| Objective:1.Explore the method of separation,culture and purification of adipose-derivedmesenchymal stem cells(ADMSC)in vitro and identify them according totheir phenotypes,so as to provide a broader choice for the source ofmesenchymal stem cells.2.Establish and evaluate animal models of small intestinal ischemia-reperfusion injury in rats.3.To explore the repair effect and mechanism of adipose mesenchymal stemcells on small intestinal ischemia-reperfusion injury.Method:1.Take normal abdominal adipose tissue in male rats;use adherent culturemethod to isolate,culture and purify its ADMSC.ADMSC grow to 70-80%for passage;ADMSC obtained in the first passage is the first generation(P1),Use P3-P5 for subsequent tests.2.Establish a small intestine IR injury model in male SD rats: After 12 hours offasting in SD rats,2% pentobarbital(2mg / kg)is injected intraperitoneallyto anesthetize SD rats(200-250g),and the abdominal skin is disinfected andThe abdominal cavity was opened,the superior mesenteric artery wasisolated,and the vascular clip was removed 60 minutes after thenon-invasive small blood vessel clip was used to close the suture of theabdominal wall,and the model of small rat intestinal IR injury wassuccessfully established.3.Adopting the intraperitoneal injection treatment of ADMSC to observe therepairing effect of small intestine ischemia-reperfusion injury and theregulation mechanism;Result:1.Successful separation,cultivation,and purification of ADMSC.Observationof inverted cells by inverted phase contrast microscope showed that morecells adhered after 24 hours of primary inoculation.After 72 hours,thenumber of cells gradually increased,and the cell morphology mostly grew ina spindle shape.After that,the number of contaminated cells graduallydecreased and ADMSC was gradually purified.The CD29 and CD90 werfound to be highly expressed and the CD45?CD34 were all poorly expressedby cell surface markers,thus further identified as adipose mesenchymal stemcells.2.After the superior mesenteric artery was clamped in the experimental group,the anterior mesenteric artery pulsation disappeared,and the intestinal canalgradually turned white,spasms,and even shrunk.After restoring bloodsupply to the blood vessels,the arteries gradually resumed pulsation,and theintestinal tract gradually resumed peristalsis.The color turned to bright red.Intestinal tissues were observed under conventional pathological microscope.Most specimens were severely damaged,ulcerated and bleeding,and thelamina propria was severely damaged.At the same time,a large amount ofneutrophil infiltration was accompanied by local bleeding of the mucosa.Atrophy confirmed the successful establishment of a small intestineischemia-reperfusion injury model.3.ADMSC can significantly reduce the release of IL-6 and other inflammatoryfactors,promote tissue anti-inflammatory and small intestine blood vesselregeneration,its protective effect is related to the regulation of COX2-PGE2signaling pathway,and promote the repair of small intestine IR injury.Conclusion:1.It is feasible to use rat adipose stem cell extraction method and culturetechnology,and successfully isolate,culture and purify ADMSC,and its cellpurity is relatively high.At the same time,due to the advantages of sufficientadipose tissue source and easy access,it can provide sufficient cells forsubsequent experimental needs.2.The model of intestinal ischemia-reperfusion injury was established byclipping the superior mesenteric artery in rats,which caused thedisintegration of the innate layer of intestinal mucosa,bleeding and evenulcer formation.It can be used as an animal model of this experiment.3.Intraperitoneal injection of ADMSC can significantly reduce inflammationand promote the repair of small intestinal IR injury through the COX2-PGE2signaling pathway. |
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