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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.
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Objective To investigate the effects of 17?-estradiol (E2) on the gene expression of typeⅠA bone morphogenetic protein receptor (BMPR-ⅠA) and core-binding factor alpha 1 (Cbf?1) in rat bone marrow stromal cells exposed to the differentiation medium and to elucidate the effects of E2 on osteoblastogenesis. Methods Adherent bone marrow stromal cells were cultured in differentiation medium containing DEX (10 -7 mol/L), 1,25-(OH)2D3 (10 -9 mol/L) and different concentrations of E2. Effects of different concentrations of E2 on the gene expression of BMPR-ⅠA and Cbf?1 was quantified by RT-PCR based on the comparison with an internal reference, ?-actin expression, and identified by Northern blotting. Alkaline phosphatase (ALP) activity of cells was detected. Contents of type Ⅰ collagen were determined by Van Gieson staining. Results E2 could evidently inhibit the expression of BMPR-ⅠA and Cbf?1 mRNA during the differentiation process of bone marrow stromal cells into osteoblasts in a dose-dependent manner. These were confirmed by Northern blotting. The ALP activity increased in a concentration-dependent manner, but the amount of type Ⅰ collagen decreased in a concentration-dependent manner. Conclusion E2 can significantly inhibit the gene expression of BMPR-ⅠA and Cbf?1 in bone marrow stromal cells and inhibit osteoblastogenesis in vitro.
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Aim To investigate the effects of tamoxifen on the proliferation and DNA synthesis of cultured human pituitary adenoma cells and make a further investigation of the mechanism of the inhibitory effect of tamoxifen on the proliferation of pituitary adenoma cells. Methods The techniques of MTT colorimetry, 3H-TdR, flow cytometry, PKC activity detection and cAMP/ cGMP levels detection were used to detect or observe the effects of tamoxifen on proliferation, DNA synthesis, cell cycle, PKC activity and cAMP/ cGMP levels of cultured human pituitary adenoma cells, respectively.Results ①Tamoxifen (0.1,1 and 10 ?mol?L -1) inhibited the proliferation and DNA synthesis of cultured human pituitary adenoma cells in a dose-dependent manner.② tamoxifen (1,10 and 20 ?mol?L -1) increased the ratio of G_1 phase of pituitary adenoma cells, and decreased the ratio of S and G_2 phase markedly;②compared with control, PMA, a PKC activator, increased the activity of membrane and total PKC in human pituitary adenoma cells. However, after a 15-min treatment with tamoxifen (10 ?mol?L -1),a significant reduction of the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells was observed;③tamoxifen (1 and 10 ?mol?L -1) increased the amount of cAMP in the cytoplasm of human pituitary adenoma cells, but had no effect on that of cGMP. Conclusion These data provide an important clue to explore the molecular mechanisms of the inhibitory effect of tamoxifen on the proliferation of pituitary adenoma cells, and suggest that the modulating effect of tamoxifen on the proliferation of pituitary adenoma cells results from interactions of several cellular signaling pathways.
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AIM:To investigate the effects of 17?-estradiol (E_2) on the gene expression of typeⅠA and typeⅠB bone morphogenetic protein receptor (BMPR-ⅠA,ⅠB) in rat bone marrow stromal cells exposured to the differentiation medium and to elucidate the effects of E_2 on osteoblastogenesis. METHODS: Adherent bone marrow stromal cells were cultured in differentiation medium containing DEX(10 -7 mol?L -1 ) and 1,25(OH)_2D_3 (10 -9 mol?L -1 ) and different concentrations of E_2. The gene expression of BMPR-ⅠA,ⅠB was quantified by semiquantitative RT-PCR. RESULTS: E_2 evidently inhibited the expression of BMPR-ⅠA mRNA in bone marrow stromal cells.The suppression was dose-dependent. When examined under various concentrations of E_2 (0-10 -6 mol?L -1 ),the expression of BMPR-ⅠA mRNA were decreased from (25.7?2.5)% to(16.3?1.5)%( P
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Osteoprotegerin(OPG) ,receptoractivatorofNF -?Bligand (RANKL)andreceptoracti vatorofNF -??(RANK)areimportantmoleculesthatregulateosteoclastogenesis .OPGisdecoyreceptorof RANKL .ThebindingofOPGandRANKLblocksconnectingofRANKLandRANK ,inhibitsdevelopmentof osteoclast.ThebindingofRANKLandRANKinducesaserieskinasecascadereaction ,activatestranscription factors,introducestheproliferationanddifferentiationofosteoclastprecursors .Severalhormonesandos teotropicfactorsregulatetheformationandfunctionofosteoclastthroughtheabovemolecules .