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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 investigate the method of differentiating adipose-derived stem cells into osteoblasts and to identify the osteogenic characteristics of the induced cells. Methods: Adipose tissue was isolated from adipose tissue of patients with liposuction, type I collagenase digestion was used to culture, adherent cells were passaged, and the second passage cells were used for induction. Dexamethasone (0.01μM) Sodium β-phosphoglycerate (10 mM), 2-phosphoascorbic acid (50 μM) and 1,25- (OH) 2VitD3 (10 mM). Identification of induced osteoblasts: Quantitative detection of alkaline phosphatase (AKP), expression of osteocalcin (OCN), osteopontin (OPN) and type I collagen by immunofluorescence, Alizarn Red staining for calcium formation, The expression of AKP and OPN was detected by RT-PCR. Results: The AKP expression induced by the second generation of ADSCs was significantly different from that of the control group (P <0.05) at each test point (days 3, 7, 14, 21 and 28) The expression of OCN, OPN and type I collagen was detected by fluorescence assay. The formation of calcium nodules was observed by Alizarn Red staining. The positive expression of AKP and OPN was detected by RT-PCR. Conclusion: Human ADSCs can induce osteoblast phenotype differentiation.