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目的:研究14-3-3蛋白在心肌细胞缺氧预处理中的表达及意义。方法:实验用SD新生大鼠(1～3d龄),雌雄不拘,无菌取出乳鼠心脏,经分离附着组织,胰蛋白酶消化,纯化制成心肌细胞。在培养的第4天随机分为3组:正常对照组、缺氧/复氧(A/R)组、缺氧预处理(APC)组。各组分别进行以下指标观察:①心肌细胞搏动频率;②细胞存活率(MTT法);③培养液中乳酸脱氢酶(LDH)活性;④透射电镜观察细胞超微结构;⑤Western Blotting法检测14-3-3蛋白的表达变化。RT-PCR法检测14-3-3蛋白η、σmRNA的表达变化。结果:A/R组和APC组的14-3-3蛋白表达均上调,分别是正常对照组的(3.61±0.37)倍和(5.52±0.49)倍,A/R组和APC组与正常对照组比较、A/R组与APC组比较,均P<0.01。A/R组、APC组心肌细胞14-3-3η亚型mRNA分别为正常对照组的(1.82±0.30)倍、(2.93±0.52)倍,差异均有统计学意义(P<0.01);APC组与A/R组比较,差异亦有统计学意义(P<0.01)。A/R组、APC组心肌细胞14-3-3σ亚型mRNA表达量分别为正常对照组的(1.13±0.18)倍、(1.21±0.14倍),A/R组、APC组与正常对照组比较,A/R组与APC组比较,差异均无统计学意义(P>0.05)。结论:14-3-3η亚型mRNA可能参与心肌细胞APC后的早期保护作用。
Objective: To study the expression and significance of 14-3-3 protein in cardiomyocyte hypoxia preconditioning. Methods: SD neonatal rats (1 ~ 3 days old) were randomly divided into male and female rats. The hearts of neonatal rats were removed aseptically, and the cardiomyocytes were isolated by attachment and trypsinization. On day 4 of culture, the cells were randomly divided into 3 groups: normal control group, A / R group and APC group. The following indexes were observed in each group: ①the frequency of cardiomyocytes pulsatility; ② cell survival rate (MTT method); ③ lactate dehydrogenase (LDH) activity in culture medium; ④transmission electron microscopy observation of cell ultrastructure; ⑤Western Blotting assay 14 -3-3 protein expression changes. The expression of 14-3-3 protein η, σ mRNA was detected by RT-PCR. Results: The expressions of 14-3-3 protein in A / R group and APC group were all up-regulated, which were 3.61 ± 0.37 times and 5.52 ± 0.49 times respectively in normal control group and APC group. Compared with normal control group Group A / R group compared with APC group, P <0.01. The levels of 14-3-3η mRNA in cardiomyocytes of A / R group and APC group were (1.82 ± 0.30) times and (2.93 ± 0.52) times higher than that of normal control group (P <0.01) Compared with A / R group, the difference was also statistically significant (P <0.01). The mRNA expression levels of 14-3-3σ subtype in cardiomyocytes of A / R group and APC group were (1.13 ± 0.18) times and 1.21 ± 0.14 times respectively higher than those of the normal control group (A / R group and APC group) Comparison, A / R group and APC group, the difference was not statistically significant (P> 0.05). Conclusion: The 14-3-3η subtype mRNA may be involved in the early protective effect of cardiomyocytes after APC.