Experimental Studies On Effect Of 17β-Estradiol On Osteoblastogenesis

【Background】Osteoporosis is a progressive systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue leading to decreased bone strength, increased bone fragility and susceptibility to fracture. The primary clinical consequences of osteoporosis are fractures, which have considerable impact on mortality, morbidity and medical expense worldwide, and will increasingly affect public health as the age-specific incidence of fractures steadily rises. Loss of estrogen after menopause is the most common cause of osteoporosis. Osteoporosis is associated with an increase in the rate of bone remodeling and an imbalance between bone formation and resorption with bone resorption predominating. This fact may be explained in part by the evidence that loss of sex steroids up-regulates the formation of osteoclasts and osteoblasts in marrow by up-regulating the production and action of cytokines such as IL-6 that are responsible for osteoclastogenesis and osteoblastogenesis.However, this results almost didn't involved in the molecular mechanism of initial phase of high bone turnover in estrogen deficiency. Although the activation of osteoclast is the starting point in every cycle of bone remodeling, in which osteoblast is the center since it not only forms bone, but also regulates differentiation and mature of osteoclast and affects bone resorption indirectly. In order to investigate the effects of E2 on osteoblastogenesis and elucidate the mechanism of the initial phase of increased rate of bone remodeling in postmenopause, we must study the effects of E2 on the expression of the factors which influence osteoblastogenesis.Osteoblasts derive from precursors originating in bone marrow. Osteoblast differentiation and bone formation are regulated by many local factors, including bone morphogenetic proteins (BMPs) and their receptors(BMPRs), core-binding factor alpha 1(Cbfα1) and peroxisome proliferator activated receptor gamma2(PPARγ2). BMPs are only factors capable of initiating osteoblastogenesis from uncommitted progenitors. Cbfα1 is an osteoblast-specific transcription factor which activates osteoblast-specific genes.PPARγ2 is a crucial transcription factor in adipocytogenesis. Between Cbfα1 and PPARγ2, there is a reciprocal relationship. It is just the reciprocal relationship that determines the differentiation directs of BMSc—osteoblast or adipocyte. Studies with a clonal cell line(2T3) suggest that BMP-2 induces osteoblast or adipocyte differentiation from mesenchymal precursors, depending on whether the BMP receptor type ⅠA or ⅠB is activated. Therefore, BMP receptors may also play a critical role in both specification and reciprocal differentiation of osteoblast and adipocyte progenitors. Taken together, these findings strongly suggest there must be tight link among the three molecules.【Methods】Bone marrow stromal cells induced by 1,25-dihydroxyvitamin D3(1,25(OH)2D3) and dexamethasome(DEX) can differentiate into osteoblast. The present experiment studied the effects of estradiol on the expression of three molecules as above in bone marrow stromal cells exposured to osteoblastic differentiation medium. Adherent bone marrow stromal cells from 3-month-old female SD rat cultured in growth medium were treated at 37℃, 50ml/LCO2 for 3 days in the presence of DEX 10-7 mol/L and 1,25(OH)2D3 10-9mol/L and in the presence of different concentrations of E2(0-10-6mol/L).Total cellular RNA was isolated using total RNA kit. The effects of E2 on the expression of BMPR-ⅠA,ⅠB, Cbfα1, and PPARγ2 gene were quantified by semiquantitative reverse transcription PCR(RT-PCR), based on the comparison with an internal reference, β-actin expression, and demonstrated by Northern blot. The expression of BMPR-ⅠA and PPARγ2 protein were assayed by Western blot. Alkaline phosphatase(ALP) and type Ⅰcollagen are important markers of mature osteoblast. ALP detection was carried out by SYNCHRON CX system. Type Ⅰcollagen was shown by Van Gieson staining.【Results】...