小儿骨髓间充质干细胞生物学特性及多向分化潜能(英文)

Biological characteristics and multi-directional differentiation of bone marrow mesenchymal stem cells in children

背景：国内报道人骨髓间充质干细胞多取材于成人细胞,小儿骨髓间充质干细胞的研究较少。目的：拟分离培养小儿的骨髓间充质干细胞,观察其生物学特性以及向成骨、成脂肪、成神经分化的潜能。设计：观察对比实验。单位：华中科技大学同济医学院。材料：实验于2006-03/09在武汉同济医院骨科实验室完成,小儿骨髓间充质干细胞取自5～8岁因髋关节发育不良行骨盆截骨术的患儿,男1例,女2例,实验经过医院伦理委员会批准许可,并征得所有患儿家属知情同意。地塞米松、维生素C、β-甘油磷酸钠、甲基异丁酸黄嘌呤、胰岛素、吲哚醚辛、羟基丁酸苯甲醚均为Sigma公司产品,二甲基亚砜为Amersco公司产品。方法：经Percoll梯度分离接种获得小儿骨髓间充质细胞,倒置相差显微镜下观察细胞形态、排列分布情况。分别采用成骨细胞诱导剂（β-甘油磷酸钠,地塞米松,维生素C）、成脂肪细胞诱导液（地塞米松,甲基异丁酸黄嘌呤,牛胰岛素,吲哚美辛）及二甲基亚砜和羟基丁酸苯甲醚无血清培养基诱导剂干预细胞向成骨、脂肪、神经细胞分化,经免疫组织化学染色、PCR、免疫细胞染色方法检测成骨标志物（碱性磷酸酶、骨钙素mRNA、钙结节）、脂肪标志物（脂滴、PPARγ-2mRNA）、以及类神经标志物（尼克氏体、神经烯醇化酶、神经丝蛋白）。主要观察指标：①小儿骨髓间充质细胞形态以及增殖情况。②成骨、脂肪及类神经标志物检测结果。结果：①小儿骨髓间充质细胞贴壁容易,细胞细长梭形,增殖能力强,融合后呈旋涡状分布。②骨髓间充质细胞分别经各诱导剂诱导后,细胞形态均发生相应的变化,用化学染色、PCR、免疫细胞染色等方法检测成骨标志物、脂肪标志物及类神经标志物有明显阳性表达。结论：小儿骨髓间充质细胞生长具有稳定增殖、传代的能力,具有成骨、成脂肪、成神经等多方向的定向分化潜能。

BACKGROUND： It has been reported that in China, human bone marrow mesenchymal stem cells are mostly harvested from adults. Studies on bone marrow mesenchymal stem cells in children are few. OBJECTIVE： To isolate and expand bone marrow mesenchymal stem cells from children, and to analyze the biological characteristics of bone marrow mesenchymal stem cells and their potential of differentiating into osteoblasts, adipocytes and neural like cells. DESIGN： Observational comparative study. SETTING： Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： Experiments were performed at the Laboratory of Department of Orthopaedics of Wuhan Tongji Hospital from March to September 2006. Bone marrow mesenchymal stem cells were collected from one boy patient and two girl patients aged 5-8 years, who received pelvis osteotomy for dysplasia of the hip joint. The experimental procedures were approved by the Hospital Ethics Committee and family members of all children patients singed the informed consent. Dexamethasone, vitamin C, β -sodium glycerophosphate, 3-Isobutyl-1-methylxanthine, insulin, indometacin and butylated hydroxyanisole were bought from Sigma Company. Dimethyl sulphoxide was purchased from Amersco Company. METHODS： Bone marrow mesenchymal stem cells were cultured from mononuclear cells isolated over a Percoll gradient. Bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. Bone marrow mesenchymal stem cells could differentiate into osteoblasts, adipocytes and neural like cells with osteoblast inductor （ β -sodium glycerophosphate, dexamethasone, vitamin C）, lipoblast inductor （dexamethasone, 3-isobutyl-1-methylxanthine, bovine insulin, indometacin） and serum-free medium inductor （dimethyl sulphoxide, butylated hydroxyanisole） respectively. Osteoblast marker （alkaline phosphatase, osteocalcin mRNA, calcium node）, adipocyte marker （lipid droplet, PPAR Y-2mRNA） and neural cell-like marker （nissl body, neuron specific enolase, neurofilament protein） were respectively determined by the immunohistochemical method, polymerase chain reaction and immunocytochemical method. MAIN OUTCOME MEASURES： （1）Appearance and proliferation of bone marrow mesenchymal stem cells from children and （2）determination results of osteoblast, adipocyte and neural cell markers. RESULTS： （1）Children bone marrow mesenchymal stem cells could easily adhere to the wall, appeared fusiforrm, had high reproductive activity and arranged vortically after fusing. （2）Appearance of bone marrow mesenchymal stem cells changed after receiving inductor. Osteoblast marker, adipocyte marker and neural cell-like marker were positive after chemical staining, polumerase chain reaction and immunocyte staining. CONCLUSION： Children bone marrow mesenchymal stem cells show stable proliferation, passage and multi-direction differentiation towards osteoblasts, adipocytes and neural like cells.