不同分离方法与培养条件下大鼠骨髓间充质干细胞生长增殖情况比较  

Comparison of the growth and proliferation of rat bone marrow mesenchymal stem cells under different culture conditions by different separation methods

作　　者：王文[1] 李静[1] 王宗仁[1] 张金洲[2] 师长宏[3] 

机构地区：[1]解放军第四军医大学西京医院中医科,陕西省西安市710032 [2]解放军第四军医大学西京医院心血管外科,陕西省西安市710032 [3]解放军第四军医大学实验动物中心,陕西省西安市710032

出　　处：《中国组织工程研究与临床康复》2007年第3期482-485,489,I0003,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:应用贴壁分离法和密度梯度离心法以不同培养条件观察纯化过程中对获得的骨髓间充质干细胞的影响,试图寻找获得高质量、高活性的骨髓间充质干细胞的培养条件。方法:实验于2005-09/2006-01在解放军第四军医大学实验动物中心实验部进行。①SPF级雄性Wistar大鼠18只,贴壁分离培养取12只,根据血清和培养基的不同分为4组:进口血清+DMEM组、进口血清+F12-DMEM组、国产血清+DMEM组、国产血清+F12-DMEM组,3只/组。密度梯度离心培养实验取剩余6只,以分离液的不同分为Ficoll分离液组、Percoll分离液组,3只/组。②骨髓取材:各组大鼠断颈处死后,无菌取双下肢,剔除股骨、胫骨周围肌肉组织,剪去骨干的两端,暴露骨髓腔,穿刺两侧骨端取出骨髓。③贴壁分离法:进口血清+DMEM组、进口血清+F12-DMEM组、国产血清+DMEM组、国产血清+F12-DMEM组大鼠分别采用相应的血清和培养基冲洗骨髓,制备单细胞悬液,接种于培养瓶中,5d后首次更换培养液,弃去未贴壁细胞,此后每3～4d换液1次。④密度梯度离心法:Ficoll分离液组选用的Ficoll分离液密度为1.077;Percoll分离液组将Percoll分离液与0.1mol/L磷酸盐缓冲液按9∶1比例混匀,密度为1.073。将两组大鼠骨髓置入离心管,离心弃上清,轻轻叠加到相同体积的各自对应分离液上,再次离心收集界面层白色混浊液,采用F12-DMEM培养液重悬细胞,按1×109L-1的密度接种于培养瓶中,培养条件与贴壁分离法相同。⑤指标检测:倒置显微镜下,每天观察贴壁分离、密度梯度离心不同培养条件下细胞的生长情况和活体形态特征。进行锥虫蓝排斥试验,蓝染细胞为死亡细胞,在3min内用计数板分别计数活细胞和死细胞,未被蓝染细胞所占细胞总数的百分比即为初步得到细胞活性的数据。两种分离方法均取生长良好的第3代细胞,以时间为横坐标,细胞数为纵坐标,绘制生长曲线。AIM： To observe the effects of adherence separation and density gradient centdfugation under different culture conditions on acquired rat bone marrow mesenchymal stem cells, so as to find a suitable culture condition for acquiring high quality and activity bone marrow mesenchymal stem cells. METHODS： The experiment was conducted at the experiment department of Laboratory Animal Center, the Fourth Military Medical University from September 2005 and January 2006. （1）Twelve from the 18 SPF male Wistar rats were selected for adherence separation, which were divided into 4 groups based on different serum and- nutritive medium： import serum＋DMEM group, import serum＋F12-DMEM group, domestic serum＋DMEM group, domestic serum＋F12-DMEM group with 3 rats in each group. The other 6 rats were used in density gradient centrifugation experiment, which were divided into Ficoll separating medium group and Percoll separating medium group based on different separating medium with 3 rats in each group. （2）Marrow sampling： All rats were killed by cervical dislocation to remove both lower extremities in asepsis condition, and the muscular tissue around femur and tibia were rejected; two ends of diaphysis were cut off, medullary canal were exposed and then marrow were flushed off. （3）Adherence separation： The marrow of the import serum ＋ DMEM group, import serum ＋ F12-DMEM group, domestic serum ＋ DMEM group, and domestic serum ＋ F12-DMEM group were flushed off with corresponding serum and nutritive medium, respectively to prepare single cell suspension, which was transferred into culture flask. Five days later, the culture solution was replaced the first time to abandon the suspension cell. Then the culture solution was changed subsequently every 3-4 days. （4）Density gradient centrifugation： 1.077 Ficoll was used in the Ficoll separating medium group; Percoll separating medium stock solution were evenly mixed into 0.1 mol/L phosphate buffer saline according to the ratio of 9：1 to obtain the

关 键 词：间质干细胞 骨髓细胞 培养基 条件性 细胞培养技术 

分 类 号：R394.2[医药卫生—医学遗传学]