杜仲对去势大鼠腰椎骨和rBMSCs TGFβ与FGF2表达调控

Expression of TGFβ and FGF2 in lumber vertebrae after treatment with Eucommia Bark to ovariectomized rat and to rBMSCs

目的:观察杜仲干预去势大鼠腰椎骨及对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cells,rBMSCs)转化生长因子β(transforming growth factorβ,TGFβ)和成纤维生长因子2(fibroblast growth factor 2,FGF2)表达的调控。方法:(1)80只4月龄雌性SD大鼠随机分成4组,假手术组、阴性对照组、阳性对照组和实验组。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,阳性对照组和实验组分别用阿尔法骨化醇(ALF)和盐杜仲灌胃,每天一次,另外2组正常饲养。灌胃3个月后,解剖取各组第一腰椎骨,4%多聚甲醛固定,3天后浸入4%EDTA溶液中脱钙,4周后冰冻切片,免疫组化检测各组中TGFβ与FGF2表达;(2)取盐杜仲粉末2 g,分别加水或甲醇至15 ml,室温震荡1 h,静置过夜后,离心弃沉淀。水浸上清不经进一步处理即得水提取物;甲醇浸上清经真空浓缩,再加水至15 ml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;(3)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10-5 4个稀释度并设4个重复组,同时设一不加诱导物的对照细胞培养组。诱导6天后,免疫细胞化学法测定BMP-2表达。结果:与假手术组比较,阴性对照组腰椎骨中TGFβ、FGF2表达均下降;与阴性对照组相比,ALF阳性对照组和杜仲实验组TGFβ和FGF2表达上升;与对照细胞培养组比较,杜仲水/醇提取物诱导rBMSCs FGF2的表达显著升高,但TGFβ的表达变化不显著。结论:杜仲改善去势大鼠骨质疏松状态与调节TGFβ、FGF2表达相关,杜仲具有直接刺激细胞FGF2的表达的作用。

Objective:To examine the expression of transforming growth factor β(TGFβ) and fibroblast growth factor 2(FGF2) in lumber vertebrae after treatment with Eucommia Bark to ovariectomized(ovx) rat and to rat bone mesenchymal stem cells(rBMSCs).Methods: 80 female SD rats were divided into 4 groups,sham,negative,positive and experimental groups.All rats were subjected to ovariectomy except sham group.10 days post surgery,intragastric administration was used to rats in positive and experimental group with Alfacalcidol(ALF) and Eucommia Bark once a day respectively.Other groups were breeded normally.After 3 months,lumber vertebrae was separated and fixed in 4% paraformaldehyde for 3 days,and then the decalcification was performed in 4% EDTA for 4 weeks.Immunohistochemistry was applied to detect the expression of TGFβ and FGF2 on frozen sections.Water or methanol added to 2 g of powder of eucommia bark to 15 ml,and shaked for 1 h at room temperature.After soaked overnight,both extracts were centrifuged at 15000 rpm for 10 min.Water extract was obtained without other treatment from supernatant in water soaked powder.In MeOH soaked powder,MeOH extract was obtained by concentrated supernatant in vacuo and resolved using 15 ml water.Water and MeOH extract were then filtered by membrane of 0.22 μm,and conserved at-20℃.Bone marrow cells were obtained from SD rat of 2 months,and cultured in DMEM/F12(1∶1) medium containing 20% FBS at 37℃,5% CO2 condition.Induction of osteogenic differentiation was performed after passage 3.For induction experiments,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,and a control group of cell culture did not plus any inducer was set.At 6 days with induction,expression of TGFβ and FGF2 was detected by the method of immunocytochemistry.Results: In negative group,the expression of TGFβ and FGF2 decreased obviously compared to sham group,and increased in positive and experimental groups compared to negative group.Both water andMeOH extract stimulated expression of FGF2 on rBMSCs significantly compared to control group of cell culture,but the expression of TGFβ did not change obviously.Conclusions: Effect of Eucommia Bark on osteoporosis of ovx rat related to regulation of the expression of TGFβ and FGF2,and Eucommia Bark could stimulate expression of FGF2 on cells directly.