兔激素性股骨头坏死模型骨髓间充质干细胞的培养

Culture of bone marrow meenchymal stem cells from a rabbit model of steroid-induced avascular necrosis of the femoral head

背景:自体干细胞移植治疗已经成为目前组织工程和再生医学研究的重点。但对于兔激素性股骨头坏死模型自体骨髓移植中骨髓间充质干细胞的体外培养研究鲜有报道。目的:抽提兔激素性股骨头坏死模型的骨髓间充质干细胞,并行体外扩增和表型鉴定。方法:取成年大耳白兔10只,对照组2只不给任何药物;模型组8只每周2次臀肌注射甲基强的松龙8mg/kg制备兔激素性股骨头坏死模型。体外分离培养骨髓间充质干细胞;应用流式细胞仪分别检测第4代骨髓间充质干细胞表面抗原CD29、CD34、CD44的表达。结果与结论:经过大量激素作用后,MR灌注成像及病理切片结果显示兔激素性股骨头坏死模型组造模成功。模型组原代骨髓间充质干细胞4h内仅有少量细胞贴壁,12d左右细胞铺满瓶底的85%-90%,传代培养生长良好;第4代骨髓间充质干细胞生长曲线呈典型S型,第4-7天处于高速增殖期,与对照组生长曲线相近;经流式细胞术鉴定,第4代骨髓间充质干细胞表面抗原CD34表达呈阴性,CD29,CD44表达呈阳性,证明所培养的细胞为较纯的骨髓间充质干细胞。提示兔激素性股骨头坏死模型的骨髓间充质干细胞具有较强的生长和增殖能力,可用于干细胞标记及进一步自体移植治疗。

BACKGROUND：Autologous stem cell transplantation has become the main emphasis in tissue engineering and regenerative medicine research.In vitro culture of bone marrow mesenchymal stem cells for autologous bone marrow transplantation in a rabbit model of steroid-induced avascular necrosis of femoral head has been rarely reported.OBJECTIVE：Bone marrow mesenchymal stem cells from a rabbit model of steroid-induced avascular necrosis of femoral head were isolated for in vitro proliferation and phenotype identification.METHODS：Ten adult big-ear rabbits were divided into two groups.In the control group,two rabbits received no drugs.In the model group,eight rabbits were prepared into model of steroid-induced avascular necrosis of femoral head by intravascular administration of 8 mg/kg methylprednisolone twice a week.Bone marrow mesenchymal stem cells were in vitro isolated and cultured.Surface antigen CD29,CD34 and CD44 expression of passage 4 bone marrow mesenchymal stem cells was detected by flow cytometry.RESULTS AND CONCLUSION：MR perfusion-weighted imaging and pathological sections results showed that after treatment with a large amount of steroid,rabbit models of steroid-induced avascular necrosis of femoral head had been successfully prepared.In the model group,within 4 hours after steroid application,only a few primary bone marrow mesenchymal stem cells adhered to the culture flask wall,and at 12 days,cells covered 85%-90%of flask bottom;passage 4 bone marrow mesenchymal stem cells grew in a typical ＂S＂ shape,and they reached a peak growth pattern,which was similar to the growth curve of the control group.Flow cytometry showed that passage 4 bone marrow mesenchymal stem cells were negative for CD34 expression and positive for CD29 and CD44 expression,indicating that the cultured cells were very pure bone marrow mesenchymal stem cells.These findings suggest that bone marrow mesenchymal stem cells of rabbit models of steroid-induced avascular necrosis of femoral head show strong growing and proliferative capability and can be used for stem cells labeling and further autologous transplantation therapy.