Protective Effect Of HO-1 And A20 Protein Against The Apoptosis Induced By TNF-α In Rat Islets In Vitro

Background:Diabetes mellitus is a very common and dreadful disease in recent decades. Type 1diabetes mellitus (T1 DM) is a common metabolic disease in children and adolescence,and exogenous insulin substitution is the traditional therapeutic method. Heretoforepancreatic tissue transplantation is the only known therapy to. reliably reestablishendogenous insulin secretion. Several protocols of pancreatic tissue transplantationhave acquired amazingly successful outcomes, among which, islet transplantation caneffectively restore endogenous insulin secretion in T1DM, and therefore can retard,prevent, even reverse diabetic complications. Edmonton protocol, published in 2000,prompted enthusiasm that islet transplantation could move into the clinic as a viableoption for T1DM. But there are still many challenges to be faced in the realm of islettransplantation, one of which is the islet cell death, primarily the beta cells areeliminated by apoptosis soon after transplantation. The loss of beta-cell mass could beimplicated in the failure of islet transplantation and requires further investigation.Genetic engineering of a suboptimal islet graft with cytoprotective gene, A20 or HO-1,could probably preserve beta cell mass and maintain their function.Objectives:1. To develop a simple, rapid, economical and Ficoll-free method to purifySprague-Dawley rat islets by filtration sequentially through two cell strainers indifferent size. 2. To establish techniques for transferring human A20 gene and humanHO-1 (heme oxygenase-1) gene into the isolated rat islets using lentiviral transfectionsystem, and assess the expression of A20 and HO-1 protein in the cultured islets invitro. 3. To investigate the vitality of the gene transfected islets. 4. To study theprotective effect of A20 and HO-1 protein against the apoptosis induced by CHX andTNF-α, and survey the mechanism of such effect briefly. Methods:The islets were isolated from the rat pancreata using the standard collagenasedigestion procedure and were purified with the generally used Ficoll density gradientmethod or the innovative two-step sequential filtration method. The purification of theisolated islets was visualized by the DTZ staining, and the vitality was assessed withAO/PI staining and insulin concentration in the supernatant detected with ELISAassay. The A20 gene and HO-1 gene were cloned and inserted into the lentiviraltransfection system which was used to transfer the genes to the isolated islets in vitro.The efficacy of gene transfer was measured by the intensity of the EGFPfluorescence-positive islets transfected with lenti-EGFR Western blot was applied toverify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, theisolated islets were pre-incubated with CHX (cyclohexamide 50μtg/ml) for 2 h andthen induced with TNF-α(5000 U/ml) for 48 h. The function of the islets wasassessed with the determination of the insulin concentration in the culture media.Glucose-stimulated insulin-secretion test during subsequent static incubations for 2hours was performed using the ultra-sensitive rat insulin ELISA kit. TUNEL and theFACS methods were used to evaluate the apoptosis of the islet cells and Western blotwas used to detect caspase-3 activation.Results:The conventionally used Ficoll density gradient islet purification method and theinnovative Ficoll-free two-step sequential filtration method were both used in thisstudy and the islet yields were compared. In the Ficoll method, the yield of islets was598±135 islets/pancreas, the islet purity was approximately 65～85% and the vitalityabout 85～95%, while in the two-step filtration method, the islet yield, the purity andthe vitality of the isolated islets were 782±115, 90～100%, and more than 95%,respectively. Considering the islet yield, the two-step filtration method is muchoptimized one (P＜0.01). Introduced into the isolated islets by lentiviral transfectionsystem, A20 and HO-1 genes could be highly expressed. The expression of A20 andHO-1 genes could preserve the function of the isolated islet induced with CHX and TNF-αin vitro. The A20 and HO-1 protein could prevent the islet cells from CHXand TNF-αinduced apoptosis; co-transfection of A20 and HO-1 genes led to betterprotective effect. The protective effect of A20 and HO-1 against the CHX and TNF-αinduced apoptosis is due to their inhibitory effect on caspase-3 activation.Conclusion:In this study we developed a two-step sequential filtration method for isletpurification which was simple, rapid, economical and Ficoll-free, and the vitality andpurity of the islets acquired with this method were very high. The two-step sequentialfiltration method could be widely applied to rodent islet purification. The lentiviralgene transfer system was an efficient and stable gene transfer vector, and could beused in genetic engineering of islet graft. The over-expressed A20 and HO-1 proteincould preserve the function of the islets treated with CHX and TNF-α.