| Objective:To investigat the effects of the allogeneic bone marrow mesenchymal stem cells on rats with severe acute pancreatitis (SAP) and the change of inflammatory factors, and to further analyze the mechanism of the treatment with mesenchymal stem cells to SAP.Methods:The whole bone marrow cells were harvested from 14 days old male SD rats,and then were isolated, purified, cultured.BMSCs grown to passages 3 were identified for the fllowed experimental; 84 healthy female Sprague-Dawley rats (weight:200±20g),were randomly divided into sham operation group (sham operation group, S group), severe acute pancreatitis model group (severe acute pancreatitis group, SAP group) and bone marrow mesenchymal stem cell transplantation 24 hours after modeling (bone marrow mesenchymal cells transplant group, B group). SAP model could be established by using the pancreatic subcapsule slow uniform injection with lml 4% of the slow sodium taurocholate. Twenty-four hours later, S group and B group respectively used tail intravenous injection of lml stem cells (1ml contains 1.0×106 cells).Meantime SAP was injected lml PBS. Rats were killed by abdominal aorta exsanguinations after operation At three time points of 1st,7th,14th d after injection the rats (six rats of each group at each time point). The levels of serum amylase were measured, (Part of the serum specimens to prepare for measure of TNF-a, IL-10);In the meanwhile, pancreatic tissue were fixed in 10% neutral formalin to observe the pathological changes of pancreatic tissue and for pathological score; we measured the serum levels of TNF-a and IL-10 by ELISA assay and observed the effects of allograft mesenchymal stem cells on the above indexes.Results:1.A small amount of the whole bone marrow cells which were harvested from childhood bone marrow were adherent after inoculation for 8 hours, and after 7th day bone marrow cells extracted can reach 90% confluence.When cultured to the third-generation,spindle cell-based cell populations which arranged neatly in uniformity of morphology were observed in the light microscope.2.Determined by flow cytometry,the 3rd generation of cultured cells expressed CD29+99.02%, CD45+0.97%, CD44+80.86%.3. Twenty-four hours after transplantation, fragments of Sry gene could be detected in rat pancreatic tissue of the transplantation group and the sham group. After 7 days or 14 days transplantation expression of Sry gene of the transplantation group rats significantly increased,by contrast,the sham group had no significant change.4.Serum amylase changes:After mode, serum amylase levels of SAP, B group at each time point was obviously higher than the S group,which had significant statistical difference (P<0.01); 24 hours after transplantation, serum amylase levels of B group and SAP group, although decreased, had not statistically significant (P> 0.05); 7 days after transplantation, serum amylase levels of B group and SAP group was decreased with statistical significance (P<0.01).5.Pancreatic histological score:①S group score is low with no significant changes at each time point.②The score of S group,SAP and B group score at each time point had significant statistical difference (P<0.01).③24 hours or 7 days after transplantation,the histological score of B group was lower than SAP group with no significant difference (P<0.05); 14 days after transplantation, group B which was compared with SAP group were significantly different (P<0.05.)6. Changes of serum TNF-a level:①At different time points after model S group serum TNF-a level had no significant change and was significantly lower than SAP, B group serum TNF-a level with statistical significance at each counterpart time point (P<0.01).②The difference between the serum TNF-a levels of B, SAP group at 1st day was not significant (P> 0.05); but the difference at the 7th day was significant (P<0.05); Compared with the serum level of SAP group, the serum level of B group at 14th day had significant lower(P<0.01).7.Change of serum IL-10 levels:①Serum IL-10 levels of S group was extremely low and had no significant change at different time points after model and was also significantly lower than serum IL-10 levels of SAP and B group at each counterpart time point(P<0.01).②The difference between serum IL-10 levels of B, SAP group at first day was not significant (P> 0.05); but the difference at the 7th day was significant (P<0.05); The serum IL-10 level of B group at 7th or 14th day was significantly lower than SAP group (P<0.01).Conclusion:1. Bone marrow mesenchymal stem cells of rats were successfully isolated and cultured through screening adheren.2.Bone marrow mesenchymal stem cells by transplantation could migrate to specific tissue damage in the pancreas.3. Allogeneic bone marrow mesenchymal stem cells could alleviate pancreatitis and promote repair of pancreatic damage.4. Allogeneic bone marrow mesenchymal stem cells could down-regulate the level of serum proinflammatory cytokines TNF-a on rats with severe acute pancreatitis, but up-regulate the level of anti-inflammatory cytokine IF-10. |
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