骨髓骨片法体外培养大鼠骨髓间充质干细胞和PKH26标记

Bone marrow mesenchymal stem cells isolated from rats by bone marrow and slice adherent culture in vitro and PKH26 labeling

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5 d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(95.04±0.11)%,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5 d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 to isolate and culture rat bone marrow mesenchymal stem cells(BMSCs)and apply PKH26 to label them in vitro to explore the effect of PKH26 labeling on their biological characteristics and achieve in vitro tracing.Methods Hind limb bones of 5-day-old rats were separated,surrounding muscle and fascia were removed,and the bones were cut into small pieces for culture.BMSCs were purified by fluid exchange and passaging.Cell surface antigens of third passage cells were analyzed by flow cytometry.Under the same culture conditions,third passage BMSCs were labeled with PKH26.Cell morphology and proliferation of labeled and unlabeled groups were observed under a fluorescence microscope.Adipogenic induction characteristics and identification of labeled and unlabeled groups were compared.Results The bone marrow slice method was used to separate hind limb bones of 5-day-old mice.The BMSC shape was slender,spindle,and uniform.A large number of BMSCs was rapidly obtained in a short time.Flow cytometry showed CD29 expression in(9118±129)%of cells,CD90 expression in(9504±011)%of cells,and CD45 expression in(174±036)%of cells.PHK26 labeling had no significant effect on the morphology or proliferation of BMSCs(P>005)or induction of osteogenesis or adipogenesis.Conclusions A large number of high-purity BMSCs was rapidly cultured by the method from 5-day-old rat bone marrow slices,which can be used as seed cells for bone tissue engineering.PKH26 can also label rat BMSCs in vitro.