中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2014年
4期
315-319
,共5页
谢琼慧%赵超莉%叶子青%杨飞%阮琼芳%谢卫国
謝瓊慧%趙超莉%葉子青%楊飛%阮瓊芳%謝衛國
사경혜%조초리%협자청%양비%원경방%사위국
烧伤%心肌缺血%微小RNA-21%程序性细胞死亡因子4
燒傷%心肌缺血%微小RNA-21%程序性細胞死亡因子4
소상%심기결혈%미소RNA-21%정서성세포사망인자4
Burns%Myocardial ischemia%MicroRNA-21%Programmed cell death 4
目的 观察大鼠严重烫伤早期心肌组织中微小RN A-21以及程序性细胞死亡因子4 (PDCD4)的表达变化,在细胞水平验证两者关系,初步探讨微小RN A-21参与大鼠严重烫伤早期心肌损害的分子机制. 方法 (1)将40只SD大鼠按随机数字表法分为假伤组8只(致假伤)和烫伤组32只(背部致30％ TBSAⅢ度烫伤).假伤组伤后不予补液,lh后处死取左心室组织;烫伤组伤后经腹腔注射乳酸林格液,于伤后3、6、12、24 h分别处死8只大鼠取左心室组织.实时荧光定量RT-PCR法检测心肌组织中微小RNA-21的表达水平,蛋白质印迹法检测心肌组织中PDCD4蛋白表达.(2)取大鼠心肌细胞株H9C2按随机数字表法分为微小RNA-21拮抗剂组(转染微小RNA-21拈抗剂)和转染对照组(转染微小RNA拮抗剂阴性对照).转染后48 h实时荧光定量RT-PCR法和蛋白质印迹法分别测定心肌细胞PDCD4的mRNA和蛋白表达水平.对数据行单因素方差分析、ISD-t检验、两独立样本t检验,对大鼠心肌组织微小RNA-21表达与PDCD4蛋白水平行直线相关分析. 结果 (1)假伤组伤后lh及烫伤组伤后3、6、12、24 h大鼠心肌组织微小RNA-21的相对表达量分别为0.96±0.13、0.44±0.08、0.42 ±0.10、0.33±0.07、0.61±0.10(F=27.331,P＜0.001).与假伤组伤后1h比较,烫伤组伤后3、6、12、24 h心肌组织微小RNA-21水平均明显下降(t值为4.558～9.410,P值均小于0.01).假伤组伤后1h及烫伤组伤后3、6、12、24 h大鼠心肌组织PDCD4蛋白的相对表达量分别为0.44±0.05、0.60±0.09、0.92±0.15、0.86±0.11、0.57±0.10(F=8.622,P=0.003),其中烫伤组伤后6、12 h表达量显著高于假伤组伤后1 h(t值分别为4.968和4.122,P值均小于0.01).烫伤组大鼠各时相点心肌组织微小RN A-21表达水平与PDCD4蛋白表达水平呈明显负相关(r=-0.572,P=0.026).(2)微小RNA-21拮抗剂组心肌细胞PDCD4的mRNA和蛋白的相对表达量分别为1.73±0.29和0.38±0.08,明显高于转染对照组的0.95±0.14和0.23±0.03(t值分别为4.857和3.356,P＜0.05或P＜0.01). 结论 大鼠严重烫伤早期心肌组织微小RNA-21表达下降,PDCD4表达升高,微小RNA-21可能通过负向调节PDCD4表达而参与烫伤早期心肌损伤.
目的 觀察大鼠嚴重燙傷早期心肌組織中微小RN A-21以及程序性細胞死亡因子4 (PDCD4)的錶達變化,在細胞水平驗證兩者關繫,初步探討微小RN A-21參與大鼠嚴重燙傷早期心肌損害的分子機製. 方法 (1)將40隻SD大鼠按隨機數字錶法分為假傷組8隻(緻假傷)和燙傷組32隻(揹部緻30％ TBSAⅢ度燙傷).假傷組傷後不予補液,lh後處死取左心室組織;燙傷組傷後經腹腔註射乳痠林格液,于傷後3、6、12、24 h分彆處死8隻大鼠取左心室組織.實時熒光定量RT-PCR法檢測心肌組織中微小RNA-21的錶達水平,蛋白質印跡法檢測心肌組織中PDCD4蛋白錶達.(2)取大鼠心肌細胞株H9C2按隨機數字錶法分為微小RNA-21拮抗劑組(轉染微小RNA-21拈抗劑)和轉染對照組(轉染微小RNA拮抗劑陰性對照).轉染後48 h實時熒光定量RT-PCR法和蛋白質印跡法分彆測定心肌細胞PDCD4的mRNA和蛋白錶達水平.對數據行單因素方差分析、ISD-t檢驗、兩獨立樣本t檢驗,對大鼠心肌組織微小RNA-21錶達與PDCD4蛋白水平行直線相關分析. 結果 (1)假傷組傷後lh及燙傷組傷後3、6、12、24 h大鼠心肌組織微小RNA-21的相對錶達量分彆為0.96±0.13、0.44±0.08、0.42 ±0.10、0.33±0.07、0.61±0.10(F=27.331,P＜0.001).與假傷組傷後1h比較,燙傷組傷後3、6、12、24 h心肌組織微小RNA-21水平均明顯下降(t值為4.558～9.410,P值均小于0.01).假傷組傷後1h及燙傷組傷後3、6、12、24 h大鼠心肌組織PDCD4蛋白的相對錶達量分彆為0.44±0.05、0.60±0.09、0.92±0.15、0.86±0.11、0.57±0.10(F=8.622,P=0.003),其中燙傷組傷後6、12 h錶達量顯著高于假傷組傷後1 h(t值分彆為4.968和4.122,P值均小于0.01).燙傷組大鼠各時相點心肌組織微小RN A-21錶達水平與PDCD4蛋白錶達水平呈明顯負相關(r=-0.572,P=0.026).(2)微小RNA-21拮抗劑組心肌細胞PDCD4的mRNA和蛋白的相對錶達量分彆為1.73±0.29和0.38±0.08,明顯高于轉染對照組的0.95±0.14和0.23±0.03(t值分彆為4.857和3.356,P＜0.05或P＜0.01). 結論 大鼠嚴重燙傷早期心肌組織微小RNA-21錶達下降,PDCD4錶達升高,微小RNA-21可能通過負嚮調節PDCD4錶達而參與燙傷早期心肌損傷.
목적 관찰대서엄중탕상조기심기조직중미소RN A-21이급정서성세포사망인자4 (PDCD4)적표체변화,재세포수평험증량자관계,초보탐토미소RN A-21삼여대서엄중탕상조기심기손해적분자궤제. 방법 (1)장40지SD대서안수궤수자표법분위가상조8지(치가상)화탕상조32지(배부치30％ TBSAⅢ도탕상).가상조상후불여보액,lh후처사취좌심실조직;탕상조상후경복강주사유산림격액,우상후3、6、12、24 h분별처사8지대서취좌심실조직.실시형광정량RT-PCR법검측심기조직중미소RNA-21적표체수평,단백질인적법검측심기조직중PDCD4단백표체.(2)취대서심기세포주H9C2안수궤수자표법분위미소RNA-21길항제조(전염미소RNA-21념항제)화전염대조조(전염미소RNA길항제음성대조).전염후48 h실시형광정량RT-PCR법화단백질인적법분별측정심기세포PDCD4적mRNA화단백표체수평.대수거행단인소방차분석、ISD-t검험、량독립양본t검험,대대서심기조직미소RNA-21표체여PDCD4단백수평행직선상관분석. 결과 (1)가상조상후lh급탕상조상후3、6、12、24 h대서심기조직미소RNA-21적상대표체량분별위0.96±0.13、0.44±0.08、0.42 ±0.10、0.33±0.07、0.61±0.10(F=27.331,P＜0.001).여가상조상후1h비교,탕상조상후3、6、12、24 h심기조직미소RNA-21수평균명현하강(t치위4.558～9.410,P치균소우0.01).가상조상후1h급탕상조상후3、6、12、24 h대서심기조직PDCD4단백적상대표체량분별위0.44±0.05、0.60±0.09、0.92±0.15、0.86±0.11、0.57±0.10(F=8.622,P=0.003),기중탕상조상후6、12 h표체량현저고우가상조상후1 h(t치분별위4.968화4.122,P치균소우0.01).탕상조대서각시상점심기조직미소RN A-21표체수평여PDCD4단백표체수평정명현부상관(r=-0.572,P=0.026).(2)미소RNA-21길항제조심기세포PDCD4적mRNA화단백적상대표체량분별위1.73±0.29화0.38±0.08,명현고우전염대조조적0.95±0.14화0.23±0.03(t치분별위4.857화3.356,P＜0.05혹P＜0.01). 결론 대서엄중탕상조기심기조직미소RNA-21표체하강,PDCD4표체승고,미소RNA-21가능통과부향조절PDCD4표체이삼여탕상조기심기손상.
Objective To explore the molecular mechanism of microRNA-21 in myocardial damage of rats in the early stage of severe scald injury by observing the expression of microRNA-21 and programmed cell death 4 (PDCD4) in myocardial tissue of rat and to validate the relationship between them in cell model.Methods (1) Forty SD rats were divided into sham injury group (n =8,sham injured) and scald injury group (n =32,inflicted with 30％ TBSA full-thickness scald on the back) according to the random number table.The left ventricular tissue was collected from rats in sham injury group at post injury hour 1 without any fluid infusion.Rats in scald injury group were given an intraperitoneal injection of lactic acid Ringer's solution and 8 rats were respectively sacrificed at post injury hour 3,6,12,24 to harvest left ventricular tissue.The expression of microRNA-21 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR.The protein expression of PDCD4 in myocardial tissue was assessed by Western blotting.(2) Rat myocardial cell line H9C2 was divided into microRNA-21 inhibitor group (cells were transfected with microRNA-21 inhibitor) and negative transfection control group (cells were transfected with negative control of microRNA inhibitor) according to the random number table.At post transfection hour 48,real-time fluorescent quantitative RT-PCR and Western blotting were performed respectively to determine the mRNA and protein expression levels of PDCD4 in cells.Data were processed with one-way analysis of variance,LSD-t and two independent samples t test.The relationship between microRNA-21 expression and PDCD4 protein level in myocardial tissue of rats was assessed by linear correlation analysis.Results (1) The expression levels of microRNA-21 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3,6,12,24 were respectively 0.96 ± 0.13,0.44 ± 0.08,0.42 ±0.10,0.33 ± 0.07,and 0.61 ± 0.10 (F =27.331,P ＜ 0.001).Compared with that in myocardial tissue of rats in sham injury group at post injury hour 1,expression level of microRNA-21 was significantly decreased in scald injury group at post injury hour 3,6,12,24 (with t values from 4.558 to 9.410,P values below 0.01).The protein expression levels of PDCD4 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3,6,12,24 were respectively 0.44 ± 0.05,0.60 ± 0.09,0.92±0.15,0.86±0.11,and 0.57 ±0.10 (F =8.622,P =0.003).Compared with that in sham injury group at post injury hour 1,protein expression level of PDCD4 was significantly increased in scald injury group at post injury hour 6 and 12 (with t values respectively 4.968 and 4.122,P values below 0.01).A significant negative correlation between the expression of microRNA-21 and PDCD4 protein in myocardial tissue of rats of scald injury group was observed at each time point (r =-0.572,P =0.026).(2) The mRNA and protein expression levels of PDCD4 of myocardial cells in microRNA-21 inhibitor group were respectively 1.73 ± 0.29 and 0.38 ± 0.08,which were significantly higher than those in negative transfection control group (0.95 ±0.14 and 0.23 ±0.03,with t values respectively 4.857 and 3.356,P ＜0.05 or P ＜ 0.01).Conclusions Expression of microRNA-21 was decreased,while expression of PDCD4 was increased,in myocardial tissue of rats in the early stage of severe scald injury.MicroRNA-21 might participate in myocardial damage in the early stage of scald injury by negatively regulating expression of PDCD4.
