葛根素抑制乙醇诱导骨髓基质细胞成脂分化的实验(英文)

Inhibition of puerarin in the differentiation of marrow stromal cells into adipocytes induced by alcohol

背景:动物实验证明乙醇可引起股骨头内脂肪积聚,导致骨坏死。分子生物学实验证明乙醇能够诱导骨髓基质细胞成脂分化。若某种药物能够对抗乙醇诱导骨髓基质细胞的成脂分化作用,则可能防治酒精性骨坏死。目的:从细胞生物学角度观察葛根素对抗酒精诱导骨髓基质细胞成脂分化作用,探讨葛根素对酒精性骨坏死的防治作用。设计:随机对照实验。单位:郑州大学第一附属医院骨科和郑州大学基础医学院生物化学与分子生物学教研室。材料:实验于2000-04/2002-12在郑州大学基础医学院生物化学与分子生物学教研室实验室完成。选用清洁级健康昆明小鼠50只,6～8周龄,雌雄不拘。获取双侧股骨骨髓细胞,细胞接种密度1.5×106/cm2,置入6孔及24孔培养板内,随机分组,每孔作为1个样本,每组为24个样本。方法:分离培养小鼠骨髓基质细胞,随机分为3组:模型组(给予乙醇0.09mol/L处理细胞),实验组(乙醇0.09mol/L和葛根素终浓度为0.01g/L),对照组(无乙醇和葛根素)。苏丹Ⅲ染色,光镜下脂肪细胞计数;测定细胞内三酰甘油含量、碱性磷酸酶活性和细胞培养液中的骨钙素含量。主要观察指标:①3组小鼠骨髓基质细胞分化为脂肪细胞结果。②3组小鼠骨髓基质细胞内三酰甘油含量。③3组小鼠骨髓基质细胞内碱性磷酸酶活性值。④3组细胞培养液中骨钙素含量。结果:①处理细胞并培养21d后,实验组、对照组中脂肪细胞均比模型组显著为少。模型组中脂肪细胞数是实验组的8.9倍,是对照组的15.6倍[(319.17±19.92),(35.92±23.77)(20.42±12.15)个/cm2,P<0.001]。②处理细胞并培养21d后,实验组和对照组中三酰甘油含量均明显低于模型组[(4.15±1.92)和(3.42±1.60),(11.55±4.42)μg/孔,P<0.001]。③处理细胞并培养12d后,实验组和对照组细胞内碱性磷酸酶活性均明显高于模型组[(9.51±2.96),(11.18±3.11),(4.84±2.23)μkat/g,P<0.001]。④处理细胞并培养14d后,实验组和对照组培养液中的骨钙素含量分别是模型组的2.2倍和2.7倍,与模型组间的差异均非常显著[(11.11±4.57),(13.43±5.29),(4.95±2.31)μg/g,P<0.001]。结论:葛根素能够抑制酒精诱导骨髓基质细胞分化为脂肪细胞,维持其成骨分化,可能预防骨坏死。

BACKGROUND： Animal tests verified that alcohol could cause lipid accumulation in femoral head, leading to osteonecrosis. Molecular biology tests verified that alcohol could induce differentiation of marrow stromal cells （MSCs） into adipocytes. If a drug can resist the differentiation of MSCs into adipocytes induced by alcohol, it can prevent and treat alcoholinduced osteonecrosis. OBJECTIVE： To observe the effects of inhibition of puerarin on differentiation of MSCs into adipocytes induced by alcohol from the aspect of cell biology. DESIGN： Randomized controlled experiment. SETTING： Department of Orthopaedics, First Affiliated Hospital, Zhengzhou University and Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University. MATERIALS： The experiment was conducted at the Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University from April 2000 to December 2002. A total of 50 clean healthy Kunming mice aged 6-8 weeks, of either sex, were selected. Bone marrow cells of the bilateral femur were obtained, with the inoculated density of 1.5×10^6/cm^2, implanted into 6 wells and 24 wells cultured plate, randomized into groups, a well as a specimen and 24 specimens in each group. METHODS： MSCs were separated, cultured and assigned into 3 groups at random： model group （cells were treated with 0.09 mol/L alcohol）, experimental group （0.09 mol/L alcohol and 0.01 mg/mL final concentration of puerarin） and controlled group （no alcohol and puerarin）. Sudan Ⅲ staining was performed and adipose cells was counted under light microscope. Content of triacylglycerol, activity of alkaline phosphatase and content of osteoealcin in cell culture fluid were measured. MAIN OUTCOME MEASURES： （1）Result of differentiation of MSCs into adipocytes in mice of the three groups, （2）content of triacylglycerol in MSCs of mice of the three groups, （3）activity of alkaline phosphatase in MSCs of mice of the three groups, （4）content of osteocalcin in cell culture fluid in the three groups. RESULTS： （1）After being disposed and cultured for 21 days, number of adipoeytes in the experimental group and control group decreased markedly compared with the model group. The number of adipocytes in the model group was 8.9 times of that in the experimental group, and 15.6 times of that in the control group [（319.17±19.92）, （35.92±23.77）（20.42±12.15 ）per cm^2, P 〈 0.001]. （2）After being disposed and cultured for 21 days, content of triacylglycerol in the experimental group and control group was remarkably lower than that in the model group [（4.15±1.92） and （3.42±1.60）, （11.55±4.42） μg in a well, P 〈 0.001]. （3）After being disposed and cultured for 12 days, activity of alkaline phosphatase in the experimental group and control group was distinctly higher than that in the model group [（9.51±2.96）,（ 11.18±3.11 ）, （4.84±2.23） μkat/g,P 〈 0.001]. （4）After being disposed and cultured for 14 days, content of osteocalcin in the experimental group and control group was 2.2 times and 2.7 times of that in the model group, respectively, and the difference was very. significant corn pared with the model group [（11.11±4.57）,（13.43±5.29）, （4.95±2.31） μg/g, P 〈 0.001]. CONCLUSION： Puerarin can inhibit the differentiation of MSCs into adipocytes induced by alcohol, and maintain the osteogenic differentiation, which may prevent the development of osteonecrosis.