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The first part AdBMP10 AdBMP13' dissertation">BMP13 and AdGFP amplification and C of 3 the H10T / 2 stem cells infected purpose: use of HEK293 cells amplified collect high titer adenovirus AdBMP10, AdBMP13 well AdGFP infection C 3 the H10T / 2 stem cells was BMP10 BMP13 high expression. Methods: HEK293 cells collected after repeated amplification to obtain high titer adenovirus. HEK293 cells fused to a density of 70% to 80% were added AdBMP10, AdBMP13 and AdGFP observed after 24 hours the amount of the expression of green fluorescence was observed under a microscope. And so forth until the high titer virus. C 3 H10T 1/2 stem cell growth and integration of the density of 70% to 80% AdBMP10 AdBMP13 AdGFP three viruses infected stem cells and observe the amount of green fluorescent protein. Results: AdBMP10 the AdBMP13 AdGFP by HEK293 cells repeatedly amplified high titers of virus. Three viruses were infected with C to 3 the H10T / 2 stem cells and get the ideal rate of infection. Conclusion: After repeated amplification in HEK293 cells can be obtained the high titer AdBMP10, AdBMP13, as well as AdGFP and can efficiently infect C 3 the H10T / 2 stem cells to make stem cells within high expression of BMP10, BMP13, and GFP. The second part of BMP10 and BMP13 in C 3 the H10T / 2 stem cells into cardiomyocyte-like cells in the differentiation process, the role purpose: the study of bone morphogenetic protein 10 (Bone morphogenetic proteins 10 is formed , BMP) and bone morphogenetic protein 13 (Bone morphogenetic proteins 13) to promote C 3 H10T 1/2 stem cells into cardiomyocyte-like role in cell differentiation process. : C 3 the H10T / 2 stem cells were randomly divided into four groups: BMP10 group, BMP13 GFP control group and blank control group. 4 groups of cell the myocardial specific proteins of cTnT were detected by Western blot and immunofluorescence techniques at 7, 14, 21, 28 days four time points, Cx43, β-MHC expression changes; using qPCR technology to detect cardiac-specific genes GATA4 , MEF2C expression of β-MHC; 28 days, electron microscopy and masson staining of cardiac-specific micro-structure. Simultaneous detection of the expression of cardiac-specific functions, using the whole-cell patch clamp technology groups on the cell membrane outward potassium currents, inwardly rectifying potassium current, as well as T-type calcium current, a small amount of sodium current. Results: 1 inverted microscope observation, uniform cell morphology after recovery was class round. Adherent after 3 to 4 hours, followed by cell began to elongate, shape like a triangle, and spindle. Induced cell spreading, refraction enhanced cell development to long fusiform or triangular cell more tightly connected cells arranged consistency. Western Blot and immunofluorescence techniques to detect the expression of cardiac-specific proteins of cTnT, Cx43, β-MHC expression. Experimental results show that in the 21 and 28 day BMP10, BMP13 group has been The cTnT content in serum, Cx43, β-MHC expression, GFP control group and blank control group cells 14, 21 and 28 days had no cardiac-specific protein cTnT, Cx43, β-MHC expression. (P lt; 0.05) 3, qPCR detection of cardiac-specific gene expression. BMP10, BMP13, GFP, and blank control group cells cardiac-specific genes GATA4, MEF2C, and β-MHC expression were detected in 14, 21 and 28 days, four time points. GATA4, MEF2C and β-MHC has its expression in the four groups, but BMP10, BMP13 expression levels are higher than the control group. (P lt; 0.05) 4, electron microscopy and masson staining was detected in each experimental group C 3 H10T 1/2 stem cells in myocardial specific ultrastructure. 28 days of electron microscopy detection BMP10 BMP13 group myocardial the myofilament like well intercalated disc-like structures masson staining more than two sets of cells appear filament-like structure of the cardiac muscle. GFP, blank control group, 14, 21 and 28 days were not found the heart muscle silky and intercalated disc-like structures. 5, whole cell patch clamp detection in each experimental group C 3 the H10T / 2 Stem Cell membrane current expression. BMP10, BMP13, GFP, blank control group membrane currents were measured at 7, 14, 21, 28 days 4. BMP10, BMP13 group 28 days can detect outward currents: ultrarapid delayed rectifier K + current (I kur ); inward currents: inward rectifier potassium current (I kir ). T-type calcium current (I T-Ca ), a small amount of sodium current expression (the I Na ) and can be detected in a small number of cells in the GFP, blank control The group of four time points have failed to have any current expression. Conclusion: BMP10 and BMP13 can promote C 3 the H10T / 2 stem cells into myocardial cells differentiation.
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