PDF《近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性》

中南大学学报(医学版) J Cent South Univ (Med Sci) 2015, 40(3) http://www.

csumed.org

261 近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性 向杨1 ,邢健生1 ,白志明1 ,高元慧2 ,曹卉2 ,王顺兰2 ( 中南大学湘雅医学院附属海口医院 1. 泌尿外科;

2. 中心实验室,海口 570208) [摘要] 目的:建立近交系五指山小型猪(inbreed line miniature pig of Wuzhishan,ILMW)骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BM-MSCs)体外分离培养和鉴定方法,比较我国不同种系猪来源BM-MSCs的生物 学特性,为下一步研究BM-MSC对ILMW单侧输尿管梗阻所致肾损伤的修复机制提供细胞来源.方法:选取4月龄及 10月龄健康ILMW各4头,从距髂骨翼边缘1 cm处穿刺,抽出骨髓约5 mL;

经密度梯度法分离单个核细胞,分别用含 10%,12%和15%胎牛血清(fetal bovine serum,FBS)的完全培养基于体外培养;

并取第1代、第3代、第5代、第11代细胞 分别行细胞形态学、表型、分化能力、生长曲线、细胞周期等检测.结果:分离培养的BM-MSCs在体外贴壁,呈成 纤维样或旋涡状生长;

原代细胞18 h出现贴壁,第6天进入对数增长期,约9 d后80%融合,传代后细胞状态未出现明显 异常.细胞表面抗原检测显示CD29,CD44,CD90表达呈阳性;

CD34和CD45表达呈阴性;

且各代间差异无统计学意 义(均P>

0.05).成骨诱导18 d后,茜素红染色阳性;

成软骨诱导21 d后,阿利新蓝染色阳性.细胞周期检测显示G0/G1 的细胞百分比约为81.45%.结论:ILMW的BM-MSCs贴壁时间早,细胞增殖活跃,传代周期短,传代后仍能保持稳定 的生物学特性,是中国大陆地区猪来源BM-MSC的最佳选择之一. [关键词] 猪;

骨髓间充质干细胞;

生物学特性;

慢性肾损伤 Separation culture and biological characteristics analysis for bone marrow-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan XIANG Yang1 , XING Jiansheng1 , BAI Zhiming1 , GAO Yuanhui2 , CAO Hui2 , WANG Shunlan2 (1. Department of Urology;

2. Department of Central Laboratory, A?liated Haikou Hospital, Xiangya School of Medicine, Central South University, Haikou 570208, China) ABSTRACT Objective: To establish a method to isolate, culture and identify bone marrow-derived mesenchymal stem cells (BM-MSCs) from inbreed line miniature pig of Wuzhishan (ILMW) in vitro, to compare the biological characteristics of BM-MSCs derived from different pigs, and to supply BM-MSCs for investigating the repair mechanisms of renal injury in ILMW after unilateral 收稿日期(Date of reception):2014-08-01 第一作者(First author):向杨,Email: [email protected];

邢健生为并列第一作者,Email: [email protected] 通信作者(Corresponding author):白志明,Email: [email protected] 基金项目(Foundation item):海南省重大科技项目(2013-SZK-01-039).This work was supported by the Majoy Science and Technology?Project?of Hainan Province, P. R. China (2013-SZK-01-039). DOI:10.11817/j.issn.1672-7347.2015.03.005 www.csumed.org/xbwk/fileup/PDF/201503261.pdf

262 ureteral obstruction. Methods: Four or 10-months old ILMW (n=4 per group) were selected and

5 mL of bone marrow fluid was extracted at

1 cm position from iliac wing edge. Mononuclear cells were isolated by density gradient?method, then were cultured in the complete medium containing

3 different kinds of fetal bovine serum (FBS) (10% FBS, 12% FBS or 15% FBS). The cells of Passage 1, Passage 3, Passage

5 or Passage