摘要
目的:观察中药杜仲在干预去势大鼠骨质疏松过程中对骨细胞BMP-2表达的调节作用,以及杜仲水提取物和醇提取物对大鼠骨髓间充质细胞(mesenchymal stemcells,MSCs)BMP-2表达的影响。方法:80只4月龄雌性SD大鼠随机分成4组,假手术组、模型组、阿尔法骨化醇(法能)组和杜仲实验组。除假手术组打开腹腔,暴露卵巢后即缝合外,其他3组均摘除双侧卵巢。大鼠手术10天后,阿尔法骨化醇组和杜仲实验组分别用阿法骨化醇软胶囊和盐杜仲灌胃,另外2组正常饲养。3个月后,解剖取股骨,4%多聚甲醛固定,三天后浸入4%EDTA溶液中脱钙,4周后进行冰冻切片,免疫组化测定BMP-2表达。取盐杜仲粉末2g,分别加水/甲醇至15ml,室温震荡1小时,静置过夜后,经15000rpm离心10min,弃沉淀。水浸上清不经处理即得水提取物;甲醇浸上清经真空浓缩,再加水至15ml溶解剩余物后,得醇提取物。水/醇提取物均经0.22μm膜过滤,-20℃保存。取2月龄SD大鼠骨髓细胞,置含20%小牛血清的DMEM/F12培养基中,37℃,5%CO2培养,传代3次后进行杜仲水/醇提取物诱导,诱导物分为10-2、10-3、10-4、和10-54个稀释度并分为4个重复组。诱导6天后,免疫组化测定BMP-2表达。结果:杜仲实验组大鼠骨切片中的BMP-2的表达量明显高于模型组,杜仲水/醇提取物对大鼠MSCs的BMP-2表达具有显著刺激作用,醇提取物的作用大于水提取物,并且10-5稀释度的作用大于其他稀释度。结论:在体和离体实验均证实杜仲有干预去势大鼠骨质疏松的作用与调节BMP-2表达相关。
Objective： To investigate expression of bone morphogenctic protein2 （BMP-2） in bone cells of ovariectomized （ovx） rat after administered eucommia bark and influence of expression of BMP-2 on rat bone mesenchymal stem cells （MSCs） induced by water and MeOH extract of eucommia bark.Methods： 80 femal SD rats were divided into 4 groups, sham operation, negative, positive, and experimental groups.The rats in each group were ovariectomized except sham opration group.10 days after operation, intragastrie administration was used to rat in positive and experimental group with Alfacalcidos Soft Capsules and Eucommia Bark respectively.After 3 months, femur was separated and fixed in 4% paraform for 3 days, and then the decalcification was performed in 4% EDTA for 4 months and frozen section was pefformed.Immunohistechemistry was used to detect expression of BMP-2. Water or methanol added to 2g of powder of eucommia bark to 15ml, and shaked for lh at rom temperature.After soaked overnight, both extracts were centrifuged at 15000rpm for 10min. Water extract was obtained without other treatment from supematant in water soaked powder.In MeOH soaked powder, MeOH extract was obtained by concentrated supernatant in vacuo and resolved using 15ml water.Water and MeOH extract were then filtered by membrane of 0.22μm, and conserved at - 20℃ .Bone marrow cells wre obtained from SD rat of 2months, and cultured in DMEM/F12 （1 ： 1 ） medium containing 20% FBS at 37℃, 5% CO2 condition.Induction was performed after passage 3 and both water and MeOH extract were diluted to 10^-2, 10^-3, 10^-4 and 10^-5 respectively, and 4 repeated groups were set for every dilution.At 6 days after induction, detection of expression of BMP-2 was performed by the method of immunohistochemistry.Results： Expression of BMP-2 in experimental group increased compared to negative group. The water and MeOH extract stimulated expression of BMP-2 on MSCs significantly. The MeOH extract has a stronger effect than water extract and 10^-5 dilut
出处
《四川中医》
2009年第8期24-27,共4页
Journal of Sichuan of Traditional Chinese Medicine
基金
无锡中医医院骨伤科的无锡市科技局2007年社会发展项目(CSE00713)
