The Effect And Significance On Expression Of E-selectin In Lung Tissue Of Rat With Severe Acute Pancreatitis Transplanted By Bone Marrow Mesenchymal Stem Cells

Severe acute pancreatitis（SAP） is a refractory disease with complicated disease risks,heavy complications and high fatality rate. In the acute phase,SAP could induce acutelung injury（ALI） with capillary membrane diffusion failure.ALI may result in pulmonaryedema and pulmonary atelectasis, even acute respiratory distress syndrome（ARDS）.Atpresent clinical treatment still has no specificity and validity. Thus,ALI becomes one ofthe most common and severe diseases among SAP. Lung contains a large number ofcapillaries with rich blood flow,Excessive release of inflammatory mediators in bloodcauses lung to be the main target injury organ.The pulmonary vascular endothelialcells（PVEC）were important target cells with ALI.Activated PVEC enable leukocytes toextravascular migration, which is the key link to ALI.E-selectin（Es） mediates thisprocess.With the increased activity of PVEC, Es expression increased.Elevation of PVECactivity is the important stage of vascular lesions.Therefore, the high expression of Eswith PVEC contributes to the suggestive of pulmonary vascular disease early endothelialcell activation, a further sign of endothelial cell injury.According to he latest sthdy,forALI adult tissue derived mesenchymal stem cells （BMSCs） has such functions as toregulate immunity, to repair microenvironment or replace damaged lung tissue cells. Thisstudy aims to explore the dynamic change characteristics of Es in lung tissue in SAPanimal model. Discusses the distribution of BMSCs in lung tissues, and the impact on Esin lung tissue and serum inflammatory mediators after treatment with allogeneic BMSCs.[Objectives]1. Establish a reliable, convenient, reproducible SAP animal model with obviousALI performances, and understand the course laws and pathological changes with SAPrat，which will provide a good animal model for further study.2. Analyze the severity of rat lung injury induced by SAP, and explore Es expression rule of the lung tissue in this model, which will provide the basis for the nextstudy on SAP-associated lung injury intervention.3. Establish the scientific method of in vitro separation, purification and rapidexpansion of BMSCs with SD rat to obtain sufficient BMSCs and identify BMSCsaccording to suffice markers, which will provide an experimental basis material for thenext sthdy on BMSCs intervention therapy of SAP-associated lung injury.4. Test Es expression in lung tissue, level of TNF-α and IL-1β through BMSCs onSAP-associated lung injury. Explore whether BMSCs can have good effects on injurylung by inhibiting Es expression and release of inflammatory mediators.[Methods]1. SAP model was induced by retrograde injection of5%tauroursodeoxycholic acid.Clean male SD rats were randomized into the experimental group（SAP） and the shamoperation group（SO）.Rats were intraperitoneally injected with10%of chloral hydrateanesthesia, exposed pancreas region, and identified pancreatic duct. SAP model wasprepared through retrograde injection of5%tauroursodeoxycholic acid,the pancreaticduct was ligated. Rats in SO group were only flipped pancreas. Examined SAP rats forany pancrease tissue damage at3,6,12and24h,and observed changes of serum amylase，albumin and calcium.2. Clean male SD rats were randomized into the experimental group（SAP） and thesham operation group（SO）. SAP model was prepared through retrograde injection of5%tauroursodeoxycholic acid.Observed all rats in the two groups for any lung tissue damageand Es expression in lung tissue at3,6,12and24h.3. Cultured BMSCs by chosing whole bone marrow adherent culture. In sterilitycondition,both lower limbs of the SD rats were removed and femur medullary cavitieswere flushed with Dulbecco’s modification of Eagle’s medium/F12（DMEM/F12）. Thedouche was centrifuged. Then resuspended the deposition with DMEM/F12,10%fetalbovine serum（FBS） and1%doubie-antibiotics. The cells suspension of2×10⁵/m1wasinnoculated in25cm²cell culture flask and cultured in the incubator at37°C in a saturatedhumidity atmosphere with5%CO₂. Morphology and growth of BMSCs were observedevery day. BMSCs were subcultured and amplificated at a rate of1:2or1:3. After passage 3,CD90,CD29,CD34,CD45antigen on the surface of BMSCs were detected with flowcytometry analyses（FACS）.4. Prepared SAP model through retrograde injection of5%taurocholic acid. Takeonly4for BMSCs tracer observation,and the rest were randomized into the Control-SAPgroup,BMSCs-SAP group and MP-SAP group. The passage3BMSCs were infused intothe rats by caudal vein with1ml/100g（1×10⁶cells/ml）1h after SAP model induction（BMSCs tracer rats were infused the CM-DiI stained BMSCs）.MP-SAP rats were dealedwith methylprednisolone sodium succinate（MP）. Control-SAP animals were withouttreatment only injected with saline. Examined SAP rats of every groups for any lung andpancrease tissue pathology, serum index and Es expression in lung tissues at3,6,12,and24h. Watch the distribution of CM-DiI positive cells（BMSCs） in lung tissue of SAP rats.[Results]1. ALI or ARDS and other clinical manifestations occurred in SAP rats. Pathologicalinjury of pancreas aggravated gradually.Showed bloody ascites, coagulation necrosis ofthe pancreas jelly-like plaques and fat saponification and so on. The serum amylase andhistological, pancrease tissue damage of SAP group rats were significantly higher thanthat of SO group rats（p<0.05）.48-hour survival rate of animals in SAP group dropped.2. SAP group rats,as the disease develops, pulmonary edema, ALI or ARDS andother clinical manifestations occurred.Pathological damage of lung tissue aggravatedgradually.It showed varying degrees of interstitial lung edema,inflammatory cellsinfiltration, alveolar exudation and hemorrhage, widened alveolar cavity collapse andatelectasis, reality of the lung tissue and so on.Es expression and W/D of lung tissue weresignificantly higher than that of SO group rats,and Es protein distributed in membraneand cytoplasm of PVEC.3. Adherent cells were seen when primarily cultured for24hours and the shapeswere morphologically diversified,BMSCs showed colony-like distribution at the7th-8thday. Cells were purified by changing the medium and subculturing. In passage3, BMSCshad uniform shapes,mostly slightly wide long spindle,fibroblast-like shape andwhirlpool-like distribution.FACS showed that the percentage of CD29⁺CD90⁺CD34^-CD45^-BMSCs was over91.3%.Phenotype-specific antigen was in line with BMSCs surface characteristics，which provide seed cells for the intervening study of Es in injurylung induced by SAP.4. In BMSCs-SAP group, mentality and breathing of the rats improved,48-hoursurvival rate significantly increased. CM-DiI positive cells（BMSCs） could be seen in lungtissue of BMSCs tracer rats by fluorescent microscope. Real-time RT-PCR analysis andimmunohistochemical analysis showed that Es expression of the BMSCs-SAP group ratssignificantly decreased compared with Control-SAP group. W/D in lung tissue,lung andpancreas pathological damage and serum amylase,TNF-α,IL-1β were significantly lowerthan that of Control-SAP group rats. Serum albumin was markedly higher than that ofControl-SAP group rats. However, the effects in BMSCs-SAP group at each time pointwere almost significantly worse than that of MP-SAP group rats（p<0.05）.[Conclusions]1. The SAP model with respiratory symptoms of ALI in rats is establishedsuccessfully through retrograde injection of5%tauroursodeoxycholic acid. It resultes insevere pathological damage of pancreas and symptoms of ALI in rats. A goodexperimental animal model is provided for the study of SAP-associated lung injury.2. SAP early cause irreversible pulmonary disease reason damage and thecorresponding lung function damage performance.When lung injury is induced by SAP,the expression of Es in PVEC rapidly increases and then slowly dropped. Es mediates inthe rolling and adhesion of leukocyte with blood vessel linings,then the migration andseepage through blood vessel. The early Increase of Es reflects early the activation degreeof PVEC and endothelial barrier function imbalance.3. Adherent culture of whole bone marrow can gain highly purified BMSCs. Purityof P3BMSCs is over91.3%. FACS results show that BMSCs are positive for CD29andCD90and negative for CD45and CD34.Purity of passage3BMSCs is over91.3%. Itconfirmes that this method is simple, applicable and efficient.4. Allogeneic BMSCs can transfer to and settle at damaged lung tissue, and showthe beneficial effects in lung injury induced by SAP. The results show that BMSCs areable to regular immune response caused by SAP and alleviate lung immune damages byinhibiting over-releasing of inflammatory mediators. Simultaneously, BMSCs decrease Es expression so as to reduce intravascular leukocyte rolling, adhesion and migrationbehavior,block the leukocyte activation and release of damage material. And they helpto stabilize and protect endothelial cells, promote the complete pulmonary vascularendothelial barriers function, effectively prevent intravascular material leakage, andimprove lung function. Early emergency efficacy of methylprednisolone sodiumsuccinate in SAP rats is obvious, but the rich biological functions of BMSCs play a moreimportant role in tissue repair in the later period of SAP.