雌二醇对大鼠骨髓基质细胞过氧化物酶增殖活化受体γ2基因表达的影响

背景:雌二醇能否改变骨髓基质细胞分化过程中过氧化物酶增殖活化受体γ2-mRNA的表达进而影响其向成骨细胞分化有待研究。目的:观察骨髓基质细胞在向成骨细胞分化的介质中,17β-雌二醇对其过氧化物酶增殖活化受体γ2mRNA及蛋白表达的影响。设计:对比观察实验。单位:成都军区总医院中心实验室,德阳市人民医院检验科,成都百奥生物技术有限公司。材料:选用1只雌性SD大鼠,3月龄,清洁级,体质量(200±20)g,由成都中医药大学实验动物研究中心提供(动物许可证号码11)。DMEM培养液购自Hyclone,1,25一双羟维生素D3、地塞米松和雌二醇购自Sigma公司,引物用Jellyfish软件设计,由大连宝生物技术有限公司合成。RT-PCR试剂盒和RNA提取试剂盒均由大连宝生物提供。兔抗山羊多抗(北京中山生物技术有限公司),山羊抗鼠PPARγ2抗体和Western Blotting增强化学发光试剂均购自Santa Cruz公司。方法:实验于2001-04/2002-07在成都军区总医院中心实验室和成都百奥生物技术有限公司完成。①无菌条件下分离大鼠骨髓基质细胞,1,25一双羟维生素D3和地塞米松诱导大鼠骨髓基质细胞向成骨细胞分化,观察细胞生长情况。②用0,0.1,10和1000nmol/L不同浓度雌二醇对细胞分化过程进行干预3d。培养细胞用Tris-Triton X-100缓冲液加超声粉碎裂解细胞,在Beckman CX-7生化分析仪上测定碱性磷酸酶的活性,观察不同浓度雌二醇条件下细胞产生碱性磷酸酶的情况。③100g/L中性甲醛固定培养于24孔板的骨髓基质细胞,Weigert苏木素染液染色10min,自来水冲洗,5g/L盐酸乙醇分化,Van Gieson染色,体积分数为0.95乙醇分化脱水,二甲苯透明,显微镜下观察并照相,观察细胞胶原合成情况。④应用半定量RT-PCR及Northern blot、Western blot技术,观察不同浓度雌二醇对骨髓基质细胞分化过程中PPAR-γ2mRNA及蛋白表达的影响。主要观察指标:①骨髓基质细胞生长情况、细胞胶原合成情况。②不同浓度雌二醇条件下细胞产生碱性磷酸酶的情况。③不同浓度雌二醇对骨髓基质细胞分化过程中PPAR-γ2mRNA及蛋白表达的影响。结果:①骨髓基质细胞接种后4h开始贴壁,24～72h可见贴壁细胞明显增大且分裂增殖,细胞呈三角形,多角形或梭形。3d后首次换液,贴壁细胞体积增大,呈集落生长,10d左右融合成遍。多次传代的骨髓基质细胞呈有序的成纤维细胞样分布。②随着雌二醇浓度的增加,胞浆染色越淡,由鲜红、淡红到黄色。胞浆呈红色说明有胶原合成,红色越深,表明胶原越多。③不同浓度雌二醇条件下细胞均能产生碱性磷酸酶,其活性随雌二醇浓度增加而降低,雌二醇浓度从0nmol/L增加到1000nmol/L时,碱性磷酸酶从(710.1±41.7)nkat/g降至(60.0±11.7)nkat/g(t=29.0,P<0.01)。④雌二醇浓度为0.1,10和1000nmol/L时,PPAR-γ2mRNA的表达量均明显高于雌二醇0mol/L组(t=6.1,7.2,11.5,P<0.01),Northern blot结果显示雌二醇浓度为0.1,10和1000nmol/L时PPAR-γ2mRNA表达量分别为(4.0±0.4)%,(1.7±0.2)%,(2.8±0.2)%,明显高于雌二醇0mol/L组[(1.5±0.1)%,t=5.4,105.0,14.2,P<0.01]。⑤Western Blot检测结果显示雌二醇能明显增加骨髓基质细胞PPAR-γ2蛋白的表达。雌二醇浓度为0.1,10和1000nmol/L时,PPAR-γ2蛋白的表达量分别为(2.2±0.2)%,(2.6±0.2)%,(4.1±0.2)%,明显高于雌二醇0mol/L组[(1.2±0.10)%,t=6.6,8.5,13.2,P<0.01]。结论:雌二醇能明显抑制体外培养的骨髓基质细胞碱性磷酸酶的表达,同时促进骨髓基质细胞内过氧化物酶增殖活化受体γ2mRNA和蛋白的表达。

Effect of estradiol on the gene expression of peroxisome proliferator-activated receptor gamma-2 in rat bone marrow stromal cells

BACKGROUND: Whehter estradiol (E2) can change the expression of peroxisome proliferator-activated receptor gamma-2(PPAR-γ2) during differentiation of bone marrow stroma cells should be studied further.OBJECTIVE: To investigate the effects of 17β-estradiol (E2) on the gene expression of PPAR-γ2 mRNA and PPAR-γ2 protein.DESIGN: Contrast observational trial.SETTING: Laboratory Department of People's Hospital of Deyang City; Laboratory Center of Chengdu Military Command General Hospital and Chengdu Bai'ao Biol-Tech Limited Company.MATERTALS: A 3-month-old female SD rat (200±20) g used to isolate bone marrow stromal cells was obtained from Animal center of Chengdu Chinese Medicine University (Medical Experimental Animal Number 11). Dulbecco's mimimum essential medium (DMEM) was obtained from Hyctone Compnay. 1α, 25(OH)2D3, dexamethasome (DEX) and E2 were purchased from Sigma Company. Total RNA kits were obtained from Omga. One step RNA PCR kit (AMV) was obtained from Takara Shuzo Co, Ltd. Northern direct HRP labeling and detection kit was purchased from PIERCE. Western blotting luminol reagent was obtained from Santa cruz.METHODS: The experiment was carried out from April 2001 to July 2002 in the Laboratory Department of People's Hospital of Deyang City, Laboratory Center of Chengdu Military Command General Hospital and Chengdu Bai'ao Biol-Tech (0, 0.1, 10, 1 000 n) was used to interfere cell differentiation for 3 days. Cultured cells were crushed with Tris-Triton X-100 PBS; activity of alkaline phosphatase was detected with Beckman CX-7 biochemical analytical device; effect of 100 g/L formalin, stained with Weigert-hematoxylin for 10 minutes, rinsed with water, differentiated with 5 g/L hydrochloric ethanol, stained with Van Gieson, desiccated with ethanol of the fractional volume of 0.95, cleared with dimethylbenzene with RT-PCR, Northern blot and Western blot during cell differentiation.protein.proliferated within 24-72 hours. Cells shaped as triangle, multiple angles and fusiform. Three days later, volume of adherent cells was increased and colony, and 10 days later, they confluenced. Bone marrow stroma cells in many generalight red to yellow). Red plasma presented synthesis of collagen. The deeper the red was, the more the collagens were.pression of PPAR-γ2 mRNA was (4.0±0.4)%, (1.7±0.2)% and (2.8±0.2)% (t=6.1, 7.2, 11.5, P＜ 0.01), which was higher crease the expression of PPAR-γ2 protein. When concentration of E2 was 0.1, 10 and 1 000 nmol/L, expression of PPAR-γ2 protein was (2.2±0.2)%, (2.6±0.2)% and (4.1 ±0.2)%, which was higher than that in 0 nmol/L E2 group [(1.2±0.10)%, t=6.6, 8.5, 13.2, P＜0.01].CONCLUSTON: E2 can inhibit expression of alkaline phosphatase and promote differentiation of bone marrow stromal cells and expression of PPAR-γ2 mRNA and PPAR-γ2 protein.