Effects Of Thyroid Stimulating Hormone On Male Bone Metabolism And Mechanism Research

Osteoporosis is a common disease for middle-aged and old people,which specific pathogenesis is still not very clear.In recent years,the study found that thyroid stimulating hormone(TSH) may be associated with osteoporosis.This study is divided into two parts:clinical analysis and in vitro experiments, aiming at the effects of TSH of male bone metabolism and its further research on the possible mechanisms of osteoblast.Experiment One:Changes of serum hormones and BMD in men with normal thyroid functionObjective:To observe the changes of TSH and BMD in men with normal thyroid function.Methods:We chose 1865 individuals who aged 20 to 82 years living in Yangguang community of Jinan from October to November 2009.In strict accordance with the exclusion criteria,a total of 114 male incorporated in the analysis.Using roche to detect serum FT3,FT4,TSH level,dual-energy X-ray absorptiometry respondents forearm one-third lower BMD.And BMD between high-level serum TSh group and low-level serum TSH group was compared.Results:Two groups of objects in age,BMI,FT3,FT4 photogenic matching,serum TSH was positively correlated with BMD in men.Conclusions:TSH and BMD were positively correlated in male with nomal thyroid function serum,which suggest TSH have direct regulating effect on bone mass.Experiment Two:Effects of TSH on osteoblast proliferation and differentiation.Objective:To study the effects of TSH on osteoblast proliferation and differentiation on related genes mRNA and protein expression.Methods:Calvarial osteoblasts were separated from 8 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identied by HE,alkaline phosphatase (ALP) staining and alizarin red staining, also by drawing the seven days’growth curve.The cells at passage 3 were cultured with TSH culture medium at concentrations of 0,1,10,100 mu/mL (0,1,10, 100mu/mL groups),then on the 12h,24h and 48h they were determined by MTT colorimetric test.Osteoblasts’gene expression associated with differentiation, such as ALP,type I collagen (COL1), Runx2 and BMP2,was detected by RT-PCR at 48h.To use ELISA method to detect cellular changes in ALP and the expression of BMP2 level.The protein expressions of Runx2 and osterix were determined by Western Blot at different concentrations.Results:1.The cells extracted from Rat calvarial were identied as primary osteoblast.2.The cell proliferation ability of groups 10,100 mu/ml was increased to different extent,concentration of 100 mu/ml in the most obvious.3.The differentiation and regulation gene of OB,such as ALP, COI1, BMP2, Runx2 mRNA expression showed a trend of increasing in experimental group and TSH could dose dependently increased the gene mRNA expression.4.ELISA result showed that compared with control group,ALP,BMP2 protein expression levels were increased in experimental group, consistenting with the results of rt-pcr.5.The protein expressions of Runx2 and osterix were increased in a dose-dependent manner determined by Western Blot.Conclusion:Our results confirmed that TSH promotes osteoblast proliferation and differentiation, and guessed that in which BMP2 may play an important role,but the specific mechanism and signal transduction pathways still needed our further exploration.