基于自组装纳米多肽及脂肪间充质干细胞3D打印组织模型的研究

发布时间：2018-05-19 21:09

本文选题：3D生物打印 + 功能性自组装纳米多肽　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景我们可以在多种组织中找到间充质干细胞,它是一种拥有多向分化潜能的多功能干细胞,其具有容易分离扩增、多向分化和低免疫原性等特性。研究发现脂肪来源的间充质干细胞(Ad-MSCs)相对于其他干细胞,具有来源充足,取材方便,低创即得,易分离培养,生物学性状稳定等特征,因此研究者普遍认为其在组织工程学和再生医学中是一种更为理想的种子细胞。构成组织工程学支架的材料需要具备良好的生物相容性,较强可塑性,抗张强度大和无免疫原性等特点。目前制备支架的常用材料包括胶原蛋白酶、聚乳酸、海藻酸盐等材料。但这些生物材料都其相应的局限性,现实实验条件下对这些生物材料进行可控研究难度较大。近年来,以多肽为基本组成结构单元,依靠纳米多肽分子自组装得到的纳米组织工程学框架材料已成为组织工程领域新的研究热点,这类纳米多肽生物材料具有良好的生物兼容性、降解性,且最为突出的优点是其具有生物活性。随着3D打印技术的兴起和快速发展,3D打印已开始广泛应用于医学领域,为精准化、个性化医疗提供条件,现阶段对组织工程学高精密度打印也已进入深入的探索和研究。以自组装多肽纳米溶液负载Ad-MSCs为"生物墨汁"利用3D生物打印技术打印出三维结构,通过体外培养3D打印模型内的Ad-MSCs,并给予不同诱导分化条件,观察其内细胞的生长状态及分化程度,研究功能性自组装纳米多肽作为负载Ad-MSCs的3D打印材料的可行性,探寻3D打印新的发展方向。研究目的本实验岂在研究以自组装纳米多肽与人脂肪间充质干细胞(Ad-MSCs)为"生物墨汁",利用3D打印技术,制造组织工程学模型,观察其内细胞生长状态及多向分化能力。希望借此找到新的组织工程学材料。研究方法1.分离、培养原代Ad-MSCs,传代至P2、P3后用于后续实验;使用流式细胞仪对细胞进行细胞免疫表型鉴定并软件分析;对细胞进行鬼笔环肽染色,观察细胞微丝形态;对细胞分别成内皮、成骨、成脂诱导分化,并进行鉴定。2.用10%蔗糖-去离子水溶液溶解三种多肽(RADA16-I、RGD、KLT),分别按所需浓度和体积比混合并原子力显微镜(Olympus公司)观察;将混合溶液放入Transwell成胶,成胶后进行扫描电镜观察;将三种多肽与细胞混合并成胶后进行培养,并观察多肽内细胞形态和生长状态。3.以自组装纳米多肽、Ad-MSCs混合溶液为"生物墨汁"利用3D生物打印技术打印组织工程学模型:打印后切片进行鬼笔环肽荧光染色并观察;诱导3D模型中的Ad-MSCs成骨和成脂分化,诱导完成后分别进行茜素红S、油红0染色,并与对比组进行染色比较。研究结果1.倒置相差显微镜下可见Ad-MSCs为梭形,整体呈现出漩涡状生长趋势;流式细胞仪鉴定可见分离、培养的细胞CD166,CD90,CD29表达阳性,不表达CD31、CD34、CD45和HLA-DR,符合Ad-MSCs免疫表型。鬼笔环肽染色后可见细胞内微丝结构;成骨、成脂诱导分化后可见细胞内出现相关染色剂着色现象。2.原子力显微镜观察纳米多肽溶液,可见其内有纳米纤维结构;纳米多肽水凝胶无色透明,含水量丰富;扫描电镜观察水凝胶,见其内纳米纤维结构并有孔隙;细胞与水凝胶混合成胶并培养后观察可见其内细胞排列紧密,生长状态良好。3.利用"生物墨汁"打印出组织工程模型,切片染色后可见其内的细胞生长状态良好,贴壁紧密。对模型内的Ad-MSCs进行诱导分化,并分别对实验组和对照组进行相关染色,茜素红S染色可见成骨诱导实验组中钙结节形成,油红0染色可见成脂诱导实验组Ad-MSCs内脂滴着色,两对照组不着色。结论以自组装纳米多肽、脂肪间充质干细胞混合溶液为"生物墨汁" 3D打印出的组织工程学模型内Ad-MSCs生长状态良好,仍具有较强的分化能力。
[Abstract]:Research background we can find mesenchymal stem cells in a variety of tissues. It is a multifunctional stem cell with multidirectional differentiation potential. It has the characteristics of easy separation and amplification, multidifferentiation and low immunogenicity. It is found that fat derived mesenchymal stem cells (Ad-MSCs) are abundant in source and are obtained from other stem cells. It is widely believed that it is a more ideal seed cell in tissue engineering and regenerative medicine. The material of tissue engineering scaffold needs good biocompatibility, strong plasticity, strong tensile strength, and no immunogenicity. The commonly used materials for the preparation of scaffolds include collagenase, polylactic acid, alginate and other materials. However, these biomaterials have their respective limitations. Under the actual experimental conditions, it is difficult to study these biomaterials. In recent years, polypeptides are the basic structure units and self-assembled by nanoscale peptides. Nanoscale tissue engineering frame material has become a new research hotspot in the field of tissue engineering. This kind of nano peptide biomaterial has good biocompatibility, degradability and the most outstanding advantage is its biological activity. With the rise and rapid development of 3D printing technology, 3D printing has been widely used in the medical field and is accurate. At the present stage, the high precision printing of tissue engineering has also been deeply explored and studied. With the self assembled polypeptide nano solution load Ad-MSCs as "biological ink", the three-dimensional structure is printed by 3D biologic printing technology, and the Ad-MSCs in the 3D printing mold type is cultured in vitro, and the different inducible differentiation strips are given. To observe the growth state and degree of differentiation of the cells in the Ad-MSCs, the feasibility of functional self-assembled nano peptide as the 3D printing material of the load is studied, and the new development direction of 3D printing is explored. The purpose of this study is to study the use of 3D to print the self assembled nano peptides and human fat mesenchymal stem cells (Ad-MSCs) as "biological ink" and to print the 3D Technology, make tissue engineering model, observe the cell growth state and multidirectional differentiation ability. Hope to find new tissue engineering materials. Study method 1. isolation, culture of original Ad-MSCs, generation to P2, P3 for follow-up experiment; use flow cytometry to identify cell immunophenotype and software analysis; The morphology of cell microfilament was observed by PHA, and the cells were divided into endothelium, bone and fat induced differentiation, and three kinds of polypeptides (RADA16-I, RGD, KLT) were dissolved in 10% sucrose deionized solution (RGD, KLT). The mixed solution was observed by the mixture of the required concentration and volume ratio and the atomic force microscope (Olympus company), and the mixed solution was put into Transwell. The glue was observed by scanning electron microscope. The three kinds of peptides were mixed with cells and cultured, and the morphology and growth state of the cells in the polypeptide were observed and the.3. was self-assembled. The Ad-MSCs mixed solution was used as "biological ink" to print the tissue engineering model using 3D biologic printing technology. Color and observation; induced Ad-MSCs osteogenesis and lipid differentiation in the 3D model. After the induction, alizarin red S and oil red 0 were stained respectively. The results were compared with those in the contrast group. The results of the 1. inverted phase contrast microscope showed that Ad-MSCs was spindle shape, and the overall trend was swirling growth trend. Flow cytometry identified the visible separation and culture of cell CD16. 6, CD90, CD29 were positive, and did not express CD31, CD34, CD45 and HLA-DR, which conformed to the immunophenotype of Ad-MSCs. The peptide hydrogels were colorless and transparent and rich in water content; the hydrogels were observed by scanning electron microscopy, and the structure and pore of nanofibers in the gel were observed. The cells and hydrogels were mixed into glue and cultured to observe that the cells arranged closely and the growth state of.3. was printed out of the tissue engineering model using "biological ink". The cell growth was visible after the slice staining. The Ad-MSCs in the model was induced and differentiated, and the experimental group and the control group were stained, and the alizarin red S staining showed the formation of calcium nodules in the experimental group of osteogenesis induced, the oil red 0 staining showed that the lipid droplet in the lipid induced experimental group was stained in Ad-MSCs, and the two control group did not stain. Adipose tissue derived mesenchymal stem cells (MSCs) mixed solution was printed in the "biological ink" 3D tissue engineering model, Ad-MSCs grew well, and still had strong differentiation ability.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;TP391.73

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