miR-675促进脂肪干细胞构建表型稳定的可注射性工程软骨修复喉软骨缺损的作用及机制研究

Tissue engineering technology is one of the effective methods to solve the problem of cartilage defect. Ideally, engineered cartilage should contain appropriate amounts of the primary extracellular matrix components, proteoglycans and collagen type II, differentiation/phenotypic maintenance, and resemble native cartilage structurally and functionally.But how to promote stem cell differentiate into chondrocyte and construct tissue engineered cartilage and keep its characteristic is still a tough problem.Adipose derived stem cells (ADSCs) have the ability to proliferate extensively and maintain the ability to differentiate into multiple cell types for the ues of tissue engineering to repair tissue defects.Our previous study have demonstrated that ADSCs may provide a clinical option for repairing the cartilage defect of larynx, induced by cytokines, such as TGF-β1 and bFGF. miR-675 and SOX9 might changed with the course of the induction. It is shown that COL2A1 expression is highly dependent on SOX9 in normal articular chondrocytes. miRNAs have been shown to be essential for normal cartilage development and multi-directional differentiation of stem cells. Recently, a research found that SOX9 can activate COL2A1 indirectly via a previously unreported miR-675 mediated mechanism, because COL2A1 levels can be rescued in SOX9-depleted cells specifically by overexpression of miR-675. So it is indentified H19-derived miR-675 as a highly expressed, cartilage-specific, SOX9-dependent positive regulator of COL2A1, the most abundant and functionally important cartilage matrix protein. Thus, this represents a new mechanism regulating cartilage matrix expression. However, it is not clear if the underlying new regulating mechanism could produce a marked effect on differentiation of stem cell into chondrocyte and construction of tissue engineered cartilage, and the role of miR-675 in keeping phenotypic maintenance is completely unexplored. So in this future research, the effect of miR-675 on regulating of differentiation and phenotypic maintenance of ADSCs into chondrocyte and construction of tissue engineered cartilage for laryngeal cartilage repair will be observed, through experimental methods, such as immunohistochemistry, western blot, RT-PCR, small interfering RNA and gene transfection. In this way, the role of miR-675 could be identified and might providing a potential new target for cartilage repair.

软骨组织工程中如何促进种子干细胞向软骨分化构建工程软骨，同时维持其软骨特性、防止退化是目前研究的热点和难点。我们前期研究可用脂肪干细胞(ADSCs)构建出工程化软骨修复喉软骨缺损，同时发现miR-675和调控软骨特异性标志蛋白COL2A1关键转录因子SOX9可随细胞软骨分化或退化而表达增高或减少。近期国外研究发现，SOX9可通过上调miR-675促进COL2A1表达，miR-675在促进关节软骨细胞分化和维持其表型稳定中起到核心调控作用，认为miR-675是一个有希望的软骨修复治疗的新靶点。然而miR-675能否在促进干细胞构建工程化软骨并维持其表型中发挥作用有待证实。为此，本项目拟通过RNA干涉和基因转染等干预手段，在细胞水平和在体层面，探讨miR-675在ADSCs向软骨细胞分化并构建表型稳定的工程化软骨修复喉软骨缺损中的作用及机制，为应用干细胞进行软骨修复提供新的治疗靶点和理论依据。

软骨组织工程中如何促进种子干细胞向软骨分化构建工程软骨，同时维持其软骨特性、防止退化是目前研究的热点和难点。我们前期研究可用脂肪干细胞(ADSCs)构建出工程化软骨修复喉软骨缺损，同时发现miR-675和调控软骨特异性标志蛋白COL2A1关键转录因子SOX9可随细胞软骨分化或退化而表达增高或减少。近期国外研究发现，SOX9可通过上调miR-675促进COL2A1表达，miR-675在促进关节软骨细胞分化和维持其表型稳定中起到核心调控作用，认为miR-675是一个有希望的软骨修复治疗的新靶点。然而miR-675能否在促进干细胞构建工程化软骨并维持其表型中发挥作用有待证实。本项目通过RNA干涉和基因转染等干预手段，在细胞水平和在体层面，观察到成软骨诱导后的ADSCs细胞H19及 miR-675表达明显上调，H19对miR-675具有正向调节作用，证实了H19及 miR-675能促进ADSC成软骨分化，机制可能与H19竞争性拮抗miRNA对成软骨分化有关的mRNA的抑制，进而促进ADSC向成软骨细胞的分化。本课题为应用miR-675调节脂肪干细胞进行软骨损伤的修复提供了理论基础和实验依据。