Preparation Of Bone Marrow Mesenchymal Stem Cells And The Effect Of MSCs On Experimental Severe Acute Pancreatitis

Acute pancreatitis is a common clinical acute abdomen.Ten to twenty percent of the patients develop into severe acute pancreatitis(SAP).The mortality rate of SAP is about fifteen to twenty percent,and the prognosis is unfavorable.Hemorrhagic necrosis in pancreas is the main pathological character of SAP.Usual occurrence with that is systemic inflammation response syndrome(SIRS) and infection in later stage.So far,inhibition of pancreatic exocrine,prophylactic antibiotic and nutrition supports are predominant therapy principals for SAP.No specific measures are available until now.Recent paper reports and our previous studies suggest mesenchymal stem cells(MSCs) may have a role in the pathophysiologic process of SAP.Mobilization of MSCs can attenuate SAP,and improve the prognosis.In this study,we try to utilize rat acute pancreatitis model to observe the effects of bone marrow intervention on SAP.Then engraft homogenic MSCs to treat SAP,and analyze homing,migration,planting and differentiation of MSCs,and identify the role of bone marrow mesenchymal stem cells in pancreas self-restoration and pathological regeneration.1.Induction of acute pancreatitis and evaluation of modelObjectives:To induce SAP and MAP model in rats and investigate the biological and histologic characteristics of SAP model.Methods:Experimental SAP was induced by intra pancreatic duct injection of sodium taurocholate(STC).Male SD rats,weighting 250-300g, anesthetized by pentobarbital,were fixed on the operating plat.Hemorrhagic necrotizing pancreatitis was induced by the injection of STC as previously described.We injected 3%STC into the biliopancreatic duct at a rate of 12ml/h using a microinfusion pump.Edematous pancreatitis was induced by the intraperitoneal injection of cerulein(20ug/kg) 4 times with 1-hour intervals.Blood samples in STC pancreatitis rats and controls were collected from the heart,respectively,at 0.5,2,4,8,12 and 24 hours after the induction of pancreatitis(n≧5 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 0.5h after STC injection and reached a peak value at 24h(9980.6±2328.3U/L), compared with control levels(1431.2±368.5U/L).Serum alanine aminotransferase (AAT) activity were significantly increased at 8h after the STC injection and serum creatinine were significantly increased at 24h after the STC injection.The survival rate was 90%in MAP and 40%in SAP. In MAP,pancreas interstitial edema and inflammatory infiltrate was observed, and these changes were maximal at 12h.In SAP,a number of small vesicles within the acinar cells were seen 6h after STC injection.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.Electron microscope photos show phagocytosis of bacteria in pancreas implying infection of necrosis tissue in SAP rats.Conclusion:The STC model of experimental SAP is reliable with good reproducibility and reproducibility.Because of its severe pathological damage in pancreas,STC model can be used for therapy research.2.Primary culture and serial subcultivation of MSCs and identifycation of MSCsObjectives:To primary culture,purify MSCs,and identify MSCs according to surface markers.Methods:Bone marrow samples were collected from healthy adult donor rats in sterility condition.Both lower limbs of the rats were removed and the femur medullary cavities were flushed with 10%FBS until the diaphysis became white.The douche was centrifuged for 5 min at 800 rpm. Then removed the supernatant,and washed the deposition 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, 100mg/ml streptomycin,and 2mmol/l L-glutamine.The cells were plated at a density of 5×10⁵cells/cm².Cultures were maintained at 37℃in a humidified atmosphere with 5%CO₂.Adherence of MSCs was observed in the first 24 h.After 24 h,non-adherent cells were removed by changing the medium.Regular observation was performed every 12 h thereafter.MSCs were subcultured according to the density.After passage 3,surface markers were detected with flow cytometry.MSCs were separated into 5 tubes.CD44,CD45,CD29 antigen on the surface of MSCs were detected. MSCs were induced to differentiate to osteocyte,chondrocyte and adipocyte respectively in corresponding induction medium.After full induction period,MSCs were stained with specific dye for osteocyte,chondrocyte and adipocyte respectively.Results:Average time taken by MSCs to be adherent was 24-72h. The shapes of adherence cells were triangle and fusiform.MSCs tended to grow into colony.After several passages,MSCs were purified.In passage 3 MSCs had uniform shapes.Flow cytometry showed primary culture MSCs included CD29+CD44+CD45-,CD29+CD44-CD45+ and CD29+CD44-CD45-.The CD29+CD44+CD45- group was MSCs group. After serial passages,percentage of MSCs enhanced.MSCs could be stained by alizarin red,oil red and typeâ…¡collagen after oriented induction.Conclusion:After serial passages,MSCs are purified.Purity of P3 MSCs is over 95%.MSCs keep differentiation capacity during culture.3.Analysis of the functional status of MSCs during SAPObjectives:To analyze the functional status of MSCs during SAP.Methods:Experimental SAP was induced by intra pancreatic duct injection of sodium taurocholate(STC) as part 1 described.Animals were sacrificed 12,24 and 36h after model induction.Bone marrow cells were implanted in DMEM+F12+10%FBS medium.Adherence of MSCs was observed in the first 24h and per 12h in the following.MSCs were subcultured according to the density.After passage 3,they were detected with flow cytometry as part 2 described.Colony formation rate was calculated by CFU-F method.MSCs after P3 were induced to differentiate to osteocyte, chondrocyte and adipocyte respectively in corresponding culture medium.After full induction period,MSCs were stained with specific dye for osteocyte,chondrocyte and adipocyte respectively.Control group underwent sham operation without induction of pancreatitis. Results:Average time taken by MSCs of SAP rats to be adherent was shorter than controls.Colony formation rate of SAP group was higher than that of control.Percentage of Lin-CD29⁺CD44⁺CD45^- cells of SAP 36h group was lower than that of other groups.MSCs of all groups could be stained by alizarin red,oil red and typeâ…¡collagen after oriented induction.Conclusion:During SAP,surface marker CD29,CD44 and CD45 do not changed,and MSCs keep their differentiation capacity.Compared to control group,number and reproductive ability of MSCs during SAP changed.4.Influence of bone marrow pretreatment on SAPObjectives:To observe the influence of bone marrow pretreatment on SAP.Methods:Bone marrow mobilization group was treated with intraperitoneal injection of granulocyte-colony stimulating factor(G-CSF) 2h before pancreatitis induction at a dose of 100μg/kg.Bone marrow suppression group was treated by intraperitoneal injection of busulfan 6h before model induction at a dose of 1mg/100g.The control group did not receive bone marrow pretreatment.Two hours after bone marrow intervention, some animals were killed and blood samples,pancreas and medulla were obtained.Blood films and medulla films were made and stained with Gimsa.Blood slides and bone marrow slides were observed under immersion objective to count karyotes.Blood samples in pretreatment-SAP rats and controls were collected at 12 hours after the induction of pancreatitis.The pancreas samples were histologically examined by light microscope,and the levels of amylase were examined by HITACHI-7150. Survival rate was calculated at 72h.Results:No significant difference in serum amylase was found among mobilization group,suppression group and control group.Karyotes could hardly be seen on blood films of bone marrow suppression group. Karyotes of medulla films of bone marrow suppression group were less than the other two groups.No significant difference in serum amylase was found among mobilization-SAP group,suppression-SAP group and con- trol-SAP group.Total pathologic scores of three group were equivalent,but hemorrhage necrosis score was higher in suppression-SAP group than the other two groups.Survival rate of suppression-SAP group was lower than that of other groups at 72h.Conclusion:G-CSF and busulfan were effective to influence the status of bone marrow.Hemorrhage necrosis score was higher in suppression-SAP group than the other two groups and the survival rate of suppression-SAP group was lower than that of other groups at 72h.5.Therapeutic effects of homogenic MSCs transplantation on SAP and the mechanismObjectives:To observe the therapeutic effects of homogenic MSCs transplantation on SAP and to analyze the mechanism.Methods:MSCs used in this part were obtained from male SD rats and method of primary culture was described in part 2.SAP model was induced in female SD rats as described in part 1.At 24h after pancreatitis induction,5-7×10⁷ MSCs of P3 were infused.Twenty four hours later,recipient mice were sacrificed.Organs were taken out,including pancreas, liver,spleen,lung and heart.Tissue DNA was extracted by phenol-chloroform extraction,and PCR was performed to determine the presence of SRY region of Y chromosome from male donor.In some rats, MSCs were stained by CFSE before infusion,then CFSE positive cells were detected with flow cytometry 24h after infusion.Bone marrow cells suspension was observed under fluorescence microscopy to find CFSE positive cells.Tissue RNA of lung and pancreas was extracted,and detected for TNF-α,IL-1βmRNA expression with real time RT-PCR method. Adherence MSCs,pathological section of SAP pancreas before and after infusion were analyzed with immunohistochemistry method for SHH antigen. We also detected SHH,CFSE double labeled or CK19,CFSE double labeled cells on pancreas sections.Survival rate was calculated at 72h.Results:Y chromosome SRY region was present in all those tissues as shown by PCR,and the positive rate was 100%.CFSE stained cells could be found in bone marrow cells suspension.No CFSE cells were found by flow cytometry in blood sample.Expressions of TNF-αand IL-1β mRNA were lower in infusion group than control group.Survival rate of infusion group was higher than that of control group.Both adherence MSCs and pancreas of SAP without infusion group did not express SHH antigen,but after MSCs infusion,SHH could be found in pancreas sections. SHH and CFSE positive areas overlapped in pancreas pathological sections of infusion group.Double labeled CK19 and CFSE cells were not found.Conclusion:MSCs transplantation could improve prognosis of SAP rats,and enhance the survival rate.Engrafted MSCs have homing,migration and planting capacity.Migration or differentiation of MSCs may have a correlation with SHH signal pathway.SUMMARY:Bone marrow mesenchymal stem cells(MSCs) play an important role in severe acute pancreatitis.MSCs transplantation could improve SAP rats' conditions,and enhance the survival rate.Participation of MSCs in pancreatitis is correlated with SHH signal pathway.