JNK信号通路调控体外软骨再生机制及其应用

In vitro construction was a major trend of cartilage regeneration. Based on the compound of growth factors, we had built the key techniques of in vitro cartilage construction. Nevertheless, the high cost and short half-life period of the growth factors had particular disadvantages on clinical translation. Our recent study found that the quality of regeneration cartilage could be remarkably enhanced by inhibiting the JNK pathway, and the effect of cartilage regeneration was nearly equal to that of the growth factors. However, the exact roles, molecular mechanisms, and bio-safety were not clear yet. This study aimed at the following researches: 1. Comprehensively evaluate and define the roles of the JNK pathway inhibitor on in vitro cartilage regeneration based on the established evaluation system of three-dimensional cartilage regeneration; 2. At the cellular level, screening the effects of the JNK inhibitor on the downstream signal pathway, and using the activation or inhibition to make it clear that JNK pathway regulates cartilage regeneration; 3. Using the karyotype analysis, telomerase activity, and proto-oncogene expressing level comprehensively evaluate the biological safety of the JNK inhibitor; 4. Establishing new cartilage regeneration system or developing new controlled release scaffold based on the JNK inhibitor to explore the possibility of regulating cartilage regeneration. The study was expected to establish the new techniques of cartilage regeneration and promote its clinic translation.

体外构建是软骨再生临床转化研究重要方向。项目组前期基于生长因子组合已建立体外软骨再生核心技术，但费用昂贵、半衰期短对临床转化极为不利。项目组新近研究发现，抑制JNK通路能显著提升体外软骨再生质量，其效果甚至等同于生长因子，但JNK通路调控体外软骨再生的具体作用、分子机制及生物安全性等系列问题仍未阐明。本项目拟基于上述科学现象及已有基础开展以下研究：1）基于已建立的三维软骨再生评价体系全面评估和明确JNK抑制剂对体外软骨再生的具体调控作用；2）二维细胞水平筛查JNK抑制剂对软骨再生相关信号通路的影响，结合JNK下游相关通路的激活/抑制，明确JNK通路调控软骨再生的分子机制；3）通过核型分析、端粒酶活性、原癌基因激活水平等全面评估JNK抑制剂调控软骨再生的生物安全性；4）应用JNK抑制剂优化软骨再生体系或研制新型缓释支架，调控体内外软骨再生。本项目有望建立新型软骨再生技术体系，推动其临床转化。

软骨缺损是临床常见疾病，体外构建稳定的再生软骨是组织工程软骨临床转化的关键。项目组现已建立的基于生长因子组合的体外软骨再生培养体系虽然可以明显促进软骨分化和基质合成，但对细胞增殖有着一定的负面影响，且费用高昂，不利于体外构建软骨的稳定再生和临床转化。项目组前期研究发现，抑制JNK通路可以抑制炎症、降低软骨基质降解，从而稳定软骨再生。本项目在此基础上，深入开展系列研究，进一步明确了JNK抑制剂对体外软骨再生的调控作用，证实了JNK抑制剂显著促进软骨细胞增殖，甚至可以逆转现有成软骨诱导液对细胞增殖的不利影响，同时抑制软骨基质合成与降解，但其对软骨基质合成的抑制作用可被成软骨诱导液所逆转。对软骨形成相关通路因子的表达进行筛查与测定，明确了JNK抑制剂通过下调软骨形成上游信号通路从而抑制软骨基质合成，通过下调前炎症因子从而抑制软骨基质降解。联合成软骨诱导液构建不同模式的体外软骨再生培养体系，明确了长期联合应用JNK抑制剂和成软骨诱导液可获得最稳定的体外软骨再生及体内转归。随后在此基础上，项目组通过开展支架材料制备、活体软骨再生及缺损模型修复等系列研究，为后续结合基于JNK抑制剂优化软骨再生培养体系提供了体内外研究模型及核心技术，为实现活体软骨再生技术优化及其临床转化奠定了重要的研究基础。