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Effects of 17β-E2 and chOPG on Proliferation, Apotosis, Cell Cycle, and mRNA Expression of BGP and Col Ⅰ of Ostoblasts Induced from BMSCs in Chicken Embryos

Author: WangZhiMei
Tutor: HouJiaFa
School: Nanjing Agricultural College
Course: Clinical Veterinary Medicine
Keywords: entrogen chOPG BMSCs osteoblast BGP ColⅠ chicken embryo
CLC: S858.31
Type: Master's thesis
Year: 2010
Downloads: 5
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Abstract


Osteoporosis is characteristic of loss in bone mass and disruption of cancellous bone architecture, resulting in the impairment of bone strength and the augmentation of fracture risk. OPG (osteoprotegerin) which is a TNF receptor family member and is mainly produced by BMSCs (bone marrow mesenchymal cells) and OB (osteoblasts) can inhibit OC (osteoclasts) formation. Both the BMSCs and OB have the receptors of ERa and ERβ, so estrogen enhances osteoblasts differentiation of BMSCs via an estrogen-receptor-dependent pathway. BMSCs are multipotent cells and can be differentiate into adipose, osteoblasts, and chondrocytes. The cells were induced to differentiate into osteoblasts in a differentiation induction medium. It is indicated that the close relationship and interaction among estrogen, OPG, and BMSCs can provide theoretical bases for probing into the machenism of medullary bone formation, pathogenesis and prevention and treatment of osteoporosis in laying hens.Experiment I Culture and Identification of Osteoblasts Induced from BMSCs in Chicken EmbryosObjective:To investigate the methods of culturing of 18-day chicken embryos’BMSCs in vitro, the growth regularity and the differentiation of BMSCs into OB, the functions of the inducd OB. Method:Chicken BMSCs were flushed out with DMEM/F12 from the tible bone marrows and purified with whole marrow adherent method and subcultured in vitro. The third generation of BMSCs was chosen to induce into OB. The growth and morphological changes of the cells were observed by inverted phase contrast microscope. The rate of cell proliferation was detected with methyl-thiazol-tetrazolium (MTT) colorimetry at different time points. The changes in cell cycle of cells were measured by flow cytometry after Annexin V-FITC and PI staining. Alkaline phosphatase (ALP) was detected with BCIP/NBT, Collagen I was detected by Van-Gieson staining and calcified nodules were stained with Alizarin red. Result:The results showed that both the BMSCs and the induced OB had good reproductive activity; The induced osteoblasts’ALP and calcified nodules’staining were strongly positive. Conclusion:Thus it is shown that BMSCs separated from chick embryos’bone marrow can be induced into OB.ExperimentⅡEffect of 17β-E2 on ALP Activity, mRNA Expression of BGP and ColⅠof OB Induced from BMSCs in Chicken EmbryosObjective:Investigate the effects of 17β-E2 on chicken BMSCs induced OB, to provide the theoretical base for the mechanisms of medullary bone formation and pathogenesis of osteoporosis in laying hens. Methods:BMSCs were isolated from chicken tible marrow and purified, amplified in vitro. The third passages of BMSCs were induced into osteoblasts and digested. The induced OB were divided into 5 experimental groups and cultured respectively with osteogenic differentiated medium containing different concentrations (10-7,10-8,10-9,10-10,10-11 mol·L-1) of 17-E2. Cells cultured without 17β-E2 as blank control group. MTT growth curves were obtained at the range of concentrations. The ALP level of the OB was examined by PNPP method and the optimization concentration was determined. Culture the induced OB in the most effective concentration, and then the percentages of apoptotic cells were measured by flow cytometry. The mRNA expression of OPG, BGP (bone r-carboxyglutamic protein), ColⅠ(collagenⅠ) were determined by RT-PCR semi-quantitatively during the differentiation. Results:compared with control group,10-9 mol·L-1 17β-E2 experimental group showed 17β-E2 can promote proliferation of induced OB and ALPsecrete; and could promote the mRNA expression of BGP and ColⅠ,showed a signifcant (P<0.05). Conclusion:10-9 mol·L-1 17β-E2 can enhance the function of BMSCs induced OB. ExperimentⅢThe Effect of chOPG on ALP Activity, mRNA Expression of BGP and ColⅠof OB Induced from BMSCs in Chicken EmbryosObjective:To observe the effect of chicken-osteoprotegerin (chOPG) on BMSCs dinduced OB and provide the theoretical base for the mechanisms of medullary bone formation and pathogenesis of osteoporosis in laying hens. Methods:BMSCs were isolated from chicken tible marrow and purified, amplified in vitro. The third passages of BMSCs were induced into osteoblasts and digested. The induced OB were divided into 3 experimental groups and cultured respectively with osteogenic differantiated medium containing different concentrations (4.0,0.4,0.04 mg·mL-1) of chOPG. Cells cultured without chOPG as blank control group. MTT growth curves were obtained at the range of concentrations. The ALP level of the OB was examined by PNPP method and the optimization concentration was determined. Culture the induced OB in the most effective concentration, and then the percentages of apoptotic cells were measured by flow cytometry. The mRNA expression of BGP, ColⅠwere determined by RT-PCR semi-quantitatively during the differentiation. Results:Both of the BMSCs and induced OB, the survival condition was good, the biochemical indexes were stable. Compared 0.4 mg·mL-1 experimental group with control groupthe ALP secretion of induced OB decreased significantly, BGP and ColⅠmRNA levels decreased significantly (P<0.05). Conclusion:chOPG inhibited the function of induced OB, the mRNA expression of BGP and ColⅠgradually decreased.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Chicken
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