The Research Of Apoptosis And Function Of The Rat Islet Which Was Transfected Akt1 Gene Via Adenovirus Vector

As 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 has the advantages including;safer,se conimpler, morvenient for the modifying in vitro,lower rate of side-effect and easier to repeat than the pancreas transplant.But the high number of islets are required(needed 2 adults' pancreatic gland),and the low percentage of the graft long-term survival posttransplant is still the "the bottleneck" to hinder the development islet transplantion. Presently,about 70% islets lose their function after transplant(7-10days).which results from such factors as allo-immunological rejecting reaction and primary nonfunction of islet(PNF).The apoptosis of islet'βcells nonspecific inflammatory response,"Anoikis" ischemia and hypoxia are related to PNF.If we can overcome above factor to reduce the nonfunction of islet maybe we can achieve the aim to enhance transplant successratio and reduce nesessory for the isletsAKT,also called protein kinase B,is a member in the family of Serine/Threonine prokinase,the AKT molecular weight is 57KD,AKT acts as the signal to antiapoptosis in the many kinds of cells,in addition plays a key role in the cell living,proliferation, metabolism and apoptosis.In summary,if can increase the Akt expression in the islets cell,thus reduces islet apoptosis and to enhances its secretion function,then the need for islet will be reduced and the survival of graft will be significant lengthened.ObjectivesTo research the survival and the function of the Islet cells which have been transduted D Akt1 gene via adenovirus vector we can determine whether Akt1 gene can enhance the beta cell survival percentage and strengthen its secretion functionMethods1.Constructed the recombinant replication-deficient adenovirus vector named Ad5-Akt1(1)Construction shuttle plasmid pDC316-Akt1;â‘ rat liver tissue procured for the RNA isolation.â‘¡The first cDNA strain was produced by reverse transcriptâ‘¢AKT1 gene PCR expanding;Take the rat cDNA as the template,PCR expands this gene the opening reading frame.The directing sequence was designed by the software Primer 3.0â‘£expression apo carder was confirmed by the EcoRI and Hindâ…¢bi-enzyme electrophoresis and sequencing plasmids.After the electrophoresis cuts the rubber recycling goal genefragment and the carder fragment,connected bythe T4 DNA enzymeconnection,passed night in 16℃.Transformed feeling conditionfungus DH5 alpha,by contains the ammonia aminipenicillin the LBselective solid medium raise, next day choice the masculine clone,withdraws the material particle,the enzyme cuts the appraisal reorganization⑤Massively withdraws really nuclear expresses carder DC316-Akt1(2)constructed the recombinant replication-deficient adenovirus vector named Ad5-Akt1;â‘ 293 cells was vaccinated in six orifices,5×10⁵ cell each cave.the culturemedium is DMEM+10%FBS,sets at 37℃,5% CO2 passed night.â‘¡takeed pDC316-Akt1 and pBHglox,transfected 293 cells according to the instruction using the Lipofectamine2000.â‘¢Reaped the adenovirus vector when the 293 cell get pathological majority changes and falls off from the base.â‘£Goal gene appraisal;the result of RT-PCR and Western blot showed the AKT1 gene,mRNA and protein expression in the 293 cells2.the research of apoptosis and function of the rat islet which was transfected AKT1 gene via adenovirus vector(1)According to different processing the islets will divide three groups,Akt1 islets group were transfected with Ad5-Akt1;control group EGFP,with Ad5-EGFP;and control group PBS,with PBS.Experimental group and the blank transfection group was cultured with the virus for lhour after cultured for 24 hours in 37℃,5%C O₂,replaced the 1640 and go on cultured in 37℃,5%C O₂..After 48 hours the blank transfection group was observed the EGFP expression under the fluorescencemicroscope,the Experimental groupwas examined the Akt1expression situation with Western blot.(2)The transfection efficiency was identified by flow cytometry(3)Examination to the survival of the islets adopts;â‘ AO/EB fluro-stain showed the viability of cell.â‘¡The apoptotic cells were identified by TUNEL method(4)The examination to the function of the islets adopts;â‘ the amount of insulin excretion was measured by radioimmunoassy.â‘¡calculated the insulin stimulation index with the insulin release test;Counts 10IEQ islet, which was cultured in the low density glucose(2.8mmol/L)Kreb' S-Hank' The s fluid (includes 10mmol/L HEPES and 0.25%FBS)for 2 hour,then cultured in the highly concentrated glucose(the 16.7mmol/L glucose)Kreb' S-Hank' for 1hour,collects 2h and the 3h nutrient fluid,uses radioimmunoassy to measured the amount of insulin,the insulin stimulation index(SI);SI=3h(high sugar environment)insulin content/2h(low sugar environment)insulin content.In the experiment supposes 5 duplicate holes.â‘¢observe the islet's morphology change by insulin-6 immunohistochemical stain. Statasticis analysis;all the data expressed in mean±SD.T test was used and P＜0.05 was considered having statitical significance.Result1.600IEQ islet could be extracted from every rat,the DTZ stain shows purity more than 90% and islet viability more than 98%.2.successful AKT1 expression apo carrier was confirmed by theBamHâ…¢and EcoRI bi-enzyme electrophoresis and sequencing plasmids,293cells was modified by via Lipofectamine 2000,the result of RT-PCR and Western blot showed the AKT1 gene,mRNA and protein expression in the 293cells.3.293 cells transfected with Ad5-Akt1 can express Akt1.4.After 48 hours green fluorescence was observed in the islets which was co-cultured with Ad5-EGFP5.islets transfected with Ad5-Akt1 can express Akt1,moreover the quantity of Akt1 of Akt1 group was higher than the two control groups.6.In the processing cultured in vitro,islets overexpressing Akt1 showed obviously decrease of the apoptosis and the life get lengthen,and the serum insulin concentration was significantly higher in group Akt1Conclusions1.The Ad5-Akt1can successful transfected islet cell and Goal gene can expressed in the islet cells2.Aktlcan enhanced rat islets antiapoptotic function lets and prolong survival in vitro.3.Akt1 can promoted insulin release of rat islets...