【作者】 张文君；

【导师】 王常勇；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目前,慢性阻塞性肺疾病(COPD)、哮喘以及肺纤维化(PF)都已成为高死亡率的肺脏疾病。伴随着气候和环境的改变,人类现在面临的肺脏疾病呈明显增长的趋势。近半个世纪以来,随着人们对于肺脏的基础理论研究的不断深入,以及针对一些肺脏疾病发病机理研究所取得的积极进展,氧气疗法、手术治疗和肺脏疾病康复疗法等治疗手段有了长足进步。尽管如此,肺脏移植仍是肺脏疾病晚期的主要治疗手段。然而,由于缺少肺脏器官来源,目前很多患者根本无法得到有效治疗。寻找新的治疗方法和手段业已成为肺脏疾病治疗的当务之急。组织工程技术的不断发展已为肺脏疾病的治疗带来新的希望。组织工程是融合了生命科学、材料科学以及工程科学的原理和方法,旨在再造人体组织器官的新兴交叉学科。近年来,组织工程研究先后在皮肤、软骨、神经、血管及心肌组织再造中取得了突破性进展,并有部分组织工程产品进入临床应用。肺脏具有通气和换气功能,肺实质为肺内支气管的各级分支和其末端的肺泡。近年来肺脏组织工程研究进展迅速,先后有研究人员成功在体外再造了组织工程化肺泡和支气管粘膜。尽管如此,针对肺脏体外再造,仍存在如何优选细胞支架材料,如何优化细胞-支架复合体体外培育环境以及进一步提高再造肺脏组织质量等一系列问题。本论文采用组织工程技术开展了肺泡和支气管粘膜体外再造研究。其中,在肺泡体外再造方面,以小鼠胚胎期肺脏细胞(FPCs)为种子细胞,利用胶原/Matrigel为细胞提供三维立体生长环境,并针对目前国际上尚无有效手段控制体外再造肺泡结构现状,首次应用海藻酸钠(APA)微囊发挥空间占位作用,通过APA微囊控制肺泡样结构的形成,成功在体外再造了肺泡样结构,取得了预期结果。在支气管粘膜体外再造方面,以胶原/Matrigel为胚胎期肺脏成纤维细胞和支气管上皮细胞支架,成功在体外再造了支气管粘膜样结构,并研究了Matrigel对人支气管上皮细胞再生的影响。目前,针对Matrigel对支气管上皮细胞再生的影响尚未见报道。本论文基于肺脏体外再造所需支架(APA微囊)以及肺脏再造不同结构(肺泡及支气管粘膜样结构),主要研究内容共分三个部分,概括介绍如下:第一部分:制备海藻酸钠(APA)微囊及其物理与生物学性能的评价微囊是一种可以形成免疫隔离屏障的半透膜结构,它的物理和生物学特性通过控制制备过程中的物理参数可以加以调控。本研究的目的是,在已有的研究基础上,制备出适合用于肺泡样结构再造中保持肺泡大小与形状的APA微囊,并对其物理和生物学特性进行评估。我们在已有的研究基础上,选定以下参数制备微囊:静电场为6kV/1.5cm,泵推进速度为15mL/h,针头内径为0.09mm,海藻酸钠浓度为2%,多聚赖氨酸浓度为0.05%,作用时间为6min,制备粒径为200-300μm。人的肺泡平均直径在250μm左右。因此在这些参数控制下,制备的微囊更适宜用于肺泡样结构的构建。在物理性能评价方面,研究结果表明,APA微囊具有较好的机械强度;制备的APA微囊可以使4kD的小分子物质自由扩散进微囊内,70kD的中等分子物质可部分通过,而150kD的大分子物质不能或大部分不能通过微囊膜。在生物学性能方面,观察APA微囊腹腔移植后的形态表明,其具有较好的生物相容性。在本部分研究中,研制了APA微囊,掌握了APA微囊研制的关键技术,摸索了微囊研制的系列条件,并对其物理和生物学性能进行评价。研究发现,研制的APA微囊大小适用于肺泡样结构构建研究,并且具有良好生物相容性。第二部分:以胶原/Matrigel-海藻酸钠微囊为支架材料体外构建组织工程化肺泡样结构的实验研究对于远端肺脏组织的构建,肺泡样结构的实现至关重要。本研究的目的是以FPCs为种子细胞,采用胶原/Matrigel为细胞提供三维生长环境,采用APA微囊这种材料来发挥空间占位作用,进而通过改变制备过程的物理参数来控制肺泡大小和形状,从而更好地实现组织工程化结构的构建。大体观察发现,FPCs与胶原/Matrigel-APA微囊复合可以形成组织片层,随着培养时间的延长,片层逐渐收缩;培养14d后观察发现片层收缩更加明显,继续培养至21d,最终形成蝴蝶状外观。倒置显微镜下观察,发现在三维支架材料中FPCs在微囊周围生长,APA微囊保持良好的完整性。H.E.染色结果显示,体外构建7d和14d的组织片层中的囊泡样结构和小鼠正常肺脏的组织结构很相似。通过免疫组织化学染色可以看出,在体外培养7d和14d的肺脏组织片层中,均有广谱细胞角蛋白(pan-CK),波形蛋白(Vimentin)和细胞表面活性物质C(SpC)阳性表达。通过透射电子显微镜观察体外培养7d和14d的组织片层可以观察到板层小体,表明Ⅱ型肺泡细胞在囊泡状空间结构的环境下能够保持未分化状态。扫描电子显微镜观察发现肺脏组织片层纵剖面有球形中空的囊状结构。本部分研究,成功在体外构建了肺泡样结构,使微囊发挥空间占位作用,并能够保持Ⅱ型肺泡细胞的未分化状态。第三部分:以胶原/Matrigel为支架材料体外构建组织工程化支气管粘膜的实验研究支气管粘膜在肺脏导气部发挥着重要的作用。我们采用支气管上皮细胞(Beas-2B)和胚胎期肺脏成纤维细胞(MRC-5)为种子细胞,以胶原/Matrigel为支架材料,应用气液界面培养的方法体外构建支气管粘膜。其中,上皮细胞的接种位于成纤维细胞与胶原/Matrigel混合并凝胶化之后。在研究中,还通过是否在接种上皮细胞之前添加Matrigel的方式,来探讨了Matrigel对上皮细胞再生的影响。倒置显微镜下观察,发现构建的支气管粘膜表面有支气管上皮细胞的聚集、融合,其下层成纤维细胞在胶原/Matrigel支架材料中,可以相互连接并形成网络样结构。H.E.染色结果表明,在成纤维细胞和胶原/Matrigel复合后,如果添加Matrigel后再接种上皮细胞层,可以形成表面完整的上皮细胞层。相反,没有添加Matrigel的支气管粘膜上皮细胞层不完整。免疫组织化学染色结果显示,添加Matrigel的支气管粘膜pan-CK阳性表达细胞较多,说明Matrigel可以促进气道上皮细胞的生长;没有添加Matrigel的支气管粘膜pan-CK阳性表达细胞较少。添加Matrigel对成纤维细胞的生长基本没有影响。扫描电子显微镜观察体外构建的支气管粘膜,发现添加Matrigel后,有纤毛样结构的形成,说明Matrigel可以促进气道上皮细胞的进一步分化。本部分研究,在体外成功构建了支气管粘膜,并证实Matrigel可以促进气道上皮细胞的再生。综上所述,本研究中,在支架研制方面,掌握了APA微囊研制的关键技术,摸索了微囊研制的系列条件,成功制备了适用于肺泡样结构构建的APA微囊,且具有良好的物理性能和生物相容性。在肺泡构建方面,以FPCs为种子细胞,胶原/Matrigel为支架材料,充分发挥APA微囊的空间占位作用,在体外构建了肺泡样结构,其中,通过调节微囊的大小和形状可以有效控制再造肺泡样结构中肺泡的大小和形状,实现了对肺泡样结构的初步控制。在支气管粘膜构建方面,以Beas-2B和MRC-5为种子细胞,胶原/Matrigel为支架材料,在体外构建了支气管粘膜,并证实Matrigel可以影响气道上皮细胞的再生。组织工程化肺脏的构建,可以为肺脏疾病治疗提供新的技术和手段,此外,上述再造组织还可以作为肺脏疾病研究模型或者治疗肺脏疾病药物筛选、毒性检测的模型。更多还原

【Abstract】 Currently, chronic obstructive pulmonary disease (COPD), Asthma and pulmonary fibrosis (PF) have become lung disease with high mortality. Accompanied by changes in climate and environment, human lung diseases were now facing significant growth trend. Nearly half a century, as people’s basic theory for the study of lung deepening, as well as the launch mechanism of lung disease, oxygen therapy, surgery and rehabilitation treatment of lung disease has progressed. Nevertheless, lung transplantation remains the treatment of advanced lung disease. However, the lack of lung organ sources, many patients simply can not currently be effectively treated. Find new ways and means to treat lung diseases have become a top priority. The continuous development of tissue engineering has been a new hope for the treatment of lung disease.Tissue engineering to reconstruct human tissue and organ is an interdisciplinary that integrates life science, materials science and engineering principles. In recent years, tissue engineering has made breakthrough on the skin, cartilage, nerves, blood vessels and heart tissue. In the meantime, some products of tissue engineering have been put into the clinical application.Pulmonary parenchyma consists of the lung bronchial and its terminal branches at all levels of alveolar. Lung tissue engineering has a rapid development in recent years. Researchers have successfully reconstructed tissue engineered alveolar and bronchial mucosa in vitro. Nevertheless, for the reconstruction of lung in vitro, there are still many problems, such as how to choose cell scaffold material, how to optimize the cell-scaffold nurturing environment in vitro and to further improve the quality of the reconstructed lung tissue or other issues and so on.This paper carried out reconstruction of the alveolar and bronchial mucosa in vitro using tissue engineering. About reconstructing alveolar in vitro, we used mouse fetal pulmonary cells (FPCs) as seeding cells and collagen/Matrigel provided three dimensional growth environments for cells. And for now there is no effective means to control the reconstruction of alveolar structures in vitro situation. We present the first application of alginate-poly-L-lysine-alginate (APA) microcapsules to play the role of space occupying and control the formation of alveolus-like structures. In the meantime, we successful reconstructed alveolus-like structures in vitro, which achieved expected results. About engineered bronchial mucosa, we used collagen/Matrigel as embryonic lung fibroblasts and bronchial epithelial cells as scaffolds. We successful reconstructed the bronchial mucosa-like structure in vitro and studied regeneration of human bronchial epithelial cells with Matrigel. At present, there is no report on regeneration of the bronchial epithelial cells with Matrigel.This paper based on scaffold of reconstructed lung tissue in vitro (APA microcapsules), and different structures of reconstructed lung tissue (alveolar and bronchial mucosa-like structures), the main research contents contained three parts, outlined as follows:Part I: Preparation of APA microcapsules and evaluation of physical & biological propertiesMicrocapsules can be formed as a semipermeable membrane immunoisolation barrier structure; its physical and biological characteristics of the preparation process by controlling the physical parameters can be changed. The purpose of our research was to prepare APA microcapsules used to reconstruct alveolus-like structure, and assess their physical and biological characteristics. We used electrostatic field was 6kV/1.5cm, the speed of the pump was 15mL/h, needle inner diameter was 0.09mm, the concentration of sodium alginate was 2%, concentration of poly-lysine was 0.05%, the reaction time was 6min, diameter of APA microcapsules were 200-300μm. The average diameter of human alveolar about 250μm. Therefore, under the controlling of these parameters is more suitable for preparation of microcapsules for reconstructing alveolus-like structure.In the physical performance evaluation, the results show that APA microcapsules had good mechanical strength. About microcapsule permeability, we prepared APA microcapsules can 4kD free diffusion of small molecules into the microcapsules, 70kD middle molecular substances can be some passed, and 150kD macromolecular material can not be or most can not be encapsulated membrane. Performance in biology observation of APA microcapsules form after abdominal transplantation showed good biocompatibility.In this part of the study, we prepared the APA microcapsules, mastered the key technologies of developing APA microcapsules, and groped a series of conditions developing APA microcapsules, and its physical and biological properties were evaluated. We found that the size of the prepared APA microcapsules was suitable for construction of alveolar structure, and had good biocompatibility. Part II: Study of reconstructing tissue engineered alveolus-like structures in vitro used collagen/Matrigel-APA microcapsules as scaffoldsConstruction of the distal lung tissue, alveolar-like structure to achieve is important. The purpose of this study is to use FPCs as seeding cells, using microcapsules play a space occupying effect and prepare biological materials and then by changing the preparation process of the physical parameters to control the size and shape of the alveolar to achieve the construction of tissue-engineered structures well.Generally observed, FPCs and collagen/Matrigel-APA microcapsules can be formed tissue sheets, and then the sheets were gradually shrinking. Observed 14 day-cultured significantly shrink and continued to train to 21 days. We could observe the final formation of butterfly-like appearance. Under inverted microscope, we found that cells in three-dimensional scaffold materials formed vesicle-like structure. APA microcapsules maintained good integrity, the seed cells grew around the microcapsules. H.E. staining showed that the vesicle-like structures of 7 day-cultured and 14 day-cultured sheets were very similar to the structure of normal lung tissue in mice. By immunohistochemical staining pan-CK, Vimentin, and SpC-positive cells could be seen in 7 day-cultured and 14 day-cultured sheets. Observed by transmission electron microscopy lamellar bodies gained in 7 day-cultured and 14 day-cultured sheets, indicating typeⅡalveolar cells in alveolus-like structures maintained the undifferentiated state. Scanning electron microscope found that the longitudinal section of lung tissue sheets had cystic structure.This part of the study, successfully constructed the alveolar structures in vitro, microcapsules played space occupying effect, and typeⅡalveolar cells could maintain the undifferentiated state.Part III: Study of reconstructing tissue engineered bronchial mucosa in vitro used collagen/Matrigel as scaffolds Bronchial mucosa in the lungs plays an important role in the department. Weintend to use human embryonic lung fibroblast cells (MRC-5) and human bronchial epithelial cells (Beas-2B) as seed cells as well as employed collagen/Matrigel as scaffolds, meanwhile applied air-liquid interface culture method to reconstruct airway mucosa in vitro. We also want to study the effect of Matrigel on differentiation of epithelial cells. Among them, the epithelial cells inoculated on the fibroblasts after collagen/Matrigel mixture gelation. In the study, also by Matrigel was added before epithelial cells inoculation way to explore the effect of Matrigel on epithelial cell regeneration.Under inverted microscope, we found that fibroblasts in collagen/Matrigel scaffolds formed a network like structure in collagen/Matrigel scaffold and built bronchial mucosal surface of airway epithelial cell fusion. H.E. staining showed that in Beas-2B cells formed intact epithelial cell layer under the surface of Matrigel was added. On the contrary, not dropping Matrigel, airway epithelial cell layer was incomplete. Immunohistochemical staining showed that Matrigel under the dropping of bronchial mucosa pan-CK positive cells were more shows that Matrigel promoted the growth of epithelial cells; without dropping Matrigel, pan-CK positive cells were few. Dropping Matrigel under the Beas-2B cells had no effect on the growth of fibroblasts. Scanning electron microscopy of bronchial mucosa in vitro found that dropping Matrigel under the Beas-2B cells had ciliated-like structure formation, which indicated Matrigel can promote epithelial cell differentiation.This part of the study, reconstructed the bronchial mucosa in vitro successfully, and confirmed that the Matrigel can promote epithelial cell regeneration.In summary, this study, about scaffolds development, we mastered key technologies developed APA microcapsules, and explore a series of development conditions for microcapsules, successfully prepared APA microcapsules for the alveolar structure, and with good physical properties and biocompatibility. Construction of the alveolar, we used FPCs as seeding cells, collagen/Matrigel as scaffolds. And APA microcapsules played full role of space occupying in vitro to construct the alveolar structure. By regulating the size and shape of microcapsules can effectively control size and shape of alveoli in alveolus-like structure. We achieved initial control of alveolar structure. About bronchial mucosa, we took Beas-2B and MRC-5 as seeding cells, collagen/Matrigel as scaffolds and successfully reconstructed the bronchial mucosa in vitro, and confirmed that Matrigel can influence bronchial epithelial cell regeneration.Construction of the engineered lung tissue supply new technologies and means for treatment of lung diseases. In addition, above mentioned engineered lung tissue also can serve as a model of lung disease or the treatment of lung disease research, drug screening and toxicity testing.更多还原