Protective Effect Of Mesenchymal Stem Cells On Cardiac Muscle In Rats With Sepsis

Chapter I Culture and identification of bone marrow mesenchymal stem cellsObjective:To separate, culture and purify rat bone marrow mesenchymal stem cells by whole bone marrow adherent culture method in order to develop a sufficient amount of P3 MSCs and its supernatant to meet the needs of the experiment. The changes of the number of mesenchymal stem cells in 3 storage temperatures were detected.Methods:The 60-80g SPF level of SD rats were put to death by cervical dislocation and their bilateral femorals and tibias were separated.Their cavitas medullaris were douched in the super-clean bench.After bone marrow fluid was centrifuged and discarded its thesupernatant, it was cultured with the completed medium of rat bone marrow mesenchymal stem cells in CO2 gas incubator. The growth was observed by inverted microscope, the P3 generation of bone marrow mesenchymal stem cells was detected by flow cytometry.The changes of the number of mesenchymal stem cells at 37 ?,25 ? and 4 ? were observed via Countstar cell counter.Results:Bone marrow mesenchymal stem cells were detected by flow cytometry, and they growed in good condition under the microscopic.There was no significant decrease in the number of cells in a short period of storage at 37 ?,25 ? and 4 ?.Conclusion:Bone marrow mesenchymal stem cells can separate by the whole bone marrow adherent method, its purity can meet the experimental requirements.Chapter II Protective effect of mesenchymal stem cells on cardiac muscle in rats with sepsisObjective:To investigate the effects of mesenchymal stem cells and their conditioned medium on myocardial protective mechanism in rats with sepsis.Methods:55 SPF level SD male rates,were randomly divided into four groups:sham group,septic group,mesenchymal stem cells group and mesenchymal stem cells-conditioned medium group.Sepsis was induced by cecal ligation and puncture method(CLP) except sham group.After operation,tail intravenous 0.5ml 0.9% saline solution was injected via tail vein in sham operation group and sepsis group,0.5ml mesenchymal stem cells-conditioned medium which collected from P3 generation mesenchymal stem cells was injected via tail vein in mesenchymal stem cells-conditioned medium group,2 x 106 P3 generation of bone marrow mesenchymal stem cells that resuspended in 0.5ml 0.9% saline solution was injected via tail vein in mesenchymal stem cell group.Serum was collected after 24h via heart.TNF-??IL-10 and Tn-T were detected by double-antibody sandwich ELISA method.The heart is placed in the 4% poly formaldehyde liquid 48-72h, conventional paraffin embedded, HE staining.Results:TNF- ? value:Compared with septic group,the levels of TNF- ? of mesenchymal stem cells group and mesenchymal stem cells-conditioned medium group were significantly lower,there were statistical significant differences(P<0.01).IL-10 value:Compared with septic group,the levels of IL-10 of mesenchymal stem cells group were higher,there were statistical differences(P<0.05).Among septic group,mesenchymal stem cells-conditioned medium group and sham group,there were no statistical differences(P>0.05).Tn-T value:Compared with septic group,the levels of Tn-T of mesenchymal stem cells group and mesenchymal stem cells-conditioned medium group were significantly lower,there were statistical significant differences(P<0.01).Among mesenchymal stem cells group,mesenchymal stem cell-conditioned medium group and sham group,there were no ststistical differences(P>0.05).Conclusion:Mesenchymal stem cells and their conditioned medium had a protective effect on myocardial injury i rats with sepsis,which may be regulated by inflammatory reaction.