干细胞体外培养微环境对肌腱分化及修复作用的研究

发布时间：2018-05-25 21:44

本文选题：肌腱组织工程 + 诱导多能干细胞　；参考：《浙江大学》2017年博士论文

【摘要】：肌腱韧带损伤占运动损伤的50%,但其自我修复能力差,严重影响患者的生活质量,是骨科和运动医学治疗中公认的难题。目前,临床上对肌腱损伤的治疗手段主要包括理疗、手术缝合以及组织移植,然而修复后肌腱的组织形态和功能特性与正常肌腱相差甚远,且力学性能差,伴随肌腱粘连、疤痕组织形成。因此,肌腱再生修复新手段亟待开发。近年,随着干细胞与组织工程的发展,组织修复进入了再生的新阶段。然而,肌腱组织工程应用仍需要解决三个关键问题:1)明确促进干细胞向肌腱系分化的物理环境;2)找到体外种子细胞表型维持及肌腱系分化的调控手段;3)构建生物诱导活性的三维支架。因此,本论文针对待解决问题,以诱导多能干细胞为模型,开展了成体系的研究,旨在促进肌腱修复和再生。本研究发现:(1)程序性替换基质物理特性能将诱导多能干细胞分阶段定向分化为肌腱细胞。体外将iPSCs从MEF饲养层转移到光滑平面可以诱导iPSC分化为间充质干细胞样多能细胞(iPSC-MSCs),该细胞在克隆形成、三系分化和表面标志物方面与MSCs极为类似。随后,将iPSC-MSCs种植在平行纳米支架上可成功被诱导向肌腱细胞分化,表达肌腱特异性基因。进一步分析发现平行支架可以通过integrin介导的力学通路促进iPSC-MSCs体外肌腱系分化并抑制其骨系分化。大鼠原位跟腱修复实验证明平行纳米支架复合iPSC-MSCs构建的组织工程肌腱对肌腱损伤修复具有促进作用。(2)在追踪肌腱干细胞体外培养表型丢失的过程中发现肌腱干细胞体外培养存在连续的去分化和失功能过程,组蛋白去乙酰化在此过程中发挥了作用。在P3及以前通过HDAC(histonedeacetylase)抑制剂TSA或VPA可以显著逆转Sex表达并维持肌腱干细胞的表型和功能,且此效果通过提高细胞中组蛋白乙酰化水平及抑制HDAC1/3表达起作用。(3)结合以上结果构建HDAC小分子抑制剂修饰的平行纤维支架,发现该复合支架可协同调控细胞组蛋白乙酰化水平,促进肌腱干细胞腱系分化与成熟,促进肌腱再生。本研究的成果和发现为肌腱组织工程的种子细胞来源、质量和诱导性生物材料提供了新的理论依据和技术手段,为肌腱损伤治疗提供了新的种子细胞和支架。
[Abstract]:Tendon ligament injury accounts for 50% of sports injuries, but its self-repair ability is poor, which seriously affects the quality of life of patients. It is a recognized problem in orthopedics and sports medicine treatment. At present, the clinical treatment of tendon injury mainly includes physiotherapy, surgical suture and tissue transplantation. However, the tissue morphology and functional characteristics of the repaired tendon are very different from the normal tendon, and the mechanical properties of the tendon are poor, accompanied by tendon adhesion. Scar tissue is formed. Therefore, tendon regeneration and repair of new means need to be developed. In recent years, with the development of stem cells and tissue engineering, tissue repair has entered a new stage of regeneration. However, The application of tendon tissue engineering still needs to solve three key questions: 1) the physical environment to promote the differentiation of stem cells into tendon system clearly.) to find out the regulation of seed cell phenotype maintenance and tendon differentiation in vitro and to construct a three-dimensional scaffold for biological induction activity. Therefore, in order to promote tendon repair and regeneration, a systemic study was carried out on the model of induced pluripotent stem cells. It was found that the physical properties of the induced pluripotent stem cells could be differentiated into tendon cells in stages. In vitro transfer of iPSCs from MEF feeder layer to smooth plane could induce iPSC to differentiate into mesenchymal stem cell-like multipotent cells (iPSC-MSCs), which were very similar to MSCs in clone formation, three-line differentiation and surface markers. Subsequently, iPSC-MSCs implanted on a parallel nano-scaffold could be successfully induced to differentiate into tendon cells and express tendon-specific genes. Furthermore, it was found that parallel scaffolds could promote the differentiation of tendon system and inhibit the differentiation of bone system of iPSC-MSCs through the mechanical pathway mediated by integrin. In situ Achilles tendon repair experiment in Rats demonstrated that the tissue engineered tendon constructed by parallel nano-scaffold combined with iPSC-MSCs could promote tendon repair. (2) the thin tendon stem was found in the process of tracking the phenotype loss of tendon stem cells cultured in vitro. Cell culture in vitro has a continuous process of dedifferentiation and defunction. Histone deacetylation plays a role in this process. The expression of Sex and the maintenance of phenotype and function of tendon stem cells were significantly reversed by TSA or VPA at P3 and before by HDA histonede acetylase inhibitor. By increasing the level of histone acetylation and inhibiting the expression of HDAC1/3, we constructed the parallel fiber scaffold modified by HDAC small molecule inhibitor. It was found that the composite scaffold could regulate the level of histone acetylation in combination with the above results. Promote the differentiation and maturation of tendon stem cells and promote tendon regeneration. The results and findings of this study provide a new theoretical basis and technical means for the source, quality and inductive biomaterials of tendon tissue engineering, and provide new seed cells and scaffolds for the treatment of tendon injury.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R873;R318.08

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