Study Of The Therapeutic Effect Of Vitamin A And Vitamin E On Rat’s Hepatic Fibrosis In Vitro

ObjectThis experiment is intended to explore the nonperfusion method toextract the hepatic stellate cells of rat feasibility, and to discuss thetherapeutic effect with both VA and VE separate and joint acted on rat’shepatic stellate cells in vitro research preliminary.Method1. The nonperfusion method to extract the rat’s hepatic stellate cells:Take out the rat’s liver under aseptic condition, cut it up, to bedigested with chain enzymes protease and trypsin, collect the digestivejuices to further fluid density gradient centrifugation to get hepaticstellate cells.2. Research for the morphology of HSC:Identify the rat’s hepatic stellate cells extracted by the method ofLipid oil red O staining.3. The dyeing identification of rat hepatic stellate cells:To dye the specific protein of hepatic stellate cells, such as a-SMAand GFAP, by the method of Immunohistochemical staining, and toidentify whether the extracted cells is hepatic stellate cells or not.4. Dose and time-effect research of VA and VE acted on rat’s hepaticstellate cells in vitro:The rat’s hepatic stellate cells in the logarithmic phase wasvaccinated in96-well plates with5x103cells each hole, VA, VE and thejoint were made into concentrations as10mg/l,1mg/l and0.1mg/l. Thenput them into plates with hepatic stellate cells, select0,12,24,48,72has five time points to analyze the drug’s fuction at the same time.5. The study on the effect of VA, VE and combination acted on HSC’s type I collagen, TGF-beta1, TIMP2factorThe cells in the logarithmic growth phase was treated with drugs(1mg/l) for24hours, and then texted the changing of type I collagen,TGFβ1and TIMP-2by using corresponding kits, analyzing thedifferences between each group and the control group.6. Immunohistochemical stainingThe cells in the logarithmic growth phase were climbed on pieces,then treated with VA, VE and the combination for futherimmunohistochemical staining, to analyze the expression of TGF-β1andTIMP-2.Results1. The analysis and identification of the nonperfusion method to extractrat’s hepatic stellate cells: the rat’s hepatic stellate cells extracted bynonperfusion method present sample as stars in morphology, and thepurity can reach more than90%, GFAP and α-SMA protein weresignificant expressed in results, at the same time, cells which werechoosed by P2and P5generation for sample showed no difference in theexpression of specific proteins.2. Research for effect in fibrosis related factors while treated with VA, VEand the combination: it concluded that the most effcetive concentration is1mg/L while treated for24hours with VA, VE and the combination.Under this condition, it showed that VA can significantly inhibit theexpression of TGF-β1, VA and VE can significantly inhibit theexpression of TIMP-2, VA, VE and the combination all can significantlyinhibit the expression of type I collagen, but the combination showedmore significant therapeutic effect controled with the others. Conclusion1. The nonpersion method to extract rat’s hepatic stellate cells hascertain feasibility, and it is much easier to operate. It showed moreadoptability than the extraction method ever before. It can contribute tothe research of HSC’s experiments as a practical basis.2. VA, VE can inhibit the preliferation of hepatic stellate cellseffectively under the condition with concentration1mg/L, treated for24hours. And it showed that the combination of VA and VE was moreeffective. VA can inhibit the expression of TGF-β1significantly, while,VA combined with VE can inhibit the expression of TIMP-2significantly,all the three drugs can inhibit the expression of Type I collagensignificantly, and the combination was more significant, too.