人髌下脂肪垫干细胞的鉴定及成软骨分化  

Identification and chondrogenic differentiation of human infrapatellar fat pad derived stem cells

作　　者：李聪聪 姚楠[1,2,3] 黄丹娥 宋敏 彭莎 李安安 卢超 刘文刚 Li Congcong;Yao Nan;Huang Dane;Song Min;Peng Sha;Li Anan;Lu Chao;Liu Wengang(The Fifth Clinical College of Guangzhou University of Chinese Medicine,Guangzhou 510095,Guangdong Province,China;Guangdong Second Traditional Chinese Medicine Hospital(Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine),Guangzhou 510095,Guangdong Province,China;Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine,Guangzhou 510095,Guangdong Province,China)

机构地区：[1]广州中医药大学第五临床医学院,广东省广州市510095 [2]广东省第二中医院(广东省中医药工程技术研究院),广东省广州市510095 [3]广东省中医药研究开发重点实验室,广东省广州市510095

出　　处：《中国组织工程研究》2021年第19期2976-2981,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金项目(2018A030313584),项目负责人:姚楠;广东省中医药局科研项目(20183001),项目负责人:刘文刚;广东省医学科学技术研究基金项目(A2020071),项目负责人:卢超。

摘　　要：背景:人髌下脂肪垫是在全膝关节置换手术中经常被切除的废弃物,如果充分加以利用可以作为间充质干细胞的主要来源用于软骨组织工程。目的:探索人髌下脂肪垫干细胞体外分离、培养及鉴定方法,并在特定条件下诱导其成软骨分化,探讨其作为软骨组织工程种子细胞的可行性。方法:获取8例人工膝关节置换术患者废弃的髌下脂肪垫,综合运用胰蛋白酶和Ⅰ型胶原蛋白酶消化法及差速贴壁法分离培养人髌下脂肪垫干细胞,体外培养至第3代后进行细胞形态学观察、细胞生长曲线以及细胞表面抗原分析,然后进行成软骨诱导分化,通过免疫组化和甲苯胺蓝染色以及RT-qPCR方法对人髌下脂肪垫干细胞成软骨分化能力进行鉴定。结果与结论:①分离纯化及培养出的人髌下脂肪垫干细胞呈梭形贴壁生长,第3代细胞生长曲线呈“S”形,CD44、CD105呈阳性表达,而CD45呈阴性表达;②成软骨诱导14 d后,诱导分化组免疫组织化学染色和甲苯胺蓝染色均呈阳性,并且ACAN、BMP-2、COL2A1和SOX-9的基因相对表达量明显高于空白对照组;③该研究证实了从髌下脂肪垫中可分离出具有分化潜能的间充质干细胞,其具有较强的体外增殖能力以及成软骨分化能力,有望成为软骨组织工程理想的种子细胞。BACKGROUND:Human subpatellar fat pad is a kind of waste,which is often removed in total knee arthroplasty.If it is fully utilized,it can be used as a source of mesenchymal stem cells.OBJECTIVE:To explore the methods of isolation,culture and identification of human infrapatellar fat pad derived stem cells in vitro,to induce chondrogenic differentiation under specific conditions,and to explore the feasibility of being used as seed cells for cartilage tissue engineering.METHODS:The discarded infrapatellar fat pad from eight patients undergoing the total knee arthroplasty was obtained and human infrapatellar fat pad derived stem cells were isolated and cultured according to trypsin digestion,type I collagenase digestion and differential adherence methods.After being cultured to the third generation in vitro,cell morphology observation,cell growth curve and cell surface antigen analysis were performed.In addition,chondrogenic differentiation was induced,and human infrapatellar fat pad derived stem cells were identified by immunohistochemistry,toluidine blue staining and RT-qPCR.RESULTS AND CONCLUSION:(1)Isolated,purified and cultured human infrapatellar fat pad derived stem cells showed spindle-shaped adherent growth.The growth curve of the third generation of human infrapatellar fat pad derived stem cells was in the shape of“S”.Cells were positive for CD44 and CD105,while negative for CD45.(2)After 14 days of induction,immunohistochemical staining and toluidine blue staining were positive in the differentiation group,and the relative gene expression levels of ACAN,BMP-2,COL2A1 and SOX-9 were significantly higher than those in the blank control group.(3)This study confirmed that mesenchymal stem cells with differentiation potential could be isolated from infrapatellar fat pad,which had strong proliferation ability and chondrogenic differentiation ability in vitro,and were expected to become ideal seed cells for cartilage tissue engineering.

关 键 词：干细胞 髌下脂肪垫干细胞 骨关节炎 鉴定 成软骨分化 种子细胞 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]