The Preliminary Study About The Effect Of 5-azacytidine Combined With Appropriate Fluid Shear Stress On Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells In Vitro

part 1 the separation, identification,and cultivation of BMSCs.Objective: To separate, culture and identify the BMSCs in vitro. Methods: BMSCs were isolated from rats marrow mononuclear cells by attaching growth. the cell morphology was Observed with inverted phase contrast microscope. CD29 ,CD34 ,CD44 ,CD105 and Cell Cycle were measured by flow cytometry .Results:⒈The cell morphology of primary cultured cells were polygonal, as colony growth trend.While the passage cells were fusiform, arranged in a direction.⒉The result of detection on the 4th generation of BMSCs showed, CD29 expression was 99.8%, CD44 expression was 99.9%, CD105 expression was 99.4%, while the CD34 expression rate was 1.1%. It indicated that the 4th generation of experimental BMSCs was purified cells.⒊The measurement of cell cycle on the 4th generation BMSCs showed that, G1 phase cells was 80.049%, S phase was 14.832%. That indicated most cells were quiescent, and these were a group of undifferentiated non-targeted cells.Part 2 5-azacytidine(5-Aza) induced differentiation of BMSCs into cardiomyocyte-like cells in vitroObjective: Differentiation of BMSCs into cardiomyocyte-like cells induced by 5-Aza in vitro. Methods: The 4th generation BMSCs were cultured in culture solution in which the density of 5-azacytidine were 3,5,10,15,20μmol/L, and reactive time were 12,24,48h respectively.α-actin, desmin and cTnI were identified by immunofluorescence cytochemistry at 21 days after induction.Consider the 5-Aza's cytotoxicity, combined with cell survival, statistical analysis of striated muscleα-actin expression, then 10 umol/L for 24 hours was established as optimal induction conditions. Experiment was under the optimal concentration of 10μmol /L for 24h. The expression ofα-actin,desmin and cTnI were identified by immunofluorescence cytochemistry in 7,14,21,28 days.Results:1.Consider the survival of the cells andα-actin positive rate, 10 umol/L for 24 hours was established as optimal induction conditions.⒉The cell volume and nucleus increased, and stretched out tubers with different size,and different directions, similar to cardiac-like cell growth after 5-Aza induced .⒊Under condition of 10 umol / L for 24 hours, 7 days identified by immunofluorescence staining technique, there was no expression ofα-actin, desmin, cTnI at the 7th day. While a small amount of expression ofα-actin, desmin exist at the 14th day, but no cTnI.Expression ofα-actin, desmin increase in the number of cells at the 21th day and a small number of cells were visible expression of cTnI. The number of cells with expression ofα-actin, desmin, cTnI increased further at the 28th day.Part 3 Effect of fluid flow-induced shear stress on differentiation of BMSCs into Cardiomyocyte-like Cells induced by 5- AzacytidineObjective: To explore the role of the fluid flow-induced shear stress in the differentiation of BMSCs into Cardiomyocyte-like Cells induced by 5- AzacytidineMethods: The 4th generation BMSCs were seeded in the slide, with a final concentration of 5-Aza was10μmol/L. Slides were fixed in parallel plate flow chamber in the Clean Benches placed after 24h of the intervention.the fluid shear stress loading device was conducted by changing the flow of circulating fluid. Groups: fluid shear stress were 5 dyne/cm2, 15 dyne/cm2, 25 dyne/cm2 reactive for 24h respectively, the control group did not receive mechanical stimulation.After the intervention of mechanical loading, morphological changes and orientation change was observed by inverted microscope and. The positive rate of cTnI was identified by immunofluorescence cytochemistry at the 21th day.Nkx2.5 ,a myocardial expression of growth related gene was determined by RT-PCR at the 7th day after the intervention. Gene expression of cTnI was determined at the 21th day after the intervention.Results:1.After the intervention of mechanical loading, the cell gap widened, shape got length, the cells extend along the long axis of flow chamber .Cells arranged parallel to the direction of the force.⒉After the intervention of mechanical stimulation, the positive expression rate of cTnI increased, the most obvious effect was in the group of 15 dyne/cm2.⒊Nkx2.5, cTnI were positive, but after stimulation of mechanical positive bands is more obviously, the group of 15 dyne/cm2 had most obvious effect, but the effect of the group of 25 dyne/cm2 did not enhance as the force increased.Conclusion:1.BMSCs can be isolated from bone marrow and cultured in vitro.2.5-Aza can induce BMSCs to differentiate into Cardiomyocyte-like Cells.3. 5-Aza combined with appropriate fluid shear stress induced BMSCs into cardiomyocyte-like cells, and it was more effective than single use of 5-Aza.