人骨髓间充质干细胞的贴壁分离与体外培养  

Adherent separation and culture of human bone marrow-derived mesenchymal stem cells

作　　者：赵凌云[1] 钱淑琴[1] 赵廷宝[1] 

机构地区：[1]解放军济南军区总医院脊髓修复科,山东省济南市250031

出　　处：《中国组织工程研究与临床康复》2007年第28期5649-5651,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:验证贴壁方式分离人骨髓间充质干细胞,并进行体外扩增培养的可行性。方法:实验于2006-02/12在解放军济南军区总医院脊髓修复科完成。①实验材料:骨髓来源于解放军济南军区总医院脊髓修复科收治的脊髓完全性损伤患者,对本实验知情同意。基础培养液由含体积分数为0.15胎牛血清和低糖α-MEM配置。②实验方法:无菌条件下髂后上棘穿刺抽取骨髓组织6mL,进行细胞培养,观察细胞生长情况,待细胞融合成片、长满培养瓶底部后,用质量浓度为2.5g/L的胰蛋白酶流过所有细胞表面。倒置显微镜下观察细胞变圆、部分脱壁后,立即加入有血清培养液终止消化。③实验评估:取第3代生长状态良好的细胞,胰蛋白酶消化制成细胞悬液,接种,以细胞数为纵坐标,时间为横坐标,绘制细胞生长曲线。同时每隔2h进行细胞贴壁率检测。结果:①骨髓间充质干细胞的形态学观察:倒置显微镜下,骨髓间充质干细胞接种1d即贴壁,去除悬浮细胞后继续培养3d贴壁细胞开始增殖,伸展为椭圆型、短梭型、多角型及不规则型等。至14d细胞密集在集落中心,基本铺满瓶底,第3～5代细胞呈均匀一致的长梭型,排列成旋涡状或放射状。②骨髓间充质干细胞的生长曲线:细胞传代后3d内处于潜伏期,3d后进入生长期,7d后进入平台期。③骨髓间充质干细胞的贴壁率:随着培养时间的延长,骨髓间充质干细胞贴壁率逐渐升高。传代后2,4,6,8,10,12,14,16,18,20h细胞贴壁率分别为(20.20±0.25)%,(33.00±0.29)%,(46.50±0.32)%,(69.20±0.30)%,(76.60±0.34)%,(86.50±0.27)%,(90.30±0.20)%,(96.10±0.28)%,(98.50±0.12)%,(99.00±0.07)%。结论:贴壁法分离骨髓间充质干细胞操作简便,经体外扩增培养后细胞增殖活性强,传代周期为7d,是比较理想的骨髓间充质干细胞培养方法。AIM： To investigate the feasibility of segregating and culturing bone marrow-derived mesenchymal stem cells （MSCs） in vitro by adherence method. METHODS： The experiment was conducted at Department of Spinal Cord Repairing, General Hospital of Jinan Military Area Command of Chinese PLA from February to December 2006.①The bone marrow was derived form patients suffered from fracture dislocation with spinal cord damaged completely, who were treated at Department of Spinal Cord Repairing, General Hospital of Jinan Military Area Command of Chinese PLA, and consented to this experiment. Fundamental culture solution comprised fetal bovine serum with volume fraction of 0.15 and low carbohydrates α-MEM. ②6 mL bone marrow was isolated sterilely from posterior superior iliac spine for cell culture. Cell growth was observed until cells coalesced and covered the bottom of the culture flask the cells were infiltrated by trypsin with concentration of 2.5 g/L. Fundamental culture solution was added to terminate concoction when cells turned into circle and detached partly. ③The third generation well-grew cells were digested into cell suspension and inoculated. Cells growth curve was drawn taking amount of cells as Y-axis and time as X-axis. Simultaneously, adhesion rate was determined every 2 hours. RESULTS：①Morphological observation of MSCs： Under inverted microscope, the MSCs began to adhere to the wall at day 1. MSCs began to proliferate and become into ellipse, Fusiform shape, polygon and irregularity at day 3. MSCs concentrated in the center of colony and covered the bottom of culture flask at day 14. The 3^rd-5^th generation MSCs presented Iong-fusiform shape and arranged into whirlpool or radiation. ②Growth curve of MSCs： MSCs were in latency in 3 days, converted into growing period after 3 days and entered into stationary phases after 7 days. ③Adherence rate of MSCs： The adherence rate increased gradually with time goes by. The adherence rates were （20.20±0.25）%, （33.00± 0.29）%, （46

关 键 词：骨髓间充质干细胞 贴壁 分离培养 细胞活性 

分 类 号：R394.2[医药卫生—医学遗传学]