Role Of Bone Marrow Mesenchymal Stem Cells In Experimental Severe Acute Pancreatitis

Acute pancreatitis is a common clinical acute abdomen, and ten to twenty percent of the patients develop into severe acute pancreatitis (SAP).SAP is a life-threatening disease with a mortality rate of fifteen to twenty percent, and the prognosis is unfavorable. Hemorrhagic necrosis of pancreas is the main pathological character in SAP. Usual occurrence with that is systemic inflammation response syndrome (SIRS) and infection in later stage. Despite recent improvements in our understanding of the disease process and the development of a range of supportive measures,today's treatment approaches for SAP are still less than ideal. It has been demonstrated recently that mesenchymal stem cells (MSCs) can exhibit tremendous functional plasticity in the pathophysiologic process of SAP. Mobilization of MSCs can attenuate SAP and improve the prognosis.The present study is designed to utilize rat acute pancreatitis model to observe the effects of MSCs intervention on SAP. Then engraft homogenic MSCs to treat SAP, and analyze migration, planting and differentiation of MSCs, and in attempt to develop new protocols for the improvement of SAP therapy.1.Isolation and culturing of MSCsObjectives:To primary culture, purify MSCs, and analysis of cell surface molecules using flow cytometry.Methods:Femru of healthy adult donor rats were excised under sterile conditions. Bone marrow plugs were extracted from the bones by flushing the bone marrow cavity with 10% FBS until the diaphysis became white. The douche was centrifuged for 5 min at 1000 rpm. Then removed the supernatant, and washed twice with PBS solution. The washed cells then were resuspended in MSC medium, consisting of Dulbecco's modified Eagle's medium (DMEM), low glucose supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin,100 mg/ml streptomycin, and 2 mmol/1 L-glutamine. The cells were plated at a density of 5×105cells/cm2, and cultured at 37℃in a humidified atmosphere containing 5% CO2. The mesenchymal population was isolated on the basis of its ability to adhere to the culture plate.The hematopoietic cells, which constituted the majority of the cells, did not stick to the culture plate and were removed with subsequent medium changes. When the cultures reached 90% of confluence, cells were recovered by 0.25% trypsin and passages followed. Proliferation and growth were observed with phase contrast microscope in primary and passage culture. After passage 3, surface markers were detected with flow cytometry.Results:Three to four days after MSCs seeding in culture plates, a contrast phased microscope revealed adherent cells in small colonies with typical fibroblast-shaped morphology. The primary cells reached confluence in single layer after plating for seven to ten days and were passaged for the first time. The cells were noted to have a large expansive potential after subculture. The fibroblast-like morphology was also maintained after cell passages and throughout the culture period. Flow cytometry showed culture MSCs included CD29+CD44+CD45-,CD29+CD44-CD45+ and CD29+CD44-CD45-. The CD29+CD44+CD45- group was MSCs group.Conclusion:After serial passages, MSCs were purified. Purity of Passage 3 MSCs is over 95%.The differentiation capacity of MSCs was maintained during culture.2.Establishment of severe acute pancreatitis modelObjectives:To induce SAP model in rats and examine both grossly and microscopically for the observation of pathological changes of the abdominal viscera.Methods:SD rats weighting 250-300g were divided randomly into 2 groups. Group A (n=6) was normal rats,Group B (n=30) was SAP rats. Group B rats was induced by intraperitoneal injections with L-arginine 2.5g/kg body weight twice. The pancreas and lung were collected at 4,12,24,48 and 72 hours after the induction of pancreatitis (n=6 for each time point). The pancreas samples were histologically examined by light microscope, and the changes of amylase was examined by HITACHI-7150.Results:Serum amylase activity was significantly increased at 4h after L-arginine intraperitoneal injections and reached a peak value at 24h (9981±2901.6U/L), compared with control levels.In SAP group, a number of small vesicles within the acinar cells were seen 4h after L-arginine intraperitoneal injections.At 24h, the acinar architecture was partially destroyed with focal acinar cell necrosis; interstitial edema and inflammatory infiltrate were greater in degree than that at 12h.Conclusion:The model of experimental SAP is reliable with good reproducibility and reproducibility. Because of its severe pathological damage in pancreas, the way of L-arginine intraperitoneal injections indused rats SAP model can be used for therapy research.3.Therapeutic effects of homogenic MSCs transplantation on SAPObjectives:To observe the therapeutic effects of homogenic MSCs transplantation on SAP.Methods:MSCs used in this part were obtained from male SD rats and the method of primary culture was described in part 1.SAP model was induced in female SD rats as described in part 2. At 24h after pancreatitis induction,5-7×107 MSCs of P3 were infused intravenously. Twenty four hours later, recipient rats were sacrificed. Organs were taken out, including pancreas, liver and lung. Tissue RNA was extracted and detected for TNF-a, IL-1βmRNA expression with real time RT-PCR method.The morphological changes of pancreas between the two groups were examined. Sruvival rate was calculated at 72h.Results:Serum amylase activity was decreased after MSCs transplantation. Expressions of TNF-a and IL-1βmRNA were lower in infusion group than control group. The pancreas pathematology score was significantly decreased after MSCs transplantation. The 72h of rats survival rate was significantly increased after MSCs transplantation.Conclusion:MSCs transplantation could improve prognosis of SAP rats and enhance 72h of the survival rate.4. Migration and differentiation of intravenously transplanted bone marrow mesenchymal stem cells to impaired pancreases in ratsObjective:To observe whether the intravenously transplanted homologous bone marrow mesenchymal stem cells (MSCs) can migrate to the impaired pancreas tissue in the rats with sever acute pancreatitis (SAP), and to establish experimental basis for improving stem cell therapy in pathologic state of sever acute pancreatitis.Methods:MSCs were isolated from bone marrow of male Sprague-Dawley (SD) rats and adhered to the culture plate in vitro. Female rats were divided randomly into 3 groups (n=10). Group A was normal rats with MSCs infusion through tail vein, Group B was SAP rats received MSCs infusion via tail vein 24 h after SAP, Group C the SAP rats were given culture medium infusion via tail vein. SAP was induced in Group B and C rats by intraperitoneal injections with L-arginine 2.5g/kg body weight twice. The pancreas was harvested 72h after transplantation. The morphological changes were examined, and the characteristics of migration of MSCs to pancreas were detected with the expression of sry gene of Y chromosome by using chromogenic in situ hybridization (CISH).Results:72h after transplantation, the SAP pathological score was significantly lower in Group B compared with Group C (P<0.05). In addition, sry positive cells were observed in the pancreatic tissue in both Group A and Group B, these cells in Group B were more than in Group A under microscope, and some of these had been differentiate into acinar cells and pancreatic cells. No sry positive cells were observed in the pancreases of Group C.Conclusions:The inflammatory pancreatic tissue have the ability of recruiting MSCs in vivo; the transplanted MSCs can migrate towards the impaired pancreas, and differentiate into pancreatic cells partially, which can locally alleviate inflammation.SUMMARY:MSCs transplantation could improve the prognosis of SAP rats and enhance 72h of the survival rate. The inflammatory pancreatic tissue have the ability of recruiting MSCs in vivo; the transplanted MSCs can migrate towards the impaired pancreas, and differentiate into pancreatic cells partially, which can locally alleviate inflammation.