| ObjectiveWith the aging society becoming more and more serious,the fracture caused by osteoporosis has become a health concern all over the world.Mounting studies of osteoporosis has demonstrated that one of the major causes is over production of inflammatory factors.Inflammatory cytokines IL-17A and IL-17F,secreted by TH17 cells,can stimulate bone marrow mesenchymal stem cells(BMSCs)to migrate and differentiate into osteoblasts(OB).So far,the underlying mechanism of the effect of IL-17F on osteoporosis is unclear.Based on the preliminary study on osteoblast metabolism in osteoporosis rats,we researched the effects of IL-17F on the proliferation of osteoblasts,and measured the levels of protein and mRNA expressions of IL-17F receptor(IL-17RA and IL-17RC),Bone Morphogenetic Protein 2(BMP-2)and its inhibitor Noggin.Methods1.Rat primary osteoblasts cell isolation,culture and identificationUnder the aseptic conditions,six new-born(younger than 24 h)Wistar rats were sacrificed and took skulls.After repeated washing,the osteocommas were cut into pieces.Primary osteoblasts were isolated by trypsin,cultured with low glucose DMEM medium containing 10%fetal bovine serum and purified by differential adhesion of subculture.We selected the fourth purified generation osteoblasts for further experiment.2.Experimental grouping and stimulation of IL-17FThe cells were divided into the control group and different IL-17F stimulating groups.The control group used normal medium and the experiment group used medium added with 1,10,20,50,100 ng/ml IL-17F respectively.3.Detecting indexesProliferation activity of osteoblasts were tested by MTT assay.The mRNA transcriptions of IL-17RA,IL-17RC,BMP-2 and Noggin were investigated with real time fluorescent quantitative,and protein expressions of IL-17RC,BMP-2 and Noggin were also detected by western blotting.Results1.Rat primary osteoblasts cell isolation,culture and identificationThe characters and growth status of adherent osteoblasts accorded with the feature of osteoblasts,and they were stable during culture.The results of ALP staining demonstrated that the positive cells,which were osteoblasts,were full of black dyeing precipitation.2.Effect of IL-17F on proliferation activity of osteoblastsThe MTT results showed that the rate of osteoblasts proliferation was dose-dependent and time-dependent to some degree stimulated by the same concentration of IL-17F.In the first,third and fifth days,the OD value of 20,50 and 100 ng/mL were significantly higher than control group(P<0.05).And at the concentration of 100ng/mL on the fifth day,IL-17F had the greatest influence on the proliferation of osteoblasts.3.Effect of IL-17F on IL-17RA and IL-17RC mRNA expressionsOn the first,third and fifth day,the mRNA transcription of IL-17RA at 20,50 and 100 ng/mL were significantly increased with dose-dependent(P<0.05).However.only at the concentration of 100 ng/mL of IL-17F,the expression of IL-17RC was markedly raised(P<0.05).4.Effect of IL-17F on BMP-2 and Noggin mRNA expressionsCompared with the control group,at the dose of 20,50 and 100 ng/mL of IL-17F,the levels of BMP-2 mRNA expression began to rise from the first day(P<0.05)with a dose-dependent manner.However,the expression levels of Noggin mRNA were significantly decreased from the first day stimulated by 20,50 and 100 ng/mL of IL-17F in a dose dependent manner.5.Effect of IL-17F on IL-17RA,BMP-2 and Noggin protein expressionsCompared with the control group,at the dose of 20,50 and 100 ng/mL of IL-17F,the levels of BMP-2 and IL-17RA expressions significantly raised(P<0.05)with a dose-dependent manner.However,the expression level of Noggin significantly decreased stimulated by 20,50 and 100 ng/mL of IL-17F in a dose dependent manner.(P<0.05).ConclusionThe dose of 20,50 and 100 ng/mL of IL-17 can stimulate osteoblasts proliferation of rat in vitro,improve the BMP-2 mRNA and protein expressions,and decrease Noggin mRNA and protein expressions in dose-dependent and time-dependent manners.Therefore,we proposed that IL-17F at the low dosage promoted the proliferation of osteoblast mainly via IL-17RA but IL-17RC,while IL-17F at the high dosage promoted the proliferation of osteoblast by both IL-17RA and IL-17RC. |
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