Tenomodulin调控间充质干细胞向肌腱源性分化的作用及机制

Tissue engineering provides a new approach for clinlical tendon defects. Though mensenchymal stem cell (MSC) is extensively used as a seed cell in tendon engineering due to its potency of multi-lineage differentiation, there still lacks a well-defined tenogenic differentiation method. Recent studies in developmental biology demonstrate that the differentiation of MSC to tenocyte is the initiation of tendon development. Tenomodulin（Tnmd）is considered as a specific biomarker for tendon and is highly correlated with the formation of tendon primordium, and can also promote the proliferation of tennocyte and collagen maturation. Based on these evidences, we propose that Tenomodulin can regulate the tenogenic differentiation of MSC. In this study, C3H10T1/2 cell, Tnmd-overexpression mice and Tnmd-knockout mice are used to testify the aboved hypothesis. On this basis, plasmids of different areas of Tnmd are transfected into C3H10T1/2 cell to identify the different functions. Then the western blot and GST Pulldown tenchnique are used to demonstrate that Jak-Stat signaling, TGF-βSmad signaling and cytoskeltal proteins are involved. At last, the tendons are engineered with MSC transfected with the functional domain of Tnmd. This study can provide scientific evidence for the tendon engineering with MSC.

间充质干细胞（MSC）因其具有多向分化潜能而被广泛应用于肌腱组织工程，但目前仍然缺乏明确的成肌腱分化方法。近年来发育生物学研究表明，MSC向肌腱细胞分化是肌腱发育的起始阶段。Tenomodulin（Tnmd）是肌腱特异分子标志，与肌腱始基的形成高度相关，并能促进肌腱细胞增殖和胶原成熟。因此我们推测Tnmd能够调控MSC向肌腱源性分化。本课题利用C3H10T1/2为细胞模型，Tnmd过表达小鼠以及基因敲除小鼠为动物模型，从多个层面证实Tnmd促进MSC向肌腱源性分化的能力；在此基础上，构建转染Tnmd不同区段的质粒，采用western blot和GST蛋白沉降技术，揭示Tnmd不同区段能通过Jak-Stat信号通路、TGF-βSmad信号通路和细胞骨架蛋白而发挥不同的功能。最后，通过Tnmd转染小鼠骨髓MSC组织工程肌腱构建进行应用验证，从而为更好地利用MSC构建组织工程肌腱提供科学依据。

间充质干细胞（MSC）因其具有多向分化潜能而被广泛应用于肌腱组织工程，但目前仍然缺乏明确的成肌腱分化方法。近年来研究表明, Tenomodulin（Tnmd）是肌腱特异分子标志，与肌腱始基的形成高度相关，并能促进肌腱细胞增殖和胶原成熟。因此我们推测Tnmd能够调控MSC向肌腱源性分化。本课题利用C3H10T1/2为细胞模型，通过质粒转染，发现Tnmd能显著促进C3H10T1/2细胞增殖和肌腱相关分子如Scleraxis、1型胶原、3型胶原、decorin等的表达，抑制成脂、成软骨和成骨分化。裸鼠体内移植实验显示Tnmd过表达C3H10T1/2细胞能形成肌腱样新生组织。利用Tnmd条件性过表达小鼠动物模型，通过多西环素诱导后，同样发现Tnmd能促进小鼠骨髓MSC增殖和肌腱相关分子的表达。从而从多个层面证实了Tnmd能促进MSC向肌腱源性分化的能力。在此基础上，通过构建转染Tnmd不同区段的质粒，发现ΔshTnmd段能上调磷酸化Smad2/3的蛋白表达，提示是通过激活TGF-βSmad信号通路发挥作用。此研究为更好地利用MSC构建组织工程肌腱提供了科学依据。