| Objective:To explore a practical method of isolation and culture of the rat BMSCs in vitro and to observe their living characteristics and to offer an experimental foundation for their further differentiation and actual application Methods:MSCs were isolated from adalt SD rate and then were cultured and cloned by density gradient method and adhesive cultiration.the cell surface antigen of CD44and CD34 in the third generation of MSCs were detected with flow cytometry and immunofluorescenceResults:The isolated MSCs belonged to the mononuclear cells of marrow , the living characteristic was quite stable in L-DMEM medium containing 100ml/ L newborn bovine serum , mostly subcultured cells adhered the wall in 24-48hs , Presented the colony type growth,exhibiting a large expansive potential and high suitability , a typical fibroblastlike morphology , and 80±4.56%expressing CD44 by immunofluorescence,78±5.34%expressing CD44 by flow cytometry technique.Conclusion:The BMSCs of CD44+/CD34-could be effectively isolated and purified by density gradient centrifugation and adhesive cultured in vitro. PartⅡEffects of MRPS on the apoptosis of rat bone marrow mesenchymal stem cells induced by AdriamycinObjective:To determine the effect of Milkvetch Root Parenteral Solution(MRPS) on the apoptosis of bone marrow mesenchymal stem cells (BMSCs)induced by Adriamycinin(ADR) in vitro, Whether thus is the BMSCs treatment cardiovascular disease brings the better clinical practice prospect for the MRPS provide the experiment basisMethods:extracted BMSCs from the thighbone,shinbone marrow and appraised ,Under serum culture media, BMSCs were incubated with ADR and different doses of MRPS for 12h,24h,48h,the experiment include six groups:blank control group, 10μg/l ADR group,10μg/l ADR and MRPS(4μg/l,40μg/l,400μg/l)groups and MRPS 400μg/l group,and then MTT examinate cell multiplication situation, the apoptosis of BMSCs was measured by TUNEL assays and flow cytometry assays.Results:After treated with ADR and MRPS at different concentrations of 40μg/l,400μg/l for 48h, the MTT result shows, the ADR group with ADR+MRPS(40μg/L,400μg/L), the group extinction value comparison has the significance difference (n=3, F=83.149, p<0.01), the apoptosis rates measured by FCM shows , the ADR group, with ADR+MRPS (40μg/l, 400μg/l) group has the significance difference (n=3, F=791.65, p<0.01). Under the microscope,TUNEL examination shows the apoptosis cells number of the ADR+MR (40μg/l, 400μg/l) group was obviously lower than the ADR group, the statistical result shows the apoptosis rate comparison to have the significance difference (n=3, F=612.484, p <0.01) Regarding the ADR+MRPS40μg/L group, 48 hour group OD value with 12 hour, 24 hour group is quite even has the significance difference (N=3, F=12.7, P<0.01) Flows cell technique examination shows the apoptosis rate comparison also to have the significance difference (N=3, F=14.127, P<0.01或0.05) The TUNEL examination shows the apoptosis rate to be consistent with the Flows results, has the significance difference (N=3, F=14.127, P<0.01或0.05) .Conclusion: MRPS can dosage dependently inhibit the apoptosis of BMSCs induced by Adriamycin in vitro, and lengthens along with the response time, anti- apoptosis is enhanced . Chongwei Hao (pediatrics)... |
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