成人脂肪间充质干细胞的定向成骨诱导

背景：脂肪分离可获得大量的间充质干细胞并成功向骨、软骨、脂肪、心肌等多个方向诱导分化。 目的：建立成人脂肪间充质干细胞体外分离培养、成骨分化方法，并探讨脂肪间充质干细胞作为骨组织工程的种子细胞的前景。 方法：通过胶原酶消化法从成人脂肪中分离间充质干细胞，进行体外培养，流式细胞仪检测细胞表面标记物， CCK8检测细胞活性，成骨诱导液诱导干细胞向骨细胞分化，BCIP/NBT比色法染色检测碱性磷酸酶，茜素红染色检测钙结节形成，RT-PCR检测碱性磷酸酶，骨桥蛋白表达变化。 结果与结论：利用脂肪抽吸液成功培养出脂肪间充质干细胞，且能稳定传代，增殖能力旺盛；流式细胞仪检测证实有特定的间充质干细胞表面标记物表达；成骨诱导脂肪间充质干细胞后呈典型的成骨细胞形态；碱性磷酸酶染色阳性，茜素红染色后可见钙结节形成，成骨诱导培养0，3，7，14，21，28 d，RT-PCR定量检测结果证实碱性磷酸酶、骨桥蛋白阳性表达。说明用酶消化法可以从人脂肪中分离得到脂肪间充质干细胞；脂肪间充质干细胞可向骨细胞诱导分化，阳性表达骨桥蛋白，碱性磷酸酶，是一种优良的骨组织工程的种子细胞。

Osteogenic induction of human adipose-derived stem cells

BACKGROUND：A great amount of mesenchymal stem cels can be successfuly derived from fat tissue and induced to differentiate into osteoblasts, chondrocytes, adipocytes and myocardial cels.
OBJECTIVE：To establish the method of isolating, culturing and osteogenic differentiation of adipose-derived stem celsin vitro, and to explore the potential of adipose-derived stem cels as seed cels for bone tissue engineering.
METHODS：Colagenase enzymatic digestion was used to isolate adipose-derived stem cels from human fat tissue which were then culturedin vitro. Flow cytometry was used to detect cellsurface markers. cellcounting kit-8 assay was performed to examine cellviability. Adipose-derived stem cels were induced by osteogenic induced reagent to differentiate into bone cels. In addition, we also performed BCIP/NBT method to detect alkaline phosphatase activity. Alizarin red staining was used to detect the formation of calcium node. RT-PCR was performed to examine alkaline phosphatase and osteopontin expression.
RESULTS AND CONCLUSION：We successfuly obtained adipose-derived stem cels from fat aspirated liquid. Adipose-derived stem cels obtained could passage stably and proliferate highly. Flow cytometry data showed the expression of stem cellsurface markers. Adipose-derived stem cels showed typical osteoblast morphology after osteogenic differentiation. Alkaline phosphatase staining was positive and alizarin red staining displayed the formation of calcium node. Furthermore, we found that alkaline phosphatase and osteopontin mRNA was expressed after differentiation 0, 3, 7, 14, 21, 28 days. These findings indicate that adipose-derived stem cels can be obtained from fat tissue through enzymatic digestion, differentiate towards bone cels, and express alkaline phosphatase and osteopontin, which can become potential seed cels for bone tissue engineering.