小鼠密质骨间充质干细胞的培养及多能性研究  

Culture and Pluripotentiality of Murine Compact Bone-derived Mesenchymal Stem Cells

作　　者：陈广华[1] 杨婷[1] 乔淑敏[1] 田竤[1] 刘慧文[1] 乔曼[1] 顾斌[1] 冯宇锋[1] 朱子玲[1] 吴德沛[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,卫生部血栓与止血重点实验室,江苏苏州215006

出　　处：《中国实验血液学杂志》2012年第2期448-452,共5页Journal of Experimental Hematology

基　　金：江苏高校优势学科建设工程资助项目;新药研究开发关键技术研究项目(2009ZX09503);福建省科技厅重点项目(编号2009Y0022;2009J1004)

摘　　要：本研究培养小鼠密质骨间充质干细胞(MSC)并分析其免疫功能和分化潜能。分离小鼠双侧股骨和胫骨,反复冲洗,取出骨髓细胞,用胶原酶消化骨碎片,分离有核细胞并接种于6孔板。对第3代MSC进行免疫表型测定、免疫抑制功能分析及进行成脂细胞、成骨细胞和成软骨细胞诱导分化实验。结果表明,培养3周内可自小鼠密质骨分离高纯度的MSC,后者具有向成脂细胞、成骨细胞和成软骨细胞分化的潜能,具有抑制混合淋巴细胞反应的功能。BALB/c T细胞经C57BL/6 T细胞刺激后对照组液体闪烁计数仪检测每分钟计数(CPM)值为(2.56±0.31)×104,而与细胞比例为100∶1和10∶1的C57BL/6第3代密质骨MSC共孵育组的CPM值分别为(0.47±0.12)×104和(0.28±0.09)×104,MSC处理组CPM值与对照组CPM值相比差别有统计学意义(P<0.001),MSC抑制功能呈剂量依赖性。结论:小鼠密质骨富含MSC,原代培养MSC造血细胞污染程度低,具有抑制混合淋巴细胞反应功能,具有更广泛的实验研究应用价值。This study was purposed to culture murine compact bone-derived mesenchymal stem cell（MSC） and analyze the immunological and trilineage differentiation potential.Tibia and femur were extracted.Bone marrow cells were flushed out and compact bone fragments were digested with collagenase.The digested cells were cultured in 6-well plates.The immunophenotype,immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry,mixed lympocyte reaction and Oil red O,von Kossa and alcian blue straining,respectively.The results indicated that the pure compact bone MSC could be isolated with in 3 weeks.The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction.The count per minute（CPM） value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was（2.56±0.31） ×104, while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100∶1 and 10∶1 were（0.47±0.12） ×104 and（0.28±0.09） ×104. The CPM value of control group was higher than those of MSC cocultured group（P0.001）.Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner.It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells.Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential.Compact bone-MSC have promising experimental study value.

关 键 词：密质骨 间充质干细胞 细胞分化 免疫表型 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]