Cardiomyocyte-like Cells Differentiation Of BMMSCs Induced By PFT-α And BMP-2in Rat

BackgroundHeart failure induced by Ischemic heart disease showed an upward trend day by day.If the one did not receive treatment timely, large-area myocardial cells will degenerationand necrosis, leading to serious complications and consequences, a serious threat to humanlife. Generally speaking, myocardial cells are terminal cells and it can’t regenerate afterinjury, even if it could, the renewal rate annual is less than1%. Once the myocardial tissueis damaged badly, drug therapy can only control symptoms, but can not repair the injuredmyocardial cells. Heart transplantation is a kind of effective method but can not widelyused in clinical, because it’s exspensive and could not find a donor easily. So looking for anew therapy has been our urgent need. In recent years, cell transplantation has become ahot spot in the treatment of IHD, and develop rapidly. Stem cell differentiation and for theclinical study on the treatment of heart disease is in hand, but the main problem is that thelow differentiation rates remain to be solved at present. Objectives1. Research on the methods of isolation, cultivation and purification of BMMSCs invitro and find a way to do it better.2. Research on cardiomyocyte-like cells differentiation from BMMSCs induced by PFT-α and BMP-2in rat.Methods1. Isolation and culture BMMSCs in vitro and then identification of surface antigenof them: The experiment adopted differential velocity adherent and density gradientcentrifuging togetherly isolate the BMMSCs from SD rat limb long bone, and purify thecell by passaging. Identification of surface antigen CD45(-), CD44(+),CD29(+) of theBMMSCs by Flow cytometry. Observation, record and photo of growth status of cellsunder the microscope.2. The assay of cardiomyocyte-like cells differentiated from BMMSCs induced byPFT-α and BMP-2: We induce the fourth passage BMMSCs and the experimental groupswere as follows: the Control group (complete medium), the PFT-α group (20μ mol/L),the BMP-2group (10μ g/L) and the PFT-α+BMP-2group (PFT-α:20μ mol/L,BMP-2:10μ g/L). Replace the culture of the former3groups after induction for24hours and thePFT-α+BMP-2group after induction for48hours. Assay the cell apoptosis by flowcytometry at the end of induction. Immunocytochemistry and Western blot assay of cellCx43, cTn I expression of cardiomyocytes and the differentiation rate was measured withby flow cytometry.Results1. Morphological characteristics of BMMSCs cultured in vitro: primary cells separatedimmediately are small, transparen and in possession of refractive index. On the third daythe cells grew against the wall of culture flask, the cells varied forms and showed asfollow:oval, stellate, spindle and irregular in shape. After culture the cells1w theyarranged regularly and owe a colony growth. The cells were showed long fusiform andlined up tightly. The results of the positive rate of BMMSCs antigen measured by flow cytometry were as follows: CD45(1.97±0.74)%; CD44(93.03±1.10)%; CD29(93.77±2.44)%.2. The effect of cardiomyocyte-like cells differentiated from BMMSCs induced byPFT-α and BMP-2: The results of cell apoptosis after induction: Q3of the Control group,the PFT-α group, the BMP-2group and the PFT-α+BMP-2group were respectively(89.34±3.18)%,(55.55±5.32)%,(80.44±4.34)%and (70.33±2.61)%. There is nosignificant differences between the BMP-2group and the Control group (P>0.05).Thereis significant differences exist between the other groups (P <0.01).Western Blot results:Cx43expression of the Control group, the PFT-α group, the BMP-2group and the PFT-α+BMP-2group in a week were respectively (0.153±0.068),(0.313±0.036),(0.263±0.024),(0.394±0.054). The Control group was lower than that of the induction groups (P<0.01)and the PFT-α+BMP-2group was higher than that of other groups (P <0.05).There is no significant differences between the PFT-α group and the BMP-2group (P>0.05). Cx43expression of the Control group, the PFT-α group, the BMP-2group and thePFT-α+BMP-2group in4week were respectively (0.342±0.038),(0.763±0.053),(0.627±0.083),(1.065±0.048). The Control group was lower than that of the inductiongroups (P <0.01) and the PFT-α+BMP-2group was higher than that of otherexperimental groups (P <0.05). cTn I expression of the Control group, the PFT-α group,the BMP-2group and the PFT-α+BMP-2group in a week were respectively (0.034±0.001),(0.263±0.026),(0.193±0.033),(0.394±0.045), the Control group was lowerthan that of the induction groups (P <0.01) and the PFT-α+BMP-2group was higher thanthat of the other groups (P <0.05). cTn I expression of the Control group, the PFT-α group,the BMP-2group and the PFT-α+BMP-2group in4week were respectively (0.072±0.014),(0646±0.043),(0.546±0.029),(0.877±0.037). The Control group was lowerthan that of the induction groups (P <0.01) and the PFT-α+BMP-2group was higher thanthat of the other groups (P <0.05). The immunocytochemistry results: expression of Cx43(1w): the Control group had no expression and the induced grou express weakly. After4weeks, the Control groups of had a weak expression, and the induction groups had anenhanced express. expression of cTn I(1w), the Control group had no expression and the induced group express weakly. After4weeks, the Control groups of still have noexpression, and the induction groups have an enhanced express. The differentiation rateof the Control group, the PFT-α group, the BMP-2group and the PFT-α+BMP-2group in4week were respectively (2.04±1.63)%,(26.96±1.90)%,(16.53±2.63)%,(38.48±2.44)%. The Control group was lower than that of the induction groups (P <0.01). ThePFT-α+BMP-2group was higher than that of the other experimental groups (P <0.01).Conclusion1. It is a method to obtain BMMSCs combined with differential adherence method anddensity gradient centrifugation and we can get BMMSCs of high purity when they passageto the fourth generation.2. In fact, the PFT-α+BMP-2group can improve cardiomyocyte-like cellsdifferentiation rate， and the cells owe the similar biological properties as normalmyocardial cells. The cell differentiation rate was significantly higher than that of theBMP-2group or the PFT-α group alone induced. and cell survival rate was significantlyhigher than that of the PFT-α group alone induced, so, it is an efficient and safe method forinducing of BMMSCs.