物代谢和药物筛选的体外模型,显示出较好的体外和体内结果相关性。利用光刻和磁控溅射工艺制得了图案化接收板,结合普通静电纺丝设备制备了图案化纤维膜,方法简单,操作性强；图案的形状、尺寸和排列方式可控；制得的图案化纤维膜沟脊结构明显,图案区域的纤维具有较高的定向性。成纤维细胞在不同尺寸的图案化纤维支架上培养,发现支架能有效调节细胞的空间分布,使其在预定的图案区域生长,并渗透进入纤维支架内部；同时细胞沿纤维取向生长,分泌的细胞外基质呈现定向分布等特征。合成了乳糖改性聚乳酸,发现混纺比例为5：5的乳糖改性聚乳酸／聚乳酸-聚乙二醇嵌段共聚物(PELA)复合支架能有效维持肝细胞的活力和功能,原代肝细胞形成了直径约为60μm的肝细胞球体。将原代肝细胞和成纤维细胞分别接种在上述复合纤维、PELA纤维的图案化支架上,通过图案化纤维的层层锲合,实现了两种细胞的共培养。在15天的培养中,与肝细胞单独培养相比,共培养肝细胞能较好地维持肝细胞活力、白蛋白分泌、尿素合成和P450酶活力等。在此基础上,将原代肝细胞、成纤维细胞和内皮细胞分别接种于三种不同的图案化纤维支架中,通过层层锲合组装的方式,成功构建了类似体内肝小叶的基质结构。三种细胞的生长状态较好,均能分泌各自的特征性蛋白,肝细胞球体内生成了胆小管,成纤维细胞渗透生长进入图案化纤维支架内部,内皮细胞表现出较好的毛细血管生成能力。以7-苄氧基-4-三氟甲基香豆素、7-甲氧基-4-三氟甲基香豆素和7-羟基香豆素等作为底物,检测了图案化共培养肝细胞中药物代谢相关酶的活性,发现共培养肝细胞中CYP3A4. CYP2C9及Ⅱ相代谢酶的活力均能维持在较高水平。采用咪达唑仑、睾酮、甲苯磺丁脲、华法林和对乙酰氨基酚药物等研究了共培养肝细胞的药物代谢行为,发现上述药物在共培养体系中的代谢清除率接近体内数据,说明能较准确地反映体内肝脏的药物代谢情况。以利福平和谷胱甘肽作为诱导剂,酮康唑和丙磺舒作为抑制剂,证明了共培养肝细胞能敏感地响应药物的诱导和抑制作用。上述结果显示出图案化共培养肝细胞体系适合在体外进行药物代谢和毒性的评价。制备了PELA/碳纳米管的图案化纤维支架,纤维呈现核壳结构,具有一定的导电性。在图案纤维支架上接种原代心肌细胞、原代心肌成纤维细胞,将内皮细胞接种到PELA图案纤维支架上,通过图案化纤维的层层锲合组装,构建了心肌细胞平面共培养的微环境。该体系不仅模拟了心肌组织中不同细胞的排列分布,更表现出与体内心肌相似的生物化学和电生理特性。针对目前组织工程化心肌的厚度不够,为了使所培养的心肌细胞更接近于体内的三维结构,设计了具有众多孔道结构的图案化纤维支架,并模拟心肌细胞在体内生长所需的力学微环境,构建了三维心肌组织,三种细胞均能较好地生长,共培养心肌细胞能长时间维持自发的跳动,孔道结构促进了细胞在支架内部的渗透生长和排列,内皮细胞也表现出了血管化能力。采用实时拍摄记录共培养支架上心肌细胞的搏动信号,在体外构建的共培养心肌细胞体系开展了药物筛选的研究。通过观察奎尼丁、红霉素和甲磺胺心定等对心肌细胞的作用效果和起效时间,对比体内和体外测定的半效应浓度(EC50)值,发现共培养心肌细胞能准确地反映药物在体内作用的量效和时效关系。进一步通过氟哌啶醇的多次药物洗脱实验,验证了共培养心肌细胞体系在药物筛选中表现出的相关性、快速性、重复性、稳定性和灵敏性。综上所述,本论文系统地研究了三维微图案纤维支架的构建、原代肝细胞共培养体系、原代心肌细胞共培养体系,在体外获得了接近于体内组织的生理结构和功能,并应用于肝药物代谢及心肌药物筛选,为药物研究和应用提供了有效的体外筛选手段。
[Abstract]:Most tissues in the body consist of a variety of cells, and these cells and their secreted extracellular matrix are arranged in an orderly manner in the tissue to exert specific tissue functions. With the development of tissue engineering and regenerative medicine, researchers have been committed to simulating and reconstructing these complex tissue structures in vitro, in which the patterning tissue engineering scaffold has obvious effects on regulating cell morphology, cell distribution and multi-cell co-culture. The electrostatic spinning fiber scaffold has a high specific surface area and a structure similar to that of the extracellular matrix, and has great advantages as a tissue engineering scaffold. However, the patterning technology is combined with the electro-spinning technology to build a patterned fiber scaffold, and the spatial distribution of different kinds of cells is regulated, and the research on the reconstruction of the micro-environment of the tissue structure is still relatively small. According to this, the lithography mask is designed, and the patterned receiving plate is fabricated by lithography and magnetron sputtering. The patterned fiber support is obtained by using ordinary electrostatic spinning equipment. In order to simulate the structure of liver lobules and myocardial slices, the co-culture of other relevant cells, cardiac myocytes and other relevant cells in liver tissue and cardiac muscle tissue was achieved by patterning the fiber scaffold, and the viability of hepatocytes and myocardial cells was better maintained. On the basis of this, the co-cultured hepatocyte system and myocardial cell system were used as the in vitro model of drug metabolism and drug screening, which showed better correlation between in vitro and in vivo results. The patterned receiving plate is manufactured by a photoetching and magnetron sputtering process, the patterned fiber film is prepared by combining the common electrostatic spinning equipment, the method is simple, the operability is strong, the shape, the size and the arrangement mode of the pattern are controllable, and the prepared patterned fiber film groove ridge structure is obvious, the fibers of the pattern region have a higher orientation. the fibroblasts are cultured on a patterned fiber support of different sizes, the space distribution of the cells can be effectively adjusted by the support, the cells grow in a predetermined pattern area and penetrate into the interior of the fiber support, and meanwhile, the cells grow along the fiber orientation, The secreted extracellular matrix exhibits characteristics such as directional distribution. Lactose modified poly (lactic acid) was synthesized. It was found that the blend ratio of lactose modified polylactic acid/ polylactic acid-polyethylene glycol block copolymer (PELA) with a blend ratio of 5: 5 could effectively maintain the viability and function of hepatocytes, and primary hepatocytes were formed with hepatocytes with a diameter of about 60. m the primary liver cells and the fibroblasts are respectively inoculated on the patterned support of the composite fiber and the PELA fiber, and the two cells are co-cultured through the layer-by-layer bonding of the patterned fibers. In the 15-day culture, the co-cultured hepatocytes could better maintain the viability of hepatocytes, albumin secretion, urea synthesis and P450 enzyme activity, as compared to the individual culture of hepatocytes. On this basis, the primary liver cells, fibroblasts and endothelial cells were seeded in three different patterned fiber scaffolds. The growth status of the three cells is good, can secrete each characteristic protein, the liver cell ball body generates the bile canaliculus, the fibroblast permeates into the inside of the patterned fiber scaffold, and the endothelial cells show better capillary generating capacity. The activity of drug metabolism-related enzymes in the patterned co-cultured hepatocytes was detected with 7-triethoxy-4-trifluoromethyl coumarin, 7-methoxy-4-trifluoromethyl coumarin and 7-hydroxycoumarin, and CYP3A4 was found in co-cultured hepatocytes. The activity of the metabolizing enzymes of CYP2C9 and 鈪，
本文编号：2273590
[Abstract]:Most tissues in the body consist of a variety of cells, and these cells and their secreted extracellular matrix are arranged in an orderly manner in the tissue to exert specific tissue functions. With the development of tissue engineering and regenerative medicine, researchers have been committed to simulating and reconstructing these complex tissue structures in vitro, in which the patterning tissue engineering scaffold has obvious effects on regulating cell morphology, cell distribution and multi-cell co-culture. The electrostatic spinning fiber scaffold has a high specific surface area and a structure similar to that of the extracellular matrix, and has great advantages as a tissue engineering scaffold. However, the patterning technology is combined with the electro-spinning technology to build a patterned fiber scaffold, and the spatial distribution of different kinds of cells is regulated, and the research on the reconstruction of the micro-environment of the tissue structure is still relatively small. According to this, the lithography mask is designed, and the patterned receiving plate is fabricated by lithography and magnetron sputtering. The patterned fiber support is obtained by using ordinary electrostatic spinning equipment. In order to simulate the structure of liver lobules and myocardial slices, the co-culture of other relevant cells, cardiac myocytes and other relevant cells in liver tissue and cardiac muscle tissue was achieved by patterning the fiber scaffold, and the viability of hepatocytes and myocardial cells was better maintained. On the basis of this, the co-cultured hepatocyte system and myocardial cell system were used as the in vitro model of drug metabolism and drug screening, which showed better correlation between in vitro and in vivo results. The patterned receiving plate is manufactured by a photoetching and magnetron sputtering process, the patterned fiber film is prepared by combining the common electrostatic spinning equipment, the method is simple, the operability is strong, the shape, the size and the arrangement mode of the pattern are controllable, and the prepared patterned fiber film groove ridge structure is obvious, the fibers of the pattern region have a higher orientation. the fibroblasts are cultured on a patterned fiber support of different sizes, the space distribution of the cells can be effectively adjusted by the support, the cells grow in a predetermined pattern area and penetrate into the interior of the fiber support, and meanwhile, the cells grow along the fiber orientation, The secreted extracellular matrix exhibits characteristics such as directional distribution. Lactose modified poly (lactic acid) was synthesized. It was found that the blend ratio of lactose modified polylactic acid/ polylactic acid-polyethylene glycol block copolymer (PELA) with a blend ratio of 5: 5 could effectively maintain the viability and function of hepatocytes, and primary hepatocytes were formed with hepatocytes with a diameter of about 60. m the primary liver cells and the fibroblasts are respectively inoculated on the patterned support of the composite fiber and the PELA fiber, and the two cells are co-cultured through the layer-by-layer bonding of the patterned fibers. In the 15-day culture, the co-cultured hepatocytes could better maintain the viability of hepatocytes, albumin secretion, urea synthesis and P450 enzyme activity, as compared to the individual culture of hepatocytes. On this basis, the primary liver cells, fibroblasts and endothelial cells were seeded in three different patterned fiber scaffolds. The growth status of the three cells is good, can secrete each characteristic protein, the liver cell ball body generates the bile canaliculus, the fibroblast permeates into the inside of the patterned fiber scaffold, and the endothelial cells show better capillary generating capacity. The activity of drug metabolism-related enzymes in the patterned co-cultured hepatocytes was detected with 7-triethoxy-4-trifluoromethyl coumarin, 7-methoxy-4-trifluoromethyl coumarin and 7-hydroxycoumarin, and CYP3A4 was found in co-cultured hepatocytes. The activity of the metabolizing enzymes of CYP2C9 and 鈪，
本文编号：2273590
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