The Investigation Of Isolation, Culture, Characterization And Activation Of Pancreatic Stellate Cells

Objective:To establish a simple collagenase perfusion method through abdominal aorta cannulation to isolate rat pancreatic stellate cell and demonstrate the procedures of its culture and characterization;To investigate initially why PSC was activated.Method:Via a cannula iserted into adominal aorta,Gey's balanced salt solution containing 0.025%Collagenase P was perfused into rat normal pancreas;After digestion with Collagenase P and Protease,Nycodenz density gradient centrifugation was performed to isolate PSC;PSC was identified by its morphology,cytoplasmic lipid droplets and immunocytochemical staining for desmin,GFAP andα-SMA;RT-PCR and WestBlot were performed to measure the expression level of Smad2/3 in activated PSC.Results:The production,viability and purity of isolated PSC by this method were:15.3±4.6×10~3/g body weight,95.0±3.5%,and＞80% respectively;After 24 hours in culture,most PSC attached to culture dishes,showed angular or stellate appearance;PSC became positive for desmin and GFAP after 48 hours in culture but did not expressα-SMA until 96 hours in culture;The mRNA levels of Smad2/3 increased in the early stage followed by a decreas after reaching the peak point(After 48 hours in culture),and there protein levels showed a similarity to the phenomena.Conclusions:The collagenase perfusion method through abdominal aorta cannulation facilitated PSC isolation,and for high production,viability and purity the isolated PSC was able to meet the need of in vitro experiments;Smad2/3 played a central role in the early activation of PSC.