The Protective Effect Of Endometrial Stem Cells On Myocardial Ischemia Reperfusion Injury In Mice

Objective: To elucidate the relationship between the myocardial protective effect of EnSCs derived cytokine cocktail(EdCC)and MEK/ERK signaling pathway in endometrial stem cells(EnSCs).Methods: The culture medium was incubated in 37℃,5% CO2 incubator,and the culture medium was changed after24 hours.After each day,the culture was up to about 70%.The culture medium was cultured to the third generation and identified by flow cytometry EnSCs express mesenchymal stem cell surface markers,and then EdCC was obtained from the cultured EnSCs supernatant by Millipore technique,and the protein was quantified for trial or-80°C.The model of myocardial ischemia-reperfusion injury was prepared by ELISA.The model of myocardial ischemia-reperfusion injury was established by ligating the anterior descending coronary artery.According to the experiment,we need to inject EdCC and the corresponding control group with different dose or different cryopreservation time.After 24 hours of reperfusion,TTC/Evans blue is used to stain the mouse I/R myocardium,and the infarct size is calculated by image pro software.The expression of ERK1/2 and cleaved caspase-3 were detected by Western blot.The expression of ERK1 / 2 was detected by immunohistochemical staining with TUNEL staining and immunofluorescence staining.Mice were divided into normal group,control group and EdCC group.Oxidative stress was induced by hydrogen peroxide(H2O2).The activity of lactate dehydrogenase(LDH)in supernatant was detected.Apoptosis was also detected by TUNEL staining.DCFH-DA was used to detect intracellular reactive oxygen species(ROS)concentration.Results:1,In vitro culture of EnSCs adherent growth,was spindle-shaped,fusion formed after the vortex-like structure.The expression of CD90 on CD34 and CD45 hematopoietic cells was observed by flow cytometry.Enpis expressed mesenchymal stem cells on the surface markers.The Millipore protein ultrafiltration technique was used to trap EGF protein in supernatant of nearly 90%EnSCs.2,The concentration of EnCls supernatant was(200.5± 11.2)μl and the protein concentration was(5.57± 0.95)μg / μl,and the extraction efficiency of EdCC was increased every 24 hours(222.4 ±29.3)μg/106 cells.3,-80 ℃ long-term storage can lead to EdCC protein concentration and the total gradually reduced.4,EdCC can protect the mouse myocytes induced by oxidative stress-induced cell injury and apoptosis.5,EdCC to reduce the ischemia and reperfusion injury in mice injected100μg as the best dose.6,EdCC can promote myocardial ERK1/2 phosphorylation.7,EdCC-mediated ERK1/2 phosphorylation can be blocked by PD98059.8,EdCC through MEK1-ERK signaling pathway to reduce myocardial ischemia-reperfusion injury.9,EdCC through the MEK1-ERK signal pathway inhibition of apoptosis.10,EGFR is partially involved in the myocardial protection of EdCC.Conclusion: 1,The culture of EnSCs supernatant successfully prepared for the direct use of EdCC.2,The results of the study of adult stem cell therapy from the cell to the cytokine has important theoretical significance.3,The protective effect of EdCC on myocardial ischemia-reperfusion injury was verified by three aspects: tissue necrosis,apoptosis and apoptosis protein expression.