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  

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

作　　者：宋赛赛 夏国宏 尹峰[3] 汤妍 SONG Sai-sai;XIA Guo-hong;YIN Feng;TANG Yan(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China;Shanghai Saiao Biotechnology Co.,Ltd.,Shanghai 200062,China;Department of Orthopedics,East Hospital,Tongji University,Shanghai 200120,China)

机构地区：[1]复旦大学基础医学院生物化学与分子生物学系,上海200032 [2]上海赛傲生物技术有限公司,上海200062 [3]同济大学附属东方医院骨科,上海200120

出　　处：《复旦学报（医学版）》2022年第3期345-352,359,共9页Fudan University Journal of Medical Sciences

基　　金：上海市科委高校Ⅳ类高峰学科建设。

摘　　要：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IPFPASCs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。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 osteophyt

关 键 词：皮下脂肪干细胞(Sc-ASCs) 髌下脂肪垫干细胞(IPFP-ASCs) 骨关节炎 

分 类 号：R34[医药卫生—基础医学]