TGFβ/Smad3抑制lncRNA-ANCR表达在干细胞错误分化及钙化性肌腱炎中的作用及机制研究

Errors differentiation of endogenous tendon stem cells into cartilage cells is the pathological basis of calcified tendinitis. Our previous researched demonstrated the lncRNA – ANCR is low expressed in calcified tendinitis tissues, and found that it may function as the downstream of TGF- beta/Smad3 pathways in regulating the differentiation of stem cell, however, the specific mechanism is still unknown. The hypothesis is: The expression of lncRNA-ANCR may be inhibited by TGF-beta/Smad3 pathway, then induces error differentiation of tendon derived stem cells into cartilage cells, and finally results in calcified tendinitis. This topic proposed: 1) Analyze the effect of lncRNA-ANCR in differentiation of stem cell in vitro. 2) Analyze the effect and mechanism of TGF beta/Smad3 on lncRNA-ANCR through the ChIP, the dual luciferase report, methylation specific PCR and so on. 3) Explore the downstream targets of lncRNA-ANCR through the RNA Pull Down technique, RNA - RIP, deletion mapping, dual luciferase report and so on. 4) Explore the role of lncRNA-ANCR in calcified tendinitis in vivo. The present study will help to identify the role and mechanism of lncRNA-ANCR in tendon derived stem cells differentiation and in developing calcified tendinitis, which may provide new target in the diagnosis and treatment of calcified tendinitis.

内源性肌腱干细胞错误分化为软骨细胞是钙化性肌腱炎的病理基础。前期研究通过基因芯片筛选出在钙化性肌腱炎中低表达的lncRNA-ANCR，并发现其可能作为TGF-β/Smad3通路的下游，参与干细胞分化调控，但具体机制尚不明了。本课题假设：TGFβ/Smad3通路抑制ANCR表达，诱使肌腱干细胞错误分化为软骨细胞，从而导致钙化性肌腱炎。本课题拟：1）体外实验验证ANCR的干细胞分化调控作用。2）通过ChIP、双荧光素酶报告、甲基化特异性PCR等方法验证TGFβ/Smad3对ANCR的调控作用及机制。3）通过RNA Pull Down、RNA-RIP、缺失作图、双荧光素酶报告等方法寻找ANCR的下游作用靶点。4）体内实验进一步明确ANCR在钙化性肌腱炎中的作用。通过本研究，将明确lncRNA-ANCR对肌腱干细胞的分化调控作用，明确其在钙化性肌腱炎中的作用及具体作用机制，为临床发掘新的诊疗靶点。

内源性肌腱干细胞错误分化为软骨细胞是钙化性肌腱炎的病理基础，然而干细胞分化的机制尚不明了。前期研究通过基因芯片筛选出在钙化性肌腱炎中低表达的lncRNA-ANCR，并发现其可能作为TGF-β/Smad3通路的下游，参与干细胞分化调控。本课题假设：TGFβ/Smad3通路抑制ANCR表达，诱使肌腱干细胞错误分化为软骨细胞，从而导致钙化性肌腱炎。本课题首先通过体内实验明确lncRNA ANCR在肌腱钙化标本中低表达，尤其在软骨异形变早期;然后通过体外实验验证lncRNA ANCR的干细胞分化调控作用，当敲低lncRNA ANCR表达时，肌腱干细胞成软骨分化能力明显增强，当lncRNA ANCR高表达时，成软骨分化能力明显降低;最后通过RNA Pull Down实验明确了lncRNA ANCR的作用靶点为EZH2，同时当过表达EZH2后，lncRNA ANCR的促软骨分化能力明显减弱。通过本研究，明确了lncRNA-ANCR可能作为维持干细胞干性的闸门，其低表达促使干细胞在特殊微环境下成软骨分化，从而构成钙化性肌腱炎的发病基础之一。