中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (29): 4654-4659.doi: 10.3969/j.issn.2095-4344.2017.29.011

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

两种方法培养骨质疏松症小鼠脂肪干细胞的比较

黄成龙1，黎 庆2，王 雷2，黄 馗2，罗道文2，肖金刚1，2   

  1. 西南医科大学附属口腔医院，1口腔颌面外科，2口颌面修复重建和再生实验室，四川省泸州市  646000
  • 修回日期:2017-05-01 出版日期:2017-10-18 发布日期:2017-11-08
  • 通讯作者: 肖金刚，教授，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
  • 作者简介:黄成龙，男，1988年生，重庆市人，汉族，2015年西南医科大学附属口腔医院毕业，硕士，主要从事脂肪干细胞与口腔颌面部组织再生相关研究。
  • 基金资助:

    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]

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

Huang Cheng-long1, Li Qing2, Wang Lei2, Huang Kui2, Luo Dao-wen2, Xiao Jin-gang1, 2   

  1. 1Department of Oral and Maxillofacial Surgery, 2Orofacial Reconstruction and Regeneration Laboratory, Hospital of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Revised:2017-05-01 Online:2017-10-18 Published:2017-11-08
  • Contact: Xiao Jin-gang, Professor, Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Huang Cheng-long, Master, Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81371125; the Project of Sichuan Science and Technology Department, No. 2014JY0044; the Project of Sichuan Education Department, No. 10ZB030; the Project of Sichuan Health Department, No. 80170; the Major Project of Southwest Medical University, No. 201207; the Science and Technology Strategic Cooperation Project of Luzhou Municipal People’s Government-Southwest Medical University, No. 2015LZCYD-S05(2/12); a grant from the Hospital of Stomatology of Southwest Medical University, No. (2017)20

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摘要：

文章快速阅读：

文题释义：
组织块培养法：将组织剪成1 mm3左右的组织块；用PBS清洗组织块3次；将组织块按照一定间距转入培养瓶内；轻轻将培养瓶翻转过来，将适量培养液加到非细胞生长面上，注意翻瓶时勿令组织小块流动，塞好瓶塞置36.5 ℃温箱培养2 h左右(勿超过4 h)，使小块微干涸；从微箱中取出培养瓶，开塞，46°斜持培养瓶，箱瓶底脚部轻轻注入培养液少许，然后缓缓再把培养瓶翻转过来，让培养液慢慢覆盖附于瓶地上的组织小块置温箱中静止培养；待细胞从组织块游出量增后，再补加培养液。
胶原酶消化法：就是用胶原酶处理消化组织达到分离细胞的目的，所用胶原酶有不同类型：胶原酶Ⅰ用于上皮、肺，脂肪和肾上腺组织细胞的分离。用于消化组织中连接部分使其成为单个细胞，用于哺乳动细胞的分离；胶原酶Ⅳ包含至少7种蛋白酶成分，相对分子量从68 000-130 000不等，能消化多种组织；胶原酶Ⅴ包含至少7种蛋白酶成分，相对分子量从68 000-130 000不等，可用于胰腺小岛组织的分离，将结缔组织分离成单个细胞；胶原酶Ⅱ适用于肝脏、骨、甲状腺、心脏和唾液腺组织。

 

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。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-6933-8312(黄成龙)

关键词: 干细胞, 脂肪干细胞, 骨质疏松症, 组织块培养法, 胶原酶消化法, 成脂分化, ；成骨分化, 国家自然科学基金

Abstract:

BACKGROUND: To obtain enough adipose-derived stem cells (ADSCs) is the premise of repairing osteoporotic bone defects by autograft transplantation; therefore, how to efficiently, simply and economically harvest primary autologous ADSCs 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 culture and collagenase digestion, and to compare the efficacies of these two methods.
METHODS: Adipose tissues were isolated from the inguen of C57BL/6 mice, from which OP-ADSCs were obtained by tissue explant culture and collagenase digestion respectively. Then, the cells were subjected to primary culture and subculture. The surface specific antigens of passage 3 cells were observed using flow cytometry, while the yields and proliferation abilities of passage 3 were compared at 7 days of culture. The adipogenic and osteogenic differentiation of OP-ADSCs at passage 4 was detected.
RESULTS AND CONCLUSION: (1) The expression levels of CD34, CD146, Sca-1 in passage 3 cells 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 adipose tissue by tissue explant culture was significantly higher than that by collagenase digestion (P < 0. 05). (3) The proliferation rate of cells in the tissue explant culture group was higher than that in the collagenase digestion group after 24 hours 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 (both P > 0.05). These experimental findings indicate that the tissue explant culture is more suitable for obtaining OP-ADSCs in vitro as compared with collagenase digestion, which contributes to provide adequate cell sources for studies on ADSCs treatment of osteoporotic fractures and bone defects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Osteoporosis, Cell Culture Techniques, Tissue Engineering

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