生物工程学报 Chin J Biotech 2010, December 25; 26(12): 1645 1651 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn 2010 CJB, All rights reserved. 干细胞专栏 牛脂肪间充质干细胞的分离 培养与鉴定 任宇 1, 吴海青 1, 马玉珍 1,2, 仓明 1, 王瑞 1, 刘东军 1 010021 2 010017 摘要 : 为了给组织工程提供种子细胞, 对牛间充质干细胞 (Adipose-derived stem cells,adscs) 进行体外分离培养 首先应用胶原酶消化法分离牛 ADSCs, 进行体外培养 连续传代, 并观察细胞的形态变化, 通过细胞计数绘制生长曲线, 细胞压片进行染色体分析, 采用细胞免疫荧光化学方法检测细胞表面标记, 利用成骨分化和成脂分化检测其分化能力 结果显示牛 ADSCs 体外培养时细胞形态呈成纤维细胞样, 增殖稳定 ;Vimentin CD49d CD13 表达呈阳性, CD34 表达呈阴性 ; 成骨诱导条件下的细胞碱性磷酸酶活性高, 茜素红染色呈阳性 ; 成脂诱导条件下细胞周围脂滴明显, 油红 -O 染色呈阳性 结果证明牛 ADSCs 体外生长稳定 增殖速度快 定向分化能力强, 简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础 关键词 : 脂肪间充质干细胞, 体外分离培养, 细胞鉴定, 细胞分化 1 Isolation, cultivation and identification of adipose-derived stem cell in bovines Yu Ren 1, Haiqing Wu 1, Yuzhen Ma 1,2, Ming Cang 1, Rui Wang 1, and Dongjun Liu 1 1 Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology, Inner Mongolia University, Huhhot 010021, China 2 Inner Mongolia Hospital, Hohhot 010017, China Abstract: To obtain bovine adipose-derived stem cells (ADSCs), bovine ADSCs were digested in collagenase type I solution. The growth curve of ADSCs was checked by cell counting. Chromosome analysis was checked. The molecular markers of ADSCs were detected with immunofluoresence staining. The morphology of ADSCs was identical to fibroblast 1ike and the cells showed active proliferative ability. Vimentin, CD49d and CD13 antigens were detected, but CD34 antigen was negative. Alkaline phosphatase activity was greater in ADSCs during calcification, and Alizarin Red staining was positive. Lipid droplets were apparent around cells during adipogenesis, and Oil Red-O staining was positive. The results demonstrated that ADSCs could be used as seed cells for tissue engineering due to the simple isolation, differentiation and stable and active growth. Keywords: adipose-derived stem cell, cell separation in vitro, cell identification, cell differentiation (Mesenchymal stem cells MSCs) [1] (Bone marrow stromal Received: July 7, 2010; Accepted: October 13, 2010 Supported by: Major Projects in Inner Mongolia Natural Science Foundation (No. 20090505). Corresponding author: Yuzhen Ma. Tel/Fax: +86-471-6619236; E-mail: mayz@imnu.edu.cn 内蒙古自然科学基金重大项目 (No. 20090505) 资助
1646 ISSN1000-3061 CN11-1998/Q Chin J Biotech December 25, 2010 Vol.26 No.12 stem cells BMSCs) BMSCs [2] (Adipose-derived stem cell ADSCs) ADSCs 3 1 材料与方法 1.1 材料 1.1.1 1.1.2 DMEM/F12 PBS ( Hyclone ) Ⅰ β- Alizarin Red BSA IBMX Oil Red-O ( Sigma ) ALP ( ) 1.2 方法 1.2.1 ADSCs PBS 2 50 ml PBS ( 1 BSA 0.1 Ⅰ ) 37 60 min 20 min 1 200 r/min 5 min 3 1 200 r/min 5 min 1.2.2 2 10 5 /cm 2 60 mm 5 ml 37 5 CO 2 48 h 80 0.25 ADSCs 0.5 10 6 /ml (10 DMEM 10 DMSO 80 FBS) 80 24 h 1.2.3 ADSCs ADSCs 1 10 5 /ml 24 80 4 30 min (0.1 TritonX-100) 1 h (PBS+2 BSA+2 +2 +0.15 MGlycine) 37 2 h 1 200 Vimentin ( ) CD49d ( ) CD34 ( ) CD13 ( ) 37 2 h 1 50 FITC 37 2 h PBS+BSA PI 15 min PBS (BX61
: 1647 ) 1.2.4 5 10 1 10 4 /ml 24 1 ml 2 3 (d) ( 10 4 /ml) 1.2.5 5 10 ADSCs 0.1 μg/ml (Sigma) DMEM-F12 4 h 0.25 37 0.075 mol/l KCl 5 ml 37 20 min ( = 3 1) 1 ml 1 min 1 000 r/min 10 min 30 min 2 30 min 1.5 ml 20 1 9 (Sigma) 15 min 1.2.6 24 h 3 (DMEM/ F12+10 FBS+1 +10 mmol/l β- +20 nmol/l +50 μg/ml ) 3 21 d 1.2.7 24 h (DMEM/F12+3 FBS+1 +33 μmol/l +17 μmol/l +1 μmol/l +1 μmol/l +0.5 mmol/l IBMX+5 μmol/l + 5 ) 3 d IBMX 3 3 21 d 1.2.8 21 d 150 mmol/l NaCl 3 4 70 1 h 2 (NaOH ph 4.1~4.3) 10 min (Alkaline phos phatase ALP) 21 d PBS 2 4 100 1 h NBT/BCIP ALP PCR (Osteocalcin) TaKaRa RNAiso reagent RNA DEPC RNA cdna PCR ( 1) PCR 94 4 min 94 30 s 56 30 s 72 30 s 35 72 10 min 4 表 1 本研究中所用引物 Table 1 Primers used in this study Gene GAPDH Forward primers (5 3 ) TGAACGGGAAG CTCACTGG Osteocalcin GGTGCAGACCT AGCAGACA PPARγ2 GAGCATGGTGC CTTCGCTGA Reverse primers (5 3 ) TCCACCACCCTG TTGCTGTA GTGCCGTCCATA CTTTCG AGCAAGGCACTT CTGAAACCGA Length (bp) 360 424 564 1.2.9 -O 10 20 min -O 20 min PCR PPARγ2 1 ( PCR Osteocalcin ) 2 结果 2.1 形态学观察 ADSCs 4~6 h 48 h 4 d ( 1) 8~10 d 80 ~85 ADSCs ( 2)
1648 2.2 ISSN1000-3061 CN11-1998/Q Chin J Biotech 牛 ADSCs 鉴定 FITC 荧光信号呈绿色 激发波长 488 nm 在波 December 25, 2010 呈阴性 以 PBS 代替一抗和牛成纤维细胞为阴性对 照 以上抗体染色均呈阴性 (图 3) 图1 图2 图3 原代牛 ADSCs 形态观察 (100 ) Morphology of primary bovine ADSCs (100 ). No.12 Vimentin CD49d 和 CD13 均匀染色呈阳性 CD34 长 530 nm 以 上 观 察 第 5 代 牛 ADSCs 细 胞 Fig. 1 Vol.26 Fig. 2 第 5 代牛 ADSCs 形态观察 (100 ) Morphology of P5 bovine ADSCs (100 ). 牛 ADSCs 免疫组织化学染色结果 Fig. 3 Results of ADSCs immunofluoresence staining. (A-D) Bovine ADSCs. (E-H) Bovine fibroblasts. (A, E) Vimentin staining. (B, F) CD49 staining. (C, G) CD13 staining. (D, H) CD34 staining. (A1-H1) A-H corresponding to excited without excitation of the original map.
1649 任宇等: 牛脂肪间充质干细胞的分离 培养与鉴定 2.3 生长曲线 从图 4 中可以看出 第 5 10 代的牛 ADSCs 生 长曲线基本符合细胞生长曲线规律 经历了潜伏期 对数期 平台期 分别为 1 d 2~6 d 6~8 d 后 说 明牛 ADSCs 在体外增殖稳定 生长状态无异常 图5 Fig. 5 图4 Chromosomes of F5ADSCs (1 000 ). 牛 ADSCs 生长曲线 Fig. 4 2.4 F5ADSCs 染色体 (1 000 ) Growth curve of bovine ADSCs. 染色体分析 经消化收取细胞进行染色体分析 第 5 代牛 ADSCs 有 90 (27/30) 细胞的染色体具有正常二 倍 体 倍 性 (图 5) 第 10 代 牛 ADSCs 有 84 (25/30) 细胞的染色体仍具有正常二倍体倍性 (图 图6 Fig. 6 F10ADSCs 染色体 (800 ) Chromosomes of F10ADSCs (800 ). 6) 说明牛脂肪间充质干细胞在体外生长染色体正 常 (图 5) 2.5 诱导分化 2.5.1 成骨诱导分化 牛 ADSCs 成骨诱导后 第 10~14 天已具有较强 成骨活性 21 d 后实验组细胞结节中心的细胞逐渐 融合失去细胞结构 骨化结形成明显 经茜素红染 色呈暗红色 (图 7A 箭头所指处) 对照组细胞经过 21 d 培养后仍然呈成纤维细胞样生长 细胞无堆积 现象出现 茜素红染色后无暗红色骨化结出现 (图 图7 牛 ADSCs 成骨诱导 21 d 后茜素红染色 (100 ) Fig. 7 Alizarin Red Staining of bovine ADSCs after osteogenic induction on 21th day (100 ). (A) Experimental group. (B) Control group. 7B) ALP 染色实验组细胞呈阳性 细胞着色程度 不一 呈蓝紫色 且细胞密集处着色更深 (图 8A 箭 头所指处) ALP 阳性率可到 85 以上说明有大量 成骨细胞形成 而对照组细胞经 ALP 染色后细胞无 明显着色 呈阴性 (图 8B) 骨钙素是骨组织的特异 性蛋白 是骨细胞分化成熟的标志 在牛 ADSCs 中 不表达 (如图 9A 中泳道 3 所示) 在成骨细胞中表 图8 达 (如图 9B 中泳道 3 所示) 以上结果均说明牛间充 Fig. 8 ALP Staining of bovine ADSCs after osteogenic induction on 21th day (100 ). (A) Experimental group. (B) Control group. 质干细胞在成骨诱导体系下已经向成骨细胞分化 牛 ADSCs 成骨诱导 21 d 后碱性磷酸酶染色 (100 )
1650 ISSN1000-3061 CN11-1998/Q Chin J Biotech December 25, 2010 Vol.26 No.12 图 9 牛 ADSCs 电泳结果 Fig. 9 Electrophoresis of bovine ADSCs. (A) Electrophoresis of GAPDH. (B) Electrophoresis of osteoblasts. (C) Electrophoresis of adipocyte. 1: 200 bp marker (TaKaRa); 2: GAPDH (360 bp); 3: Osteocalcin (424 bp); 4: PPARγ2 (564 bp); 5: negative control. 2.5.2 ADSCs -O ( 10A ) 21 d -O ( 10B) (PPARγ2) PPARγ2 ADSCs ( 9A 4 ) ( 9C 4 ) 图 10 牛 ADSCs 成脂诱导 21 d 后油红 -O 染色 (100 ) Fig. 10 Oil Red-O Staining of bovine ADSCsafter adipogenesis introduction on 21th day (100 ). (A) Experimental group. (B) Control group. 3 讨论 21 2001 Zuk [3] ADSCs [4-6] [7] [8] [9] [10] ADSCs ADSCs NH 4 Cl NH 4 Cl ADSCs Vimentin CD49d CD13 CD34 Vimentin 5 ADSCs Vimentin ADSCs CD49d CD49d CD106 [11-12] CD34 CD34 ADSCs ADSCs ADSCs
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