软骨干/祖细胞移植治疗大鼠骨性关节炎

Chondrogenic stem/progenitor cells transplantation in the treatment of osteoarthritis in rats

目的:探究大鼠软骨干/祖细胞(cartilage stem/progenitor cells,CSPCs)对骨性关节炎(osteoarthritis,OA)的治疗作用。方法:分离、鉴定、培养大鼠CSPCs,利用白细胞介素(interleukin,IL)-1β刺激大鼠软骨细胞诱发炎症反应,后与软骨干细胞共培养。采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-PCR)检测炎症相关因子的表达,脱氧核苷酸末端转移酶(terminal deoxynucleotidyl transferase,TdT)介导的dUTP缺口末端标记(TdT-mediated dUTP nick-end labeling,TUNEL)技术检测细胞凋亡数目变化,Western Blot检测凋亡基因表达。采用半月板失稳术(destabilization of the medial meniscus,DMM)法建立大鼠OA模型,预防组术后3 d膝关节腔内注射CSPCs,治疗组术后8周注射CSPCs。采用HE染色、番红固绿染色、Pelletier评分、Mankin′s评分、骨关节炎软骨组织病理学评价系统(osteoarthritis research society international,OARSI)评分评价关节内软骨损伤情况。结果:CSPCs与软骨细胞共培养后,肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-6、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)基因表达下降,TUNEL阳性凋亡细胞的数量减少,凋亡标志物Cleaved caspase3和Bax的蛋白水平明显降低。CSPCs关节内注射明显改善OA软骨缺损情况,Pelletier评分、Mankin′s评分、OARSI评分均显著下降。结论:CSPCs能够有效地预防和治疗OA。

Objective:To explore the therapeutic effect of rat cartilage stem/progenitor cells(CSPCs)on osteoarthritis(OA).Methods:Rat cartilage-derived progenitor cells were isolated,identified and cultured.Rat chondrocytes were stimulated with interleukin(IL)-1βto induce inflammation,and then co-cultured with CSPCs.The inflammatory factor expression was measured by real-time quantitative polymerase chain reaction(RT-PCR),its changes in the number of apoptotic cells were measured by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL),and its gene expression was measured by Western Blot.The rat OA model was established by the destabilization of the medial meniscus(DMM)method.CSPCs were injected into the knee 3 days after the operation in the prevention group,CSPCs were injected into the knee 8 weeks after the operation in the treatment group.Cartilage damage was evaluated by HE staining,Safranine O-Fast Green staining,Pelletier score,Mankin′s score and osteoarthritis research society international(OARSI)score.Results:After co-cultured with CSPCs,the expression level of tumor necrosis factor-α(TNF-α),IL-6 and matrix metalloproteinase-13(MMP-13)were significantly decreased in chondrocytes.Meanwhile,the number of TUNEL-positive apoptotic cells and the protein levels of Cleaved caspase3 and Bax were also decreased.After intra-articular injection of CSPCs,the cartilage defects were significantly improved in the preventive and treated groups.Pelletier score,Mankin′s score and OARSI score were all decreased.Conclusion:CSPCs can effectively prevent and treat OA.