论文部分内容阅读
目的观察并比较两种小鼠骨髓间充质干细胞分离培养方法及选取GFP-BMSCs作为移植实验示踪的种子细胞可行性。方法通过骨片培养法(A法)和全骨髓贴壁法(B法)培养扩增小鼠骨髓间充质干细胞,并参照不同的培养方法优化其换液方式;观察两种培养方法原代及传代GFP小鼠骨髓MSCs形态变化;取第3代细胞进行生长曲线测定及多向分化功能检测;观察GFP小鼠骨髓MSC经多次传代及诱导分化后绿色荧光的稳定性。结果 1)A法原代培养可见贴壁细胞增殖形成大小不等的克隆集落,并向周围进一步扩展、融合,而B法在原代培养时由于混杂较多血液系统细胞,传代至3、4代即可获得较纯的BMSCs;2)观察其生长曲线,A法细胞扩增速度快,纯度高,B法细胞由于受杂细胞的影响,扩增难度大;3)BMSCs可被诱导成脂肪细胞,油红O染色可见红色脂肪颗粒;在成骨分化过程中可见骨结节结构形成;4)GFP-BMSCs传代后生物学特性稳定,传代至5代及诱导分化后其GFP表达仍为强阳性。结论与传统全骨髓贴壁法相比,骨片培养法可在原代或1代就获得高浓度的足量干细胞,GFP-BMSCs经多次传代后荧光不减退,可作为体内细胞示踪。
Objective To observe and compare the isolation and culture methods of two kinds of mouse bone marrow mesenchymal stem cells and the feasibility of using GFP-BMSCs as seed cells traced by transplantation experiment. Methods Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and expanded by bone chip culture method (A method) and whole bone marrow adherent method (B method), and the method of fluid exchange was optimized with reference to different culture methods. And passaged GFP mice bone marrow MSCs morphological changes; take the third generation of cells growth curve determination and multidirectional differentiation function detection; GFP mouse bone marrow MSC after multiple passages and induction of green fluorescence stability. Results 1) In primary culture of A method, adherent cells proliferated to form colonies with different sizes and further expanded and fused to the surrounding area. However, in the primary culture, method B was passaged to passages 3, 4 due to more mixed blood system cells BMSCs can be obtained more pure BMSCs; 2) observe the growth curve, A method of cell expansion speed, high purity, B cells due to miscellaneous cells, expansion difficult; 3) BMSCs can be induced into adipocytes , The red fat granules were observed by Oil Red O staining; the formation of bone nodules was observed during osteogenic differentiation; 4) The biological characteristics of GFP-BMSCs after passage were stable, and their GFP expression was still strong after passage 5 and after differentiation . Conclusion Compared with the traditional whole bone marrow adherence method, the bone marrow culture method can obtain high concentration of sufficient stem cells in the primary or first generation. The GFP-BMSCs do not subside after the multiple passages and can be used as the in vivo cell tracing.