Effects Of High Concentration Of FK506 On Dynamic Insulin Secretion And Viability From Isolated Mice Pancreatic Islets

Objective Investigae the effects of high concentration of FK506 on dynamicinsulin secretion and viability from isolated mice pancreatic is1etsMethods BaIb/c mice l8-20g,kiIIed by broken neck..The lower end of the commonbile duct of each donor was ligated in place tO prevent passage of col1agenase solutioninto the duodenum. 26 gape needle was inserted into the conunon biIe duct abovethe region of entry of the pancreatic ducts, l.5 miIliliters of ice-cold Hanks-HEPESsolution(PH7.4) containing 2mg/mI collagenase IV were infused into eachpancreatic duct. The pancreas was then quick1y excised and incubated inHanks-WS solution at 37t .After various periods(30-40min),using cold HBSSwith HEPES stop the digestion, filter the solution. Using ice-cold Hanks-HEPESsoIution centrifuggd at l520rpm for wishing twice. The digested tissue was vortexedwith Ficoll(sigma) at a densfy of 25%,overlaid with density of 23%,20%,ll%consecutively, and centrifuged at l820rpm for 5 minutes. Most pure islets are in theifltCrface of 20%-23%, some in the 20%-l l%,and 23%-25%.pick up islets, cold HBSSwash 2-3 times. recombined, and resusPended to RPMI-1640 cultUre mediumcontaining 10% FCS,l00lU/m1 penicillin and streptomycin. Then islets were distributedas an average in 24 well plate. The number of islets were assessed included in eachdish.Isolated islcts were cultured 24 hours in a humidified atInosphere of 95% air and 5oC C02 at 37 oC. Group l The cu1ture of is1ets were incubated withFK506(l00, 1000, 10000ng/tn1)for 24 hours, before adding glucose at theconcentration 2. 8all into islet insu1in test media, cu1ture for 4hour, change 2. 8all g1ucose media for is1et, cu1ute for 2 hour, taking mostof SN for insulin re1ease at low g1ucose stimu1us. Group 2 The cu1ture ofis1ets were incubated with FK506(10000ng/m1) and CsA{100, l000, l0000ng/ml), respective1y, for 24 hours, to assessed the number ofislets included in each dish,to determined the survive rate. Group 3 The cu1ture ofislets were incubated with FK506(10000ng/ml) and CsA (1000, 10000ng/m1)respective1y, for 24 hours. Taking out lml SN of RPMI--1640, detCrmined theinsul in re1ease in p1ate 2. Picking up the islets in p1ate l, dying A0--EB, usefluorometric method for viabi11ty assessment.Results Groupl Pretreatment with FK506(100, 1000, 10000ng/ml) improved theinsulin secretion in a dose--dependent fashion. Group 2, 3 Compared withislets pretreated by CsA, not only both the viabi1ity and survive rateare high in is1ets by FK506, but a1so improved the insu1in secretion. Isletspretreated by CsA directly suppress insulin secretion, Compared withislets pretreated by FK506. There is no comparative studies, Which comparedthe effect and viability of high concentration of the fk506 on low g1ucose-stimu1ated insu1in re1ease from the iso1ated mice pancreatic is1et, havebeen reported.Conclusion The high concentration of the FK--506 can improve the viabi 1 ity'the survive rate and the insu1 in secretion of the iso1ated mice pancreaticislet.