An improved Method for the Isolation of Hepatic Stellate Cells in Rats with Hepatic Fibrosis
Objective:To establish a simple,economical and efficient method of isolating hepatic stellate cells from rat with liver fibrosis for lay a foundation in the study of hepatic fibrosis.Methods:The liver fibrosis model of SD rats was induced by intraperitoneal injection of carbon tetrachloride(CCL4).After hepatic fibrosis of SD rats through portal vein heparinization,the inferior vena cava was cut out,and D-hanks fluid was infused into the liver to the earthy yellow color.Then closed the inferior vena cava,slowly injected 5mL 37℃ containing 0.100％pronase,0.050％Ⅳ collagenase D-Hanks fluid,let stand for 10 min.And continue to slowly remove liver after injected 5mL of the fluid.After in 37℃ incubator standing for another 30 min,adding 0.050％Ⅳcollagenase,0.020％pronase and 0.010％DNases Ⅰ solution digest liver homogenate 30 min.HSCs were obtained by 18％Nycodenz density gradient centrifugation.Cell viability was determined by trypan blue rejection assay,and cell purity was determined by spontaneous fluorescence,Desmin and α-SMA immunocytochemistry stain.Results:the rate of HSCs was about 5x107/rat,and the cell activity was more than 90％.Hepatic stellate cells spontaneously fluoresce blue-green at 328 nm.The purity was up to 90％by Desmin and α-SMA immunocytochemistry staining.Conclusion:a simple,economical and efficient method for isolating HSCs in rats with hepatic fibrosis was established.
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