The Research Of Effect For Testicular Sertoli Cells On Islets Xenotransplantation

Research BackgroundDiabetes has become a kind of global common disease which badlyimpair people health and life quality,despite all that insulin had beendiscovered 80 years and mortality of diabetes decreased year afteryear ,the complication of diabetes yet can not be cured during thecourse of disease diagnosis and treatment .In 1992,an open authoritydebate has been performed in the 14th international organtransplantation academic meeting ,then resulted that transplantationmethod of islets will instead of that of simple pancreas.This meetingestablished the position of islet transplantation in curing diabetes.Itwill be a relatively full way in treating all of typeⅠand the part of typeⅡdiabetes patients through systemic evaluation and analysis.In development and improvement of transplantation,exploitationand application of new immunosuppressant can energeticallycontribute in solving graft rejection and prolonging graft life time.Butlong time even life time in applicatation of immunosuppressant willlead very extremely unfavorable influence to recipient through drugsitselves'side effect,despite of heavy economy burden.therefore,peopleare devoting themselves to search for another available method inorder to transplantated organ can functionally survival in long timewithout any immunosuppressants.This ideal method isimmunotolerance.At present,that cell programme death mediated by Fas/Fasl systemhas become hot spot and attracted attention of transplantationcommunity .FasL expression can lead lymphocytes that express Fas toapoptosis.Scholars try to attain immunotolerance through transgeneway or cotransplantation of functional testicle cells.Some scholars hadgotten positive results,but the others obtained opposite conclusion.Thisis possibily concerned with their adoptive way such as directexpression FasL by functional cells.There have not been reported inislets xenotransplantation.We assume if testicle sertoli cell superioritywhich can keep highly expressing Fasl is applied into isletsxenotranplantation and bring into full play their function of inducinglymphocytes into apoptosis maybe partly resolve graft rejectivereaction problem which constantly pule us,and establish the base ofdiabetes treatment.ObjectiveOnly employ cotransplantation of testicle sertoli cells andxeno-islets to cure diabetes to induce immunotolerance, but importFasL gene into islets,the experiment is divided into five parts below tocomplete:1. To improve the isolation and purification of adult pig islets andoptimize the preparation method of islets.2. Steady chemic Diabet Rat Models are set up in order to prepare forthe next pace of transplanation experiment.3. To know the best islet mass to reverse the hyperglycemia ofalloxan-induce diabetic rat and observe the results ofxenotransplantation.4. To exploit and improve the isolation of testicle sertoli cell andprovide enough cells to next step.5. To evaluate the effects of the different doses of testicle sertoli cell inpreventing islet xenograft rejection in pig to rat models.Methods1. This research adopts shaking and digesting after perfused complexcollagenase(collagenase V and DNA enzyme)into pig pancreaticduct in pancreatic body and tail and purificating in modifiedDextran discontinuous density gradient.The islet structure andfunction were evaluated by islet vitality and morphologyobservation.2. This research adopts the method that alloxan is injected in tail veinonly one time to prepare diabete Wistar rat models, thenmeasures blood sugar and urine sugar twice a week.3. Different quantities of Audlt Pig islets(APIS) were implantedunder the kidney capsule in diabetic rat, observed and evaluatedthe result of reverse the hyperglycemia of diabetic rat.4. Testicle sertoli cells are isolated through twice digestion andcultured in original generation, then morphological observationand immunohistochemical identification are performed in order todetermine their integrated construction and function.5. The alloxan-induced diabetic rats were randomly divided into fourgroups(n=8):control group, l×106 testicle sertoli cells group, 2×106testicle sertoli cells group, 4×106 testicle sertoli cells group. Thisresearch adopts isolated and purified the adult pig islets (APISs).APIS were transplanted immediately under the kidney capsule ofthe diabetic mouse. The condition of functional survival andrejective reaction are evaluated by blood glucose, pathology andimmunohistochemistry.Results1. The pancreas weight was 1 5±3.4g.Islet yield was 4130±976IEQ/gafter digest and 2320±669IEQ/g after purification.Purity of therequired islet was about 80%and insulin stimulation test showed agood islet vitality.These data were expressed in terms of isletequivalents(IEQ)as described by Ricordi . The integrity ofpancreatic cell mass was seen in islet pathological slices(HEstaining).2. 60 models of Diabete rats have been prepared, there are 53 modelsmeasure up which blood sugar maintain continually at 22.2mmol/Land urine sugar maintain continually positive (++-+++) during twoweeks experiment. Formation rate of model rat is 88.33%.3. 15000IEQ/kg APIS could not reverse the hyperglycemia indiabetic rat wholly, more than 20000IEQ/kg APIS were able torender alloxan-induced diabetic rat normoglycemic aftertransplantation . Islet could survive functionally for (4.8 ±1.1)dayswithout any immunosuppressive agents.4. Testicle sertoli cells are well appearance and integratedstructure .After 72 hours cultured, there show a great quantity ofFasL and FSHR that highly expressed in testicle sertolicells.Contrasting FSHR expressing precultured to postcultured,testicle sertoli cells show increase quantity and dominant grow.5. The graft survival time was separately (5.88±0.83)d, (20.63±2.92)d,(34.00±3.30)d, (45.38±2.92)d after transplantation.The functionalsurvival time of graft is significantly longer in experiment groupsthan in control group(P<0.01) and show does-dependent with thequantity of testicle sertoli cells.After transplantating five days,4×106 testicle sertoli cells group has much more intact APISsurvival and positive expression of insulin and Fas, throughpathology and immunohistochemistray study, while infiltrationlymphoid cells induced apoptosis.Conclusion1. The measure that adopted in this experiment of pig pancreasresection is utility and certain, the time of cold and warm ischemiapreservation is appropriate and will settle foundation for next step.2. The pig islets aggregate which required by modified method in ourresearch,showed a much better result in yield,purity and activity.3. The mangerment of isolation and purification for testicle sertolicells can reduce impairment of cells and profit functionalrestoration in order to induce immune privilege.4. Short-time culturing of sertoli cell pretransplantation can maketheir vigor restored and function better.5. Immunohisochemic approach through FasR and FasL can speciallyidentify testicle sertoli cell.6. Islets xenotransplantation may effectively improve hyperglycaemiastate of rats.7. Cotransplantation of adult pig islets and testicle sertoli cellsprovides protection for islets, graft survival time is relative tosertoli cell population, showing obviously does-dependance.8. Immune privilege of sertoli cells maintains mainly through highlypersisting in expression of FasL.9. Sertoli cells induces graft immune tolerance only regional, butspecific.