脂肪干细胞定向诱导分化为成骨细胞及其鉴定  

Inducing Human Adipose-derived Stem Cells Into Osteoblasts Directionally And Identification of Their Osteogenesis Characteristics

作　　者：刘波[1] 朱金土[1] 曹毅[1] 高寿松[1] 余土根[1] 

机构地区：[1]浙江省中医院整形外科,浙江杭州310006

出　　处：《中华中医药学刊》2011年第12期2680-2681,I0019,共3页Chinese Archives of Traditional Chinese Medicine

基　　金：浙江省自然科学基金资助项目(Y2080180)

摘　　要：目的:探讨将脂肪干细胞定向诱导分化为成骨细胞的方法并对所诱导细胞的成骨特性进行鉴定。方法:取行脂肪抽吸术患者的脂肪组织,I型胶原酶消化进行培养,贴壁细胞传代,取第2代细胞进行诱导,培养基中添加成骨诱导剂地塞米松(0.01μM)、β-磷酸甘油钠(10mM)、2-磷酸抗坏血酸(50μM)和1,25-(OH)2VitD3(10mM)。鉴定诱导细胞的成骨特性:碱性磷酸酶(AKP)定量检测,免疫荧光化学检测骨钙素(OCN)、骨桥素(OPN)、I型胶原表达,Alizarn Red染色检测钙结节形成,RT-PCR检测AKP和OPN表达。结果:第2代脂肪干细胞诱导后AKP表达自第3天起各个检测点(第3、7、14、21、28天)诱导组与对照组差异皆具有统计学意义(P<0.05),免疫荧光检测OCN、OPN及I型胶原表达阳性,Alizarn Red染色可见钙结节形成,RT-PCR检测诱导组AKP和OPN表达阳性。结论:人ADSCs能够向成骨细胞表型诱导分化。Objective ：To study the method of inducing human adipose -derived stem cells （ADSCs） into osteoblasts directionally and to identify osteogenesis characteristics. Methods： ADSC was isolated from liposuction aspirates using digestion method by I type collagenase and passage 2 cells were cultured in conditioned medium containing 0.01μM dexamethasone, 10 mMβ -glycerophosphate, 50μ M ascorbate-2 -phosphate and 10raM 1,25 -dihydroxyvitamin D3. The in- duced cells were chosen to check osteogenesis characteristics, including alkaline phosphatase quantitative assay, osteocalcin, osteopontin and collagen_I assay by immunoflurorescence, and calcium nodes assay with Alizarn Red staining. RT- PCR was used to assay alkaline phosphatase and osteopontin. Results ： The expression of AKP is positive at several detec ting times, and the osteopontin, collagen_I, and osteocalcin were positive by immunoflurorescence method in experimental group after induced 14 days, and calcium nodes were seen by Alizarn Red staining in induced cells. AKP and OPN expression or experimental group were detected by RT - PCR method. Conclusion ： The induced human ADSCs had typical osteogenesis characteristics.

关 键 词：组织工程骨 脂肪干细胞 成骨诱导 成骨分化 

分 类 号：R446.6[医药卫生—诊断学]