小鼠脂肪源间充质干细胞在重型再生障碍性贫血中的应用  

Application of adipose tissue-derived mesenchymal stem cells in severe aplastic anemia mice

作　　者：王萍[1,2] 房佰俊[1] 宋永平[1] 董子明[2] 

机构地区：[1]河南省肿瘤医院血液科,河南省血液病研究所,河南省郑州市450008 [2]郑州大学基础医学院病理生理教研室,河南省郑州市450052

出　　处：《中国组织工程研究与临床康复》2008年第43期8426-8430,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：河南省杰出青年基金(0612000900);河南省医学科技创新人才工程项目(200590)~~

摘　　要：背景：骨髓间充质干细胞联合造血干细胞移植是最有希望解决造血重建缓慢的策略之一，但临床实施中，抽取骨髓进而分离、扩增间充质干细胞尚不能为大部分健康供者所接受。目的：探讨小鼠脂肪源间充质干细胞对异基因重型再生障碍性贫血模型鼠造血功能的影响。设计、时间及地点：细胞学体内实验，于2007—05／11在河南省肿瘤医院中心实验室完成。材料：雄性6～8周龄BALB／c（H-2K^d）小鼠20只，雌性7～8周龄C57BL／6（H-2K^b）小鼠50只，均购于河南省实验动物中心。方法：贴壁法体外分离培养20只雄性BALB／c小鼠脂肪源间充质干细胞；反复吹打20只雌性C57BL／6（H-2K^b）小鼠骨髓细胞，加入EDTA-NH4Cl液去除红细胞，即为骨髓有核细胞。剩余30只雌性C57BL／6（H-2K^b）小鼠均给予一次全身3．0Gy^60Co-γ照射，照射后第4，5，6天皮下注射环磷酰胺及氯霉素，建立重型再生障碍性贫血模型。模型鼠随机分为3组，联合组经尾静脉输入骨髓有核细胞2×10^9个＋脂肪源间充质干细胞3×10^6个，骨髓有核细胞组单纯输入骨髓有核细胞2×10^7个，对照组输入生理盐水0．2mL，10只/组。主要观察指标：定期检测外周血和股骨有核细胞数以及巨噬细胞／粒细胞集落形成单位数的变化，PCR法鉴定Y染色体，流式细胞仪检测移植小鼠骨髓及脾脏CD3和H-2K^d阳性表达情况。结果：细胞移植后第2周，联合组外周血有核细胞数、股骨有核细胞数、巨噬细胞／粒细胞集落形成单位数均明显高于骨髓有核细胞组（P〈0．05），且随移植时间的延长，各种细胞数量逐渐恢复（P〈0．05）。细胞移植后第4周（即造血恢复期），联合组外周血可特异性地扩增Y染色体的SRY基因片段，移植小鼠骨髓及脾脏中CD3和H-2K^d双阳性细胞达6．66％，提示在受体小鼠体内有供体细胞的存在。结论：脂肪源间BACKGROUND： Bone marrow mesenchymal stem cell and hematopoietic stem cell transplantation is one of methods solving hematopoietic reestablishment. However, the method of extracting bone marrow for isolation and amplification mesenchymal stem cells can not be accepted by most healthy donors in clinic. OBJECTIVE： To explore effects of mouse adipose tissue-derived mesenchymal stem cells on hematopoietic function of allogeneic severe aplastic anemia mice. DESIGN, TIME AND SETTING： The cytology in vivo experiment was performed at the Central Laboratory, Henan Tumor Hospital from May to November 2007. MATERIALS： Twenty male BALB/c （H-2K^d） mice aged 6 8 weeks, and fifty female C57BL/6 （H-2K^b） mice aged 7 8 weeks were purchased from Henan Provincial Animal Experimental Center. METHODS： Adipose tissue-derived mesenchymal stem cells from 20 male BALB/c mice were dissociated and cultured by the adherence method. Bone marrow cells from 20 female C57BL/6 （H-2K^b） mice were blown and beaten. Erythrocytes were removed after incubating in EDTA-NH4Cl method. The remaining was bone marrow karyocytes. The remaining 30 female C57BL/6 （H-2K^b） mice received a general irradiation with 3.0 Gy ^60Co-γ. At 4, 5 and 6 days after irradiation, rats were subcutaneously treated with cyclophosphamide and chloramphenicol to establish severe aplastic anemia models. Model mice were randomly assigned into 3 groups. Mice in the combination group were injected with 2×10^7 bone marrow karyocytes and 3×10^6 adipose tissue-derived mesenchymal stem cells via the tail vein. Mice in the bone marrow karyocyte groups were simply infused with bone marrow karyocyte （2×10^7）. Mice in the control group received 0.2 mL saline. Each group contained 10 mice. MAIN OUTCOME MEASURES： Peripheral blood and femoral karyocyte number and macrophages/granulocytes colony forming unit number were regularly measured. Y-chromosome was detected by polymerase chain reaction （PCR）, and CD3 and H-2K^d were detected by flow cytometry. RESUL

关 键 词：脂肪源间充质干细胞 骨髓有核细胞 造血恢复 脂肪 

分 类 号：R394.2[医药卫生—医学遗传学]