| [Aim] Explore an economical and easy method of isolating and culturing the rat hepatic stellate cells, in order to study the pathogenesis of hepatic fibrosis at the levels of cellular and molecular in the future.[Methods] Perfuse adult healthy rats'isolated livers by two-step enzyme liquid in vitro circly, After the density gradient centrifugation of Nycodenze at the speed of 20000r/min and the low-speed centrifugation (40 x g) of cell suspension, we get the higher purity primary hepatic stellate cells, the refusal rate of cells can identify the rate of live cells in the Trypan blue test; des min and GFAP immunocytochemistry (double antibodies and single staining) identify the purity of the newly isolated hepatic stellate cells, a-SMA immunocytochemistry identify the actived rate of the hepatic stellate cells cultured for 7days.[Results] The yield of hepatic stellate cells is (3.6±0.1)×107per liver, the survival rate of hepatic stellate cells is(92.8±0.8)%, HSCs cultured for 24 hours were identified by desmin and GFAP, single staining the rate of positive cell is (96.2±0.5)%; HSCs cultured for 7 days were identified by (?)-SMA, stained by AEC, the rate of positive cell is (72.3+0.6)%.[Conclusion] The improved method increases the purity of the primary rat hepatic stellate cells on the base of promising the yield, may provide a favorable basis for the study of biologic features of hepatic stellate cells and pathogenesis of liver fibrosis at cellular and molecular level. [Aim] To observe the TGF-β1 changing of the rat primary hepatic stellate cells stimulused by endotoxin and to explore its mechanism preliminaly.[Method] Perfuse the isolated healthy adult rat'liver by the chain enzyme proteinase and collagenase fluid, centrifuge the Nycodenze density gradient fluid, then get the primary hepatic stellate cells and culture them in DMEM containing the final concentrations of endotoxin which were 0.01,0.1,1,10μg/ml, we collect the culture medium when the cells cultured at the 2nd4th,8th day, saved at-20℃, determine the amount of TGF-β1 by the Elisa. The medium of every group were changed into the medium containing the original concentration of endotoxin and 10μmol /L PDTC, and the medium of the control group were changed into new medium with original concentration endotoxin when HSCs were cultured at 2nd4th,8th day, collected the culture medium after 48 h, saved at-20℃, determine the amount of TGF-β1 by the Elisa[Results] Endotoxin stimulated the primary hepatic stellate cells directly, in every group, the expression of TGF-β1 was more than the contrast group at the 2nd,4th,8th day, and the most is the 1ug/ml group, followed are 0.1μg/ml group,10μg/ml group and 0.01μg/ml group, at the 2nd,4th,8th day added PDTC, the expression of TGF-β1 in each group was lower than the control group.[Conclusion] The amount of TGF-β1 were more when the endotoxin in a certain range concentration stimulused the hepatic stellate cells directly, suggesting that:endotoxin cause the liver fibrosis by stimulusing the HSCs directly,which maybe one of the ways how the endotoxin cause the liver fibrosis, PDTC can reduce the expression of TGF-β1 affecting the hepatic stellate cells directly, suggesting that PDTC cure the hepatic fibrosis by reducing the expression of TGF-β1 probably and the NF-κB may be one of its targets.
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