Effects Of Low Molecular Heparin On IL-8Release From Human Buccal Mucosa Fibroblasts Stimulated With Lipopolysaccharide

Objective:(1)The normal human buccal mucosa fibroblasts (HBMPs) were cultured in vitro, the oral mucosa lamina propria models were establishmented,to provided the experimental basis for further research;(2)Coincubation of different concentrations of low molecular weight heparin (LMWH), lipopolysaccharide (LPS) and HBMPs in vitro, at different times observed the changes of IL-8that secreted by HBMPs, to explored the mechanism that LMWH against the inflammatory response of the lamina propriathe.Methods:(1)Primary cultured human buccal mucosa fibroblasts, separation and purification of cells,then identification by morphological and immunohistochemical method, to establish a stable cell model in vitro.(2) Selected the fourth generation purification fibroblasts that in form shape, stable growth.The cells were randomly divided into normal groups, inflammation groups, the intervention groups. The control groups add serum-free medium, the inflammation groups add serum-free medium containing10μg/ml lipopolysaccharide (LPS),the intervention groups on the basis of the inflam--mation group joined with0.2U/ml1U/ml,5U/ml low molecular weight heparin, in each groups were incubated3h,6h,12h,24h.We dectected the content of IL-8in supernatant using enzyme-linked immunosorbent assay, and analyzed the test results using multiway ANOVA.Results:(1)Having been cultured and purified,the4th generation HBMPs were stable in the progress of growth and were morphologically good.The cells presented either long spindle or star appearance which is typically fibroblast-like cell characteristics under an inverted microscope.The antidodies against ceratin with immunohistochemistry analysis.(2) enzyme-linked immunosorbent assay results Normal control group of IL-8expression at each time point was no significant difference (P>0.05).Inflammation in the control group with low concentrations of drugs of IL-8release at each time point were significantly higher than the normal control group (P<0.05).Low concentration of the drug group compared with the inflammatory control group,12h,24h difference was significant (P<0.05).Concentrations of drugs of IL-8secretion in6h,12h,24h compared with the inflammatory group was significantly lower (P<0.05).High concentrations of the drug group compared with the normal control group,3h,6h,12h there is a significant difference (P<0.05), with inflammation in the control group at each time point differences were significant. Intervention group and time point and inflammatory control group compared to IL-8secretion in reduced dose-and time-dependent.Conclusion:(1)The cultured cells in vitro accord with normal buccal mucosa fibroblasts morphology and immunohistochemical characteristics,it is similar to the structure of the lamina propria of normal buccal mucosa, the cells can used to be model to provide experimental basis for subsequent experiments in vitro.(2)This experiment investigated the anti-inflammatory mechanism of low molecular weight heparin at the cellular level.low molecular weight heparin can significantly inhibit the lipopolysaccharide-stimulated descendants of the buccal mucosa fibroblast IL-8release, and provide a theoretical basis for clinical medication safety.