Effect Of IL-17F On MRNA Expression Of BMP-2 And Noggin In Rat Osteoblasts

ObjectiveBone cells and immunocyte not only share the same progenitors, but also are functionally connected and influenced by the same cytokines. Recent studies have shown that IL-17F which is an important member of the cytokines of IL-17 family could promote osteoblastic function in vivo, but there are few relevant theory studies. In this research, we extracted and cultured the primary osteoblasts from rats and stimulated osteoblasts by different concentration of IL-17F to investigate the effect of IL-17F on cell proliferation and expression of bone morphogenetic protein 2 (Bone Morphogenetic Protein 2, BMP-2) and Noggin (the inhibiting factor of BMP-2).Methods1. Isolation, culture and identification of the rat primary osteoblastsEight new-born Wistar rats younger than 24h were took skulls under the aseptic conditions after sacrificed. Eliminate the periosteum, vascular and connective tissue of the skull bone surface completely. The osteocommas were cut to pieces after repeated washing to translucent. Primary osteoblasts were isolated via the trypsin and collagenase digestion method and improved tissue-culture method.The primary osteoblasts were cultured in the cultivating box after adding with appropriate amount of culture media. Osteoblasts were purified by way of differential adhesion in the passage of subculture. The fourth generation purified osteoblasts were chosen for the reseach.Cell morphological observation and alkaline phosphatase (ALP) staining was used for osteoblasts identification.2. To design the experiment groups and stimulate with IL-17FThe fourth purified rat osteoblasts were divided into the normal control group and the experimental group. The normal medium was used for the normal control group. The experimental group contained five IL-17F stimulation groups which were cultured with the medium added with lng/ml, lOng/ml,20ng/ml,50ng/ml, 100ng/ml IL-17F respectively. The indexes were detected in 1,3,5 day after intervention respectively.3. Index detection(1) MTT was used to detect the proliferation ability of osteoblasts.(2) The mRNA expression of BMP-2 and Noggin of the osteoblasts was detected by RT-PCR.Results1. Culture and identification of the rat primary osteoblastsThe adherent growths of osteoblasts were stable. The cell morphological characteristics and growth status were consistent with the feature of osteoblasts. The result of the ALP staining display that a large number of positive cells whose cytomembrane or cytoplasm were filled with black particle and massive deep dyeing precipitation. The result proved that the cultured cells were osteoblasts.2. Effect of IL-17F on proliferation of osteoblastsThe result of MTT showed that, compared with the normal control group, the cell proliferation of 100ng/ml experimental group was significantly higher in the first day (P<0.05), but the cell proliferation of lOng/ml,20ng/ml,50ng/ml experimental groups had no significant difference (P>0.05). Compared with the normal control group, the cell proliferation of lOng/ml,20ng/ml,50ng/ml, 100ng/ml experimental groups were significantly higher in the third and fifth days, which had statistical difference (P<0.05), the cell proliferation of lng/ml experimental group had no significant difference in each time point (P>0.05).3. Effect of IL-17F on mRNA expression of BMP-2 and Noggin in rat osteoblastsRT-PCR results showed that, compared with the control group, the mRNA expression of BMP-2 of 20ng/ml,50ng/ml, 100ng/ml experimental groups were significantly higher from the first day (P<0.05), while the mRNA expression of BMP-2 of lOng/ml experimental group was significantly higher from the third day (P<0.05), the mRNA expression of BMP-2 of 1ng/ml experimental group had no significant difference in each time point (P>0.05). Compared with the control group, the mRNA expression of Noggin of lOng/ml,20ng/ml,50ng/ml, 100ng/ml experimental groups were significantly lower from the first day(P<0.05), while the mRNA expression of Noggin of 1ng/ml experimental group had no significant difference in each time point (P>0.05).ConclusionA certain concentration of IL-17F can promote the proliferation of rat osteoblasts in vitro, improve BMP-2 mRNA expression of osteoblasts in rats, reduce the mRNA expression of Noggin, and it present a dose-time dependence.