The Studies On Extraction,Purification And Immune Function Of Vimentin

The extracellular soluble vimentin possesses the functions as an alarmin and its extraction process optimization have not been reported internationally.The internationally literatures reported about the method of isolation vimentin were rarely and the reported methods were all complex and have long period.This study established a method of extraction and a column isolation and purification of vimentin from porcine eye lens tissue for the first time and identify the function of extracellular soluble vimentin that promotes proliferation,activation and chemotaxis of inflammatory cells.This study provided the foundation for the development and utilization of vimentin as biological medicine and its function research.The extraction temperature,solution pH,extraction time,and solid to liquid ratio of refined extraction have be optimized using single factor experiment and response surface analysis.Vimentin was isolated and purified via DEAE Sepharose fast flow anion exchange column and identificated by Western-Blot.The electrophoretogram of 10%SDS-PAGE was used to detect the purity and relative recovery rate.The proliferation of rat splenocytes stimulated with vimentin was evaluated in vitro.The lymphocyte counts and vimentin-antibody levels of peripheral blood in mice immunized with vimentin were detected in vivo.Peritoneal macrophages were collected and cultured with different concentrations of vimentin to detect the effect on phagocytosing chicken red blood cells.The chemotaxis of NIH3T3 fibroblast towards vimentin was observed in transwell chamber.The optimized conditions for the extraction included that extraction temperature,extraction time,solid to liquid ratio and solution pH of refined extract respectively were13 ?,26 min,1:34(g:ml)and 7.Followed with 36 mM NaH2PO4 buffer of 40 times volume of the colume,36-86 mM NaH2PO4 buffer of 100 times volume of the column was used to linear elution once for isolating and purifying vimentin.Through the electrophoretogram of 10%SDS-PAGE combined with Image Lab 4.0 software analysis,the purity of vimentin in purified lyophilized powder was(80.66±1.301)% and the relative recovery rate was(36.56±0.964)%.In vitro vimentin dose dependently promoted the proliferation of splenocytes.The proliferation indexes of primed and na?ve splenocytes cultured with 16?g/ml vimentin were reached up to(196.0±9.7)% and(208.9±4.6)% respectively without significant difference.In vivo vimentin significantly enhanced the lymphocytes number(109/L)of peripheral blood(5.74±0.51 vs.1.69±0.13)and the levels of vimentin specific antibody(OD value 2.31±0.06 vs.0.19±0.08)that shown no significant difference from immunization with vimentin plus CFA.In vitro vimentin dose dependently stimulated phagocytic ability of macrophages and performed the chemotactic effects on NIH3T3 fibroblasts.This method exhibits the advantage of high purity and high recovery rate.Extracellular soluble vimentin promotes the proliferation,activation and chemotaxis of concerned inflammatory cells and possesses the functions as an alarmin.