脂肪间充质干细胞的基本生物学特性及其向成骨细胞分化的特征

背景最早研究的是骨髓来源的间充质干细胞.由于脂肪组织与骨髓同为中胚层起源的组织,那么能否从脂肪组织中获得间充质干细胞,而且也具有多分化潜能呢?目的研究脂肪间充质干细胞的基本生物学特性以及在特定培养条件下向成骨细胞分化,探讨其作为骨组织工程的种子细胞的可行性.设计以细胞为研究对象的随机对照实验.单位北京大学第三医院骨科.对象实验于2002-10/2004-01在北京大学第三医院骨科完成,实验对象为从Lewis大鼠脂肪组织中提取的间充质干细胞.方法取3周龄Lewis大鼠的腹股沟脂肪垫,消化法获得脂肪间充质干细胞,分别用脂肪诱导培养基和成骨诱导培养基诱导其向脂肪细胞与成骨细胞分化,组织化学染色、免疫细胞化学染色和Westernblotting检测细胞分化的情况.主要观察指标脂肪间充质干细胞的形态学和生物学特性,以及成骨诱导后成骨细胞特异性标志.结果从成体大鼠脂肪组织中培养出脂肪间充质干细胞,体外培养呈成纤维细胞样,能大量稳定增殖传代.原代脂肪间充质干细胞能自发分化为脂肪细胞,传代细胞在胰岛素和地塞米松的作用下生成脂滴,过氧化物酶体增殖物激活受体?表达增强,向脂肪细胞分化;在地塞米松、抗坏血酸、?-甘油磷酸钠的诱导下,碱性磷酸酶活性检测显示诱导组与对照组差异有显著性意义(P＜0.01),yonKossa染色出现钙结节,骨桥蛋白免疫细胞化学染色阳性,Western blotting检测到诱导后细胞骨桥蛋白的表达.结论从脂肪组织中可获得具有多分化潜能的间充质干细胞,经诱导后可分化为脂肪细胞和成骨细胞,有可能成为骨组织工程较理想的种子细胞之一.

Basic biological characteristics of adipose mesenchymal stem cells and the characteristics in differentiation into osteoblast

BACKGROUND: Bone marrow mesenchymal stem cells(MSCs) have been investigated initially. Because both fat tissue and bone marrow are tissues originated from mesoderm, so whether MSCs could be obtained from fat tissue as well, which also have multi-lineage differentiation potential?OBJECTIVE:To investigate the basic biological characteristics of adipose MSC and its differentiation into osteoblast under given culture condition for the exploration of its feasibility as seed cell in bone tissue engineering.DESIGN: A randomized and controlled study based on cells.SETTING: Department of Orthopaedics of an Affiliated Hospital of a university.MATERIALS: The study was conducted in the Department of Orthopaedics of the Third Hospital of Peking University. Adipose MSC extracted from the fat tissue of Lewis rats was used as subject.METHODS: Adipose MSCs were obtained from inguinal fat pads of Lewis rat after digestion, which were induced into adipocytes and osteoblasts with adipose and osteogenesis induced culture mediums. The differentiations were examined with cytochemical staining, immuncytochemical staining and Western blotting.MAIN OUTCOME MEASURES: Morphological and biological characteristic of adipose MSCs, and the specific mark of osteoblast after induction.RESULTS: Adipose MSCs were obtained from rat adipose tissue culture,which appeared fibroblast-likely in the culture in vitro, and could stably proliferate and passage in vitro. Primary adipose MSCs could differentiate into adipocytes spontaneously, and passaged cells could form fat drop under the reaction of insulin and dexamethasone, and then differentiate towards adipocytes after the expression of peroxidase proliferation activated receptor ?(PPAR-?) enhanced. There was significant difference between induction group and control group in alkaline phosphatase(ALP) activity detection under the induction of dexamethasone, ascorbic acid, and ?-sodium glycerophosphate(P ＜ 0.01) . Calcium node appeared in yon Kossa staining. Result of osteopontin (OPN) immunocytochemcial staining was positive,and OPN expression was detected by Western blotting after induction.CONCLUSION: MSCs with multi-lineage potential can be obtained from rat adipose tissue, and differentiate into adipocytes and osteoblasts after inductions. Therefore, adipose MSCs can possibly be served as one of optimal seed cells for bone tissue engineering.