CAJ | 학술논문

目的探讨创伤性脑损伤(TBI)后神经细胞凋亡的变化规律及其与caspase-3基因表达的关系.方法成年健康封闭群SD大鼠120只,随机分为对照组8只、损伤组和抑制物组各56只.Feeney法致伤,抑制物组伤后脑内注射5μg caspase-3抑制剂z-DEVD-fmk.分别在伤后1,6,24,48 h和3,7,14 d处死取材(每个分析时相点8只大鼠),采集伤灶中心皮质、皮层下白质、海马、齿状回,以及对侧相应部位脑组织,应用原位末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸(dUTP)标记法(TUNEL法)和流式细胞术检测神经细胞凋亡的变化;免疫组化法、蛋白印迹(western blot)和半定量逆转录-聚合酶链式反应(RT-PCR),检测caspase-3蛋白和mRNA的表达;并借助荧光分析试剂盒检测caspase-3活性的变化.所得数据采用SIDSS 10.1统计软件包进行Sprarman等级相关分析和方差分析(sNK-α检验).结果伤后伤侧各脑区神经细胞凋亡指数(AI)和细胞凋亡百分率(AP)迅速增高,24～48 h达峰值,随后逐渐下降,但伤后14 d仍高于正常(P＜0.01).伤后caspase-3蛋白和mRNA的表达明显增加,caspase-3活性迅速上升,峰值在24～48 h.其中伤后24 h伤侧海马区caspase-3蛋白谱密度与对照组相比增加1484%,caspase-3 mRNA的表达量增加1043%,caspase-3活性增加148%;伤后48h伤灶皮层下白质caspase-3蛋白谱密度增加1690%,caspase-3 mRNA的表达量增加1181%,caspase-3活性增加183%.Western blot显示,伤后caspase-3原酶及p17活性亚单位的表达均增强.抑制物组caspase-3蛋白和mRNA的表达均明显下降,caspase-3活性明显降低;同时,AI值和AP值也明显降低.统计学相关分析发现.伤后神经细胞凋亡与caspase-3 mRNA和蛋白的表达间呈正相关(r=0.821和r:0.638,P＜0.01),伤后caspase-3在mRNA和蛋白水平的表达间呈正相关(r=0.945,P＜0.01).结论急性TBI后神经细胞凋亡的发生与caspase-3的激活有关;神经细胞凋亡与其调节基因caspase-3的表达间具有一致性,TBI对caspase-3的调节发生在转录水平前的某一环节.caspase-3抑制剂能有效地阻断TBI后的神经细胞凋亡. 目的探討創傷性腦損傷(TBI)後神經細胞凋亡的變化規律及其與caspase-3基因錶達的關繫.方法成年健康封閉群SD大鼠120隻,隨機分為對照組8隻、損傷組和抑製物組各56隻.Feeney法緻傷,抑製物組傷後腦內註射5μg caspase-3抑製劑z-DEVD-fmk.分彆在傷後1,6,24,48 h和3,7,14 d處死取材(每箇分析時相點8隻大鼠),採集傷竈中心皮質、皮層下白質、海馬、齒狀迴,以及對側相應部位腦組織,應用原位末耑脫氧覈糖覈痠轉移酶介導的脫氧尿苷三燐痠(dUTP)標記法(TUNEL法)和流式細胞術檢測神經細胞凋亡的變化;免疫組化法、蛋白印跡(western blot)和半定量逆轉錄-聚閤酶鏈式反應(RT-PCR),檢測caspase-3蛋白和mRNA的錶達;併藉助熒光分析試劑盒檢測caspase-3活性的變化.所得數據採用SIDSS 10.1統計軟件包進行Sprarman等級相關分析和方差分析(sNK-α檢驗).結果傷後傷側各腦區神經細胞凋亡指數(AI)和細胞凋亡百分率(AP)迅速增高,24～48 h達峰值,隨後逐漸下降,但傷後14 d仍高于正常(P＜0.01).傷後caspase-3蛋白和mRNA的錶達明顯增加,caspase-3活性迅速上升,峰值在24～48 h.其中傷後24 h傷側海馬區caspase-3蛋白譜密度與對照組相比增加1484%,caspase-3 mRNA的錶達量增加1043%,caspase-3活性增加148%;傷後48h傷竈皮層下白質caspase-3蛋白譜密度增加1690%,caspase-3 mRNA的錶達量增加1181%,caspase-3活性增加183%.Western blot顯示,傷後caspase-3原酶及p17活性亞單位的錶達均增彊.抑製物組caspase-3蛋白和mRNA的錶達均明顯下降,caspase-3活性明顯降低;同時,AI值和AP值也明顯降低.統計學相關分析髮現.傷後神經細胞凋亡與caspase-3 mRNA和蛋白的錶達間呈正相關(r=0.821和r:0.638,P＜0.01),傷後caspase-3在mRNA和蛋白水平的錶達間呈正相關(r=0.945,P＜0.01).結論急性TBI後神經細胞凋亡的髮生與caspase-3的激活有關;神經細胞凋亡與其調節基因caspase-3的錶達間具有一緻性,TBI對caspase-3的調節髮生在轉錄水平前的某一環節.caspase-3抑製劑能有效地阻斷TBI後的神經細胞凋亡.
목적 탐토창상성뇌손상(TBI)후신경세포조망적변화규률급기여caspase-3기인표체적관계.방법 성년건강봉폐군SD대서120지,수궤분위대조조8지、손상조화억제물조각56지.Feeney법치상,억제물조상후뇌내주사5μg caspase-3억제제z-DEVD-fmk.분별재상후1,6,24,48 h화3,7,14 d처사취재(매개분석시상점8지대서),채집상조중심피질、피층하백질、해마、치상회,이급대측상응부위뇌조직,응용원위말단탈양핵당핵산전이매개도적탈양뇨감삼린산(dUTP)표기법(TUNEL법)화류식세포술검측신경세포조망적변화;면역조화법、단백인적(western blot)화반정량역전록-취합매련식반응(RT-PCR),검측caspase-3단백화mRNA적표체;병차조형광분석시제합검측caspase-3활성적변화.소득수거채용SIDSS 10.1통계연건포진행Sprarman등급상관분석화방차분석(sNK-α검험).결과 상후상측각뇌구신경세포조망지수(AI)화세포조망백분솔(AP)신속증고,24～48 h체봉치,수후축점하강,단상후14 d잉고우정상(P＜0.01).상후caspase-3단백화mRNA적표체명현증가,caspase-3활성신속상승,봉치재24～48 h.기중상후24 h상측해마구caspase-3단백보밀도여대조조상비증가1484%,caspase-3 mRNA적표체량증가1043%,caspase-3활성증가148%;상후48h상조피층하백질caspase-3단백보밀도증가1690%,caspase-3 mRNA적표체량증가1181%,caspase-3활성증가183%.Western blot현시,상후caspase-3원매급p17활성아단위적표체균증강.억제물조caspase-3단백화mRNA적표체균명현하강,caspase-3활성명현강저;동시,AI치화AP치야명현강저.통계학상관분석발현.상후신경세포조망여caspase-3 mRNA화단백적표체간정정상관(r=0.821화r:0.638,P＜0.01),상후caspase-3재mRNA화단백수평적표체간정정상관(r=0.945,P＜0.01).결론 급성TBI후신경세포조망적발생여caspase-3적격활유관;신경세포조망여기조절기인caspase-3적표체간구유일치성,TBI대caspase-3적조절발생재전록수평전적모일배절.caspase-3억제제능유효지조단TBI후적신경세포조망.
Objective To observe the correlation between the changes of neural cell apoptosis arid caspase-3 gene expression in brain tissues following acute severe traumatic injury to brain(TIB).Method A total of 120 adult Spraque-Dawley rats were divided into a control group(n=8)，TIB group(n=56)and TIB with administration of caspase-3 inhibitor group(n=56).TIB models of rats were made with Feeney's method.The z-DEVDfmk(5 μg)，caspase-3 inhibitor，was administered by intracerebral infusion，and the rats were sacrificed 1，6，24，48 hours and 3，7，14 days postinjury(n=8 for each interval).The specimens of the injured cerebral cortex，suhcerticai white matter，hippocampus，dentate gyrus and contrahteral corresponding brain tissues were taken for detecting apoptesis of neural cells by the terminal deoxynucleotidyl transferase mediated DUTP nick end labeling (TUNEL)methods and flow cytomeay.Caspase-3 mRNA and protein expression were detected by using RT-PCR，immunohistochemistry and western blot analysis.The caspase-3 activity was detected by using caspase-3 fluorescent assay kit.Student t-test and Spearman correlation analysis were used to analyze the data with SPSS version 10.1 software package.Results Apoptesis indexes(AI)and the apoptesis percentage(AP)of neural cells in the injured brain regions increased quickly after injury，and reached its peak 24 to 48 hours later，then decreased slowly,but it remained at higher level above that of normal till 14 days later(P＜0.01).The levels of caspase-3 mRNA,eastme-3 protein and caspase-3 activity were increased significantly post injury，and reached its peak at 24 to 48 hours，then it gradually decreased.Compared with control group，the levels ofoptical density of caspase-3 proteins in the injured hippocampus and subcortical white matter at 24 and 48 hours post injury increased 1484% and 1690%，caspase-3 mRNA expressiom increased 1043%and 1180%，and the degreas of caspase-3 activity increased 148% and 183%，respectively.The expression of caspase-3 proenzyme and its P17 subarrit increased.After trealment with caspase-3 inhibitor z-DEVD-fmk，the levels of caspase-3 mRNA，protein expression and caspase-3 activity were significantly decreased.and AI and AP were significantly decreased as well.The correlation between caspase-3 mRNA and level of neural apoptesis was positive(r=0.821，P＜0.01)，and it was likewise between caspase-3 protein and level of neural apoptosis(r=0.638.P＜0.01).Interestingly enough，a positive correlation was found between caspase-3 mRNA and easpase-3 proteins(r=0.945，P＜0.01).Conclusions The activation of caspase-3 leads to apoptosis of neural cells after acute TIB.The expression of caspase-3 are consistent with apoptosis of neural cells following TIB.The regulation of caspase-3 induced by TIB occurs at a ceriain critical link before transduction.Caspase-3 inhibitor can efficiently inhibit apoptosis of neural cells following TIB.