| This study includes three parts, we through the establishment of SAP model of ratto study in each part. We observed phenomenon of small intestinal capillary leakagein SAP rats in the first part; explored the mechanism of small intestinal capillaryleakage in SAP rats in the second part; observed the therapeutic benefits by allogeneicBMSCs on small intestinal capillary leakage in SAP rats and to analyze the possiblemechanisms in the third part. We try to find a treatment by animal experiment toprevent small intestinal capillary leakage in SAP.1.The small intestinal capillary leakage in SAP ratsObjective: To observe phenomenon of small intestinal capillary leakage in SAPrats, to investigate the size of small intestinal capillary leakage with time and therelationship between the size of small intestinal capillary leakage and the severity ofSAP.Methods: SAP animal model was developed in adult male rats by retrogradeinjection of5%deoxy sodium taurocholate. The lanthanum nitrate and fluoresceinisothiocyanate-labeled albumin tracer was used to observe the phenomenon of smallintestinal capillary leakage in SAP rats, the Evans blue assay was used to measureintestinal capillary leakage, serum and ascites of biochemical were measured,pathological score of pancreas and small intestine were measured.Results: As compared with sham operation groups, the level of serum albumin inthe SAP rats was significantly decreased (P<0.001), the level of serum amylase,ascites amount, ascites albumin, pancreas and small intestine pathologic score in theSAP rats were significantly elevated (P<0.001).From6h to24h serum albumin decreased, the amount of ascites, ascites albumin, pancreas and small intestinepathologic score gradually increased in the SAP rats (p <0.05). As compared withsham operation groups, the level of intestinal tissue Evans Blue content in the SAPrats were significantly elevated (p <0.001), from6h to24h intestinal tissue EvansBlue content gradually increased (p <0.05). The size of the small intestinal capillaryleakage had positive correlation with the level of the small intestinal pathologicalscore (r=0.93P<0.001), pancreas pathological score (r=0.85P<0.001), ascitesamount (r=0.78P<0.001) and ascites albumin (r=0.58P<0.001), had negativecorrelation with serum albumin (r=-0.81P<0.001).Conclusion: Small intestinal capillary leakage occurred in SAP rats. The level ofserum albumin decreased, the level of ascites amount, ascites albumin increased inSAP rats. Intestinal capillary leakage gradually increased from6h to24h. The size ofintestinal capillary leakage determines the extent of the small intestinal pathologicalchanges in SAP rats. The extent of the pancreas pathological changes determines thesize of intestinal capillary leakage, pancreas pathological changes more obvious, themore serious intestinal capillary leakage.2. Explored the mechanism of small intestinal capillary leakage in SAP ratsObjective: To explore the mechanism of small intestinal capillary leakage in SAPrats.Methods: A SAP rat model was setted up. Small intestinal VASP and MMP-9expression was determined by imunohistochemistry, real-time quantitative PCR andWestern blotting. Capillary endothelial barrier changes were examined via electronmicroscopy.Results: Compared with the sham group, both VASP mRNA and proteinexpression levels increased in the SAP rats. The VASP expression in the SAP group at12h was higher than that at6h. However, the VASP expression at24h was less thanthat at12h. The differences were statistically significant (p<0.05). Compared with thesham group, the MMP-9gene and protein expression levels both increased in the SAPrats. The MMP-9expression of the SAP group at12h was significantly higher than that at6h (p<0.05). However, the MMP-9expression at24h was less than that at12h(p<0.05). Under the electron microscopy, gradually widened gaps betweenconnections of small intestinal capillary endothelial cells, dissolved membranes ofsome mitochondria, unclear mitochondrial crista, deregulation of rough endoplasmicreticulum, dissolved cell membranes of some capillary endothelial cells, and swellingand fracture of the basement membrane were observed.Conclusion: Various inflammatory mediators and cytokines which are releasedduring SAP may activate MMP-9which can degrade the capillary endothelialbasement membrane. These substances may also upregulate VASP, which could leadto cytoskeletal protein rearrangement and changes in the connections and gapsbetween capillary endothelial cells. In addition, they may directly attack capillaryendothelial cells and result in cell apoptosis and necrosis. All these changes canincrease the intestinal capillary endothelial barrier permeability in SAP, and result inintestinal capillary leakage, intestinal edema, production of ascites with increasedalbumin, and decreased serum albumin.3. Treatment of allogeneic bone marrow mesenchymal stem cell transplantationfor small intestinal capillary leakage in SAP ratsObjective: To observe the therapeutic benefits by allogeneic BMSCstransplantation on small intestinal capillary leakage in SAP rats and to analyze thepossible mechanisms. We try to find animal experimental evidence to prevent smallintestinal capillary leakage in SAP.Methods: Bone marrow samples were collected from healthy youngmale Sprague-Dawley rats in sterility condition. Culture and purification of BMSCs totreat SAP rats. Small intestinal VASP and MMP-9expression was determined byreal-time quantitative PCR and Western blotting. Capillary endothelial barrier changeswere examined via electron microscopy. The Evans blue assay was used to measureintestinal capillary leakage, serum and ascites of biochemical were measured,pathological score of pancreas and small intestine were measured. Serum IL-1β andTNF-α levels were determined by ELISA. The BMSCs in the distribution of small intestinal tissue was observed by immunohistochemical approach.Results: BMSCs were cultured and purified successfully. SAP rats were treatedby allogeneic bone marrow mesenchymal stem cell transplantation. Compared withthe SAP group, both VASP mRNA and protein expression levels, both MMP-9mRNAand protein expression levels, and intestinal capillary leakage were decrease in theBMSCs-SAP rats(P <0.05). After allogeneic BMSCs transplantation, serum IL-1β andTNF-α levels, the amount of ascites, ascites albumin, pancreas and small intestinalpathological scores decreased, serum albumin increased. BMSCs were observed in thesmall intestinal tissue by immunohistochemical approach. Under the electronmicroscopy, widened gaps between connections of small intestinal capillaryendothelial cells, swelling and fracture of the basement membrane were better thanSAP rats. The phenomenon of dissolving cell membranes of some capillaryendothelial cells were disappearance.Conclusion: By allogeneic bone marrow mesenchymal stem cell transplantation,inflammatory mediators and cytokines are released reducing in BMSCs-SAP rats. It isless that they directly attack capillary endothelial cells and result in cell apoptosis andnecrosis. MMP9which can degrade the capillary endothelial basement membrane andVASP which could lead to cytoskeletal protein rearrangement and changes in theconnections and gaps between capillary endothelial cells were decrease. Through thismechanism to maintain the integrity of capillary endothelial barrier, decrease theintestinal capillary endothelial barrier permeability in SAP by allogeneic bone marrowmesenchymal stem cell transplantation.SummaryA SAP rat model was setted up. From organs, tissues, cells and subcellular levelssmall intestinal capillary leakage were observed in SAP rats. The level of serumalbumin decreased, the level of ascites amount, ascites albumin increased in SAP rats.Intestinal capillary leakage gradually increased from6h to24h. The size of intestinalcapillary leakage determines the extent of the small intestinal pathological changes inSAP rats. The extent of the pancreas pathological changes determines the size of intestinal capillary leakage, pancreas pathological changes more obvious, the moreserious intestinal capillary leakage. Various inflammatory mediators and cytokineswhich are released during SAP may activate MMP-9which can degrade the capillaryendothelial basement membrane. These substances may also upregulate VASP, whichcould lead to cytoskeletal protein rearrangement and changes in the connections andgaps between capillary endothelial cells. In addition, they may directly attackcapillary endothelial cells and result in cell apoptosis and necrosis. All these changescan increase the intestinal capillary endothelial barrier permeability in SAP, and resultin intestinal capillary leakage, intestinal edema, production of ascites with increasedalbumin, and decreased serum albumin. By allogeneic bone marrow mesenchymalstem cell transplantation, inflammatory mediators and cytokines are released reducingin BMSCs-SAP rats. It is less that they directly attack capillary endothelial cells andresult in cell apoptosis and necrosis. MMP9which can degrade the capillaryendothelial basement membrane and VASP which could lead to cytoskeletal proteinrearrangement and changes in the connections and gaps between capillary endothelialcells were decrease. Through this mechanism to maintain the integrity of capillaryendothelial barrier, decrease the intestinal capillary endothelial barrier permeability inSAP by allogeneic bone marrow mesenchymal stem cell transplantation. |
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