Research Of Nicotinamide Inhibitory Role Of IBMIR In Islet Transplantation

PrefaceIncidence of diabetes in China showed rapid growth trend recent years.For serious harm human health of young people in particular typeⅠdiabetes mellitus(NIDDM),is used mainly treated by replacement therapy of exogenous insulin.Patients not only need to lifetime use of insulin but also often have a severe hypoglycemia reaction,even fatal hypoglycemic coma.Transplantation is currently the only way to cure the disease, and metabolic instability can be reduced to avoid the occurrence of low blood sugar coma,and can prevent a variety of long-term complications.In recent years,the pancreas transplantation are increasingly being used in clinical,but it has more drawbacks:It is a traumatic therapy,the mortality rates are still high;application immunosuppression of pancreas transplantation often cause serious side.Otherwise, islet transplantation has long been considered to be a very attractive therapy.Tt is not only simple,low mortality,but also has repeatedly operation.Therefore,for a long time, many research institutions are committed to the study.Although islet transplantation in the treatment of type 1 and type 2 diabetes have their unique advantages,its long-term efficacy is not ideal.Its main reason includes:(1) physical and chemical reason in the process of islet isolation,for example,hypothermia and hypoxia.(2)the instant blood mediated inflammatory reaction in the earlier period of the islet transplants.(3)The immunity rejects reaction.At present studies indicated,in early time of the islet transplant it often accompany with the massive islet loss,this kind of phenomenon is called "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. IBMIR is characterized by activation of the coagulation and complement systems and a rapid blinding of activated platelets to the islet surface.Recently,we have shown that the islets of Langerhans express and synthesize tissue factor(TF)which is the most important activator of the coagulation.During clinical islet transplantation,when islets come into direct contact with blood in the portal vein,islet-produced TF triggers a detrimental clottingreaction,the instant blood-mediated inflammatory reaction(IBMIR).Macrophage chemoattractant protein (MCP)-1 is also expressed in human islets.MCP-1 has a potent chemotactic activity for monocytes,and the level of this protein secreted by pancreatic islets invitro before islet transplantation correlates with the outcome of clinical islet transplantation.The IBMIR, with TF as an initiator and MCP-1 as a chemoattractant,is likely the reason for both the low success rate of clinical islet transplantation seen in previous studies and the need for islets from more than one donor to obtain normoglycemia.We hypothesized that the expression of TF and MCP-1 on islet cells may be induced by some stimulus present during the process of organ retrieval and islet isolation.This possibility is supported by the individual variations that are observed in TF and MCP-1,at both the mRNA and protein levels,between islet preparations from different donors.It is known that abnormal physiologic events that occur in patients just before death with brain death,cold storage,and hypoxia of the organ before transplantation can induce the expression of a variety of proinflammatory genes,we have shown that nicotinamide can affect TF and macrophage chemoattractant protein (MCP)-1 expression in monocytes and endothelial cells.In this study,we investigated whether compounds previously shown to affect TF and macrophage chemoattractant protein(MCP)-1 expression in monocytes and endothelial cells have the same effect in human islet cells.The occurrence of IBMIR can be supressed by pretreatment of nicotinamide.MethodAnimal:Wistar rat of either sex with weigh ranging from 250-300g(from china medical university).The first experimental groups:control group:RPMI1640 + islet, the experimental group:RPMI1640 +islet +nicotinamide;the second experimental group:the experimental group:nicotinamide pretreatment islet 500IEQ + RPMI1640 100ul +2 ml blood,and the control group:the islet of based culture 500IEQ + RPMI1640 100ul +2 ml blood,blank control group:RPMI1640 100ul +2 ml blood.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.After purifying the islet was cultured with 50 mM nicotinamide(37℃5%CO2) in incubator for 48 h.Islet activity of different culture conditions have measured by using vitro glucose stimulated insulin release test.The TF and MCP1 content have measured by using ELISA methodTubing loops as a model:this device consisted of loops made of polyvinyl chloride (diameter,6.3mm;lengh,390mm).The tubing was held together with a specially designed heparinized connector.A rocking apparatus,placed in a 37℃incubator,was used to simulate blood flow in the loop. Heparin treatment:All inside surface of tubes contacted with whole blood are furnished with heparin.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.Blood analysis:counting blood platelet,white blood cell,lymphocyte,monocyte number by blood RT.Islet activity have measured by using vitro glucose stimulated insulin release testHistomorphology test:changes in the pattern of isletis was observed by TCT examnationStatasticis 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%.After 48h mixed with nicotinamide,vitro glucose stimulated insulin release test showed that in the control group the average insulin content was 57.8±6.79μIU / ml in low- glucose and 199.45±14.80μIU / ml in high- glucose.Release index(SI)was 3.47±0.40.In experimental group the average insulin content was 58.5±7.38μIU/ml in low- glucose and 184.39±23.47μIU/ml in high- glucose.Release index(SI):3.24±0.26.In the control group TF content was 9.90±2.12pg/islet and1.42±0.42pg/ islet in the experimental group;MCP1 content was 2.22±0.58 pg / islet and 0.33±0.13 pg / islet,in the experimental group2.Blood analysis:In the control group the platelet count respectively is(n=10) 258±82.4×10~9/L,40.9±12.8×10~9/L and 232±73.5×10~9/L in the experimental group. The count of white blood cell respectively is 9.05±0.98×10~9/L,4.54±0.92×10~9/L in the control group and 7.39±1.10×10~9/L in the experimental group.In the control group the percent of lymphocyte respectively is 73.6±9.86%,49.8±9.98%and 68.63±10.34%in the experimental group.In the control group monocyte respectively is 0.52±0.16×10~9/L,0.298±0.12×10~9/L and 0.474±0.15×10~9/L in the experimental group.3.After contacting with the blood 60minites,vitro glucose stimulated insulin release test indicated that:in the control group,the average levels of insulin was 24.24±6.22μIU / ml in low- glucose conditions and 43.5±8.08μIU / ml in high- glucose conditions,the release index(SI)was 1.98±0.29.In the experimental group average level of insulin was 43.5±8.08μIU / ml in low-glucose conditions and 130.76±21.41μIU / ml in high-glucose conditions,the release index(SI)was 3.02±0.29. 4 Histomorphology test:in the control group the islet is less obviously and wrapped with the massive RBC and micro thrombus.Otherwise,in the experimental group,there is more islets which have no RBC and micro-thrombosis around.Conclusion1.Nicotinamide inhibits tissue factor and macrophage chemoattractant protein expression in isolated islets.2.IBMIR can be effectively suppressed by using nicotinamide.