The Research Of Inhibition Of Thrombin (Argatroban) Abrogates The Instant Blood Mediated Inflammatory Reaction

PrefaceAs one of the common endocrine diseases,DM can lead to kinds of complications threatening health of patients.Building a endogenous insulin-secretion system is able to cure the DM permanently.Nowadays only pancreas or islets transplant fulfills the purpose.The islet transplant is the cellular transplant,which has the advantages including:safer,simpler,more convenient for the modifying in vitro,lower rate of side-effect and easier to repeat than the pancreas transplant.Although dependents the extrogenic insulin more than the islet transplant has become,the islet transplant is a prospective method.Though the islets transplant at cure 1 type with part 2 type diabeteses contain its special advantage,not enough ideal in its long-term curative effect.Its main reason includes:(1)the technique of islet purely and isolated still not perfect,can't acquire high-quality islet,(2):The immunity rejects reaction,(3):islet hypoxic ischemic injury,(4)the instant blood mediated inflammatory reaction in the earlier period of the islet transplants.At present studies indicated,the islet transplant early time often is accompanisd by the massive islet loss,this kind of phenomenon is called have no function in the primary stage,but has nothing to do with the immunity factor.Besidies the intrinsic quality factor in the islet preparation,another important reason is the instant blood mediated inflammatory reaction when islets come in direct contact with blood.the IBMIR is characterized by activation of the coagulation and complement systems and a rapid blinding of activated platelets to the islet surface,when islets come in direct contact with blood,coagulation phenonmen appeared in five minitues.The platelets and the islet surface rapid blinding and causes the platelet rapid reduction,is following closely is the fibrin forms and revolves around the islet surface as well as the massive granular cell infiltration and the complement activation,then presents the blood coagulation,thus causes islet cells' destruction.In summary,these responses are also by the platelet activation and the clotting responded causes,therefore seek for one kind effectively the method which will suppress the platelet activation and the clotting responded to be possible to prevent IBMIR,thus reduced transplant early time islet destruction.The research indicated that the thrombin is one kind of serine proteinase,comes by the thrombinnogen,not only it is the crucial role enzyme in the clotting response, but also is one kind of effective blood platelet activator,it blinding with platelet surface PAR-1 and PAR-4 activate blood platelet,therefore the thrombin inhibitor will effectively suppress the clotting and the blood platelet activation,thus suppress IBMIR. This experiment through applies in vitro simulation circulation model discussion thrombin inhibitor(Agatroban)to the IBMIR action in the early time of islet transplant.MethodAnimal:Wistar rat of either sex with weigh ranging from 250-300g(From China Medical University).The control group(blood 2ml + RPMI-1640 150U1+islet 800IEQ),Experimental group(blood 2ml+islet 800IEQ+RPMI-1640 150 u l+argatroban40ul).Islet isolation:Retrograde perfusion of collagenase solution(prepared with cold Hank's solution,dosage is 1.5mg/ml)10-12ml into the rat pancreas duct,fully inflate the pancreas.Digestion by vibration in the water bath of 37±1℃.At the end of digestion, double centrifuge and washing followed by screen by 80.Purifying islet by Ficoll graded centrifuge at concentration of 25%,23%,20.5%and 11%.Aspirate the 11%and 20.5%Ficoll solution containing islet,ready for use after washing by RPMI-1640.DTZ stain is used to decide the purity of the isolated islet.The amount of islet cell=DTZ positive cell group of three samples÷3×20×total amount of sample(ml).AO/EB fluro-stain showed the viability of cell and the insulin secretion trial by the ex vivo glucose stimulation showed the islet activeness.Heparin treatment:All materials that came in contact with whole blood were furnished with a heparin surface.The surface concentration of heparin was 0.5μg/ cm~2,corresponding to 0.1unit/cm~2.Preparation of blood:Fresh rat blood was collected in surface heparized 20ml syringes.Tubing loops as a model:this device consisted of loops made of polyvinyl chloride (diameter,6.3mm;lengh,390mm)whose inner surface was fumished with immobilized heparin.the tubing was held together with a specially designed heparinized connector.A rocking apparatus,placed in a 37℃incubator,was used to generate blood flow insides in the loop.Blood test:counting blood platelet,white blood cell,lymphocyte,monocyte number by blood RT.Histomorphology test:after the response,filters the blood.observing islet morphology change by TCT examnation,the ex vivo glucose stimulation test islet functiona.Statasticis analysis:all the data expressed in mean±SD.T test was used and P＜0.05 was considered having statitical significance.Result1.Islet isolation:600IEQ islet could be extracted from every rat,the DTZ stain shows purity more than 90%and islet viability more than 98%.the ex vivo glucose stimulation test of fresh islet showed the average amount of insulin in the hypoglycemia solution is58.1±3.5μIU/ml,the average amount of insulin inthe hyperglycemia is 195.2±12.6μIU/ml,the Secretion Index(SI)is3.35±0.20.indicate islet function better.In the control group the average amount of insulin in the hypoglycemia solution is46.1±3.2μIU/ml,the average amount of insulin in the hyperglycemia is 103.24±11.8μIU/ml,the Secretion Index(SI)is2.25±0.18.indicate islet function injury.In the experiental group the average amount of insulin in the hypoglycemia solution is53.6±3.3μIU/ml,the average amount of insulin in the hyperglycemia is 180.2±10.8μIU/ml,the Secretion Index(SI)is3.36±0.18.indicate islet function better.2.Blood test:In the control group the platelet count is(n=10)38±14×10~9/L,In the experimental group the platelet count is 255±85×10~9/L,During two groups has the obvious statistics difference(p＜0.05).In the control group the white blood cell is 4.2±0.96×10~9/L,In the experimental group the white blood cell is 8.14±1.19×10~9/L, During two groups has the obvious statistics difference(p＜0.05).In the control group the monocytes count is(n=10)0.25±0.10×10~9/L,In the experimental group the monocytes count is 0.41±0.13×10~9/L,During two groups has the obvious statistics difference(p＜0.05).In the control group the percent of lymphocyte is 51.54±8.5%,In the experimental group the percent of lymphocyte is 66.24±9.8%,During two groups has the obvious statistics difference(p＜0.05).Histomorphology test:in the control group the islet was broken obviouely,the surroundings is wrapped the massive micro thrombus which around the islets.in the experimental group the islets morphology is integrated,not obvious inflammatory cell infiltration,also has not seen the micro thrombus formation.Conclusion1.The primary course of islet damage is IBMIR in the early time of islet transplant.2.IBMIR can be effectively suppress by using inhibition of thrombin (Argatroban).