ABSTRACT
Objective To study the effect of dynamic pressure on the expression of parathyroid hormone-related protein (PTHrP)mRNA in metaphyseal cartilage stem cells of rats so as to further explore whether fiber actin (F-actin)is involved in the mechanical signal transduction process.Methods We isolated and cultured metaphyseal cartilage stem cells of rats by immunomagnetic beads.The third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups:0%,3%,6%,and 12% deformed groups according to the size of dynamic pressure strength.We used a self-prepared dynamic tonic culture device to exert different intensity of pressure on each group of cells for 24 hours.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.The expression of PTHrP mRNA in each group was detected by Rea-l time quantitative PCR. Furthermore,the third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups:control group,simple pressure group (6% deformation),pressure+cytoskeleton relaxin D group,and simple cytoskeleton relaxin D group according to whether or not to apply pressure and cytoskeleton relaxin D.F-actin fibers in each group of cells were stained with phalloidin and placed under a laser scanning confocal microscope.The expression of PTHrP mRNA in each group was detected by Real-time quantitative PCR.Results The results of flow cytometry showed no significant difference in G0/G1,G2/M and S phases between 0%,3%,6% and 12% deformed groups (P＞0.05).There was no significant difference in the apoptosis rate between 3% and 6% deformed groups compared with 0% deformed group (P＞0.05).The apoptosis rate was significantly higher in 1 2 % deformed group than in control group (P＜0.05).The results of laser confocal microscopy showed that the arrangement of F-actin fibers in the pressure group was neat and parallel compared with that in the control group, which was consistent with the direction of force.The intracellular F-actin fiber structure in pressure+cytoskeleton relaxin D group and simple cytoskeleton relaxin D group was destroyed and aggregated into clusters.Real-time quantitative PCR results showed that PTHrP mRNA expression did not significantly differ between 3% and 0% deformed groups (P＞0.05).The expression of PTHrP mRNA in 6% and 12% deformed groups was significantly higher than that in 0% group (P＜0.05).The expression of PTHrP mRNA in the cells of simple pressure group was significantly higher than that in the control group (P＜0.05).There was no significant difference in the expression of PTHrP mRNA between simple cytoskeleton relaxin D group and control group (P＞0.05).The mRNA expression of PTHrP was higher in pressure+cytoskeleton relaxin D group than that in control group,but lower than in simple pressure group (P＜0.05).Conclusion The dynamic pressure of proper intensity can increase the mRNA expression of PTHrP in chondrocytes of metaphyseal hypertrophy in rats,and F-actin is involved in the mechanical signal transduction process.