The Effect On Expression Of MMP8 And Mgp In Human Periodontal Ligament Fibroblasts Under Simulated Microgravity

The biggest challenge of space flights is a tremendous impact on astronauts' health under weightless environment. With the development of space medicine, the increases of in orbit flight time, put forward higher requirements of the astronauts' periodontal tissue health, the integrity of dentition, teeth stability. Previous studies have demonstrated that weightness envrioment can cause varying degrees of influence for periodontal tissue, such as alveolar bone remodeling, proliferation and differentiation changs in periodontal ligament cells, changes in the cytoskeleton and the biological characteristics change in preperiodontal ligament cells, which suggesting weightlessness causes damage on periodontal tissue. Periodontal ligament tissue decided important functions of metabolism and alterations, the main component of periodontal ligament is periodontal ligament fibroblasts, which has characteristics either of fibroblasts or osteoblasts, have osteogenic differentiation potential and can creat a quick response for mechanical stimulation. Studying the changes of human periodontal ligament cells under microgravity, can provide a foundation for molecular mechanism of damge in periodontal tissue under weightless environment.Cell proliferation, differentiation, migration and other life activities, as a result of internal and external interaction. Cellular signaling molecules and external cellular microenvironment together causs cell range of life activities. The current aerospace medicine mainly emphasis on change and control mechanism of cell signaling molecules under weightless environment, but the changes of environment of the extracellular matrix under simulated microgravity have not been reported.In this study, set up a vitro model of hPDLFs under simulated microgravity.Combined with our previous microarray data, using bioinformatics method for screening for signal molecules of bone-related functions, observing the changes under simulated microgravity. Enriching candidate sensitive genes to microgravity, provide laboratory basis for oral health protection measures of long-term space flight in future.This study established hPDLFs cultured model under simulated microgravity, dealing hPDLFs wiht osteogenic inducer, observe the change of ALP and osteocalcin. of in Analysis and comparise gene microarray data of mouse dental follicle tissue of mandibular first molar in two development phases, screening significant differences changes of gene expression, find a relationship wieh hPDLFs and weightlessness;determine purposed genes for research; by RT-PCR Change and western-blot and other semi-quantitative detection method, detect the expression of the genes in hPDLFs under simulated microgravity, the results are as follows:1. using the modified enzyme digestion tissue method, successfully isolated hPDLFs,and useing immunohistochemical methods for their identification; hPDLFs established in vitro model under simulated microgravity, hPDLFs in simulated microgravity culture model grew well, identify research time point of 24h, 48h and 72h;2. The establishment of drug-induced model hPDLFs osteogenic differentiation, and the effect of simulated micro gravity on hPDLFs the ALP and OC expression.Osteogenic inducer in normal gravity environment and simulated microgravity environment hPDLFs can induce differentiation of bone and simulated microgravity group decreased expression of ALP and OC, bone-inducing agent to antagonize this decline;3. Define gene metalloproteinase 8 (Matrix metalloproteinases 8, MMP8) and matrix Gla protein (Matrix Gla protein, mgp) as target gene. Observed the changes of MMP8 and mgp under simulated microgravity: in simulated microgravity, MMP8 expression of mRNA and protein in hPDLFs higher than normal gravity group, the most obvious difference is in the third day, with statistical significance, and MMP8 expression increased in osteogenic induced group compared with non-treated group; in not induced group, mRNA and protein expression of mgp in simulated microgravity group increased of compared with expression in normal gravity group, the most obvious differences in the third day, have statistical significance. But in osteogenic induced group, mRNA and protein expression of mgp doesn't show significantly change.The results of this study show that: 1. successfully established a culture model of hPDLFs under simulated micro gravity; 2. The effect of simulated microgravity will reduce the secretion of ALP and OC in hPDLFs, but osteogenic inducer can weaken the decreased trend; 3. Define MMP8 and mgp as the taget gene for research;increased expression of MMP8 and mgp under simulated microgravity compare with in normal gravity group, indicating MMP8 and mgp gene involved in regulating the differentiation of hPDLFs' osteogenesis under simulated microgravity; 4. metabolic activity of periodontal ligament increased in weightless environment which may lead to further reduction of alveolar bone density, accelerated progress of periodontitis and increased tooth mobility, suggesting that long-term space flight cause injury of astronauts stomatognathic system. The findings can provides laboratory evidence.for the future development of astronauts stomatognathic system health protection.