1.North China University of Technology;2.Affiliated Hospital of North China University of Technology
General funded project of Hebei Natural Science Foundation (H2019209154)
目的 旨在建立一种高效的方法分离和培养大鼠骨髓间充质干细胞 (Bone marrow mesenchymal stem cells , BMSCs) 的方法，并应用PKH26进行体外标记，探讨PKH26标记对BMSCs生物学特性的影响，以及体外示踪情况。方法 将大鼠5d乳鼠双后肢骨进行分离，去除周围的肌肉和筋膜，并将其剪成小块进行培养，利用换液、传代提纯BMSCs，应用流式细胞仪测定第3代细胞表面抗原。在培养条件相同的情况下，取第3代BMSCs应用PKH26进行标记，标记组与未标记组在荧光显微镜下对细胞形态学、增殖状态进行观察，比较标记组与未标记组成骨成脂诱导特点及鉴定。结果 骨髓骨片法分离乳鼠双后肢骨，培养BMSCs呈细长梭形，形态均一，短时间内可迅速获得大量BMSCs; 经流式细胞仪鉴定结果表明，表达CD29为(91.18±1.29)%，表达CD90为(91.18±1.29)%, 表达CD45为(1.74±0.36)%； PHK26标记对BMSCs细胞形态，增殖无明显影响(P>0.05),对成骨成脂诱导无影响。结论 采用大鼠5d乳鼠骨髓骨片法能够快速培养出大量高纯度的BMSCs，该细胞可作为种子细胞用于骨组织工程；PKH26可对体外大鼠BMSCs进行标记。
Objective To establish an efficient method for isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs), and apply PKH26 to label them in vitro to explore the effect of PKH26 labeling on the biological characteristics of BMSCs, as well as the in vitro tracing. Methods The bone of both hind limbs of 5d suckling rats were separated, the surrounding muscle and fascia were removed, and cut into small pieces for culture. BMSCs were purified by fluid exchange and passage, and the third generation cell surface antigen was determined by flow cytometry. Under the same culture conditions, the third generation BMSCs were labeled with PKH26. Cell morphology and proliferation status were observed under fluorescence microscope in the labeled group and the unlabeled group, and the adipogenic induction characteristics and identification of the labeled group and the unlabeled group were compared. Results The bone marrow slice method was used to separate the hind limb bones of suckling mice. The BMSCs were slender spindle shaped and uniform in shape. A large number of BMSCs could be rapidly obtained in a short time; The results of flow cytometry showed that the expression of CD29 was (91.18 ± 1.29)%, the expression of CD90 was (91.18 ± 1.29)%, and the expression of CD45 was (1.74 ± 0.36)%; PHK26 labeling had no significant effect on the morphology and proliferation of BMSCs cells (P>0.05), and had no effect on the induction of osteogenesis and adipogenesis. Conclusions A large number of high-purity BMSCs can be rapidly cultured by the method of 5-day rat bone marrow slices, which can be used as seed cells for bone tissue engineering; PKH26 can label rat BMSCs in vitro.