绿色荧光蛋白小鼠骨髓间充质干细胞培养及其示踪的可行性

Culture and labeling of bone marrow mesenchymal stem cells from green fluorescence protein mice: A comparative study

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 obverse and compare the two isolation and culture methods for bone marrow mesenchymal stem cells （BMSCs） and study the feasibility to use green fluorescence protein（GFP）-BMSCs as a labeling method in transplantation experiment. Methods Proliferation of BMSCs cultured with compact bone culture method（A） and whole bone marrow culture method（B） was induced and other culture methods were used to optimize fluid-changing measures. Morphological changes in GFP-BMSCs cultured with methods A and B were observed. Activity of BMSCs in GFP mice was identified according to the growth curve and multi-differentiation functions of BMSCs after the third passage. The stability of BMSCs in GFP mice after several passages and green fluorescence-induced differentiation was observed. Results 1）BMSCs were proliferated into clone colonies with different sizes, and then further expanded around and integrated with the adjacent clone colonies after primary culture with method A. The cells after the third or forth passage and the purified BMSCs were obtained after primary culture with method B because they were mixed with many blood cells. 2）The growth curve of BMSCs was observed after cultured with methods A and B. The BMSCs proliferated rapidly and their purity was high after cultured with method A. However, the BMSCs were difficult to proliferate due to the influence of other cells. 3）The BMSCs with a higher activity cultured with methods A and B could be induced to adipocytes with oil O staining. The bone structure of some aggregated BMSCs was observed during osteogenesis differentiation. 4）The GFP-BMSCs after several passages were characterized by stable biological properties and the GFP was strongly expressed after 5 passages and induction of differentiation. Conclusion More GFP-BMSCs with a high concentration can be obtained with bone slice culture method than with traditional method after primary or first passage. Their fluorescence is not diminished after several passages and can thus be used as a labeling method in vivo.