中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2013年
3期
267-271
,共5页
郑军%黄跃生%黄晓元%范鹏举%贺伟峰%张小容
鄭軍%黃躍生%黃曉元%範鵬舉%賀偉峰%張小容
정군%황약생%황효원%범붕거%하위봉%장소용
烧伤%血清%缺氧%p38丝裂原活化蛋白激酶类%肌细胞,心脏%细胞凋亡
燒傷%血清%缺氧%p38絲裂原活化蛋白激酶類%肌細胞,心髒%細胞凋亡
소상%혈청%결양%p38사렬원활화단백격매류%기세포,심장%세포조망
Burns%Serum%Anoxia%p38 mitogen-activated protein kinases%Myocytes,cardiac%Apoptosis
目的 研究反义p38α MAPK(以下简称p38α)对缺氧及烧伤血清复合处理心肌细胞的作用. 方法 取30只成年SD大鼠制备烧伤血清(背部40％ TBSAⅢ度烧伤).取80只SD大鼠的乳鼠分离、培养心肌细胞,按随机数字表法将细胞分为4组:正常对照组,心肌细胞未经任何处理,常规培养；烧伤血清+缺氧组,心肌细胞中加入体积分数10％烧伤大鼠血清后行缺氧培养；烧伤血清+缺氧+感染组,采用反义p38α转染的腺病毒感染心肌细胞,再加入体积分数10％烧伤大鼠血清后行缺氧培养；烧伤血清+缺氧+空载感染组,用空载腺病毒感染心肌细胞,再加入体积分数10％烧伤大鼠血清行缺氧培养.后3组于缺氧1、3、6、12h后,分别采用RT-PCR法和蛋白质印迹法检测各组心肌细胞p38α mRNA和蛋白表达,采用噻唑蓝法检测细胞活力,检测乳酸脱氢酶(LDH)含量；于缺氧1、6、12h后,采用膜联蛋白V单染法检测心肌细胞凋亡率.正常对照组行同上检测.每组各时相点设3个复孔.对数据进行单因素方差分析及LSD-t检验. 结果 (1)缺氧1、3、6h,烧伤血清+缺氧组p38α mRNA表达水平与正常对照组和烧伤血清+缺氧+感染组比较均升高,且差异有统计学意义(t值为2.725 ～4.375,P值均小于0.05).(2)缺氧1、3、6h,烧伤血清+缺氧组p38α蛋白表达水平与正常对照组和烧伤血清+缺氧+感染组比较均显著升高(t值为5.351～7.981,P值均小于0.01).(3)缺氧3、6、12h,烧伤血清+缺氧组心肌细胞活力分别为0.115±0.007、0.104 ±0.006、0.094±0.005,与正常对照组的0.141 ±0.014和烧伤血清+缺氧+感染组的0.136±0.009、0.124±0.010、0.112±0.007比较均降低,且差异有统计学意义(t值为2.357 ～6.812,P值均小于0.05).(4)缺氧后各时相点,烧伤血清+缺氧组LDH含量与正常对照组和烧伤血清+缺氧+感染组比较均显著升高(t值为22.753～ 201.273,P值均小于0.01).(5)缺氧1、6、12 h,烧伤血清+缺氧组心肌细胞凋亡率分别为(5.4±0.7)％、(8.7±1.1)％、(13.6±1.7)％,与正常对照组的(3.1±0.3)％和烧伤血清+缺氧+感染组的(4.3±0.5)％、(5.1±0.7)％、(7.2±0.9)％比较均升高(t值为2.345 ～9.700,P＜0.05或P＜0.01). 结论 反义p38α对烧伤血清+缺氧损伤条件下心肌细胞具有保护作用.
目的 研究反義p38α MAPK(以下簡稱p38α)對缺氧及燒傷血清複閤處理心肌細胞的作用. 方法 取30隻成年SD大鼠製備燒傷血清(揹部40％ TBSAⅢ度燒傷).取80隻SD大鼠的乳鼠分離、培養心肌細胞,按隨機數字錶法將細胞分為4組:正常對照組,心肌細胞未經任何處理,常規培養；燒傷血清+缺氧組,心肌細胞中加入體積分數10％燒傷大鼠血清後行缺氧培養；燒傷血清+缺氧+感染組,採用反義p38α轉染的腺病毒感染心肌細胞,再加入體積分數10％燒傷大鼠血清後行缺氧培養；燒傷血清+缺氧+空載感染組,用空載腺病毒感染心肌細胞,再加入體積分數10％燒傷大鼠血清行缺氧培養.後3組于缺氧1、3、6、12h後,分彆採用RT-PCR法和蛋白質印跡法檢測各組心肌細胞p38α mRNA和蛋白錶達,採用噻唑藍法檢測細胞活力,檢測乳痠脫氫酶(LDH)含量；于缺氧1、6、12h後,採用膜聯蛋白V單染法檢測心肌細胞凋亡率.正常對照組行同上檢測.每組各時相點設3箇複孔.對數據進行單因素方差分析及LSD-t檢驗. 結果 (1)缺氧1、3、6h,燒傷血清+缺氧組p38α mRNA錶達水平與正常對照組和燒傷血清+缺氧+感染組比較均升高,且差異有統計學意義(t值為2.725 ～4.375,P值均小于0.05).(2)缺氧1、3、6h,燒傷血清+缺氧組p38α蛋白錶達水平與正常對照組和燒傷血清+缺氧+感染組比較均顯著升高(t值為5.351～7.981,P值均小于0.01).(3)缺氧3、6、12h,燒傷血清+缺氧組心肌細胞活力分彆為0.115±0.007、0.104 ±0.006、0.094±0.005,與正常對照組的0.141 ±0.014和燒傷血清+缺氧+感染組的0.136±0.009、0.124±0.010、0.112±0.007比較均降低,且差異有統計學意義(t值為2.357 ～6.812,P值均小于0.05).(4)缺氧後各時相點,燒傷血清+缺氧組LDH含量與正常對照組和燒傷血清+缺氧+感染組比較均顯著升高(t值為22.753～ 201.273,P值均小于0.01).(5)缺氧1、6、12 h,燒傷血清+缺氧組心肌細胞凋亡率分彆為(5.4±0.7)％、(8.7±1.1)％、(13.6±1.7)％,與正常對照組的(3.1±0.3)％和燒傷血清+缺氧+感染組的(4.3±0.5)％、(5.1±0.7)％、(7.2±0.9)％比較均升高(t值為2.345 ～9.700,P＜0.05或P＜0.01). 結論 反義p38α對燒傷血清+缺氧損傷條件下心肌細胞具有保護作用.
목적 연구반의p38α MAPK(이하간칭p38α)대결양급소상혈청복합처리심기세포적작용. 방법 취30지성년SD대서제비소상혈청(배부40％ TBSAⅢ도소상).취80지SD대서적유서분리、배양심기세포,안수궤수자표법장세포분위4조:정상대조조,심기세포미경임하처리,상규배양；소상혈청+결양조,심기세포중가입체적분수10％소상대서혈청후행결양배양；소상혈청+결양+감염조,채용반의p38α전염적선병독감염심기세포,재가입체적분수10％소상대서혈청후행결양배양；소상혈청+결양+공재감염조,용공재선병독감염심기세포,재가입체적분수10％소상대서혈청행결양배양.후3조우결양1、3、6、12h후,분별채용RT-PCR법화단백질인적법검측각조심기세포p38α mRNA화단백표체,채용새서람법검측세포활력,검측유산탈경매(LDH)함량；우결양1、6、12h후,채용막련단백V단염법검측심기세포조망솔.정상대조조행동상검측.매조각시상점설3개복공.대수거진행단인소방차분석급LSD-t검험. 결과 (1)결양1、3、6h,소상혈청+결양조p38α mRNA표체수평여정상대조조화소상혈청+결양+감염조비교균승고,차차이유통계학의의(t치위2.725 ～4.375,P치균소우0.05).(2)결양1、3、6h,소상혈청+결양조p38α단백표체수평여정상대조조화소상혈청+결양+감염조비교균현저승고(t치위5.351～7.981,P치균소우0.01).(3)결양3、6、12h,소상혈청+결양조심기세포활력분별위0.115±0.007、0.104 ±0.006、0.094±0.005,여정상대조조적0.141 ±0.014화소상혈청+결양+감염조적0.136±0.009、0.124±0.010、0.112±0.007비교균강저,차차이유통계학의의(t치위2.357 ～6.812,P치균소우0.05).(4)결양후각시상점,소상혈청+결양조LDH함량여정상대조조화소상혈청+결양+감염조비교균현저승고(t치위22.753～ 201.273,P치균소우0.01).(5)결양1、6、12 h,소상혈청+결양조심기세포조망솔분별위(5.4±0.7)％、(8.7±1.1)％、(13.6±1.7)％,여정상대조조적(3.1±0.3)％화소상혈청+결양+감염조적(4.3±0.5)％、(5.1±0.7)％、(7.2±0.9)％비교균승고(t치위2.345 ～9.700,P＜0.05혹P＜0.01). 결론 반의p38α대소상혈청+결양손상조건하심기세포구유보호작용.
Objective To study the effects of antisense p38α mitogen-activated protein kinase (hereinafter referred to as p38α) on myocardial cells exposed to hypoxia and burn serum.Methods Thirty adult SD rats were inflicted with 40％ TBSA full-thickness burn on the back to obtain burn serum.The myocardial cells were isolated from 80 neonatal SD rats and cultured,then they were divided into 4 groups according to the random number table:normal control group (N,ordinary culture without any treatment),hypoxia + burn serum group (HB,exposed to hypoxia after being treated with 10％ burn rat serum),hypoxia + burn serum + infection group (HBI,exposed to hypoxia and 10％ burn rat serum after being infected with antisense p38α gene-carrying adenovirus),hypoxia + burn serum + empty vector infection group (exposed to hypoxia and 10％ burn rat serum after being infected with adenovirus empty vector).At post hypoxia hour (PHH) l,3,6,and 12,mRNA and protein expression levels of p38α in the latter 3 groups were determined by RT-PCR and Western blotting,cell viability was determined by methylthianolyldiphenyl-tetrazolium bromide assay,and lactate dehydrogenase (LDH) activity was assayed at the same time point.At PHH 1,6,and 12,apoptosis rate of myocardial cells was assessed by annexin V staining method.The indexes of group N were determined with the methods mentioned-above.Three wells were set at each time point in each group.Data were processed with one-way analysis of variance and LSD-t test.Results (1) At PHH 1,3,and 6,the p38α mRNA level was higher in group HB than in group N and group HBI (with t values from 2.725 to 4.375,P values all below 0.05).(2) At PHH 1,3,and 6,the p38o protein level was higher in group HB than those in group N and group HBI (with t values from 5.351 to 7.981,P values all below0.01).(3) At PHH3,6,and 12,the cell viability in group HB (0.115 ±0.007,0.104±0.006,0.094 ± 0.005) was lower than that in group N (0.141 ± 0.014) and group HBI (0.136 ± 0.009,0.124±0.010,0.112 ±0.007,witht values from 2.357 to 6.812,Pvalues all below 0.05).(4) The LDH activity was up-regulated in group HB as compared with that in group N and group HBI at each time point (with t values from 22.753 to 201.273,P values all below 0.01).(5) At PHH 1,6,and 12,the apoptosis rate of myocardial cells in group HB [(5.4 ±0.7)％,(8.7 ± 1.1) ％,(13.6 ± 1.7)％] was higher than that of group N [(3.1 ±0.3)％] and group HBI [(4.3 ±0.5)％,(5.1 0.7)％,(7.2 ± 0.9)％,with t values from 2.345 to 9.700,P ＜0.05 orP ＜0.01].Conclusions Antisense p38α can protect the myocardial cells from the injury of hypoxia and burn serum.
