脂肪源性干细胞体外成骨特性的研究

目的探讨兔脂肪源性干细胞体外成骨分化能力。方法取3个月龄新西兰白兔颈背部皮下脂肪组织,型胶原酶消化获得细胞。绘制细胞生长曲线,采用免疫细胞荧光法检测细胞表面抗原CD44、CD45;分别加入条件培养液对所获得细胞进行成骨成脂诱导,并分别进行形态学、碱性磷酸酶、钙盐沉积和油红O染色相关检测;将脂肪源性干细胞分为成骨诱导组和非成骨诱导组,分别于诱导后1、2、3、4周检测两组的碱性磷酸酶和钙离子浓度并作统计分析。结果 a）所获细胞CD44阳性表达,CD45阴性表达;所绘制的生长曲线成典型的＂S＂型。b）在诱导条件下,碱性磷酸酶及钙盐沉积均呈阳性表达,油红O染色为阳性。c）碱性磷酸酶浓度在诱导组与非诱导组之间存在组间差别,在各个时间点诱导组碱性磷酸酶浓度均高于非诱导组（P〈0.05,n=5）;诱导组碱性磷酸酶浓度在不同诱导时相其浓度变化趋势不同（P〈0.05,n=5）;钙离子浓度在诱导组与非诱导组之间存在组间差别,在各个时间点诱导组钙离子浓度高于非诱导组（P〈0.05,n=5）。诱导组钙离子浓度在不同诱导时相其浓度变化的趋势不同（P〈0.05,n=5）。结论脂肪干细胞的易分离培养、增殖快和良好的成骨诱导活性完全符合对种子细胞的要求,是一种理想的骨组织工程种子细胞。

Osteogenic Differentiation of Adipose-Derived Stem Cells from Rabbits in Vitro

Objective To explore the differentiation of adipose-derived stem cells（ADSCs）from rabbits into osteogenic cells in vitro.Methods In this study,ADSCs were gained by collagenaseⅠdigestion of subcutaneous fat from the nape of New-Zealand white rabbit.Cell surface antigen CD44、CD45 were identified by immunofluorescence and cell auxodrome were drawn.The ADSCs were performed osteogenic and adipogenic differentiation respectively,following with texts related morphology,alkaline phosphatase assay,VonKossa and rathonum red O stain.ADSCs were divided into osteogenic induced-group and osteogenic noninduced-group.Alkaline phosphatase assay and calcium concentration of these two groups were checked and statistics from the check were analyzed at 1,2,3,4w after these two groups were both induced.Results This study got following results：a）ADSCs CD44 expression were positive and ADSCs CD45 expression negative;cell auxodromes were S shaped.b）Alkaline phosphatase assay,VonKossa and rathonum red O stain of ADSCs were all positive under the influence of osteogenic and adipogenic differentiation mediums.c）Alkaline phosphatase assays（ALP）were different of these two groups-ALP of induced-groups was higher than that of non-induced group at each different time（P〈0.05,n=5）and ALP of the induced-group was distinct in different tendency under different induction（P〈0.05,n=5）.Calcium concentration were different of these two groups-calcium concentration of induced-groups was higher than that of non-induced group at each different time（P〈0.05,n=5）and calcium concentration of the induced group was distinct in different tendency under different induction（P〈0.05,n=5）.Conclusion This study suggests that ADSCs can be isolated and cultured easily and have the property of fast generation and favourable osteogenic differentiation,which makes ADSCs ideal seed cells of bone tissue engineering.