The Effect Of BMP10 And BMP13 Inmyocardiocytes Differentiation To C₃ H10T_1/2 Stem Cell In Vitro

PART ONE: AMPLIFICATION OF AdBMP10, AdBMP13 AND AdGFP, THE INFECTION OF C₃H10T_1/2 STEM CELLSObjective: Amplified and collected Adenovirus AdBMP10, AdBMP13, AdGFP with HEK293 cells, then got the high-titer adenovirus. C₃H10T_1/2 stem cells were infected with the adenovirus AdBMP10, AdBMP13 and AdGFP, and got the higher expression of BMP10, BMP13 and GFP.Methods: HEK293 cells could be used to amplify and collect the high-titer adenovirus. HEK293 cells defined under the microscope till the cells cultivated about 70%～80%, the HEK293 cells could be thansfected with the adenovirus AdBMP10, AdBMP13 and AdGFP, the expression of the green fluorescence of the HEK293 cells observed. The project should repeated until the access to get high-titer virus. Then C₃H10T_1/2 stem cells infected with the high–titer adenovirus AdBMP10, AdBMP13, AdGFP, expression of the green fluorescence of the stem cells observed.Results: the adenovirus AdBMP10, AdBMP13 and AdGFP was amplified to high-titer, and the infection efficiency of the C₃H10T_1/2 stem cells have been ideal.Conclusion: The adenovirus AdBMP10, AdBMP13, AdGFP could got high-titer with HEK293 cells amplified repeatedly. And the C₃H10T_1/2 stem cells could infected by the adenovirus efficiently and then detected the high expression of the BMP10, BMP13 and GFP in C₃H10T_1/2 stem cells.PART TWO: THE FEEECT OF BMP10 AND BMP13 IN MYOCARDIOCYTES DIFFERENTIATION OF C₃H10T_1/2 STEM CELLS IN VITROObjective: To investigate the effect of BMP10 (Bone morphogenetic proteins 10) and BMP13(Bone morphogenetic proteins 13) on C₃H10T_1/2 stem cells differentiation into myocardiocyte-like cells in vitro.Methods: the C₃H10T_1/2 stem cells divided into four groups:BMP10, BMP13, GFP and control group. The four cardiac-specific proteins include cTnT, Cx43,β-MHC of the C₃H10T_1/2 stem cells of the four groups were detected by western blot technique and immunofluorescence technique at 7, 14, 21 and 28d. At the same time, the cardiac-specific gene GATA4 and MEF2C,β-MHC expression were deteced by Real-time quantitative PCR after the C₃H10T_1/2 stem cell transfected with AdBMP10, AdBMP13 and AdGFP plasmid. The ultrastructure of the cells were detected by electron microscope and masson staining technique at 7, 14, 21, 28d. The expression of the cardiac-specific structure of cells after transfected by adenovirus AdBMP10, AdBMP13, AdGFP, but the expression of the cardiac-specific functions also detected, after C₃H10T_1/2 stem cells infected, stem cell membrane outward potassium current, inward potassium current and T-type calcium current and a small amount of sodium current were detected at 7, 14, 21 and 28d by whole-cell patch clamp technology.Results:1. After resuscitation, the C₃H10T_1/2 stem cells morphology uniform and became class round under the inverted microscope. Adherent began after 3 to 4 hours, followed by cells elongate, and the cell morphology like similar triangles and spindle. The C₃H10T_1/2 stem cells after infected began to stretch the length; refraction enhanced; cells to the development of long fusiform and triangular; the cell connections between cells began closer and cells were arranged consistency after transfected.2. The expression of the cardiac-specific proteins the C₃H10T_1/2 stem cells cTnT, Cx43,β-MHC were detected by western blot and immunofluorescence. The cardiac-specific proteins expression had already begun at 21, 28d in group BMP10 and BMP13, but not any cardiac-specific proteins expression at 7,14d. And there was not any expression of the cardiac-specific proteins cTnT, Cx43,β-MHC at 7, 14, 21, 28d in GFP group and control group at all.(p<0.05)3. The cardiac-specific gene expression was detected by qPCR technique. at 7, 14, 21, 28d the cardiac-specific gene GATA4 expression of all group include AdBMP10, AdBMP13 and AdGFP and control group,and MEF2C expressed from 14d, thenβ-MHC only indeted at 28d. At the above four time points, the expression of the there genes were more in group BMP10 and BMP13 than in GFP group and control group.4. The cardiac-specific ultrastructure of all experimental groups of the C₃H10T_1/2 stem cells was detected with electron microscope and masson staining techquine. The cardiac-specific ultrastructural myofilament-like and intercalated disc-like structure could be found only in group BMP10 and BMP13 at 28d with electron microscope,and myofilament-like could be found only in group BMP10 and BMP13 at 28d with masson staining techquine.But in GFP and control group could not found any cardiac-specific ultrastructure.5. In this study, the expression of the membrane currents in each C₃H10T_1/2 stem cells experimental group were detected with whole-cell patch clamp. The membrane current of the C₃H10T_1/2 stem cells that transfected by adenovirus AdBMP10, AdBMP13, AdGFP and control group were detected at day 7, 14, 21 and 28. Some currents could found in group BMP10 and BMP13, that includes some outward currents: a super outward activation delayed rectifier K⁺ current(I_Kur); some inward currents: a inward rectifier potassium K⁺ current(I_Kir), T-type Calcium Cacurrent(I_T-Ca), and in a few cells ,the expression of a small amount of sodium current(I_Na), but there was not any membrane currents could be detected at day 7, 14, 21. But the expression of the membrane currents could not be detected at 7, 14, 21 and 28d in GFP group and control group.Conclusion: BMP10 and BMP13 probably promote the differentiation of the C₃H10T_1/2 stem cells to cardiomyocytes in vitro.