基于CRISPR/Cas9技术探讨补肝肾强筋骨中药促进BMSCs软骨分化的网络机制研究

The disease that is represented by osteoarthritis and own the core pathology of injury in the articular cartilage,so far,have not ideal drugs and therapies to repair cartilage that lack the normal mechanical properties by self-healing.Compared with other cells,Bone Marrow Mesenchymal Mtem Cells(BMSCs)have better characteristics of chondrogenic differentiation.Traditional Chinese Medicine inducing BMSCs into Chondrogenic.Differentiation has certain characteristics and advantages,but Traditional Chinese Medicine have very little research on chondrogenic differentiation.The early studies have shown that serum containing Achyranthes can promote BMSCs to express protein biomarkers (Sox9,Proteoglycan,type II collagen) of cartilage cells ,which Achyranthes can induce BMSCs into chondrogenic differentiation in Vitro，but Its specific mechanism needs further study.Using BMSCs from rabbit cultured in vitro to separate,culture,multiply,and Co-cultivation of BMSCs that serum containing effective component of Achyranthis together with the the classic inducer of chondrogenic differentiation to Culture cells promote differentiation of BMSCs in vitro into cartilage.And using gene chips,proteomics technology screen major differences in gene,protein,and a major role in signaling pathways of Achyranthis inducing BMSCs into chondrogenic differentiation,and that Using CRISPR/Cas9 technology to knock out or transcriptional activating function of the target gene validation results reveal effect,mechanism of Achyranthis inducing BMSCs into chondrogenic.Whereby,these will confirm the theory of traditional chinese medicine of achyranthis which is good at “tonifying the liver and the kidney to strengthen the tendons and the bones”,provide a theoretical basis on solving clinical articular cartilage damage and repairing degenerative.

以关节软骨损伤为核心病理改变的骨关节炎疾病，自然修复软骨组织缺乏正常力学性能，尚无理想药物和疗法修复软骨，成为治疗难点。骨髓间充质干细胞（BMSCs）能优于其他来源细胞分化为软骨细胞，中药干预下的干细胞软骨分化具有特色和优势，但促软骨分化的研究较少。前期研究发现，牛膝含药血清培养BMSCs能促使其明显表达软骨细胞标志性Sox9、蛋白聚糖、Ⅱ型胶原蛋白，表明BMSCs向软骨细胞分化，但作用机制不清楚。拟通过兔BMSCs体外培养进行BMSCs和牛膝含药血清与经典软骨诱导剂对照培养，促使BMSCs体外诱导软骨形成。采用蛋白质组学、基因芯片技术筛选牛膝诱导软骨分化主要差异蛋白、基因及作用信号通路，用CRISPR/Cas9技术敲除或转录激活功能靶基因验证结果，揭示牛膝对BMSCs体外扩增和软骨细胞定向分化的机理。证实牛膝善“补肝肾、强筋骨”的中医理论，为临床解决关节软骨损伤、退变的修复提供理论依据。

以关节软骨损伤为核心病理改变的骨关节炎疾病，尚无理想药物修复软骨，成为治疗难点。骨髓间充质干细胞（BMSCs）能优于其他来源细胞分化为软骨细胞，中药干预下的干细胞软骨分化具有特色和优势，但促软骨分化的研究较少。前期研究发现，牛膝含药血清具有促进BMSCs向软骨细胞分化作用，但具体机制不清楚。.本项目通过体外培养兔BMSCs，将P3代BMSCs和牛膝有效成分制剂牛膝醇提物不同剂量含药血清进行培养，并与空白组、阳性对照组对照培养，体外诱导促使BMSCs向软骨细胞分化。采用蛋白组学iTRAQ技术、基因组高通量测序筛选牛膝诱导软骨分化主要差异蛋白、基因，并作生物信息学分析，构建生物互作网络；再用CRISPR/Cas9基因编辑技术编辑特异靶点基因验证结果。. 结果显示（1）牛膝醇提物高剂量组诱导BMSCs软骨细胞形成效果最佳；（2）蛋白组学和基因组学整合分析显示：维恩分析表明牛膝醇提物组与空白组共有14个蛋白/基因，阳性对照组与空白组共有48个蛋白/基因，阳性对照组与牛膝醇提物组共有25个蛋白/基因；富集分析显示牛膝醇提物组与空白组共富集到7个GO功能或KEGG通路，阳性对照组与空白组共富集到21个GO功能或KEGG通路，阳性对照组与牛膝醇提物组共富集到10个GO功能或KEGG通路；网络拓扑学分析显示网络中节点3种网络拓扑性质计算得分top10内的节点基因，牛膝醇提物组与空白组是ISG15、HSPA5、HYOU1、HSP90B1、ERO1A、MANF、SLC3A2、DDX58、PRKRA、RAB39B，阳性对照组与牛膝醇提物组是LGALS3、CTSB、FABP4、ANPEP、HP、ACAN、CD9、TFRC、LCP1、NID2；（3）CRISPR/Cas9基因编辑技术编辑特异靶点基因之一RAB39B基因验证实验结果。. 结论：（1）牛膝醇提物诱导兔BMSCs成软骨分化具有多靶点、多中心的网络调控作用。（2）CRISPR/Cas9系统编辑敲低RAB39B基因后能够抑制BMSCs增殖，降低其软骨细胞分化能力，说明RAB39B基因可能对BMSCs增殖、软骨分化能力产生影响，其具体作用机制有待进一步研究。本项目揭示牛膝对BMSCs体外扩增和软骨细胞定向分化的机理；证实牛膝善“补肝肾、强筋骨”的中医理论，为临床解决关节软骨损伤、退变的修复提供了理论依据。