Changes In MRNA And Protein Expression Levels Of NCX1 Between Contractile And Synthetic Phenotype Cultured Vascular Smooth Muscle Cells

Objectives:To explore the changes in mRNA and protein expression levels of NCX1 between contractile and synthetic phenotype cultured vascular smooth muscle cells.Methods:A SD rats vascular smooth muscle cells (VSMC) was cultured according to the previous method. Cell morphological were observed using immunofluorescence. Then, the cells were transfer from contractile phenotype into synthetic phenotype using difference cultured medium. mRNA and protein expression levels of NCX1 were detected by RT-QPCR and Western blotting respectively.Results:1. Both the previous method (by20%FBS+80%DMEM medium with high glucose) and inducing cultured method(by10%FBS+45%DMEM medium with high glucose+45%Ham’s F12)turned out to be successful methods for VSMCs’culture which produced enough cells for us and the cell morphological observation indicated the cells appears a typical " peak and valley " characteristics in addition to a multiple cell morphology under microscopy.2. The VSMC cultured by inducing method shows a faster growth speed and also have more various cell morphology except for a higher sensibility to enzymatic digestion.3. The cells’ cytoplasm glow green while the cell nucleus glowed indigo when the cells were exposed to the Fluorescence microscope’s excitation light after they were combined with a specific fluorescent dye which were combined with a Goat Anti-rabbit IgG who had expecially combined itself to a Rabbit Anti-rat IgM. We can distinguish the α-SM actin clearly from their green texture appears paralleled to the long axis of the cells. The cell immunofluorescence demonstrates that the cells we cultured are Rat’s VSMC.About 483 of the 500 cells we calculated in succession at random Performed positive in the cell immunofluorescence test,it indicated the cells’ degree of purity is 96% which can meet the purity criterion in the subsequent tests.4. The conclusion generated from RT-QPCR indicated that the NCX1’s expression in mRNA levels performed a obvious rise after the VSMC were cultured by inducing method in contrast with those cultured by previous method and the growth rate is about 32%.5. The conclusion we get from the western blotting test also demonstrated that NCX1’s expression in protein levels had a obvious rise after the VSMC evolved from contractile phenotype to synthetic phenotype. And the is about 35.2%(P< 0.05)Conclusion:1. It is a successful method for VSMCs cultured in vitro.2. The inducing cultured medium can transfer VSMCs from contractile phenotype to synthetic phenotype.3. The mRNA expression of NCX1 is up-regulated significantly in contractile phenotype than that in synthetic phenotype.4.The protein expression of NCX1 is also up-regulated significantly in contractile phenotype than that in synthetic phenotype.