CAJ | 학술논문

目的评价吗啡预处理对大鼠心肌细胞缺氧复氧时微小RNA-133b-5p(miR-133b-5p)与Fas表达的影响.方法健康成年雄性SD大鼠心室肌细胞,接种于24孔板或60 mm培养皿培养,采用随机数字表法分为3组(n=24):对照组(C组)、缺氧复氧组(H/R组)和吗啡预处理组(MPC组).C组细胞正常培养,H/R和MPC组细胞缺氧90 min后复氧,MPC组于缺氧前采用含lμmol/L吗啡的无血清DMEM培养10 min,再换为无吗啡培养液培养30 min.于复氧120 min时采用MTT法测定心肌细胞活力,检测培养液乳酸脱氢酶(LDH)活性,采用Hoechst 33234染色法检测心肌细胞凋亡情况,计算细胞凋亡率;实时定量PCR法检测miR-133b-5p与Fas mRNA的表达,Western blot法检测Fas蛋白的表达.结果与C组比较,H/R组心肌细胞活力降低,培养液LDH活性和细胞凋亡率升高,miR-133b-5p表达下调,Fas mRNA及蛋白表达上调(P＜0.05);与H/R组比较,MPC组心肌细胞活力升高,培养液LDH活性和细胞凋亡率降低,miR-133b-5p表达上调,Fas mRNA及蛋白表达下调(P＜0.05).结论吗啡预处理减轻大鼠心肌细胞缺氧复氧损伤的机制与上调miR-133b-5p表达,下调Fas表达有关.
목적 평개마배예처리대대서심기세포결양복양시미소RNA-133b-5p(miR-133b-5p)여Fas표체적영향.방법 건강성년웅성SD대서심실기세포,접충우24공판혹60 mm배양명배양,채용수궤수자표법분위3조(n=24):대조조(C조)、결양복양조(H/R조)화마배예처리조(MPC조).C조세포정상배양,H/R화MPC조세포결양90 min후복양,MPC조우결양전채용함lμmol/L마배적무혈청DMEM배양10 min,재환위무마배배양액배양30 min.우복양120 min시채용MTT법측정심기세포활력,검측배양액유산탈경매(LDH)활성,채용Hoechst 33234염색법검측심기세포조망정황,계산세포조망솔;실시정량PCR법검측miR-133b-5p여Fas mRNA적표체,Western blot법검측Fas단백적표체.결과 여C조비교,H/R조심기세포활력강저,배양액LDH활성화세포조망솔승고,miR-133b-5p표체하조,Fas mRNA급단백표체상조(P＜0.05);여H/R조비교,MPC조심기세포활력승고,배양액LDH활성화세포조망솔강저,miR-133b-5p표체상조,Fas mRNA급단백표체하조(P＜0.05).결론 마배예처리감경대서심기세포결양복양손상적궤제여상조miR-133b-5p표체,하조Fas표체유관.
Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-Sp and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods Cardiomyocytes were isolated from healthy adult male Sprague-Dawley rats by using Langendorff perfusion.The cells were seeded into 24-well plates or 60 mm diameter dishes and randomly divided into 3 groups (n =24 each) using a random number table:control group (group C),group H/R,and morphine preconditioning group (group MPC).The cells in group C were cultured in normal culture atmosphere.In H/R and MPC groups,the cells were exposed to 95％ N2-5％ CO2 for 90 min followed by 120 min reoxygenation.In group MPC,the cells were cultured for 10 min in serum-free DMEM liquid culture medium containing morphine 1 μmol/L,and then were cultured for 30 min in morphine-free DMEM liquid culture medium before hypoxia.At 120 min of reoxygenation,the cells in 24-well plates were selected to detect the cell viability (by MTT),lactate dehydrogenase (LDH) activity in the culture medium,and cell apoptosis (by Hoechst 33234 staining).Apoptosis rate was calculated.Total RNA and protein were extracted from the cells in 60 mm dishes to detect the expression of miR-133b-5p and Fas mRNA (by quantitative real-time PCR) and Fas protein (by Western blot).Results Compared with C group,the cell viability was significantly decreased,LDH activity and apoptosis rate were increased,the expression of miR-133b-Sp was down-regulated,and the expression of Fas mRNA and protein was up-regulated in H/R group.Compared with H/R group,the cell viability was significantly increased,LDH activity and apoptosis rate were decreased,the expression of miR-133b-5p was up-regulatcd,and the expression of Fas mRNA and protein was down-regulated in MPC group.Conclusion The mechanism by which morphine preconditioning reduces H/R injury to rat cardiomyocytesis related to up-regulation of the expression of miR-133b-Sp and down-regulation of the expression of Fas.