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目的探讨大鼠脂肪源性干细胞(ADSCs)体外培养及诱导分化成为平滑肌细胞的可行性。方法取SD大鼠双侧腹股沟脂肪组织,酶消化法分离,体外扩增ADSCs,流式细胞仪检测ADSCs表面标志物表达及细胞周期。取第3代生长期的细胞,分别用一定浓度的维生素C、地塞米松、肝素诱导分化并培养2周,倒置相差显微镜观察细胞生长情况和形态变化,在诱导28 d左右取对照组及诱导组细胞,采用免疫荧光法及Western Blot半定量检测平滑肌α-肌动蛋白(SMA-α)表达,采用荧光半定量PCR法检测SMA-αmRNA水平。结果培养的ADSCs呈多层生长,形态多为梭形。流式细胞仪检测细胞表面标志物CD105、CD29、CD44呈阳性表达,HLA-DR、v WF、CD34呈阴性表达;细胞周期结果显示细胞为二倍体。处于生长对数期的细胞加入诱导剂诱导后细胞多为短梭形,有胞浆突起,细胞核明显,有部分细胞出现多核,相邻细胞相互融合,有向相同方向生长的趋势。免疫荧光结果显示SMA-α呈阳性表达,Western Blot结果显示SMA-α在诱导组相对含量高。荧光半定量PCR结果显示诱导组的SMA-α高表达。结论由脂肪组织分离培养的ADSCs具有干细胞特性。ADSCs在体外一定条件下可诱导分化成平滑肌细胞。
Objective To investigate the feasibility of culturing and inducing adipose-derived stem cells (ADSCs) into smooth muscle cells in vitro. Methods ADSCs were isolated from bilateral inguinal adipose tissue of SD rats by enzyme digestion, and the expression of ADSCs and cell cycle were detected by flow cytometry. The cells in the third generation of growth stage were induced to differentiate with certain concentrations of vitamin C, dexamethasone and heparin for 2 weeks respectively. The growth and morphological changes of the cells were observed under an inverted phase contrast microscope, and the control group and induced Group of cells, smooth muscle α-actin (SMA-α) expression was detected by semi-quantitative immunofluorescence and Western Blot. SMA-α mRNA was detected by semi-quantitative fluorescent PCR. Results The cultured ADSCs showed multi-layer growth with spindle-shaped morphology. The cell surface markers CD105, CD29 and CD44 were detected by flow cytometry. The expression of HLA-DR, vWF and CD34 was negative. The cell cycle was diploid. In the logarithmic growth phase, the cells were mostly short-fusiform, cytoplasm, nucleus obviously, some cells appeared multi-nucleus, adjacent cells fused with each other and had the same direction of growth. The results of immunofluorescence showed that the expression of SMA-α was positive, and the results of Western Blot showed that the relative content of SMA-α in the induction group was high. Semi-quantitative PCR results showed that the induced group of SMA-a high expression. Conclusion ADSCs isolated from adipose tissue have stem cell characteristics. ADSCs under certain conditions in vitro can be induced to differentiate into smooth muscle cells.