不同分离方法及培养条件对兔骨髓间充质干细胞生物活性的影响

Effects of separation methods and culture conditions on biological characteristics of rabbit bone marrow mesenchymal stem cells

背景:骨髓间充质干细胞常被用作骨组织工程的种子细胞和基因治疗的载体细胞,如何获得纯度较高活性较好的骨髓间充质干细胞是很多研究者关心的问题。目的:观察不同分离方法及培养条件对兔骨髓间充质干细胞生物活性的影响。方法:分别用密度梯度离心法、全骨髓培养法和红细胞裂解液法提取兔骨髓间充质干细胞,48h后观察各组获得的细胞量,记录各组首次传代时间,相差显微镜观察细胞的形态,流式细胞仪鉴定其CD44抗原表达。分别用DMEM-LG、MEM-HG和DMEM/F123种培养基培养第3代细胞和第7代老化细胞,MTT法和计数板计数法比较不同时间点3种培养基培养细胞的生长情况,相差显微镜观察老化细胞的形态变化。结果与结论:3种分离方法均能得到较均一的梭形或者多角形贴壁细胞,胞核大胞浆丰富,CD44抗原表达阳性。其中红细胞裂解液法获得的贴壁细胞的量较其他方法多,首次传代时间短(P<0.05)。用DMEM/F12培养的细胞活力更好,细胞生长更迅速,并且经过DMEM/F12培养一段时间后,部分静止期细胞开始分裂增殖。提示红细胞裂解液法能够提高骨髓间充质干细胞的贴壁效率,是一种方便、有效的提取方法;DMEM/F12更能够促进细胞的增殖,较适合于培养骨髓间充质干细胞。

BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are widely utilized as seed cells or carriers in bone tissue engineering and gene therapy. Thus, how to obtain BMSCs with high purity arose more attentions of researchers. OBJECTIVE： To observe the effects of different separation methods and culture conditions on biological characteristics of rabbit BMSCs. METHODS： BMSCs were obtained by whole bone marrow culture, density grand centrifugal and red blood cell lysis. At 48 hours after culture, the cell numbers were counted, the time of passage was recorded, in addition, the cell morphology was observed by phase contrast microscope, and the CD44 antigen expression was identified using flow cytometry. The 3rd and the 7th generation aging cells were cultured with DMEM-LG, MEM-HG, and DMEM/F12 culture medium. MTT and count cell plat were used to evaluate the growth of BMSCs. Phase contrast microscope was used to observe the morphological changes of aging cells. RESULTS AND CONCLUSION： BMSCs could be separated by each method. The adherent cells showed shuttle or multiple angle shapes, with rich cytoplasm, and positive for CD44 antigen. The more cell number and shortest primary culture time was presented in red blood cell lysis group （P 0.05）. DMEM/F12 could promote the proliferation of quiescent cells. And the cells prevented the better viability. The method of read blood cell lysis improving the efficiency of BMSCs adherent is an effective method of extraction of BMSCs. DMEM/F12 could promote the proliferation Maybe, DMEM/F12 is more suitable for BMSCs.