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Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen secretion in vitro. Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method. The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts, Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus. The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer. Cell migration was evaluted by cell wound scratch assay. Western blotting was used to detect expression of α-smooth muscle actin（α-SMA） and collagen Ⅰ（Col Ⅰ）. Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods. The multiplicity of infection（MOI） that lentivirus transduction of myocardial fibroblasts was 100. After lentivirus transduction, 88.33% myocardial fibroblasts expressed green fluorescent protein, showed that the lentivirus could significantly reduce the protein expression of Smad7. Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts, increased the protein expression of α-SMA and decreased collagen secretion. The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts, increased α-SMA protein expression and reduced ColⅠ protein expression. Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function, that is related to the pathological mechanism that lead to myocardial fibrosis
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Objective: To investigate the inhibitory effect of usnicoyinamide on the proliferation of gastric cancer cell line SGC-7901 and its mechanism. Methods: SGC-7901 cells were cultured in vitro and divided into two groups: control group and experimental group with different concentrations of usnicoyinamide. The morphology of each group of cells was observed by a microscope; Proliferation of SGC-7901 cells was measured by MTT assay; The mechanism of apoptosis was studied by AnnexinV/PI double staining and DAPI fluorescence staining; Flow cytometry was used to detect the effect of usnicoyinamide on the cell cycle; Effect of usnicoyinamide on invasion and migration of SGC-7901 cells was detected by cell scratch test. Results: After SGC-7901 cells were treated with usnicoyinamide, the cells were wrinkled, deformed and adherent cells fell off; The results of MTT showed that the inhibition of the proliferation of SGC-7901 cells was a significant dose-effect relationship and time-dependent; The results of AnnexinV/PI double staining showed that nicotine increased the late apoptosis rate of SGC-7901 cells, and DAPI staining showed obvious nuclear concentration and nuclear fragmentation of apoptosis. The results of flow cytometry showed that the cell cycle of SGC-7901 cells stagnated in S phase; Scratch test showed that the mobility of SGC-7901 cells was decreased more obviously with the prolongation of time and the increase of concentration. Conclusion: Usnicoyinamide can inhibit the proliferation of gastric cancer cell line SGC-7901, and its mechanism may be achieved by inducing late apoptosis, inducing S phase cell arrest and inhibiting the invasion and migration of SGC-7901 cells.
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Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfec-tion, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blot-ting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P47.197, P69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.