摘要
目的探讨酒精性骨坏死发病机制和葛根素的预防作用。方法分离培养小鼠骨髓基质细胞(MSCs),随饥分为3组：A组(酒精组),给予酒精0、0．03、0．09、0．15 mol/L；B组(葛根素组),酒精0．09 mol/L和葛根素终浓度为0．01 mg/ml；C组(对照组),无酒精与葛根素。苏丹Ⅲ染色,光镜下脂肪细胞计数；测定细胞内甘油三酯含量、碱性磷酸酶活性和细胞培养液中骨钙素含量。采用完整细胞斑点印迹分子杂交方法检测A组和C组细胞中422(aP2)mRNA和Ⅰ型胶原mRNA的表达。采用RT-PCR技术检测3组细胞中PPARγmRNA和osteocalcin mRNA的表达。结果酒精处理细胞后21 d,MSCs分化为脂肪细胞的数量随酒精作用时间延长及浓度增大而增多；细胞内甘油三酯含量明显增高,ALP活性降低,骨钙素含量显著减少。0．09 mol/L酒精作用细胞6 d,A组中422(aP2)mRNA表达含量显著增高,Ⅰ型胶原mRNA表达含量明显降低。B组和C组细胞中PPARγmRNA表达明显低于A组,osteocalcin mRNA表达明显高于A组,而B组与C组间差异无统汁学意义(P＞0．05)。结论酒精能诱导MSCs大量成脂分化,减少其成骨分化,这可能与酒精性骨坏死的发生机制有关。葛根素能够对抗酒精诱导MSCs成脂分化,可能预防骨坏死。
Objective To observe the pathogenesis of the alcohol-induced osteonecrosis of the femoral head and the preventive effect of puerarin which inhibits adipogenesis of marrow stromal cells induced by alco- hoL Methods The primary marrow stromal cells （MSCs） were isolated and cultured from mice bone marrow. MSCs were separated by 3 groups at random ： Group A ： the cells were treated with increasing （0, 0. 03, 0. 09, and 0. 15 mol/L） concentrations of ethanol. Group B： the cells were treated with 0. 90 mol/L ethanol and 0. 01mg/ml puerarin. Group C： the cells were treated without ethanol or puerarin as control. The cells in culture were stained with Sudan Ⅲ, and then the number of adipocytes were counted on a light microscope. The contents of intraceltular triglyceride, alkaline phosphatase activity in cells and the contents of osteocalcin in the media were determined. The expression level of 422 （aP2） and type-Ⅰ collagen mRNA＇ s in the cells treated with 0.09 mol/L ethanol and without with ethanol was investigated by means of intact cell RNA dot blot hybridization. The expression levels of the peroxisome proliferator-actived receptor-γ （PPARγ） mRNA and osteocalcin mRNA in cells were analyzed by reverse transcription-polymerase chain reaction （RT-PCR）. Results The cells in culture were treated with increasing concentrations of ethanol for 21 days. The number of adipocytes increased with longer durations of exposure to ethanol and with higher concentrations of ethanol, which showed the time- and dose-dependent relations （P 〈0. 001 ）. The level of triglyceride in the cells treated with with ethanol was markedly higher than that in the control cells （ P 〈0. 001 ）. ALP activity in the cells decreased with higher concentration of ethanol （ P 〈0.001 ）. The level of osteocalcin in culture media of the cells treated with O. 15 mol/L of ethanol greatly decreased （P 〈 0.001 ）. The 422 （aP2） mRNA contents in the group A were markedly higher than that in the group C. By contrast, the type-1 collagen mRNA contents in the group A were significantly lower than that in the group C. The expression levels of PPARγ mRNA in the cells of group B and group C were significantly lower than that in the cells of group A （ P 〈 0.05 ）, and there were not significant differences statistically between group B and group C （ P 〉 0. 05 ）. The expression levels of osteocalcin mRNA in the cells of group B and group C were significantly higher than that in the cells of group A （P〈0. 01 ）, and there were not significant differences statistically between group B and group C （P 〉 0. 05 ）. Conclusion Alcohol could induce the differentiation of marrow stromal cells into adipocytes and inhibit osteogenic differentiation by regulating gene expression, which might associate with the development of the alcohol-induced osteonecrosis. Puerarin could inhibit differentiation of marrow stromal cells into adipocytes induced by alcohol, to maintain the normal procedure of osteogenic differentiation, which may prevent the development of the osteonecrosis.
出处
《中华显微外科杂志》
CSCD
北大核心
2006年第3期209-212,i0005,共5页
Chinese Journal of Microsurgery
关键词
酒精
骨坏死
骨钙素
胶原
骨髓基质细胞
葛根素
Alcohol
Osteonecrosis
Osteocalcin
Collagen
Marrow stromal cells
Puerarin
