马的骨髓间充质干细胞诱导为软骨细胞和成骨细胞及其鉴定

Induction of Horse Bone Marrow Mesenchymal Stem Cells into Chondrocytes and Osteoblasts

本研究旨在将建立的马(Equus caballus)骨髓间充质干细胞诱导分化为成骨细胞和软骨细胞。通过原代细胞培养获取马的骨髓间充质干细胞,并对第3代(P3)纯化细胞进行干细胞特性鉴定,之后诱导其向不同细胞分化并对诱导分化的细胞进行染色和特异性基因表达的鉴定。实验结果显示,获得的马骨髓细胞表达了干细胞转录因子和间充质干细胞表面标记物,确定获得的细胞为马骨髓间充质干细胞。P3代细胞经诱导培养后由长梭形转变为“骨结节”形态的成骨细胞和“铺路石”形态的软骨细胞。茜素红将诱导的成骨细胞团染成红色,并随着时间的递增红色“骨结节”逐步增大;阿尔新蓝则将蛋白聚糖和透明质酸等含量丰富的诱导细胞染为蓝色,并且随着诱导天数的增加被染成蓝色的软骨细胞逐渐增多,而对照组细胞未见着色。实时荧光定量PCR检测发现,成骨细胞中Col和ALPL基因的表达量随诱导时间的延长发生明显变化;普通PCR结果显示,在诱导的软骨细胞中扩增获得了collagenⅡ、aggrecan和Sox9软骨特异基因,而对照组细胞不表达特异基因。综上所述,本实验建立了马骨髓间充质干细胞并成功将其诱导分化为成骨细胞和软骨细胞,为骨组织缺损修复和软骨细胞的治疗提供了实验基础。

The purpose of the experiment was to induce the differentiation of osteoblasts and chondrocytes from bone marrow mesenchymal stem cells(BMSCs)in the horse(Equus caballus).BMSCs were obtained by primary cell culture,and stem cell characteristics of the third generation(P3)of purified cells were identified.Then,the BMSCs were induced to differentiate in vitro,and the differentiated cells were stained and identified for specific gene expression.The results showed that the obtained horse bone marrow cells expressed stem cell transcription factors and mesenchymal stem cell surface markers.The cells obtained were thought to be horse BMSCs.After induction culture,P3 generation cells changed in shape from"long spindle"to"bone nodules"osteoblasts and“paving stones”chondrocytes.Alizarin red staining showed that the induced red osteoblasts,and the red"bone nodules"gradually increased with time.The number of Alsine blue stained chondrocytes,which were rich in proteoglycan and hyaluronic,increased with extended induction time,while no staining was observed in the control group.The expressions of Col and ALPL genes in osteoblasts changed significantly with the induction time.Common PCR results showed that chondrogenic genes such as collagerⅡ,aggrecan and Sox9 were amplified in the induced chondrocytes,while the control cells did not express these specific genes.In conclusion,BMSCs were successfully induced to differentiate into osteoblasts and chondrocytes,providing experimental basis for the repair of bone tissue defects and the treatment of chondrocytes.