强力学仿生细胞外基质纳米纤维支架介导DCN shRNA长效转染ASCs的肌腱缺损修复研究

Tendon defect is a common clinical disease. And tissue engineered tendon has become the ideal choice for the regeneration and repair of the damaged tendon. However, tendon tissue engineering still has many problems to be solved: limited source of seed cells, lack of scaffolds whose degradation rate matching the cell proliferation, healed tendon with the absence of large diameter collagen fibers and low mechanical properties, etc. In this project, we intent to solve these problems by the following researches: preparation of chitosan nanocapsules with the plasmid vector of Decorin shRNA, preparation of three dimensional fibrous scaffolds with the chitosan nanocapsules and silk fibroin by electrospinning, and construction of tissue engineered tendon by planting the adiopse stem cells on the scaffold and stimulating under sustained tensile force, regeneration and repair of tendon defects by transplantation of tissue engineered tendon into the rabbit Achilles tendon defect. Through these researches, we hope this three dimensional fibrous scaffold could not only provide enough mechanical strength during the tendon repairing, but also provide a extracellular matrix-like microenvironment to ASCs. And with the degradation of the scaffold, chitosan nanocapsules with Decorin shRNA vector will be released and transected into the neonatal tendon cells. In the last, the expression of DCN was inhibited and the large diameter collagen fibers were formed. And the functional regeneration and repair of injuried tendon were realized.

肌腱缺损是临床常见疾病，组织工程肌腱已成为临床肌腱缺损修复的理想选择。但目前组织工程肌腱尚存在以下不足：种子细胞来源受限、缺乏降解速率与细胞增殖相匹配的强力学生物支架、修复后肌腱因缺少大直径胶原纤维而导致力学性能低下等根本性问题。为此，我们拟将核心蛋白聚糖（Decorin）shRNA质粒包裹于壳聚糖纳米微囊中，以高压静电纺丝技术混纺壳聚糖纳米微囊-丝素蛋白制备强力学三维纳米纤维支架，并将脂肪干细胞（ASCs）种植于该支架，在持续拉伸力刺激下构建组织工程肌腱；然后将组织工程肌腱植入肌腱缺损动物模型进行肌腱缺损修复，以期在肌腱再生修复过程中，该支架不仅能提供相匹配的力学强度，为ASCs向肌腱细胞分化提供仿生细胞外基质的生长微环境，而且随着支架材料逐步降解，释放出的壳聚糖纳米微囊携带DCN shRNA进入新生肌腱细胞，抑制decorin表达，促进大直径胶原纤维形成，从而实现肌腱的功能性再生修复。

韧带/肌腱损伤是困扰运动训练和人们日常生活的常见损伤，占运动性损伤的50%以上。但是目前临床治疗效果有限，愈后组织以增生性纤维疤痕为主，使其生化特性和力学性能明显低于正常组织，常导致重复断裂。基因治疗与组织工程技术在韧带/肌腱损伤修复研究中的运用虽取得一定效果，但尚存在以下不足：种子细胞来源受限，缺乏降解速率与细胞增殖相匹配的强力学生物支架，修复后组织因缺少大直径胶原纤维而导致力学性能低下，以及基因治疗中病毒载体的潜在致癌风险与非病毒载体靶向性差等。.为此，在本项目中我们首先通过高压静电纺丝技术制备出丝素蛋白-壳聚糖（pGPU6/GFP/Neo）-胶原三维纳米纤维组织工程支架。该组织工程支架经检测具有良好的生物相容性和细胞粘附性，能够为细胞提供良好的类似于细胞外基质的生长微环境。此外，该支架在自然缓慢降解过程中，能够释放出携带核酸的壳聚糖-核酸纳米微囊，而壳聚糖-核酸纳米微囊可以将其携带的核酸导入到细胞中。因此，该组织工程支架还可以用作基因治疗的载体，发挥局部靶向、时间可控的基因治疗作用。但是，丝素蛋白经溶解高压静电纺丝力学性能降低，导致丝素蛋白-壳聚糖（pGPU6/GFP/Neo）-胶原三维纳米纤维复合支架的力学性能欠佳。其次，为了更好地通过分子调控手段促进韧带/肌腱的无疤痕再生修复，我们利用NGS技术，分析挖掘了不同胚胎发育期肌腱无疤痕再生修复的关键性基因，结果发现：E17.5与E14.5胚胎期肌腱组织间存在明显差异表达基因有377个，其中下调表达273个基因，上调表达104个。进一步通过GO和KEGG途径分析得知，这些差异表达基因中有超过120个以上直接参与细胞外基质形成，参与细胞外基质结合功能的基因有7个，参与细胞外基质组分弹性传输的基因有2个。最后，在项目执行过程中我们还分离培养了脂肪来源干细胞，发现其细胞表型与骨髓来源间充质干细胞极为接近，有很强的同源性与多向分化能力，是韧带/肌腱组织工程理想的种子细胞之一。