| Acute pancreatitis (AP) is a common clinical disorder presenting as acuteabdomen，ranging from mild self-limiting disease to severe life-threatening illness，and ten to twenty precent of the patients develop into severe acute pancreatitis(SAP).As SAP is a life-threatening disease with high rate of mortality, complicated withmany other serious diseases and prognosis is poor. At present, there is no specialtreatment approachs for SAP with sysyemic inflammation response syndrome (SIRS)in clinical. Recently it has been reported that bone mesenchymal stem cells (BMSCs)can attenuate AP and improve pancreas function, exhibit tremendous functionalplasticity in the pathophysiologic process of AP, However, the research on thepathogenesis of BMSCs to SAP has not been fully clarified yet. Therefore, the aim ofour study was to utilize SD rat SAP model, then engraft homogenic BMSCs to treatSAP model, and elucidate the underlying mechanism of BMSCs infusion in pancreasself-restoration and pathological regeneration.1、Isolation, Culture, Amplification and Identification of BoneMarrow Mesenchymal Stem Cells (BMSCs)Objectives: To purify BMSCs, primary culture, and to identify BMSCsaccording to cell surface momecules using flow cytometry for later experiment.Methods: Healthy adult donor rats were excised under sterile conditions.Flushing the bone marrow cavity with FBS until the diapysis turn into white,then thedouche was centrifuge for10min at1500rpm.The supernatant was removed.WithFicoll separation medium isolation individual nucleus cell, DMEM-LG cultivation,observation its morphology.After the first proliferation and growth were observed with phase contrast microscope,until passge3, collected surface cells were detectedwith 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 primarycells reached confluence in single layer. The result of the surface antigensidentification is that high expression of CD90and CD45and low expression CD25and CD34at the third generation cells were analyzed by flow cytometry. BMSCshave been appeared in the phenotypic characteristics of osteogenic and adipogenicrespectively.Conclusions: Using Ficoll liquid gradient centrifugation combining withattachment method can get spindle, purified and differentiation capacity of BMSCs.2、Establishment of Severe Acute Pancreatitis ModelObjectives: To establish SAP model in rat for the observation of pathlologicalchanges of the abdominal viscera.Methods: SAP model was induced by5%taurocholic acid (TCA) injection.SD rat weighting250-300g were divided randomly into2groups. The control groupwas as nomal. The model group rats were divided into five timepiont of6,12,24,48and72hours after SAP induction (n=5for each timepoint). Blood sample, pancreasand lung were collected at each timepiont.The change of serum AMY was examined.The pancreas and lung samples were examined histologically by light microscope.Results: In model group, serum AMY level 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 the control geoup. The grade ofpancreas of model group was higher than in the control group.The survival rate (72hafer SAP induction) of two groups is100%and30%respectly. In the model group, anumber of labular gap were widened associated with a number of inflammatory cells infiltration at6h. Pancreatic cell necrosis was observed at12h. Large area ofpancreatic tissue necrosis, hemorrhage were appeared obviously at24h. Much ofacinar structure were disappeared at48h. Lots of acinar damage structure andinflammatory cells infiltration were found at72h.Conclusions：The experiemental SAP rat model is reliable with5%TCAinjection. We also find that the pancreas was severe attacked at24h timepiont, soclose to the timepiont to therapy research.3、Therapeutic Effects of BMSCs Transplantation in SAP inRatsObjectives: To examine the therapeutic effects of homogenic BMSCstransplantation in SAP in rats.Methods: Adult male SD rats weighing250-300gram were used and dividedat random into3groups (each of5rats). Group1acted as normal negative controlwithout any treatment. Group2as DMEM+SAP. Group3as BMSCs+SAP. At24hafter SAP induction,1106cultured BMSCs or DMEM medium were infusedintravenously by the tail vein respectively. All animals were sacrificed72hours afterthe induction of SAP, serum amylase (AMY)、 serum alanine aminotransferase(ALT)、serum creatinine (Cr)、serum myocardial lactate dehydrogenase (LDH)、serum tumor necrosis factor-(TNF-)、 serum interferon-(INF-) andintrapancreatic mRNA expression of IL-1、 IL-6and IL-10were assayedrespectively, histological scores of pancreas and lung of these two groups wereexamined under microscopic, and immunostaining for MPO was performed also.Suvival rate was observed at72h after SAP induction.Results: Histopathological changes such as interstitial edema, inflammatorycells (mononuclear and neutrophil cells) infiltration, necrosis as well as parenchymalhemorrahages of pancreas and lungs were improved by BMSCs transplantation. In rats, inflammation (MPO positive neutrophils) was markedly less in the SAP+BMSCsgroup than in SAP+DMEM group. These levels of serum AMY、serum ALT、serumCR、serum LDH、serum TNF-and serum INF-were significantly decreased inSAP+BMSCs group than in DMEM+SAP group at each time point (P<0.05), also theSAP+BMSCs treated group inhibited intrapancreatic mRNA expression of IL-1、IL-6and IL-10compared with SAP+DMEM treated group, and improved the survivalrate72h after disease onset.Conclusions: BMSCs tansplantation could inhibite inflammatory mediatorsto block the inflammation pathway and participate into pancreatic tissue repair,enhance the survival in SAP acute phase.4、Migration of Intravenously Transplantation of BMSCs inSAP in RatsObjectives: To examine the distribution of male rat BMSC dornors to femaleSAP model recipients, and observe the therapeutic effect of BMSCs infusion in thecourse of SAP.Methods: BMSCs were obtained from male SD rats. SAP model was inducedin female rats as described in part2.1106BMSCs of P3period were infused at24h after SAP induction,24hours later, recipient rats were sacrificed and collectedpancreas、 kidney、 liver、heart and lung. Tissue DNA was performed by PCR todetermine the presence of SRY region of Y chromosome from male donors.Results: Y chromosome SRY region was present in all5kinds of tissues asshown by PCR, the expression in pancreatic tissue was higher than in other tissues.Conclusions: These results indicated that BMSCs might be capable ofdistributing to the panreas and other tissue parts and play a role in SAP pathologicalstate. SUMMARYUsing Ficoll liquid gradient centrifugation combining with attachment methodcan get a spindle, purified and differentiation capacity of BMSCs; The experimentalSAP model induced by5%TCA injection is reliable, as its severe pathologicaldamage in pancreas is close to SAP in clinical. We also find that the pancreas wasattacked severely at24h timepiont after SAP induction. BMSCs transplantation couldimprove the prognosis of SAP rats and enhance survival rate. BMSCs may migratetoward to the damaged pancreatic area, which can alleviate inflammatory cellinfiltration and promote the repair of injured pancreas. In conclusion, our findingssuggest that BMSCs participate and play an important role in the progression of SAP. |
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