豚鼠脂肪干细胞的分离培养与鉴定  

Isolation, culture and identification of adipose-derived stem cells in guinea pigs

作　　者：孙慧如[1] 王英[1] 董明敏[1] 

机构地区：[1]郑州大学第一附属医院耳鼻咽喉科,河南省郑州市450052

出　　处：《中国组织工程研究与临床康复》2007年第7期1213-1215,I0001,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:从成年雄性豚鼠脂肪中分离培养脂肪干细胞,为脂肪干细胞移植治疗感音神经性耳聋的实验研究创造条件。方法:实验于2005-12/2006-03在河南省高等学校临床医学重点学科开放实验室完成。成年雄性豚鼠,体质量500～750g。分离培养豚鼠脂肪组织,培养三四天后首次换液。倒置显微镜下观察有大量细胞贴壁和漂浮的血细胞,用磷酸缓冲液反复冲洗去除漂浮的细胞,加入DMEM基础培养基+10%胎牛血清培养液。传代前用少量磷酸缓冲液洗涤1次,加入0.25%胰蛋白酶和0.02%乙二胺四乙酸2mL,见大部分细胞胞质回缩、形态变圆,加入2mL含血清的DMEM培养液终止消化,收集细胞悬液、计数,以2×104/cm2细胞密度接种于新的培养皿内。二三天达到融合状态,继续传代培养。四甲基偶氮唑蓝测定第1、3、10代脂肪干细胞生长曲线,流式细胞仪检测脂肪干细胞表面标志。结果:①原代培养显示培养的脂肪间充质干细胞2d左右开始贴壁,7d左右可达90%融合,基本上为梭形成纤维样细胞形态。②生长曲线显示,豚鼠脂肪干细胞在1～3代增值能力较强,以第1代细胞最强,10代以后增殖能力减弱,细胞生长进入平台期。经消化传代后的第1、3、10代细胞均经历24～48h的潜伏期,此后为3～5d对数生长期,逐渐进入生长平台期。14代以前具有活跃的增殖能力。③脂肪干细胞标志CD105,CD44呈阳性表达,而造血干细胞标志CD34阴性表达。结论:成功地建立了一种分离培养豚鼠脂肪间充质干细胞的方法,其生长稳定、增殖较快,可作为干细胞移植治疗感音神经性耳聋实验研究的供体细胞。AIM： To isolate and culture the adipose-derived stem cells （ADSCs） from guinea pig so as to create bases for the experimental study on treating sensorineurel deafness by ADSCs transplantation. METHODS： The experiment was carried out between December 2005 and March 2006 in the Open and Key Laboratory of Clinical Medicine at Henan Universities. The inguinal fat pads ware excised from adult male guinea pig weighing 500- 750 g. A great quantity of adhesive and natant blood cells ware observed under inserted microscope and phosphate buffer was used to wash natant cells repeatedly. Then the cells ware cultured in DMEM with 10% fetal bovine serum. Before passage, the cells ware washed by a small quantity of phosphate buffer and then cultured in 0.25% trypsin and 0.02% EDTA （2 mL）. Then most of the ADSCs presented cytoplasm recovery and round shape, followed by digestion of 2 mL DMEM with feral bovine serum. Cellular suspension was collected to count the cells that ware incubated at the density of 2×10^4/cm^2. The morphology of ADSCs was observed constantly. Growth curve of ADSCs at passages 1, 3, 10 ware recorded and detected by MTT. Surface markers of ADSCs ware detected by flow cytometry. RESULTS： （1）The result of primary culture showed that ADSCs at day 2 began to adhesion and more than 90 % of ADSCs at day 7 were approximately fusiform or fibroblast-shapod.（2）MTT detection showed that ADSCs had the capability to proliferate successively, especially at passage 1, 2, 3. But ADSCs at passage 10 presented decreasing proliferation. After digestion, the cells at passages 1, 3, 10 experienced the latent period （24-48 hours）, Iogarithmic phase （3-5 days） and growth platform phase.（3）CD105 and CD44 ware positive by flow cytometry, whereas CD34 was negative. CONCLUSION： Due to stable growth and rapid proliferation, the ADSCs from guinea pig can be used as the donor cells in the treatment of sensorineurel deafness by stem cell transplantation.

关 键 词：脂肪组织 干细胞 细胞培养 流式细胞仪 

分 类 号：R394.2[医药卫生—医学遗传学]