| ObjectiveTo detect the cells proliferation, ALP activity, and the changes of mRNA expression of transcription factors Runx2 and Osterix at different concentrations of IL-17F intervened rat osteoblasts and different times. By discussing the influence of IL-17F on osteoblasts proliferation, differentiation, and the changes of mRNA expression of osteoblast specific transcription factor Osterix and Runx2 under the intervention of IL-17F, explore the role of inflammatory cytokines IL-17F in bone formation to provide a theoretical basis for the clinical treatment of osteoporosis and related bone metabolic diseases.Methods1. Cell extraction, cell culture and identificationEight new-born Wistar rats younger than 24 hours were taken skulls under the aseptic conditions after sacrificed. Osteoblasts were harvested from the calvarias of neonatal rats and isolated via trypsin and type II collagenase and improved tissue-culture method. Osteoblasts were incubated in 5% CO2 at 37℃ and 100% relative humidified. After the monolayer culture grew to confluence of 80% of culture flask, cells were harvested with a 0.25% trypsin solution, split, resuspended in the culture medium, and filled into new culture flasks. The osteoblasts were purified by difference-speed adherence method. After confluent monolayer cells were achieved by the fourth passage, cells were used for experiment.The identification of osteoblasts(1) Morphological characteristics and growth situation of cells were observed and photographed by an inverted phase contrast microscope.(2) Alkaline phosphatase staining verified the osteoblastic phenotype.2. Grouping and treatmentThe fourth passage osteoblasts were counted and cell concentration was adjusted to 5 × 105/ml. One day prior to cell seeding in 6-well plates, adherent cells were cultured with serum-free medium for another 24 hours in order to cell synchronization. Cells in the 6-well plates were randomly divided into the control group and the experimental group. The control group was applied with low glucose DMEM culture medium supplemented with 10% fatal bovine serum (FBS). In pre-experiment, the concentration of lng/ml,10 ng/ml,20ng/ml,50ng/ml and 100ng/ml IL-17F was used to intervene in osteoblasts, which showed that 20ng/ml,50ng/ml and 100ng/ml IL-17F affected osteoblasts significantly. Therefore the experimental groups were applied with low glucose DMEM culture medium supplemented with 10%FBS and 20ng/ml, 50ng/ml and 100ng/ml IL-17F, respectively. Cell supernatant specimens were collected on the first and the third day after the intervention of IL-17F, respectively. Cell supernatant specimens were preserved in -80℃.3. Index detection(1) CCK-8 analysis was used to detect the proliferation of osteoblasts.(2) Trace amounts of cell culture supernatant of ALP activity was detected by ELIS A method.(3) The mRNA expression changes of the osteoblast specific transcription factor Osterix and Runx2 were measured by RT-PCR.Results1. Cell extraction, cell culture and identification of osteoblastCells can be harvested with the method of trypsin-collagenase digestion and improved tissue-culture method. The morphology and growth characters of cells were consistent with the features of osteoblasts. ALP staining had proved that the cultured cells were osteoblasts which showed black particles or clumps in the cells observed by inverted phase contrast microscope.2. The proliferation of osteoblasts measured by CCK-8Compared with the control group, cell proliferation supplemented with 50ng/ml and 100ng/ml IL-17F was significantly increased on the first day, there was significant statistically difference (P<0.05); cell proliferation supplemented with 20ng/ml IL-17F was also developed, however, there was no statistical significance; cell proliferation supplemented with 20ng/ml,50ng/ml and 100ng/ml IL-17F was higher than that of control group on the third day (P<0.05); cell proliferation supplemented with 20ng/ml, 50ng/ml and 100ng/ml IL-17F was significantly developed than that of control group on the fifth day, which was significant statistically difference (P<0.05).Compared with the experimental group supplemented with 20ng/ml IL-17F, cell proliferation in the experimental group supplemented with 100ng/ml IL-17F was obviously raised on the first, third and fifth day, which was significant statistically difference (P<0.05); cell proliferation supplemented with 20ng/ml IL-17F was significantly lower than that supplemented with 50ng/ml and 100ng/ml IL-17F, there was significant statistically difference (P<0.05).Compared with the experimental group supplemented with 50ng/ml IL-17F, cell proliferation supplemented with 100ng/ml IL-17F was remarkable elevated at the first, third and fifth day, which was significant statistically difference (P<0.05).3. The ALP activity measured by ELISA assayCompared with the control group, ALP activity of the experimental group supplemented with 20ng/ml and 50ng/ml IL-17F was declined, and 100ng/ml IL-17F was conspicuously increased at the first day, there was significant statistically difference (P<0.05); ALP activity supplemented with 20ng/ml and 50ng/ml IL-17F was lower than that of the control group, but ALP activity supplemented with 100ng/ml IL-17F was outstanding raised than that of the control group and the experimental group supplemented with 20ng/ml and 50ng/ml IL-17F at the third day, which was significant statistically difference (P<0.05).4. The changes of expressions of Osterix and Runx2 mRNACompared with control group, the mRNA expression of Runx2 supplemented with 50ng/ml and 100ng/ml IL-17F was increased at the first, third and fifth day (P<0.05). The mRNA expression of Runx2 supplemented with 100ng/ml IL-17F was striking increased than that with 20ng/ml IL-17F at the first and third day (P<0.05). At the fifth day the mRNA expression of Runx2 supplemented with 50ng/ml and 100ng/ml IL-17F was increased than that of the control group (P<0.05). In comparison to experimental group supplemented with 50ng/ml IL-17F, the mRNA expression of Runx2 supplemented with 100ng/ml IL-17F was higher at the first, third and fifth day, which was prominent statistically significant difference (P<0.05).The mRNA expression of Osterix intervened in 50ng/ml and 100ng/ml IL-17F was significantly increased than that of control group at the first, third and fifth day, which was prominent statistically significant difference (P<0.05). The expression of Osterix mRNA intervened in 50ng/ml and 100ng/ml IL-17F was higher than that of the experimental group with 20ng/ml IL-17F at the first, third and fifth day, respectively, which was prominent statistically significant difference (P<0.05). The mRNA expression of Osterix intervened in 100ng/ml IL-17F was significantly higher than that with 20ng/ml and 50ng/ml IL-17F, there was prominent statistically significant difference (P<0.05).ConclusionAt the concentration of 20-100ng/ml cytokine IL-17F can promote the proliferation, differentiation, ALP activity of osteoblasts. This promotion effect may be related to that IL-17F can up regulate the mRNA expression of osteoblast-specific transcription factor Osterix and Runx2, and supplemented with 100ng/ml, the effect may be the most significant. |
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