【作者】 陈娣；

【导师】 杨光田；

【作者基本信息】 华中科技大学 ， 急诊医学， 2012， 博士

【摘要】 [目的]1.按照Utstein模式对比分析电刺激法致心脏骤停模型和窒息法致心脏骤停模型的特点。2.初步探讨H2S供体NaHS在心肺复苏大鼠脑损伤中的作用。3.研究H2S对复苏后脑损伤的保护作用与抗炎反应的关系。4.研究H2S对复苏后脑损伤的保护作用与凋亡反应的关系。5.研究H2S对复苏后脑损伤的保护作用与氧化应激反应的关系。6.研究H2S的脑保护作用是否于上调缺氧诱导因子-1(hypoxia-inducible factor-1, HIF-1)有关。[方法]第一部分分别采用经皮电刺激心外膜法制备大鼠心脏骤停模型和窒息法制备大鼠心脏骤停模型,比较两组大鼠在心脏骤停类型,诱发心脏骤停时间的差异。第二、三部分采用经皮电刺激心外膜法建立大鼠心跳骤停模型,随机(随机数字法)分为4组：(1)假手术组：仅行麻醉、气管插管及动静脉插管操作；(2)CPR组：进行模型制备及复苏；(3)CPR+硫氢化钠(NaHS)组：以NaHS作为H2S供体,在CPR开始前1分钟经股静脉给予100μg/kgNaHS,其余操作同CPR组；(4)CPR+AOAA(H2S合成酶CBS抑制剂)组：在CPR开始前1分钟经股静脉给予20mg/kgAOAA。分别对各组大鼠进行生存率、神经缺陷评分和形态学观察；用生化法测定各组大鼠在不同时间点脑组织H：S的表达水平,用Elisa方法测定各组大鼠不同时间点脑组织炎性因子TNF-α、IL-1β的表达；用PCR、免疫组化的方法测定各组大鼠脑组织凋亡相关因子Bax、Bcl-2的表达；用生化法测定各组大鼠脑组织氧化应激指标MDA、SOD的变化；用实时定量PCR、Western方法测定各组大鼠缺氧诱导因子-1基因及蛋白的表达。[结果]经皮电刺激心外膜法致大鼠心脏骤停模型心脏骤停表现类型多样,可以表现为室颤、无脉性电活动和心电静止,窒息法仅表现为无脉性电活动和心电静止,无室颤发生；经皮电刺激心外膜法致大鼠心脏骤停模型诱发心脏骤停的时间明显短于窒息法致心脏骤停模型,差异有统计学意义(P<0.05)。与假手术组相比,CPR组TNF-α水平在3h、6h、9h、12h均有明显升高,差异有统计学意义(P<0.05),其含量在6h达到高峰；与CPR组相比,CPR+NaHS组TNF-a水平在3h、6h、9h、12h均明显降低,差异有统计学意义(P<0.05)；与CPR组相比,CPR+AOAA组TNF-α水平在3h、6h均有所增加,差异有统计学意义(P<0.05),在9h、12h有增高趋势,差异无统计学意义(P>0.05)。与假手术组相比,CPR组IL-1β水平在3h、6h、9h、12h均有明显升高,差异有统计学意义(P<0.05),其含量在9h达到高峰；与CPR组相比,CPR+NaHS组IL-1β水平在6h、9h、12h明显降低,差异有统计学意义(P<0.05),在3h有降低趋势,差异无统计学意义(P>0.05)；与CPR组相比,CPR+AOAA组IL-1β水平在3h、6h、9h均有所升高,差异有统计学意义(P<0.05),12h有升高趋势,差异无统计学意义(P>0.05)。与假手术组相比,CPR组Bax表达增高,差异有统计学意义(P<0.05),与CPR组相比,CPR+NaHS组Bax表达明显下降,差异有统计学意义(P<0.05),CPR+AOAA组表达与CPR组无明显差异(P>0.05)。与假手术组相比,CPR组Bcl-2基因表达有升高趋势,差异有统计学意义(P>0.05),与CPR组相比,CPR+NaHS组Bcl-2基因表达明显升高,差异有统计学意义(P<0.05),CPR+AOAA组表达下降,差异有统计学意义(P<0.05)。与假手术组相比,CPR组Bax/Bcl-2表达增高,差异有统计学意义(P<0.05),与CPR组相比,CPR+NaHS组Bax/Bcl-2表达明显下降,差异有统计学意义(P<0.05),CPR+AOAA组表达Bax/Bcl-2表达升高,差异有统计学意义(P<0.05)。与假手术组相比,CPR组SOD活性下降,差异有统计学意义(P<0.05)；与CPR组相比,CPR+NaHS组SOD活性增高,差异有统计学意义(P<0.05)；与CPR组相比,CPR+AOAA组SOD活性降低,差异有统计学意义(P<0.05)。与假手术组相比,CPR组MDA活性增高,差异有统计学意义(P<0.05)；与CPR组相比,CPR+NaHS组MDA活性降低,差异有统计学意义(P<0.05)；与CPR组相比,CPR+AOAA组MDA活性升高,差异有统计学意义(P<0.05)。实时定量PCR和Western结果均显示CPR组HIF-1α基因及蛋白表达水平较假手术组增高(P<0.05)；与CPR组相比,CPR+NaHS组HIF-1蛋白表达水平增高(P<0.05)；与CPR组相比,CPR+AOAA组HIF-1αmRNA表达水平有降低趋势,但差异无统计学意义(P<0.05)。[结论]电刺激法所致心脏骤停模型诱导心脏骤停表现类型多样,诱发心脏骤停的时间明显短于窒息法心脏骤停,可作为心脏骤停模型制备的首选。H2S可以减轻大鼠复苏后脑损伤,给予H2S抑制剂AOAA则加重脑损伤。其脑保护机制与减轻炎症反应、减轻凋亡、减轻氧化应激反应有关,并通过上调缺氧诱导因子发挥脑保护作用。更多还原

【Abstract】 【Objective】1. To compare cardiac arrest induced by asphyxiation with cardiac arrest induced by transcutaneous electrical epicardium stimulation according to utstein style, and to find out a standardized animal cardiac arrest model which is consistant with clinical practice.2. To investigate the effects of hydrogen sulfide on the injury of brain injury induced by cardiac arrest.3. To investigate the effects of H2S on inflammatory reaction.4. To investigate the effects of H2S on apoptosis reaction.5. To investigate the effects of H2S on oxidative stress.6. To investigate whether the effects of H2S is associated with HIF-1.[Methods] In Part 1, animals were randomly divided into two groups:one group received cardiac arrest induced by asphyxiation, the other group received cardiac arrest induced by transcutaneous electrical epicardium stimulation. Animals were randomly divided into four groups:sham group, cardiopulmonary resuscitation group (CPR group), CPR+NaHS group and CPR+AOAA group. Types of cardiac arrest were observed, and time of asphyxiation or electrical stimulation to cardiac arrest were recorded. In Part 2、Part 3、Part 4, animals were randomly divided into four groups:The rats of sham group were only anesthetized and intubated. CPR group、NaHS+CPR group and CPR+AOAA group received cardiac arrest by transcutaneous electrical epicardium stimulation. For CPR+NaHS group, NaHS (100ug/kg) was administrated via the femoral venous line 1 minute before CPR. For CPR+AOAA group, AOAA (20mg/kg) was administrated via the femoral venous line 1 minute before CPR. NDS value was scored and the morphological structures of brain were also observed. TNF-a and IL-1βwere detected by ELISA method; Bax and Bcl-2 expression were detected by PCR and immunohistochemical methods; MDA and SOD were determined; the expression of HIF-1 were also determined by real-time quantitative PCR and Western blot.[Results] Cardiac arrest induced by transcutaneous electrical epicardium stimulation appeared VF, PEA, asytole. Cardiac arrest induced by asphyxiation appeared PEA, asytole. Time of asphyxiation to cardiac arrest was obviously longer than electrical stimulation to cardiac arrest(p＜0.05). Compared with sham group, the concentration of TNF-a were substantially elevated at 3h,6h,9h,12h(p<0.05), and it peaked 6h after CPR; Compared with CPR, the concentrations of TNF-a in CPR+NaHS were substantially decreased at 3h, 6h,9h,12h(p＜0.05), the concentrations of TNF-a in CPR+AOAA were substantially increased at 3h,6h(p＜0.05). Compared with sham group, the concentration of IL-1βwere substantially elevated at 3h,6h,9h,12h(p＜0.05), and it peaked 6h after CPR; Compared with CPR, the concentrations of IL-1βin CPR+NaHS were substantially decreased at 6h, 9h,12h(p＜0.05), the concentrations of TNF-a in CPR+AOAA were substantially increased at 3h,6h,9h(p＜0.05). Compared with sham group, the expression of Bax in CPR was increased(p＜0.05); Compared with CPR, the expression of Bax in CPR+NaHS group was decreased(p＜0.05); There were no obviously differences in CPR group and CPR+AOAA group. Compared with sham group, the expression of Bcl-2 in CPR was increased(p＞0.05); Compared with CPR, the expression of Bcl-2 in CPR+NaHS was obviously increased(p＜0.05); and the expression of Bcl-2 in CPR+AOAA was obviously decreased(p＜0.05). Compared with sham group, the ratio of Bax/Bcl-2 in CPR was increased(p＜0.05); Compared with CPR, the ratio of Bax/Bcl-2 in CPR+NaHS group was decreased(p＜0.05); and the ratio of Bax/Bcl-2 in CPR+AOAA group was increased(p＜0.05). Compared with sham group, the expression of SOD in CPR was decreased(p＜0.05); Compared with CPR, the expression of SOD in CPR+NaHS was obviously increased(p＜0.05); and the expression of SOD in CPR+AOAA was obviously decreased(p＜0.05). Compared with sham group, the expression of MDA in CPR was increased(p＜0.05); Compared with CPR, the expression of MDA in CPR+NaHS was obviously decreased(p＜0.05); and the expression of MDA in CPR+AOAA was obviously increased(p＜0.05). Compared with sham group, the expression of HIF-1 gene and protein was upregulated. Compared with CPR group, the expression of HIF-1 gene and protein in CPR+NaHS group was upregulated, and the expression of HIF-1 gene and protein in CPR+AOAA group has a lower trend(p＜0.05).[Conclusion] The cardiac arrest model induced by transcutaneous electrical epicardium stimulation is more close to clinical practice, the standardization of model is superior to cardiac arrest induced by asphyxiation. Hydrogen sulfide can protect brain from injury, and administration of AOAA aggravated brain damage. The mechanism is associated with antiinflammation, relieving apoptosis, reducing oxidatie stress reaction relevant and upregualating HIF-1.更多还原