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目的:探讨脂肪组织来源干细胞(adipose-derived stem cells,ADSCs)和核心结合因子(Core Binding Factor Alpha 1,Cbfa1)的成骨潜能及其应用于基因增强的骨组织工程临床治疗的可能性。方法:利用重组了Cbfa1的腺病毒载体在ADSCs中过表达Cbfa1。采用Realtime RT-PCR定量检测成骨特异性基因和成脂特异性基因的表达变化。同时采用Von Kossa染色观察Cbfa1过表达后21天ADSCs内钙盐的沉积情况。在体内实验中,裸鼠右下肢肌肉内注射Cbfa1基因修饰的脂肪来源干细胞,8周后,采用石蜡切片(HE和甲苯胺蓝染色)检测软骨和骨的生成情况。结果:Cbfa1转染后3天,ADSCs中检测到Cbfa1 RNA表达,及骨钙素、骨桥素、I型胶原表达,同时脂蛋白脂酶表达下降。Vocassa染色证明,转染Cbfa1后,ADSCs细胞基质中的钙盐沉积显著增加。体内实验8周后,注射Cbfa1基因修饰的脂肪来源干细胞的肌肉内有明显的软骨和骨组织形成。结论:Cbfa1过表达可定向诱导ADSCs体外钙盐沉积和体内成骨,可作为基因增强的骨组织工程中有潜力的治疗基因和载体细胞。
OBJECTIVE: To investigate the osteogenic potential of adipose-derived stem cells (ADSCs) and corebinding factor Alpha 1 (Cbfa1) and their potential application in gene therapy for bone tissue engineering. Methods: Cbfa1 was overexpressed in ADSCs using recombinant adenoviral vector of Cbfa1. Realtime RT-PCR was used to quantitatively detect the expression of osteogenic and adipogenesis-specific genes. At the same time, Von Kossa staining was used to observe the deposition of calcium salts in ADSCs 21 days after Cbfa1 overexpression. In vivo experiments, Cbfa1 gene-modified adipose-derived stem cells were injected intramuscularly in the right leg of nude mice. After 8 weeks, paraffin sections (HE and toluidine blue staining) were used to detect the formation of cartilage and bone. Results: Cbfa1 RNA and osteocalcin, osteopontin and type I collagen were detected in ADSCs 3 days after Cbfa1 transfection, and the expression of lipoprotein lipase decreased. Vocassa staining demonstrated a significant increase in calcium deposition in the matrix of ADSCs after transfection with Cbfa1. After 8 weeks of in vivo experiment, there was obvious cartilage and bone formation in the muscle of Cbfa1 gene-modified adipose-derived stem cells. CONCLUSION: Overexpression of Cbfa1 can induce the in vitro calcium deposition and osteogenesis of ADSCs in vitro, which may serve as a potential therapeutic gene and vector for gene engineering of bone tissue engineering.