两种方法培养骨质疏松症小鼠脂肪干细胞的比较  

A comparative study on two culture methods for adipose-derived stem cells from mice with osteoporosis

作　　者：黄成龙 黎庆 王雷 黄馗 罗道文 肖金刚 Huang Cheng-long;Li Qing;Wang Lei;Huang Kui;Luo Dao-wen;Xiao Jin-gang(Department of Oral and Maxillofacial Surgery,Hospital of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China;Orofacial Reconstruction and Regeneration Laboratory, Hospital of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China)

机构地区：[1]西南医科大学附属口腔医院,口腔颌面外科,四川省泸州市646000 [2]西南医科大学附属口腔医院,口颌面修复重建和再生实验室,四川省泸州市646000

出　　处：《中国组织工程研究》2017年第29期4654-4659,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81371125);四川省科技厅项目(2014JY0044);四川省教育厅项目(10ZB030);四川省卫生厅项目(80170);西南医科大学重点项目(201207);2015年泸州市人民政府-西南医科大学科技战略合作项目[2015LZCYD-S05(2/12)];西南医大口院[(2017)20号]~~

摘　　要：背景:获得充足的脂肪干细胞是自体移植修复骨质疏松症骨缺损的前提,因此如何高效、简便、经济的获得自体原代脂肪干细胞是治疗骨质疏松症的关键。目的:采用组织块法和胶原酶消化法体外培养骨质疏松症小鼠脂肪干细胞,并对两种培养方法进行比较分析。方法:切除雌性C57BL/6小鼠双侧卵巢,制作骨质疏松模型,造模成功后取小鼠皮下脂肪组织,分别采用组织块法和胶原酶消化法分离培养脂肪干细胞。应用流式细胞仪检测第3代脂肪干细胞表面特异性抗原;培养7 d后,检测细胞产量;检测第3代脂肪干细胞的增殖;检测第4代脂肪干细胞的成脂及成骨分化能力。结果与结论:①组织块法培养第3代细胞CD34、CD146、Sca-1表达率分别为(15.22±1.85)%、(75.55±3.36)%、(83.48±4.22)%,胶原酶消化法第3代细胞CD34、CD146、Sca-1表达率分别为(13.46±2.21)%、(76.62±2.47)%、(84.84±3.56)%;②组织块法每毫克脂肪组织获得的细胞产量高于胶原酶消化法(P<0.05);③培养24 h后,组织块培养法的细胞增殖率高于胶原酶消化法;④两种方法得到的脂肪干细胞均可向脂肪细胞和成骨细胞方向分化,两组间成脂量及成骨量比较差异无显著性意义(P>0.05);⑤结果表明,组织块法比胶原酶消化法更适合体外培养脂肪干细胞,可为骨质疏松症患者自体移植脂肪干细胞治疗骨质疏松性骨折和骨缺损研究提供充足的细胞来源。BACKGROUND:To obtain enough adipose-derived stem cells(ADSCs)is the premise of repairing osteoporotic bonedefects by autograft transplantation;therefore,how to efficiently,simply and economically harvest primary autologousADSCs is the key to the treatment of osteoporosis.OBJECTIVE:To isolate and culture ADSCs from mice with osteoporosis(OP-ADSCs)in vitro by tissue explant cultureand collagenase digestion,and to compare the efficacies of these two methods.METHODS:Adipose tissues were isolated from the inguen of C57BL/6mice,from which OP-ADSCs were obtained bytissue explant culture and collagenase digestion respectively.Then,the cells were subjected to primary culture andsubculture.The surface specific antigens of passage3cells were observed using flow cytometry,while the yields andproliferation abilities of passage3were compared at7days of culture.The adipogenic and osteogenic differentiation ofOP-ADSCs at passage4was detected.RESULTS AND CONCLUSION:(1)The expression levels of CD34,CD146,Sca-1in passage3cells were(15.22±1.85)%,(75.55±3.36)%and(83.48±4.22)%for the tissue explant culture and(13.46±2.21)%,(76.62±2.47)%and(84.84±3.56)%for the collagenase digestion method,respectively.(2)The cell yield from each milligram of adiposetissue by tissue explant culture was significantly higher than that by collagenase digestion(P<0.05).(3)Theproliferation rate of cells in the tissue explant culture group was higher than that in the collagenase digestion group after24hours of culture.(4)The OP-ADSCs cultured by the two methods could differentiate into adipocytes and osteoblasts,and the lipid accumulation and the mineralized nodules showed no significant difference between the two groups(bothP>0.05).These experimental findings indicate that the tissue explant culture is more suitable for obtaining OP-ADSCsin vitro as compared with collagenase digestion,which contributes to provide adequate cell sources for studies onADSCs treatment of osteoporotic fractures and bone defects.

关 键 词：干细胞 骨质疏松 细胞培养技术 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]