人、兔、大鼠脂肪基质细胞的生物学性状对比

Comparison of biological characteristics of human,rabbit and rat adipose tissuederived stromal cells in vitro

目的:探讨在同一质量控制条件下,人、兔、大鼠来源脂肪基质细胞(adipose tissue-derived stromal cells,ADSCs)的体外生物学性状的异同。方法:分离培养人吸脂来源ADSCs、兔及大鼠腹股沟皮下脂肪垫来源ADSCs,体外观察3种细胞原代和传代细胞的形态及生长情况,MTT法检测细胞增殖情况。以相同密度将第2代细胞接种于24孔板,并诱导其成骨及成脂向分化。成骨诱导培养基包含100 nmol/L地塞米松、50μmol/L抗坏血酸二磷酸酯和10 mmol/Lβ-甘油磷酸钠,在成骨向诱导后的第3、7、14、21天行碱性磷酸酶染色,第14、28天行茜素红染色,定量分析3种细胞体外钙沉积能力。成脂向诱导培养基包含1μmol/L地塞米松、10μmol/L胰岛素、200μmol/L吲哚美辛和0.5 mmol/L异丁基黄嘌呤,在成脂向诱导后的第7、14天行油红O染色检测细胞的成脂分化状况。结果:3种来源的脂肪均可分离培养出"成纤维细胞样"细胞,均能成脂向分化和成骨向分化,但兔ADSCs在成骨向分化时碱性磷酸酶染色呈弱阳性,无明显钙结节形成,与其他两种细胞比较,兔ADSCs的成骨向分化能力较差。结论:与人来源和大鼠来源ADSCs比较,兔皮下脂肪来源ADSCs的体外成骨能力较差,将其用于体内成骨研究时需慎重。

Objective：This study aims to investigate the difference of proliferation patterns and osteogenic and adipogenic differentiation capability of adipose-derived stromal cells （ADSCs） obtained from human lipoaspirates, rat and rabbit inguinal subcutaneous adipose tissues in vitro. Methods： Adipose tissues of healthy adults were obtained by liposuction. Human ADSCs were isolated from these adipose tissues and cultured in DMEM containing 10% fetal bovine serum （FBS）. Rat and rabbit ADSCs were obtained from inguinal subcutaneous adipose tissues and cultured with the same methods. These cells were observed under inverted microscope each day and cell growth was measured with MTT assay. Adipogenic differentiation was induced by culturing ADSCs for 1 or 2 weeks in adipogenic medium （AM） containing 1 umol/L dexamethasone, 10 umol/L insulin, 200 umol/L indomethacin, 0. 5 mmol/L isobutyl-methylxanthine （IBMX）, and assessed by Oil Red 0 staining as an indicator of intracellular lipid accumulation. Osteogenic differentiation was induced by culturing ADSCs in osteogenic medium （OM） containing 0. 1 umol/L dexamethasone, 50 umol/L ascorbate-2-phosphate, 10 mmol/L β-glycerophosphate, and examined via alkaline phosphatase （AP） activity and extracellular matrix （ECM） calcification by alizarin red S staining and quantification of matrix calcification. Results ： Fibroblast-like cells were digested from both inguinal subcutaneous adipose tissues of rabbit or rat and human lipoaspirates obtained from subcutaneous adipose tissues. Lipid-filled droplets were accumulated in human, rat and rabbit ADSCs upon treatment with adipogenic medium and were stained by Oil Red O. No lipid droplets were observed in the control undifferentiated ADSCs. After exposure to osteogenic differentiation medium, humanand rat ADSCs were found to possess greater osteogenic potentials than cells isolated from rabbit inguinal subcutaneous adipose tissues, which was evidenced by significantly different osteogenic markers including alkaline phosphatase and mineral deposition. Conclusion：Rabbit ADSCs obtained from inguinal subcutaneous adipose tissues poorly possess osteogenic potentials compared with ADSCs of human lipoaspirates obtained from subcutaneous adipose tissues or ADSCs of rat from inguinal subcutaneous adipose tissues, although they all possess comparable adipogenic capacity.