基于3D生物打印技术的微结构对组织工程表皮中干细胞行为的影响

发布时间：2018-05-21 05:26

本文选题：3D生物打印 + 组织工程表皮模型　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景:皮肤是机体最大的器官,因烧、创伤导致的皮肤大面积损伤会造成机体局部畸形、皮肤功能失调,内环境稳态失衡甚至危及生命。因此,针对创伤后皮肤的修复再生是临床上亟待解决的问题。基于组织工程技术制成的皮肤替代物一度成为有效的修复途径,但这种皮肤替代物缺少汗腺、皮脂腺、毛囊等皮肤附属器,无法恢复正常皮肤的生理功能。目前,针对皮脂腺、毛囊的再生研究已相对成熟,但针对汗腺再生的研究进展缓慢,有功能的再生汗腺有助于提高机体对外环境的适应能力和恢复排汗功能。表皮干细胞作为皮肤发育的祖细胞,在分化为皮肤和皮肤附属器时有显著的优势,是汗腺再生研究中首选的干细胞类型。本团队在前期已证实3D微结构可诱导表皮干细胞分化为汗腺细胞,但不同构架的3D微结构在诱导效应上有差别,这为后续基于3D生物打印技术的汗腺再生研究在结构选择上造成了的困难。因此探索不同构架的3D微结构对表皮干细胞行为的影响并建立稳定且具良好诱导效应的3D微结构是后续研究的重要基础。目的:基于3D生物打印技术探索不同构架的微结构对表皮干细胞行为学变化的影响并建立稳定且具良好诱导效应的3D微结构,为创伤后皮肤的修复与再生提供理论基础。方法:1、表皮干细胞的提取:胚胎期12.5天的GFP-C57BL/6小鼠,剪取其背部皮肤后,采用已成熟的表皮干细胞培养方法进行培养。2、足趾部真皮基质匀浆:选择出生后0.5天的野生型C57BL/6小鼠,剪取其足趾部研磨成真皮基质匀浆(PD)。3、3D生物打印:选择3种不同直径的打印喷头(210μm、340μm和420μm)构建微结构。4、表皮干细胞行为学检测:荧光显微镜和活/死细胞染色技术用于展示3D微结构中表皮干细胞的增殖能力和细胞活性,免疫荧光染色技术用于检测3D微结构中表皮干细胞分化为汗腺细胞的能力。5、统计学分析:采用方差分析和样本t检验等方法进行统计学分析。结果:1、三种不同构架的3D微结构均可促进小鼠表皮干细胞的有效增殖。2、在各个观察时间点上,210μm组3D微结构中的表皮干细胞活性最低,420μm组的细胞活性最高,细胞活性与剪切应力呈反比。3、三种不同构架的3D微结构均可诱导小鼠表皮干细胞分化为汗腺细胞。4、340μm组3D微结构中表皮干细胞可聚集形成类腺样球形结构。结论:本次研究构建的340μm组3D微结构具有最佳的诱导效应。这为后续基于3D生物打印技术的汗腺再生研究以及进一步构建具有生物相容性的组织工程表皮模型奠定了基础。
[Abstract]:Background: skin is the largest organ of the body. Extensive skin damage caused by burn and trauma can cause local malformation, skin dysfunction, homeostasis and even life-threatening. Therefore, the repair and regeneration of post-traumatic skin is an urgent problem to be solved. The skin substitute based on tissue engineering technology was once an effective way to repair the skin, but the skin substitute lacked sweat glands, sebaceous glands, hair follicles and other skin appendages, which could not restore normal skin physiological function. At present, the study of hair follicle regeneration has been relatively mature, but the research on sweat gland regeneration is slow. Functional regenerated sweat gland is helpful to improve the adaptability of the body to external environment and restore the function of sweat excretion. As progenitor cells of skin development, epidermal stem cells have significant advantages in differentiation into skin and skin appendages, and are the preferred stem cell types in the study of sweat gland regeneration. Our team has previously demonstrated that 3D microstructures can induce epidermal stem cells to differentiate into sweat gland cells, but 3D microstructures with different structures have different induction effects. This makes it difficult to select the structure of sweat gland regeneration based on 3D biprint technology. Therefore, it is important to explore the effects of 3D microstructures of different structures on the behavior of epidermal stem cells and to establish stable and well-induced 3D microstructures. Aim: to explore the effects of different structures on the behavioral changes of epidermal stem cells based on 3D biprint technology and to establish a stable and well-induced 3D microstructure which can provide a theoretical basis for the repair and regeneration of post-traumatic skin. Methods: 1. Extraction of epidermal stem cells: GFP-C57BL/6 mice of 12.5 days of embryonic stage were cut off and cultured with mature epidermal stem cells culture method. The dermal matrix homogenate of toe was selected as wild-type C57BL/6 mice 0.5 days after birth. Trimming the toes into dermis matrix homogenate PD-3N 3D biographic printing: selection of three different diameters of print sprayers 210 渭 m, 340 渭 m and 420 渭 m) to construct microstructures .4. Epidermal stem cell behavioral detection: fluorescent microscopy and live / dead cell staining techniques To demonstrate the proliferation and cell activity of epidermal stem cells in 3D microstructures, Immunofluorescence staining was used to detect the ability of epidermal stem cells to differentiate into sweat gland cells in 3D microstructures. Statistical analysis: variance analysis and t-test were used to analyze the ability of differentiation of epidermal stem cells into sweat gland cells. Results three kinds of 3D microstructures with different structures could promote the effective proliferation of mouse epidermal stem cells. In each observation time, the activity of epidermal stem cells was the lowest in 210 渭 m group and the highest in 420 渭 m group. The cell activity was inversely proportional to the shear stress, and three kinds of 3D microstructures with different structures could induce the differentiation of mouse epidermal stem cells into sweat gland cells. 4340 渭 m group could aggregate the epidermal stem cells to form glandular globular structures. Conclusion: the 340 渭 m group of 3D microstructures constructed in this study has the best inductive effect. This will lay a foundation for the further study of sweat gland regeneration based on 3D biprint technology and the further construction of biocompatible tissue engineering epidermis model.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R62

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