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目的:探讨β3肾上腺素能受体(β3-AR)对SD大鼠乳鼠心肌肥大的影响及其机制。方法:体外培养SD大鼠乳鼠心肌细胞,用携带β3-AR基因的慢病毒转染细胞后,用去甲肾上腺素(NE)诱导细胞48h,建立心肌细胞肥大模型。实验分4组:空白对照组(Control组)、心肌肥厚组(NE组)、β3-AR基因转染+NE组(β3-AR组)、空病毒转染+NE组(空病毒组)。用免疫荧光法鉴定心肌细胞,倒置荧光显微镜观察病毒转染组绿色荧光蛋白(GFP)表达,免疫印迹法(Western Blot)检测β3-AR、丝裂原活化蛋白激酶p38(p38MAPK)、细胞外信号调控激酶(ERK1/2)、磷酸化p38MAPK(p-p38MAPK)和ERK(p-ERK1/2)及原癌基因c-myc、c-fos蛋白水平的表达。结果:(1)慢病毒介导β3-AR基因转染心肌细胞,β3-AR表达较空白对照组明显升高。(2)用Western Blot检测各实验组细胞原癌基因c-myc、c-fos表达,其中NE组、β3-AR组、空病毒组表达均高于空白对照组,其中β3-AR组cmyc、c-fos表达明显高于NE组。(3)NE组、β3-AR组、空病毒组p38MAPK及ERK1/2的磷酸化水平较空白对照组明显上调,其中β3-AR组表达最高。结论:慢病毒介导的β3-AR基因转染使心肌细胞有效高表达β3-AR,β3-AR可能通过MAPK通路促进心肌肥厚。
Objective: To investigate the effect of β3-adrenergic receptor (β3-AR) on cardiac hypertrophy in neonatal SD rats and its mechanism. Methods: Cardiomyocytes were cultured in neonatal SD rats in vitro. After the cells were transfected with the lentivirus carrying β3-AR gene, the cells were induced by norepinephrine (NE) for 48 hours to establish cardiomyocyte hypertrophy model. The experiment was divided into 4 groups: Control group, NE group, β3-AR gene transfected group + NE group (β3-AR group) and empty virus transfected group + NE group (empty virus group). The cardiomyocytes were identified by immunofluorescence. The expression of green fluorescent protein (GFP) was observed by inverted fluorescence microscope. The expression of β3-AR, p38 MAPK and extracellular signal were detected by Western Blot. Regulated the expression of c-myc, c-fos in ERK1 / 2, p38MAPK and p-ERK1 / 2 and oncogene c-myc. Results: (1) The lentivirus-mediated β3-AR gene transfection of myocardial cells, β3-AR expression was significantly higher than the blank control group. (2) The expressions of c-myc and c-fos proto-oncogenes in each experimental group were detected by Western Blot. The expression of c-myc and c-fos in NE, β3-AR group and empty virus group were higher than those in blank control group c-fos expression was significantly higher than the NE group. (3) The phosphorylation levels of p38MAPK and ERK1 / 2 in NE group, β3-AR group and empty virus group were significantly up-regulated compared with those in blank control group, of which the expression was the highest in β3-AR group. CONCLUSION: Lentivirus-mediated β3-AR gene transfection can efficiently up-regulate the expression of β3-AR in cardiomyocytes, and β3-AR may promote cardiac hypertrophy through the MAPK pathway.