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》

11 were collected to examine biological characteristics including morphology, phenotype, differentiation ability, growth curve and cell cycle. Results: The BM-MSCs were attached to the culture dishes, which were fibroblast-like or whirlpool- like. Primary cultured cells began the adherence at

18 h and entered a logarithmic phase in the 6th day. Eighty percent of them were fused in the 9th day. There were no obvious anomalies in the subcultured cells. The expressions in cell surface?antigens of CD29, CD44 and CD90 were positive, while the expressions of CD34 and CD45 were negative. There was no statistically significant difference between cells from different generations (all P>

0.05). Under condition of osteogenic induction, alizarin red staining was positive at the 18th day, and alcian blue staining was positive at the 21th day. Cell cycle examination showed that the rate of G0/G1 was about 81.45%. Conclusion: BM-MSCs of ILMW has advantages of earlier adherent time, more active proliferation, shorter cell subculture cycle, and stable biological characteristics after subculture, which is one of the best kinds of BM-MSCs coming from swine in mainland of China. KEY WORDS swine;

bone marrow-derived mesenchymal stem cells;

biological characteristics;

chronic renal failure 慢性肾病(chronic kidney failure,CKD)已逐渐 成为影响公众健康的社会问题,且患病率和病死率 不断升高.骨髓间充质干细胞(bone marrow-drived mesenchymal stem cells,BM-MSCs)是一种来源于中 胚层系的具有多能分化潜能的细胞,主要作用是参 与自我修复、促进细胞成熟、补充造血干细胞等. 大量研究[1-8] 表明:BM-MSCs能 归巢 到损伤肾 组织,通过旁分泌及自分泌作用产生细胞因子、生 长因子等,参与抗炎、抗凋亡、抗氧化、促血管生 成、调节机体免疫及抗纤维化等作用.本研究旨在 建立近交系五指山小型猪(inbreed line miniature pig of Wuzhishan,ILMW)BM-MSCs的分离培养方法, 比较中国大陆地区其他品系猪BM-MSCs的生物学 特性,为下一步研究BM-MSCs对大动物输尿管梗 阻模型所致泌尿系损伤修复作用的实验研究提供最 佳细胞来源及理论基础.

1 材料与方法 1.1 动物、主要仪器和试剂 4月龄及10月龄健康ILMW各4头,体质量约 10?kg,雌雄不限.本实验获得中南大学湘雅医学 院附属海口医院实验动物伦理委员会批准;

所有 实验动物受到人道对待,操作符合我国《实验动 物管理条例》. 主要仪器:FACScanto6 流式细胞仪购自美 国BD公司,研究级倒置相差显微镜IX7购自日本 Olympus公司,光学显微镜TS100购自日本Nikon公司,酶标仪 X-Mark购自美国Bio-rad公司,低速离 心机KDL-40购自安徽中科生物科技有限公司,超 净工作台购自美国Baker公司,恒温CO2细胞培育 箱购自美国 Thermo公司. 试剂:青霉素链霉素双抗购自美国HyClone 公司,脯氨酸购自美国Amresco公司,猪BM-MSCs 分离液购自中国天津TBD公司,0.25%胰酶消化液 购自美国Sigma公司,KGA511购自中国南京凯基 公司,DMEM培养基、地塞米松、胰岛素-转铁蛋 白-硒添加物、FBS、谷氨酰胺、维生素C、丙酮 酸钠、转化生子因子-β

3、β-甘油磷酸酯、杜氏磷 酸盐缓冲液(Dulbecco'

s phosphate buffered saline, DPBS)均购自美国Gibco公司,CD29-APC,CD90- APC,CD44-APC,CD45-APC和IgG1等抗体均购 自英国Abcom公司. 1.2 方法 本研究分别采用10%,12%,15% 胎牛血清 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,等263 (fetal bovine serum,FBS)配置完全培养基,余成分 均相同.除特别说明,下文均以10%FBS为例,所 有图片均由奥林巴斯IX71荧光倒置高级显微镜拍 摄;