Cdkn2a示踪小鼠:研究创伤性骨关节炎软骨退变细胞衰老的新模型

Cdkn2a Tracer Mice:A Novel Model to Study Cellular Senescence in Post-traumatic Osteoarthritis

骨关节炎(osteoarthritis,OA)是临床上最为常见的老年性运动系统疾病之一。研究表明,衰老是OA发生发展的重要影响因素之一,但其具体作用及机制尚未完全清楚。本研究通过CRISPR/Cas9技术,建立了Cdkn2a-e(Luc-2A-tdTomato-2A-CreERT2-WPRE-pA)1定点敲入的杂合子小鼠模型,可在小鼠活体内追踪衰老经典标记物Cdkn2a(p16,p16INK4a)的表达情况,结合前交叉韧带横断术(ACLT)诱导OA小鼠模型,将OA病理进程中的衰老变化在体外直观呈现,明确衰老与OA之间的关系。本研究选取10~12周龄Cdkn2a小鼠,随机分为非手术对照组、假手术组和ACLT组,通过ACLT手术在小鼠中构建OA的模型,术后4周收取动物进行活体荧光成像检测显示,ACLT组术后4周的小鼠膝关节局部Cdkn2a荧光表达升高(P<0.05),小鼠膝关节组织切片的番红O固绿染色显示,4周时ACLT组膝关节软骨出现退变(P<0.05),对小鼠膝关节组织进行Cdkn2a免疫组织化学染色,相较其他2个组,ACLT组的小鼠膝关节组织软骨表面Cdkn2a染色更深。研究结果显示,通过手术诱导的OA模型在局部具有衰老的表现,这进一步验证了衰老和OA的关系。同时,该Cdkn2a示踪小鼠模型能够在活体小鼠内体现衰老的进展。结合影像学检查,可以实时观察衰老和OA发生、进展的关系,为衰老与OA疾病机制的研究提供了良好的模型,也为今后进行靶向衰老进行OA的治疗提供了很好的研究工具。

Osteoarthritis(OA)is one of the most common geriatric motor system diseases in the clinical practice.Aging,whose hallmark is cellular senescence,is an important factor leading to the occurrence and development of OA,but its exact role in the pathological development of OA is not completely clear.Studies have proved that targeting senescence can effectively treat aging-related diseases.In this study,the heterozygous mouse model of Cdkn2a-e(Luc-2A-tdTomato-2a-CreerT2-Wpre-PA)1 was established by the CRISPR/Cas9 technique.The expression of Cdkn2a(p16,p16INK4a),a classical marker of senescence,can be traced in vivo in mice.We then utilized anterior cruciate ligament transection(ACLT)to induce OA in Cdkn2a mice,and we hope to verify the relationship between aging and the occurrence and development of OA and visualize aging changes during OA development through the mice model.In this study,10-12 weeks old Cdkn2a mice were randomly divided into no surgery control group,sham operation group and ACLT group.OA model was constructed in mice by ACLT operation.After surgery for 4 weeks,animals were collected for fluorescence imaging detection in vivo,which showed that local fluorescence expression of Cdkn2a increased in knee joints of mice in the ACLT group four weeks after surgery(P<0.05).The Safranin O-Fast Green Staining of mouse knee tissue sections showed degeneration of the knee cartilage in the ACLT group at 4 weeks after surgery(P<0.05).Immunohistochemical staining of Cdkn2a was performed on the knee tissue of mice.Compared with the other two groups,Cdkn2a staining on the cartilage surface of the knee tissue of mice in the ACLT group was deeper.The results showed that the OA model induced by surgery showed local aging,which further verified the relationship between aging and OA.At the same time,the Cdkn2a tracer mouse model can reflect the aging progress of mice in vivo,with combination of imaging examinations,so that the occurrence and progress of the relationship between aging and OA can be observed in real time.This heterozygous mouse model of Cdkn2a-e(Luc-2A-tdTomato-2a-CreerT2-Wpre-PA)1 is not only useful for mechanism research of aging and OA diseases,but also beneficial for finding more potential therapy targets to OA and aging.