| Objectives:To isolate bone marrow mesenchymal stem cells(BMSCs)with multiple differentiation potential from rat bone marrow:rat bone marrow culture and adherence screening.Methods:Morphological characteristics of cells were observed under microscope.Cell surface antigen was identified by flow cytometry.Osteogenesis,adipogenesis,cartilage differentiation test to detect cell differentiation function.Results:Bone marrow mesenchymal stem cells were isolated and cultured by adherent screening method.When the third generation was close to 80%-90%,the adherent cells were observed.The morphology of the cells was similar to that of fibroblasts.,No significant changes in cell morphology.Flow cytometry showed that more than 90%of rat bone marrow mesenchymal stem cells express CD29(CD8)CD90;more than 80%of rat bone marrow mesenchymal stem cells express CD44(adhesion molecules,mediate cell-to-hyaline Acid and osteopontin adhesion),CD73.Do not express CD34(progenitor cell surface differentiation antigen)CD45 and hematopoietic progenitor cell surface marker CD11b,consistent with rat bone marrow mesenchymal stem cell characteristics.Osteogenic,adipogenic and cartilage-induced differentiation showed that the cytoplasmic staining was observed after 72 hours of adipogenic induction.After about 5 days,the lipid droplets appeared in the cells,and the number of lipid droplets increased and fused with each other,The cells are changed from long spindle to round or polygonal.After 15 days of induction,oil red O staining showed a large amount of lipid precipitation.10 days,the cytoplasm was filled with granules,the cells were clustered and aggregated,and a small amount of calcium deposition was observed between the cells.After 10 days,the cells of the cell nodule were gradually fused Loss of cell structure,calcium nodules formed significantly;16 days,the cell surface appears a lot of calcium deposition,the alizarin red staining was red nodules.After 24 hours of cartilage induction,the cells were granulated in the granules,and the surface of the cell clusters gradually became smooth.After 21 days of induction,the stained cells showed cartilage characteristics.Consistent with the differentiation of bone marrow mesenchymal stem cells.Conclusion:Rat bone marrow mesenchymal stem cells can be successfully isolated and purified by adherent screening and purification.Objectives:To establish an rat model of heterotopic heart transplantation with modified Ono techniqueMethods:The rat heart was used as donor and SD rat as the receptor,and the model of rat heterotopic heart transplantation was established by modified Ono technique.After the Doppler heart color Doppler ultrasound,HE staining model was used to establish the effect.Results:Left ventricular end diastolic diameter(LVEDd),left ventricular end-diastolic diameter(LVEDs),left ventricular ejection fraction(LVEE),and left ventricular short-axis shortening index(FS)were measured at 7 days and 15 days after heart transplantation)Showed successful establishment of rat abdominal heterotopic heart transplantation model.Transplantation of the heart shape can be seen:transplanted 7 days after the transplanted heart color and just transplanted the same color of the heart is red,intracavitary visible effusion.15 days after the heart color became dim,the surrounding bowel swelling.30 days after transplantation,the heart color darkens and sticks with the surrounding bowel.HE staining showed:7 days of transplantation of myocardial cells arranged slightly disorder around the blood vessels can see a small amount of inflammatory cell infiltration.15 days after transplantation,myocardial cells arranged disorder,blood sinus cells visible inflammatory infiltration.30 days after myocardial myocardial cell arrangement is very disorder,a large number of inflammatory cells around the vascular infiltration.Conclusion:The rat model of heterotopic heart transplantation was established by modified Ono technique,and the mortality rate was reduced.Objectives:To specifically recognize rat BMSCs secreted by Exsome to play an immunosuppressive target cell,and further validate the in vitro results in vivo.To clarify the role of Exsome secreted by rat bone marrow mesenchymal stem cells in the survival of heart transplanted.Methods:1:exosome and BMSCs were co-cultured in vitro.The exosome of BMSCs was co-cultured with T cells,and the exosome of BMSCs was co-cultured with DC cells and T cells.DC cells were cultured separately,T cells were cultured separately,and C cells were co-cultured with T cells as a negative control.The exosomes secreted by BMSCs cells were adjusted to a final concentration of 800 mg/ml,and the concentration of DC cells and T cells were increased.After 48 h,72H and 96H,the cells were counted by flow cytometry Cell and T cell surface molecular expression.2:rat abdominal ectopic transplantation heart model after the establishment of the tail vein by injection of BMSCs secreted exosome,given as Maike feeding as a drug control,do not do any treatment group as a negative control,normal transplantation group as a negative control,in the same The expression of DC cells and T cells on the surface of the cells was measured by flow cytometry after 2,4,7 days of feeding.3:rat abdominal ectopic transplantation heart model after the establishment of the tail vein by injection of BMSCs secreted exosome,given as Maike feeding as a drug control,do not do any treatment group as a negative control,under the same conditions,feeding 2,4,7 Day using Doppler heart color Doppler ultrasound and small animal live imaging to assess transplanted heart function.Results:1:The effect of exosome secreted by BMSCs on DC cells was not obvious when co-cultured with 48H and 72H,and the effect on DC cells began to change regularly when co-culture reached 96H.When the exosome secreted by BMSCs was co-cultured with T cells,the expression level of Treg cells was the highest at 48H and 72H,and decreased at 96H,but there was no significant difference.The expression of CD3 + CD4 was the lowest at 48H and 72H,and increased at 96H.CD3 + CD8 expression in 48H,72H,96H are the lowest.2:The expression of exosome on BMSCs was not significantly different from that of DC cells,and the expression level of Treg cells was the highest in each group after 2 days of feeding,but decreased after 4 days of feeding The difference was significant in the untreated group and the normal group,and the expression of the normal group was decreased after 7 days of feeding,but there was no significant difference compared with the untreated group and the untreated group.3:The results of cardiac color Doppler ultrasonography showed that the cardiac ejection fraction was significantly higher in the untreated group than that in the untreated group,and there was no significant difference between the two groups The cardiac ejection fraction was significantly higher than that of the untreated group(P<0.05).The cardiac function of the control group was increased after the exosome treatment was Secretory exosome treatment was performed 7 days after transplantation,the cardiac ejection fraction was significantly improved compared with the other groups.The results of small animal live imaging showed that the intensity of cardiac fluorescence was the strongest at 2 days,4 days and 7 days after the establishment of BMSCs ex vivo.Conclusion:The exosome of rat bone marrow mesenchymal stem cells exerts immunosuppressive target cells as T cells,which can promote exosome accumulation in the transplanted heart and improve its function. |
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