人皮下和髌下脂肪垫干细胞治疗大鼠骨关节炎的疗效比较

 目的 比较人皮下和髌下脂肪垫干细胞体外增殖、成软骨分化潜能以及体内治疗大鼠骨关节炎的疗效差异。方法 获取上海市东方医院未患代谢性相关疾病患者的皮下和髌下脂肪垫组织，从髌下脂肪垫中分离原代脂肪干细胞（adipose-derived stem cells，ASCs），体外诱导成脂、成骨、成软骨方向分化。EdU检测人皮下脂肪干细胞（subcutaneous ASCs，Sc-ASCs）和髌下脂肪垫干细胞（infrapatellar fat pad derived stem cells，IPFP-ASCs）体外增殖能力，流式细胞术检测干细胞表面标记蛋白CD34、血管相关标记蛋白CD31的表达。阿尔新蓝染色及Western blot检测Sc-ASCs和IPFP-ASCs体外成软骨潜能。体内实验采用8周SD大鼠随机分为对照（control，CON）组、内侧半月板不稳术（destabilisation of the medial meniscus，DMM）组和DMM手术后Sc-ASCs治疗组，DMM手术后IPFP-ASCs治疗组，通过大体观察、番红固绿染色及OARSI评分比较体内治疗效果。结果 人髌下脂肪垫组织中分离获得ASCs，形态呈长梭形，在体外具有成脂、成骨、成软骨分化潜能。Sc-ASCs和IPFP-ASCs表面标记蛋白CD34和血管相关标记蛋白CD31表达无显著性差异，但人IPFP-ASCs体外增殖能力较强。阿尔新蓝染色及Western blot显示IPFP-ASCs体外成软骨能力较强。体内大鼠实验大体形态学观察显示DMM组骨赘增多，软骨表面缺损，Sc-ASCs治疗组骨赘减少，IPFP-ASCs治疗组表面较光滑，无明显骨赘生成；番红固绿染色显示DMM组软骨层出现垂直裂缝且到达钙化软骨，Sc-ASCs治疗组关节面纤维化且软骨浅表层存在垂直裂隙，IPFP-ASCs治疗组软骨层较为连续，仅存在轻微纤维化；OARSI评分显示IPFP-ASCs治疗骨关节炎疗效更好。结论 人IPFP-ASCs体外增殖能力、成软骨分化潜能以及体内治疗大鼠骨关节炎的效果优于Sc-ASCs。

Comparison of human subcutaneous and infrapatellar fat pad derived stem cells in the treatment of osteoarthritis in rats

Objective To compare the proliferation and chondrogenic differentiation potential of human subcutaneous and infrapatellar fat pad derived stem cells in vitro and the therapeutic effect of osteoarthritis in rats in vivo.Methods Subcutaneous and infrapatellar fat pad tissues were obtained from Shanghai Dong fang Hospital patients without metabolic-related diseases, and primary adipose derived stem cells (ASCs) were isolated from the infrapatellar fat pad. ASCs were induced to differentiate into adipocytes, osteocytes and chondrocytes in vitro. EdU incorporation assay was used to detect the proliferation ability of subcutaneous adipose derived stem cells (Sc-ASCs)and infrapatellar fat pad derived stem cells (IPFP-ASCs) in vitro. Flow cytometry was used to detect the expression of stem cell surface marker protein CD34 and vascular-related marker protein CD31. Alcian blue staining and Western blot were used to detect the chondrogenic differentiation potential of Sc-ASCs and IPFP-ASCs in vitro. In vivo experiment, the 8-week-old Sprague-Dawley rats were randomly divided into control (CON) group, destabilisation of the medial meniscus (DMM) group, DMM plus Sc-ASCs treatment group and DMM plus IPFP-ASCs treatment group.The therapeutic effects in vivo were compared by gross observation, Safranin O-fast green staining and OARSI score.Results The morphology of ASCs isolated from human infrapatellar fat pad tissue was spindle-shaped and had the potential of adipogenic, osteogenic and chondrogenic differentiation in vitro. There was no significant difference in the expression of stem cell surface marker protein CD34 and vascular-related marker protein CD31 between human Sc-ASCs and IPFP-ASCs. However, human infrapatellar fat pad derived stem cells had strong proliferation ability in vitro. Alcian blue staining and Western blot showed that IPFP-ASCs had a stronger chondrogenic ability than Sc-ASCs in vitro. Gross morphological observation of in vivo experiments showed that osteophytes increased and cartilage surface defects in DMM group, while osteophytes decreased in Sc-ASCs treatment group. The surface of the IPFP-ASCs treatment group was smoother without obvious osteophyte formation. Safranino fast green staining showed vertical cracks in the cartilage layer in the DMM group and reached calcified cartilage. In the Sc-ASCs treatment group, the articular surface was fibrotic and there were vertical fissures in the superficial layer of cartilage. The cartilage layer in the IPFP-ASCs treatment group was relatively continuous, with only slight fibrosis. OARSI score showed that IPFP-ASCs had better efficacy in the treatment of osteoarthritis.Conclusion The proliferation and chondrogenic potential of human IPFP-ASCs in vitro and the effect of treating rat osteoarthritis in vivo were better than those of Sc-ASCs.