【作者】 李晓莉；

【导师】 刘天庆；

【作者基本信息】 大连理工大学 ， 化学工程， 2008， 硕士

【摘要】 骨髓间充质干细胞（Mesenchymal stem cells,MSCs）是一类存在于骨髓间质层的极少量细胞,不但具有强大的自我更新及多向分化潜能,还具有支持造血的重要功能,并且植入反应弱,易于被外源基因转染并稳定表达,是组织工程和细胞/基因治疗中较为理想的种子细胞,受到广泛关注。但是骨髓中的MSCs的含量非常少,并且在正常培养条件下细胞在传代过程中容易失去增殖和分化活性,这极大影响了间充质干细胞的可用性。所以在扩增过程中,如何在保证增殖数量的同时,减少在细胞增殖过程中产生的损伤,保持干细胞的特性及分泌基质的能力,这是组织工程中首先应解决的问题,也是临床利用这些细胞治疗疾病的前提。为了提高间充质干细胞的扩增倍数并保持干细胞的特性,本文在低氧条件下采用灌注式中空纤维膜生物反应器（Hollow-fiber Membrane Bioreactor）进行体外扩增MSCs。采用密度梯度离心法分离SD大鼠骨髓间充质干细胞,将第五代MSCs接种于醋酸纤维素中空纤维膜上预培养24小时后,转移至反应器中。根据骨髓中溶解氧对应的气相氧气浓度的范围是1%-7%,用三气培养箱制造低氧环境,低氧组设为5%O₂,对照组为20%O₂。连续培养7天后,收获细胞。通过计算细胞的扩增倍数、成纤维细胞-集落形成实验、多向诱导分化检测和流式细胞仪分析,考察低氧动态环境对骨髓间充质干细胞增殖的影响。结果表明,低氧条件下在灌注式中空纤维膜生物反应器内,O₂含量基本保持恒定;MSCs扩增倍数明显增加,7天后扩增倍数达到11倍,明显高于常氧组8倍的扩增倍数。在低氧动态环境下扩增的细胞具有更强的成集落能力,低氧组集落数为112,而对照组为53,并且细胞的增殖潜能和代谢活性都有所增强。多向诱导分化检测和流式细胞仪分析结果显示,骨髓间充质干细胞在低氧动态条件下培养具有更强的诱导分化能力,保持了较好的干细胞特性。因此,本文所采用的培养方法是体外培养与扩增MSCs的一种有效的方法。更多还原

【Abstract】 Rat mesenchymal stem cells （MSCs） represent a small portion of the cells in the stromal compartment of bone marrow, capable of differentiating in vitro into mature-like cells from all three germ layers. They have the potential to differentiate into cartilage cells, cardiac muscle cells, adipose cells, nerve cells and osteoblasts. They are the promising "seed cells" in tissue engineering and ideal target cells in gene therapy for their low implanting response, being easy to be transfected and expressed steadily and function of supporting hematopoiesis.However, MSCs are in limited quantity and during the process of proliferation, MSCs may lose the activity of proliferation and differentiation gradually under the normal culture conditions, which can not meet the need of autologous repairation and clinical therapy. So how to reduce the damage during the cell proliferation, and retain the character and the ability of secreting matrix of the stem cells is very important.In order to raise the expansion fold and retain the stemness, a perfusion hollow fiber membrane bioreactor was developed and used to culture and expand MSCs in vitro. SD rat mesenchymal stem cells （rMSCs） were isolated with density gradient centrifugation. The cells were seeded on cellulose acetate hollow-fiber membrane constructs and preincubated for 24 hours. Then the constructs were transferred to the bioreactor and cultured for up to 7 days. Because the partial pressure of the atmospheric oxygen （pO₂） in bone marrow is much lower than that in the air, rMSCs were grown under low-pO2 conditions （5% oxygen） and air （21% oxygen） for comparison. The cell expansion fold, colony formation, multi-differentiation were assayed and fluorescence-activated cell sorter analysis were carried out to examine the role of different pO₂ in dynamic condition on rMSC proliferation.The results showed that in hollow-fiber membrane bioreactor, O₂ concentration kept almost constant; MSCs expansion fold in 5%O₂ is 11, while 8 in control oxygen and proliferation potential and metabolic activity were higher for cultures maintained in low oxygen than those in control oxygen. In 5%O₂ condition rMSCs on hollow-fiber membranes can maintain significantly higher colony-forming unit capabilities than normoxic controls, approximately 112 and 53 respectively.Hypoxic rMSCs also expressed higher levels of CD44⁺ and CD29⁺ markers than normoxic controls. After being induced, the expanded cells still reserved the strong muti-differentiation potential. Therefore, it’s safe and effective for MSCs culture and expansion in vitro with the method used here.更多还原