| Objective To prepare MSC-MPs to observe their effect on isoproterenol-induced cardiomyocyte hypertrophy,and to analyze its preliminary mechanism.Methods Bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent method.MSCs were identified by cell morphological changes,cell growth curve and detection of specific cell surface antigens by flow cytometry.The 5th generation MSCs were starved for 48 h using MPs-free medium,then the culture supernatant was collected,and MSC-MPs were obtained by high speed gradient centrifugation.The expression of positive markers CD29 and CD90 and negative markers CD34 and CD45 in MSC-MPs were detected by flow cytometry.The protein content and number of MSC-MPs were tested by BCA protein content kit and flow cytometry.Cardiac hypertrophy model was established by treating H9C2 cardiomyocytes with 5μmol·L-1 isoproterenol for 48 h.Effects of 12-48μg·ml-1 MSC-MPs on cardiomyocytes membrane surface area,intracellular total protein content and other indexes were tested or assessed after treatment for 48 h.The protein content of cardiomyocytes in each group was determined by BCA.The m RNA levels of ANP,BNP,β-MHC in cardiomyocytes of each group were detected by q RT-PCR.Moreover,CCK-8 assay was used to detect the changes of cardiomyocytes activity.Hypertrophic cardiomyocytes were treated with microparticles derived from mesenchymal stem cells and supernatant for 48 h to observe their effects.The experiment contained 6 groups.（1）Blank control group（Control）: add culture medium and equal volume of MPsfree PBS;（2）Cardiomyocytes hypertrophy model group（ISO）: add 5μmol·L-1 isoproterenol medium;（3）MP(12μg·ml-1)group: add 5μmol·L-1 isoproterenol and 12μg·ml-1 MPs medium;（4）MP(24μg·ml-1)group: add 5μmol·L-1 isoproterenol and 24μg·ml-1 MPs medium;（5）MP(48μg·ml-1)group: add 5μmol·L-1 isoproterenol and 48μg·ml-1 MPs medium;（6）Supernatant group（S）: add 5μmol·L-1 isoproterenol and equal volume of mesenchymal stem cell supernatant medium.To further analyze the mechanism by which MSC-MPs works,change of intracellular reactive oxygen species（ROS）was detected by fluorescence microscopy and flow cytometry.The experiment had the following four groups.（1）Blank control group（Control）: add culture medium and equal volume of MPsfree PBS;（2）Myocardial hypertrophy model group（ISO）:add 5μmol·L-1 isoproterenol medium;（3）MP(48μg·ml-1)group: add 5μmol·L-1 isoproterenol and 48μg·ml-1 MPs medium;（4）Supernatant group（S）: add 5μmol·L-1 isoproterenol and equal volume of mesenchymal stem cell supernatant medium.Results 1.It was showed that mesenchymal stem cells obtained in this study had a spindleshaped and vortex-like arrangement under the microscope.The growth curve of the cells was roughly "S" shaped.The percentage of single CD29（+）and single CD90（+）MSCs were（97.66±1.30）% and（98.65±0.50）%,respectively.The percentage of single CD45（+）and CD34（+）were（0.31±0.12）% and（1.90±0.52）%,respectively.2.The protein content of MSC-MPs was（592.74±5.20）μg·ml-1.It was showed that the number of MSC-MPs was（2.69±0.21）×107 /ml.The percentage of single CD29（+）and single CD90（+）MSC-MPs were（87.61±3.43）% and（98.04±0.61）% respectively.The percentage of single CD45（+）and CD34（+）were（2.65±0.47）% and（3.43±0.42）%.3.It was showed that 1-30μmol·L-1 isoproterenol dose-dependently increased cardiomyocytes membrane surface area（P<0.001）.Different contents of isoproterenol were applied respectively to cardiomyocytes for 24 h and 48 h.The results of BCA showed that from 1 to 30μmol·L-1 isoproterenol dose-dependently and time-dependently increased the protein content of cardiomyocytes.Compared with the treatment group for 24 h,protein content of cardiomyocytes treated for 48 h increased by 23%（P<0.001）.Therefore,5μmol·L-1 isoproterenol was used for 48 h to establish a model of myocardial hypertrophy.4.It was showed the effects of MSC-MPs on the surface area of hypertrophic cardiomyocytes.Compared with the ISO group,MSC-MPs at 12,24,48 μg·ml-1 reduced the cell surface area from（1572.90±122.23）to（1215.51±107.19）,（1167.94±46.05）,（949.71±36.05）μm2（P<0.001）,respectively.Results showed that supernatant group had no significant effect on the surface area of hypertrophic cardiomyocytes.5.It was showed the effects of MSC-MPs on the protein content of hypertrophic cardiomyocytes.Compared with control group,the protein content of isoproterenol group significantly increased（P<0.001）.In the range of 1248μg·ml-1,MSC-MPs dose-dependently reduced the cell protein content.Compared with isoproterenol group,the cell protein content decreased by 24%（P<0.001）.Moreover,supernatant group had no significant effect on the protein content of hypertrophic cardiomyocytes.6.It was showed the m RNA levels of ANP,BNP,and β-MHC in cardiomyocytes,and ANP,BNP and β-MHC level increased by 3.2,1.3 and 3.2 times respectively,after isoproterenol stimulation for 48 h.It was also found that 12-48μg·ml-1 MSC-MPs dose-dependently inhibited m RNA expression in hypertrophic cardiomyocytes（P<0.05）.Changes of intracellular m RNA expression in the supernatant group were not statistically significant.7.It was showed the effects of isoproterenol on the proliferation of H9C2 cardiomyocytes.The results showed that the proliferation of H9C2 cardiomyocytes was not affected by application of 5μmol·L-1 ISO and 12-48μg·ml-1 MSC-MPs by CCK-8 assay.8.It was showed the effects of MSC-MPs on ROS level.Compared with control group,ROS level in isoproterenol group significantly increased from（10.14±0.70）% to（53.51±2.91）%（P<0.001）,and 48μg·ml-1 MSC-MPs reduced the ROS level back to（11.28±0.59）%（P<0.001）,indicating that MSC-MPs significantly inhibit isoproterenol-induced ROS increasing and reduce the intracellular ROS to almost normal level（P<0.001）.Conclusions 1.MSC-MPs were successfully induced and obtained from cultured rat bone marrow mesenchymal stem cells.2.In the range of 12-48μg·ml-1,MSC-MPs significantly inhibited isoproterenolinduced cardiomyocytes hypertrophy,and its mechanism was related to the reduction of intracellular reactive oxygen species. |
PDF Full Text Request