CAJ | 학술논문

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(1~3 d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;⑤ Western 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.
목적:연구14-3-3단백재심기세포결양예처리중적표체급의의.방법:실험용SD신생대서(1~3 d령),자웅불구,무균취출유서심장,경분리부착조직,이단백매소화,순화제성심기세포.재배양적제4 천수궤분위3조:정상대조조、결양/복양(A/R)조、결양예처리(APC)조.각조분별진행이하지표관찰:①심기세포박동빈솔;②세포존활솔(MTT법);③배양액중유산탈경매(LDH)활성;④투사전경관찰세포초미결구;⑤ Western Blotting법검측14-3-3단백적표체변화.RT-PCR법검측14-3-3단백η、σmRNA적표체변화.결과:A/R조화APC조적14-3-3단백표체균상조,분별시정상대조조적(3.61±0.37)배화(5.52±0.49)배,A/R조화APC조여정상대조조비교、A/R조여APC조비교,균P<0.01.A/R조、APC조심기세포14-3-3η아형mRNA분별위정상대조조적(1.82±0.30)배、(2.93±0.52)배,차이균유통계학의의(P<0.01);APC조여A/R조비교,차이역유통계학의의(P<0.01).A/R조、APC조심기세포14-3-3σ아형mRNA표체량분별위정상대조조적(1.13±0.18)배、(1.21±0.14배),A/R조、APC조여정상대조조비교, A/R조여APC조비교,차이균무통계학의의(P>0.05).결론:14-3-3η아형mRNA가능삼여심기세포APC후적조기보호작용.
Objective:To investigate the expression of 14-3-3 protein in the acute myocardial cells A/R injury.Method:Cultured myocardial cells of neonatal SD rats were randomly divided into three groups:control group; anoxia reoxygenation (A/R) group;anoxia preconditioning group (APC).In each group, the myocardial cells were subjected to anoxia (3 h) followed by reoxygenation (1 h) 24 h after anoxic preconditioning on the model of the cultured myocardial cells. At the end of experiment, myocardial cells beating rate,cell viability and the activity of LDH in culture were measured. Furthermore,the morphology or ultrastructure assessment of myocardial cells was observed by inverted microscope or transmission electron microscope, the expression of 14-3-3 protein was measured by western blotting techniques,and the expression of 14-3-3η,σmRNA was measured by RT-PCR. Result:①It is verified that cardiomyocytes after APC are offered more capacity to tolerate A/R damage from the level of cells.②14-3-3 protein and 14-3-3η,σmRNA showed a basic expression in myocardial cells under normal condition,14-3-3 protein and 14-3-3ηmRNA was enlarged after anoxia-reoxygenation injury.Conclusion:It is suggested that 14-3-3ηmRNA may be involved in the process of protection induced by anoxia preconditioning.