The Study Of Experiment With Bone Marrow Mesenchymal Stem Cells In Server Acute Pancreatitis

Acute Pancreatitis is a common clinical acute abdomen?and ten to twentyprecent of the patients develop into sever acute pancreatitis(SAP).SAP is a life-threatening disease with high rate of mortality,and prognosis is poor.At present,thereis no special treatment approachs for SAP in clinical.It has been report recently thatbone mesenchymal stem cells(BMSCs) can exhibit tremendous funetional Plasticity inthe PathoPhysiologle Process of SAP.BMSCs can attenuate SAP and improve thesurvival rate.However,there is a not clearly know about mechanism.The present studyis designed to utilize rat acute pancreatitis model to observe the effects of BMSCsafter intervent on SAP.Then engraft homogenic BMSCs to treat SAP,and try to discusshow to the mechanism is worked by the BMSCs thrapy SAP.1?Isolation,culture, amplification and identification of BMSCsObiectives: Isolation, culture, amplification BMSCs of rat and its antigensand the potency of multi-directional differentiation were identified for laterexperiment.Methods: Healthy adult donor rats were excised under sterileconditions.Flushing the bone marrow cavity with FBS until the diapysis turn intowhite,then the douche was centrifuge for10min at1500rpm.The supernatant wasremoved.With Ficoll separation medium isolation individual nucleus cell, DMEM-LGcultivation, observation its morphology.After the first proliferation and growth wereobserved with phase contrast microscope,until passge3,collected surface cells weredetected with flow cytometry.Results: BMSCs seeding in the culture plates after three to four days,acontrast phased mincroscope revealed adherent cells in small colonies with typicalfibroblast-shaped morphology.After several proliferation and growth,the primary cells reached confluence in single layer. The result of the surface antigens identification isthat high expression of CD90and CD45and low expression CD25and CD34at thethird generation cells were analyzed by flow cytometry. BMSCs have appeared thephenotypic character istics of osteogenic and adipogenic respectively.Conclusion: Using Ficoll liquid gradient centrifugation combining withattachment method can get spindle,purified and differentiation capacity of BMSCs.2?Establishment of severe acute pancreatitis modelObiectives: To estable reliable SAP model in rat for the observation ofpathlological changes of the abdominal viscera.Method: SD rat weighting250-300g were divided randomly into2groups.control group was nomal. Model group rats was divided into five timepiontof6,12,24,48and72hours, each timepiont had five rats were induced by5%TCA.The AMY, pancreas and lung were collected at each timepiont.The change ofAMY was examined. The pancreas and lung samples were histologically examined bylight microscope.Two groups were observed sunvival rate.Results: In model group, the level of AMY was significantly increased at6h(6526.191454.79),12h(7683.501806.41),24h(9501.002111.76U/L),48h(4516.671715.32),72h(3885.001682.67), compared with control geoup. The grade of pancreasof model was higher than control group.The survival rate of two groups is100%and30%respectly. In the model, a number of labular gap were widened, and inflammatortcells were aggressived at6h after made moedel. Pancreatic cell necrosis at12h. Largearea of pancreatic tissue necrosis, hemorrhage and inflammatort cells appearedsignificantly at24h. Much of acinar structure were disappeared.The neutrophils andlymphocytes were infiltrated at48h. At72h timepiont, Lots of acinar structure andinflammatory cells were greatly in degree than ever timepiont. Conclusion?The model of experiement is reliable with5%TCA injectioninduced rats SAP.We also find that the pancreas was severe attacked at24htimepiont, so chose this timepiont to therapy research.3?Therapeutic effects homogenic BMSCs transplantation onrats SAPObiectives: To assure the therapeutic effects of homogenic BMSCstransplantation on rats SAP.Method: SAP model rats divided randomly into control group with noinjection, SAP rats with recipient cultured solution group and SAP rats with recipientBMSCs. At24h after pancreatitis induction,5-7107cultured solution and BMSCswere infused intravenously. Twenty four hours later,all rats were sacrificed andcollected serum that were examined AMY?ALT?CR?LDH?TNF-?INF-.Pancreas one part of detected for IL-1?IL-6? IL-10expression, the other tissuewas histologically examined and estimated the grade.Suvival rate was observed at72hbetween later two group.Result: In BMSCs group, AMY?ALT?CR?LDH?TNF-and INF-were decreased significantly compared with the cultured solution, and also expressionof IL-1?IL-6? IL-10were lower than cultured solution. The72h of rats in BMSCssurvival was significantly increased than cultured solution.Conclusion: BMSCs tansplantation could improve prognosis of SAP rats andenhance the survival.4?Migration of intravenously transplantationed BMSCs in ratsObiectives: To observe the male rat BMSCs migration in the female SAPmodel, and attemp to discuss the mechanism that how to worked by the BMSCs thrapy SAP.Method: There are two groups, one of that had three nomal female rats withinject male BMSCs, the other grouphad three female SAP rats after24h made.All ratswere injected1106BMSCs. Twenty four hours later, rats were scarificed andcollected pancreas kidney liver heart and lung to detect the male Y chromosome.Result: In detected tissue, there was Sry distributed, but the pancreatic washigher than other tissues.Conclusion: Confirmed that BMSCs could located the damaged parts ofpancreas, and play a role of anti-inflammatory and repair.SUMMARYBMSCs isolated, cultured, proliferated applying density gradient centrifugation,attachment method combining with monoclone cultivation have high autoploidy.5%TCA was successfully induced SAP model. BMSCs transplantation could improve thepronosis of SAP rats and enhance72h of the survival rat. BMSCs can migrate towardthe damaged pancreatic area, which can alleviate inflammatory.