同种异体脂肪干细胞移植治疗大鼠骨关节炎的实验观察  

Transplantation of adipose derived mesenchymal stem cells alleviated osteoarthritis induced with anterior cruciate ligament transection

作　　者：周君[1] 王于[2] 崔伟[2] 谢金卫[3] 李家平[2] 杨建勇[2] 许韩师[1] 梁柳琴[1] 杨岫岩[1] 连帆[1] 

机构地区：[1]中山大学附属第一医院风湿内科,广州510080 [2]中山大学附属第一医院肿瘤介入科,广州510080 [3]中山大学中山医学院

出　　处：《中华医学杂志》2016年第13期1047-1052,共6页National Medical Journal of China

基　　金：国家自然科学基金（81102270）;广东省自然科学基金（2014A030313053,S2012010009075）

摘　　要：目的探讨同种异体脂肪干细胞（ADMSCs）移植对前交叉韧带离断法诱导的大鼠骨关节炎的治疗价值及可能机制。方法ADMSCs取自雄性SD大鼠腹膜脂肪组织，经过分离、培养并鉴定。40只SD大鼠均行膝关节前交叉韧带离断法建立骨关节炎模型，成模后一侧膝关节注射ADMSCs为治疗组，对侧膝关节注射磷酸盐缓冲液（PBS）为对照组。分别于注射后2、8周（即建模后4、10周）各取20只动物膝关节标本，观察其改变。通过QRT—PCR和Western印迹的方法初步验证ADMSCs改善大鼠骨关节炎模型的作用靶点。结果以前交叉韧带、内侧副韧带离断并切除内侧半月板的方法成功建立SD大鼠膝骨关节炎模型。膝关节腔内移植ADMSCs后大鼠全部存活，未发生明显并发症。治疗组大鼠在建模后两周关节腔内注射ADMSCs，对照组则注射PBS，注射后2、8周，治疗组大鼠骨关节炎较对照组明显好转，滑膜增生减轻、骨赘形成和软骨破坏均减少，骨赘评分及滑膜炎评分亦显著改善。QRT—PCR和Western印迹显示治疗组Ⅱ型胶原蛋白表达明显上调。ADMSCs与原代软骨细胞共培养，能抑制基质金属蛋白酶13（MMP-13）；在体外培养条件下，正常原代软骨细胞被肿瘤坏死因子仅激活后，盘状结构域受体2（DDR-2）表达上升明显，但与ADMSCs共培养后，DDR-2表达明显下调，提示ADMSCs有抑制DDR-2表达的作用；ADMSCs抑制MMP-13其上游靶点可能是DDR-2。更生霉素D（DDR-2抑制剂）可解除IL-1β抑制的ADMSCs的软骨分化。结论同种异体ADMSCs移植有助于改善大鼠骨关节炎；ADMSCs可能通过抑制MMP-13起作用，其上游靶点可能是DDR-2。Objective To explore the therapeutic potential of transplantation of adipose derived mesenchymal stem cells （ADMSCs） in rats osteoarthritis caused by anterior cruciate ligament transection. Methods Rats peritoneal adipose tissues were used to extract ADMSCs. Cell morphological appearance was documented and flow cytometric cell cycle was used to identify ADMSCs. Anterior cruciate ligament transection was used to induce knee osteoarthritis in rats. ADMSCs were injected into the knee cavities. Knee joint pathology was performed to observe the treatment effects. QRT-PCR and Western blot were used to identify the targets of ADMSCs. Results ADMSCs were successfully extracted, separated, cultured and identified. Two and eight weeks after ADMSCs transplantation, pathology showed significantly attenuation of arthritis including osteophyte and synovitis, reflecting in significantly improvement of both osteophyte and synovitis grading compared to the controls. QRT-PCR and Western blot revealed that collagen Ⅱ expression was significantly up-regulated after ADMSCs transpiantation compared to the controls. MMP-13, but not other MMP-1, MMP-3 or MMP-9 was reduced when ADMSCs were co-cultured with primary chondrocytes. DDR-2 expression in chondrocyte was heavily up-regulated when stimulated by TNF-a in vitro. However, ADMSCs could reverse the effect when co-cultured with chondrocyte, implying that ADMSCs may suppress the expression of DDR-2. IL-1β suppressed the cartilage differentiation of ADMSCs, and Actinomycin D （ DDR- 2 inhibitor） could reverse the effect. Conclusion ADMSCs can attenuate osteoarthritis induced by anterior cruciate ligament transection in rats by suppressing the expression of MMP-13, and the upstream target spot may be DDR-2.

关 键 词：骨关节炎 干细胞 基质金属蛋白酶类 盘状结构域受体2 

分 类 号：R684.3[医药卫生—骨科学]