CAJ | 학술논문

目的探讨微小RNA-133b-Sp(miR-133b-5p)在缺氧复氧诱发大鼠心肌细胞凋亡中的作用.方法大鼠H9c2胚胎心肌细胞在含10％胎牛血清DMEM/F12培养基中培养、传代.将细胞接种于96孔和6孔细胞培养板,采用随机数宁表法分为4组(n=64):空白对照组(C组)正常培养24 h;缺氧复氧组(H/R)置于缺氧小室,通入95％N2-5％CO2,37℃培养箱中缺氧5h后,用含10％胎牛血清的DMEM/F12复氧1 h;miR-133b-5p模拟物+缺氧复氧组(M+H/R组)和阴性转染+缺氧复氧组(NC+H/R)分别用miR-133b-5p模拟物(终浓度为30 nmol/L)或miR-133b-5p模拟物错义链(终浓度为30 nmol/L)孵育24 h后制备缺氧复氧损伤模型.各组处理结束后,采用实时荧光定量PCR法检测miR-133b-5p的表达水平,采用CCK-8法测定细胞活力;采用微量酶标法测定培养液中乳酸脱氢酶(LDH)的活性;采用Annexin V/PI双染流式细胞术检测细胞凋亡率.结果与C组比较,H/R组、M+H/R组和NC+H/R组心肌细胞活力降低,培养液LDH活性升高,细胞凋亡率升高,H/R组和NC+H/R组miR-133b-5p表达下调,M+H/R组miR-133b-5p表达上调(P＜0.05);与H/R组比较,M+H/R组心肌细胞活力升高,培养液LDH活性降低,细胞凋亡率降低,miR-133b-5p表达上调(P＜0.05),NC+H/R组上述指标差异无统计学意义(P＞0.05).结论大鼠心肌细胞缺氧复氧可能通过下调miR-133b-5p表达,诱发细胞凋亡.
목적 탐토미소RNA-133b-Sp(miR-133b-5p)재결양복양유발대서심기세포조망중적작용.방법 대서H9c2배태심기세포재함10％태우혈청DMEM/F12배양기중배양、전대.장세포접충우96공화6공세포배양판,채용수궤수저표법분위4조(n=64):공백대조조(C조)정상배양24 h;결양복양조(H/R)치우결양소실,통입95％N2-5％CO2,37℃배양상중결양5h후,용함10％태우혈청적DMEM/F12복양1 h;miR-133b-5p모의물+결양복양조(M+H/R조)화음성전염+결양복양조(NC+H/R)분별용miR-133b-5p모의물(종농도위30 nmol/L)혹miR-133b-5p모의물착의련(종농도위30 nmol/L)부육24 h후제비결양복양손상모형.각조처리결속후,채용실시형광정량PCR법검측miR-133b-5p적표체수평,채용CCK-8법측정세포활력;채용미량매표법측정배양액중유산탈경매(LDH)적활성;채용Annexin V/PI쌍염류식세포술검측세포조망솔.결과 여C조비교,H/R조、M+H/R조화NC+H/R조심기세포활력강저,배양액LDH활성승고,세포조망솔승고,H/R조화NC+H/R조miR-133b-5p표체하조,M+H/R조miR-133b-5p표체상조(P＜0.05);여H/R조비교,M+H/R조심기세포활력승고,배양액LDH활성강저,세포조망솔강저,miR-133b-5p표체상조(P＜0.05),NC+H/R조상술지표차이무통계학의의(P＞0.05).결론 대서심기세포결양복양가능통과하조miR-133b-5p표체,유발세포조망.
Objective To evaluate the role of microRNA-133b-Sp (miR-133b-Sp) in apoptosis hypoxia/reoxygenation (H/R) induced by in rat cardiomyocytes.Methods Rat myocardial cell line H9c2 was cultured in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum.The cells were seeded in 96-well or 6-well plates and randomly divided into 4 groups (n=64 wells each):control group (group C);group H/R;miR-133b-5p mimic + H/R group (group M+H/R);miR-133b-Sp negative control + H/R group (group NC+H/R).The cells were exposed to 95％ N2-5％ CO2for 5 h at 37 ℃ followed by 1 h reoxygenation in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum in all the groups except group C.The cells were cultured in normal culture atmosphere in group C.In M+H/R and NC+H/R groups,the cells were transfected with miR-133b-5p mimic (final concentration 30 nmol/L) and miR-133b-5p negative control (final concentration 30 nmol/L),respectively,for 24 h before H/R.Total RNA was extracted from cells to detect the expression of miR-133b-5p using quantitative real-time PCR.The cell viability (by CCK-8) and lactic dehydrogenase (LDH) activity in the culture medium were detected.Cell apoptosis was assessed by Annexin V/PI flow cytometry.Apoptotic rate was calculated.Result Compared with group C,the cell viability was significantly decreased,and the LDH activity and apoptotic rate were increased in H/R,M+H/R and NC+H/R groups,the expression of miR-133b-5p was down-regulated in H/R and NC+H/R groups,and the expression of miR-133b-Sp was up-regulated in group M+H/R.Compared with group H/R,the cell viability was significanttly increased,the LDH activity and apoptotic rate were decreased,and the expression of miR-133b-5p was up-regulated in group M+H/R,and no significant change was found in the parameters mentioned above in group NC+H/R.Conclusion H/R in rat cardiomyocytes can induce cell apoptosis possibility through down-regulating the expression of miR-133b-5p