人骨髓间充质干细胞无血清培养的实验研究

发布时间：2018-04-20 08:14

本文选题：骨髓间充质干细胞 + 细胞培养　；参考：《中南大学》2008年硕士论文

【摘要】： 目的建立一种体外无血清培养扩增人骨髓间充质干细胞的方法,为骨组织工程研究提供种子细胞。方法从5例手术病人骨髓中分离制备单个核细胞悬液,分别接种于有血清培养基及自制的无血清培养基中,比较两种培养条件下细胞生长情况,流式细胞仪分析两种培养条件下间充质干细胞表面CD分子表达情况及细胞周期。结果有血清培养2～3天后细胞开始出现贴壁生长,1周左右有细胞丛生长,12～14天细胞融合成片,呈典型的旋涡状排列。无血清培养4～5天后细胞开始出现贴壁生长,8～10天左右有细胞丛生长,16～20天细胞融合成片。单层融合的MSCs消化传代培养,有血清培养1周左右达融合,无血清培养9～10天左右达融合,可继续传代扩增。流式细胞仪检测显示,两种培养条件下扩增后的人骨髓MSCs均不表达CD14,CD34和CD45,而强表达CD29、CD54、CD105;无血清培养组G_0/G_1期细胞比例明显高于有血清培养组(P＜0.05)。结论 1.利用自行配制的无血清培养基能较好地培养、扩增人骨髓MSCs。 2.无血清培养组G_0/G_1期细胞比例明显高于有血清培养组,说明与有血清培养相比,无血清培养的细胞其干细胞特性保存更好。
[Abstract]:Objective to establish a method for amplification of human bone marrow mesenchymal stem cells (BMSCs) by serum-free culture in vitro to provide seed cells for bone tissue engineering. Methods Mononuclear cell suspensions were isolated from bone marrow of 5 patients and inoculated in serum-free medium and serum-free medium respectively. The expression and cell cycle of CD molecules on mesenchymal stem cells were analyzed by flow cytometry. Results after 2 days of serum-culture, the cells began to grow with adherent cells for 1 week or so. After 1214 days of cell growth, the cells were fused into pieces, and the cells were arranged in a typical swirl shape. After 5 days of serum-free culture, adherent growth began to occur in the cells. In monolayer fusion MSCs digestion and passage culture, serum-free culture reached fusion about 1 week, serum-free culture reached fusion about 10 days, and can continue to be subcultured and amplified. The results of flow cytometry showed that human bone marrow MSCs did not express CD14 CD34 and CD45, but strongly expressed CD29, CD54 and CD105, and the proportion of G_0/G_1 phase cells in serum-free culture group was significantly higher than that in serum-free culture group (P < 0.05). Conclusion 1. Human bone marrow MSCs could be amplified by using self-made serum-free medium. 2. The proportion of G_0/G_1 phase cells in serum-free culture group was significantly higher than that in serum-free culture group, which indicated that the stem cell characteristic of serum-free culture group was better than that of serum-free culture group.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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