【作者】 孙振涛；

【导师】 韩雪萍；

【作者基本信息】 郑州大学 ， 麻醉学， 2012， 博士

【摘要】 脑死亡是指包括脑干在内的全脑机能丧失的不可逆性病理状态,脑死亡可导致肺损伤,肺损伤引起的氧合不足将直接影响到其他器官的功能和保护,探讨脑死亡肺损伤的机制,对预防和治疗脑死亡肺损伤具有重要意义。降钙素基因相关肽(Calcitonin gene-related peptide, CGRP)广泛存在于神经系统、血管、肺脏等多种组织中,是一种具有血管舒张、心肌正性变力变时、肺保护、脑保护以及免疫调节作用等多种生物学功能的多肽。研究发现颅脑损伤后机体内CGRP含量发生改变,并且和颅脑损伤的严重程度相关,但脑死亡后CGRP的含量改变和脑死亡肺损伤发生发展的关系尚不明确。基于“脑死亡后机体CGRP含量发生改变,进而参与了脑死亡后肺损伤的发生和发展”的假设。本实验拟首先采用缓慢间断颅内加压法建立大鼠脑死亡模型,然后采用放射免疫法,RT-PCR,免疫印迹(Western blot)等实验方法观察脑死亡不同时间点大鼠血浆CGRP及内皮素-1(Endothelin-1, ET-1)含量变化及肺组织中CGRP的mRNA水平及蛋白表达,分析其与脑死亡后肺损伤的关系,探讨CGRP在脑死亡后的变化规律及其对脑死亡后肺损伤的影响与机制,为脑死亡后肺损伤的预防和治疗提供思路。进而在建立脑死亡模型时静脉给予外源性CGRP,采用放射免疫法,酶联免疫吸附实验(Enzyme-linked Immunosorbent Assay, ELISA), RT-PCR, Western blot,免疫组织化学等实验方法观察CGRP对脑死亡大鼠血浆肿瘤坏死因子-α(Tumor necrosis factor-α, TNF-α)、白细胞介素-1β(Interleukin-1β, IL-1β)、白细胞介素-6(Interleukine-6, IL-6)等炎症因子水平及肺组织中丙二醛(Malondialdehyde, MDA)含量和超氧化物歧化酶(Superoxide Dismutase, SOD)活性的影响及对肺组织γ-谷氨酰半胱氨酸合成酶(y-glutamylcysteine Synthetase,γ-GCS)、水通道蛋白-1 (Aquaporin Protein-1, AQP-1)、CGRP的mRNA水平及蛋白表达的影响。同时观察静脉给予外源性CGRP后对肺组织形态的影响,探讨CGRP的肺保护作用及其可能机制。因为外源性CGRP在体内迅速灭活,代谢快、作用时间短,而构建CGRP重组腺病毒载体气管导入具有不整合入宿主细胞基因组,安全方便,作用时间长等优点,理论上可对脑死亡肺损伤起到更好的保护作用。所以,拟在以上实验的基础上,通过气管导入带有增强型绿色荧光蛋白标记的降钙素基因相关肽重组腺病毒载体(Ad5-CGRP-EGFP),然后观察CGRP基因转染后大鼠肺组织CGRP mRNA水平和蛋白表达水平及CGRP基因治疗对肺组织γ-GCS, AQP-1等mRNA水平和蛋白表达的影响,对肺组织MDA含量、SOD活力,肺水含量、肺组织结构的影响,以及对血浆TNF-α、IL-1β、IL-6含量的影响,并探讨CGRP基因治疗在脑死亡大鼠肺保护中的作用及机制。本研究拟观察脑死亡后CGRP的变化规律及其和脑死亡肺损伤的关系,并通过CGRP基因导入观察CGRP基因治疗对脑死亡大鼠的肺保护作用并探讨其可能机制,为脑死亡肺损伤的预防及基因靶向治疗提供理论依据。本研究共分以下3个部分。第一部分脑死亡大鼠CGRP表达变化与脑死亡肺损伤1方法1.1健康Wistar大鼠20只,雌雄不限,体重250-300g,SPF级,将动物随机分为2组,即对照组(A组)、脑死亡组(B组),每组10只。B组麻醉后行股动脉股静脉插管、气管插管及颅骨钻孔置管,硬脑膜外导管颅内缓慢加压建立脑死亡模型；A组麻醉后操作同B组,但不行硬脑膜外导管颅内加压。分别在麻醉后15min(T0)、脑死亡即刻(T1)、脑死亡2h时点(T2)、脑死亡6h时点(T3)、脑死亡12h时点(T4)留取血液标本并于脑死亡12h时点留取肺组织标本。1.2采用放射免疫法分别检测不同时点血浆CGRP、ET-1含量变化。脑死亡12h时点留取肺组织,测定肺系数和肺水含量,苏木素-伊红(HE)染色观察肺脏组织结构变化。RT-PCR方法检测肺组织中CGRP mRNA; Western blot方法测肺组织CGRP蛋白。1.3统计学处理：所有数值变异均采用均数±标准差(X±s)表示,应用SPSS 16.0统计分析软件,采用重复测量数据的方差分析、单因素方差分析等分析方法进行统计学处理,显著性检验水准取α=0.05。2结果2.1血压和心率变化：A组在整个实验过程中血压和心率无明显变化(P>0.05)；B组在颅内加压过程中血压增高,心率先慢后快,B组血压在T1及以后各时点较T0时点低,B组心率在T1及以后各时点较组内T0时点快,(P<0.05)。B组在峰值时点血压增高、心率增快,在T1及以后各时点B组血压较A组低,心率较A组快,组间比较差异有统计学意义(P<0.05)。2.2血浆CGRP变化：A组在T1时点CGRP含量较TO时点增高,在T2时点CGRP含量较脑死亡时点降低(P<0.05),T2时点以后CGRP含量恢复正常；B组在T1时点CGRP含量较T0时点增高,T1后各时点CGRP含量与T0时点比较均降低,T4时点较T3时点CGRP含量有所回升,组内比较差异有统计学意义(P<0.05)。B组T1时点CGRP含量较A组增高,T1后各时点CGRP含量均较A组降低,组间比较差异有统计学意义(P<0.05)。2.3血浆ET-1变化：A组在T1时点及T2、T3时点ET-1含量较TO时点增高(P<0.05),T4时点恢复正常；B组T1后各时点ET-1含量与T0时点比较均增高,组内比较差异有统计学意义(P<0.05)。B组T1后各时点ET-1含量均较A组增高,组间比较差异有统计学意义(P<0.05)。2.4肺组织CGRP mRNA水平比较B组较A组升高,肺组织CGRP蛋白表达水平B组较A组降低,组间比较差异有统计学意义(P<0.05)。2.5脑死亡大鼠肺组织常规结构变化：HE染色结果显示A组肺脏组织结构基本正常：B组出现损伤性改变。2.6肺系数及肺水含量B组均较A组增高,组间比较差异有统计学意义(P<0.05)。第二部分外源性CGRP对脑死亡大鼠肺损伤的影响及其机制1方法1.1健康Wistar大鼠75只,雌雄不限,体重250-300g,SPF级,将动物随机分为三组,每组25只,即对照组(A组)、脑死亡组(B组)、CGRP干预组(C组)。B、C组建立脑死亡模型；A组除不行硬脑膜外导管加压外,其余处理同脑死亡组；C组为CGRP干预的脑死亡组,于脑死亡模型建立成功后经静脉用微量输注泵给予CGRP3μg/kg,随后给与CGRP 6μg/kg持续输注维持12h。分别于麻醉后15min(TO)、脑死亡即刻(T1)、脑死亡2h时点(T2)、脑死亡6h时点(T3)、脑死亡12h时点(T4)每组随机选5只共15只大鼠抽取血液标本并取肺标本。1.2采用ELISA法测定各时点血浆TNF-α、IL-1β、IL-6含量；采用放射免疫法分别检测各时点血浆CGRP、ET-1含量变化；T4时点留取肺组织,硫代巴比妥钠法测定肺匀浆中MDA含量,黄嘌呤氧化酶法测定肺组织中SOD活性,分析天平测定肺系数和肺水含量,HE染色观察肺脏组织结构变化；RT-PCR方法检测T1、T3、T4时点肺组织中γ-GCS、AQP-1、CGRPmRNA; Western blot方法测T1、T3、T4时点肺组织γ-GCS、AQP-1、CGRP蛋白表达。1.3统计学处理：所有数值变异均采用均数±标准差(X±s)表示,应用SPSS 16.0统计分析软件,采用重复测量数据方差分析、单因素方差分析等进行统计学处理,显著性检验水准取α=0.05。2结果2.1血压和心率变化：A组在整个实验过程中血压和心率无明显变化(P<0.05)；B组、C组在颅内加压过程中血压增高,心率增快,B组、C组血压在T1及以后各时点较T0时点低,B组、C组心率在T1及以后各时点较组内TO时点快,(P<0.05)。B组、C组在峰值时点血压增高、心率增快,在T1及以后各时点B组、C组血压较A组低,心率较A组快,组间比较差异有统计学意义(P<0.05)。2.2血浆TNF-α变化：A组各时点TNF-α水平差异无统计学意义；B组、C组TNF-α水平自T2开始逐渐升高,每后一时间点与前一时间点相比差异有统计学意义(P<0.05)。TNF-α水平自T2时点开始,C组高于A组,B组高于C组和A组,组间比较差异有统计学意义(P<0.05)。2.3血浆IL-1p变化：A组各时点IL-1p水平差异无统计学意义；B组、C组IL-1p水平自T2开始逐渐升高,每后一时间点与前一时间点相比差异有统计学意义(P<0.05)。IL-18水平自T2时点开始,C组高于A组,B组高于C组和A组,组间比较差异有统计学意义(P<0.05)。2.4血浆IL-6变化：A组IL-6水平T2、T3、T4时点升高与T0、T1时间点比较差异有统计学意义(P<0.05)；B组、C组IL-6水平自T2开始逐渐升高,每后一时间点与前一时间点相比差异有统计学意义(P<0.05)。IL-6水平自T2时点开始,C组高于A组,B组高于C组和A组,组间比较差异有统计学意义(P<0.05)。2.5血浆CGRP变化：A组在T1时点CGRP含量较T0时点增高,在T2时点CGRP含量较T1时点降低,组内比较差异有统计学意义(P<0.05),T2时点以后CGRP含量恢复正常；B组CGRP含量在T1时点较T0时点增高,T1后各时点与TO时点比较均降低,组内比较差异有统计学意义(P<0.05)；C组CGRP含量在T1后各时点与T0时点比较均增高,组内比较差异有统计学意义(P<0.05)。B组CGRP含量T1时点较A组增高,T2后各时点均较A组降低,C组CGRP含量T1以后各时点较A组增高,T2后各时点均较B组增高,组间比较差异有统计学意义(P<0.05)。2.6血浆ET-1变化：A组ET-1含量在T1时点及T2时点较T0时点增高(P<0.05),T3时点以后恢复正常,B组ET-1含量T1后各时点与T0时点比较均增高,C组ET-1含量T1后各时点与TO时点比较均增高,组内比较差异有统计学意义(P<0.05)。B组、C组T1后各时点ET-1含量均较A组增高, C组T2后各时点ET-1含量均较B组减低,组间比较差异有统计学意义(P<0.05)。2.7肺组织MDA含量及SOD活性：肺组织中MDA含量B组高于A组及C组,C组高于A组(P<0.05),肺组织SOD活性B组低于A组及C组,组间比较差异有统计学意义(P<0.05)。2.8肺组织γ-GCS mRNA水平B组、C组均较A组升高,C组较B组升高(P<0.05)。肺组织AQP-1 mRNA水平B组较A组降低,C组较B组、A组增高(P<0.05)。CGRP mRNA水平B组较A组升高,C组较A组降低(P<0.05)。肺组织γ-GCS、AQP-1、CGRP蛋白水平均B组较A组降低,C组较B组、A组增高,组间比较差异有统计学意义(P<0.05)。2.9脑死亡大鼠肺组织常规结构变化：HE染色结果显示A组肺脏组织结构基本正常；B组出现损伤性改变,C组出现损伤性改变较组B减轻。2.10肺系数及肺水含量B组均较A组增高,C组肺系数及肺水含量也较A组增高,但较B组降低,3组间比较差异有统计学意义(P<0.05)。第三部分CGRP基因导入对脑死亡大鼠肺损伤的影响1方法1.1 Wistar大鼠50只,雌雄不限,体重250-300g,SPF级,随机分为5组,每组10只,即对照组(A组)、脑死亡组(B组)、脑死亡CGRP干预组(C组)、脑死亡空载体导入组(D组)、脑死亡CGRP基因治疗组(E组)。B组、C组、D组、E组均建立脑死亡模型；A组开颅等处理均同脑死亡组,但不行硬脑膜外导管加压。C组为CGRP干预的脑死亡组,脑死亡模型建立后经静脉用微量输注泵给予CGRP3μg/kg,随后给与CGRP 6μg/kg持续输注维持12h；D组于脑死亡模型建立后微量输注泵给予CGRP3μg/kg,同时通过气管导入空载体；E组于脑死亡模型建立后微量输注泵给予CGRP3μg/kg同时通过气管导入携带增强型绿色荧光蛋白标记的降钙素基因相关肽重组腺病毒载体(Ad5-CGRP-EGFP)。分别在麻醉后15min(T0)、脑死亡12h时点(T1)抽取血液标本,在脑死亡12h或对照组12h时点取材肺组织标本。1.2采用ELISA方法测定各时点血浆TNF-αIL-1β, IL-6水平；采用放射免疫法分别检测各时点血浆CGRP、ET-1含量；T1时点留取肺组织,荧光显微镜下检测基因转染情况,硫代巴比妥钠法测定肺匀浆中MDA含量,黄嘌呤氧化酶法测定肺组织中SOD活性；分析天平测定肺系数和肺水含量,HE染色观察肺脏组织结构变化,免疫组化检测肺组织γ-GCS, AQP-1蛋白表达；RT-PCR方法检测肺组织中γ-GCS、AQP-1 CGRP mRNA; Western blot方法测肺组织γ-GCS、AQP-1、CGRP蛋白表达。1.3统计学处理：所有数值变异均采用均数±标准差(X±s)表示,应用SPSS 16.0统计分析软件,采用重复测量数据的方差分析、单因素方差分析等统计学方法进行统计学处理,显著性检验水准取a=0.05。2结果2.1 A组、B组、C组未见绿色荧光反应,D组、E组肺组织标本在荧光显微镜下呈较强的绿色荧光反应,证明基因转染成功。2.2血压和心率变化：A组在整个实验过程中血压和心率无明显变化(P>0.05)；B组、C组、D组、E组在颅内加压过程中血压增高,心率增快,B组、C组、D组、E组血压在脑死亡时点及以后各时点较TO时点低,B组、C组、D组、E组心率在脑死亡及以后各时点较组内TO时点快,(P<0.05)。B组、C组、D组、E组在峰值时点血压增高、心率增快,在脑死亡时点及以后各时点B组、C组、D组、E组血压较A组低,心率较A组快,组间比较差异有统计学意义(P<0.05)。2.3血浆TNF-α、IL-1β、IL-6水平：T0时点各组TNF-α、IL-1β、IL-6水平差异无统计学意义。T1时点TNF-α、IL-1β、IL-6水平B组、D组高于E组、C组和A组,E组、C组高于A组,C组高于E组,组间比较差异有统计学意义(P<0.05)。2.4血浆CGRP含量：T0时点各组血浆CGRP水平差异无统计学意义。T1时点E组血浆CGRP水平高于D组、C组、B组和A组,C组高于D组、B组和A组,A组高于D组和B组,组间比较差异有统计学意义(P<0.05)。2.5血浆ET-1含量：T0时点各组血浆ET-1水平差异无统计学意义。T1时点E组低于D组、C组、B组和A组,C组低于D组、B组和A组,A组低于D组和B组,组间比较差异有统计学意义(P<0.05)；2.6肺组织MDA含量及SOD活性：肺组织中MDA含量B组、D组高于A组、C组和E组,C组高于E组和A组,E组高于A组(P<0.05),肺组织SOD活性C组、E组高于A组、B组和D组,E组高于C组(P<0.05)。2.7肺组织γ-GCS mRNA水平B组、C组、D组、E组均较A组升高,C组较B组、D组升高,E组较C组升高(P<0.05)。肺组织AQP-1 mRNA水平B组、D组较A组低,C组、E组较A组高,E组较C组高(P<0.05)。肺组织CGRP mRNA水平B组、D组、E组较A组升高,C组较A组降低,E组较B组、D组高(P<0.05)。肺组织γ-GCS、AQP-1 CGRP蛋白表达水平均B组、D组较A组降低,C组、E组较A组高,E组较C组高,组间比较差异有统计学意义(P<0.05)。2.8肺组织γ-GCS、AQP-1免疫组化结果：γ-GCS、AQP-1蛋白表达B组、D组均较E组、C组和A组降低,E组、C组较A组增高,E组较C组增高,组间比较差异有统计学意义(P<0.05)。2.9脑死亡大鼠肺组织常规结构变化：HE染色结果显示A组肺脏组织结构基本正常；B组、D组出现损伤性改变,C组出现损伤性改变较B组、D组减轻,E组损伤性改变较C组轻。2.10肺系数及肺水含量：B组、D组均较E组、C组和A组高,E组、C组较A组高,E组较C组低,组间比较差异有统计学意义(P<0.05)。结论1、脑死亡大鼠血浆及肺组织CGRP含量下降,脑死亡后肺损伤和机体CGRP水平下降,TNF-α、IL-1β、IL-6等促炎因的释放增加,氧化应激增强,AQP-1表达下降等有关。2、外源性CGRP对脑死亡大鼠肺组织具有保护作用。3、CGRP基因转染脑死亡大鼠较静脉应用外源性CGRP血浆TNF-αIL-1βIL-6含量下降,肺组织MDA水平下降、SOD活力增高、AQP-1,γ-GCSmRNA和蛋白表达增加。4、CGRP基因转染较静脉应用CGRP对脑死亡大鼠具有更好的肺保护作用,其机制与降低炎症反应,抗氧化应激,增加AQP-1的表达有关。更多还原

【Abstract】 Brain death is an irreversible pathological state of the whole brain functioning failure which involves the brainstem. Literally, the brain death leads to lung injury, which brings oxygenation insufficiency giving rise to the dysfunction and injury of other organs. The exploration into the mechanism of brain death-related lung injury plays a key role in the prophylaxis and treatment of brain death related lung injury.The calcitonin gene-related peptide (CGRP) is a multi-tasking protein, such as vasodilator, myocardial strengthener, lung protector, brain reservoir, immunomodilator, etc.Researchers have demonstrated that the amount of CGRP in human body changed after craniocerebral injury and was related to the brain injury severity.Based on the hypothesis that the changes amount of CGRP in human body which participates in development of the brain death-related lung injury, this experiment adopted the method of slow continuous intracranial pressure to build a brain death model. The radioimmunoassay, RT-PCR, Western blot were used to study the changes of CGRP and endothelin-1 (ET-1) in rat blood plasma and lung tissue at several time points during brain death and the relationship between those changes with brain death-related lung injury. Given the research on the CGRP change pattern after brain death and the influence of CGRP to the lung injury, it initiates a novel solution for the prophylaxis and treatment of brain death-related lung injury. CGRP was administered intravenously during brain death modeling. Radioimmunoassay, ELISA, RT-PCR, Western blot and immunohistochemistry are to be employed to observe the amount of brain death rat’s tumor necrosis factor-a (TNF-α), interleukin-1β(IL-1β), interleukine-6 (IL-6) and other inflammatory factors in the blood plasma as well as that of malondialdehyde (MDA), superoxide dismutase (SOD) competence in the lung tissue and the influence on the AQP-1 and y-GCS mRNA and proteins’ expression. The lung function and morphology are simultaneously observed in order to explore the possible mechanism of CGRP’s protective effect on the lung.Since that the exogenous CGRP is inactivated, metabolized quickly and short-acting and that CGRP recombinant adenovirus vector if transferred into the trachea cells is characterized by host cell genome disintegration which is a safe convenient and durable, it is theoretically a better protector for the brain death-related lung injury. This project is aimed to construct the Ad5-CGRP-EGFP and transfer it into trachea cells to observe the CGRP expression change of CGRP gene transfected ran lung tissue and also the effect of CGRP gene therapy on the lung tissue’s AQP-1 and y-GCS, the amount of MDA, SOD competence, pulmonary water content, lung morphology as well as the content of TNF-α, IL-1β, IL-6 in blood plasma. The mechanism of CGRP gene therapy in the lung protection of brain death is also explored.This study intends to clarify the in vivo CGRP change pattern after brain death and it’s link with the brain death-related lung injury. The CGRP gene therapy effect and mechanism on the lung injury are both studied through the inference of CGRP gene transferring, with view of throwing a light theoretically on the prophylaxis and treatment of brain death-related lung injury.This study includes the following three parts.Part one CGRP expression in brain death rat and related lung injury1 Methods 1.1 Twenty adult Wistar rats, weighted between 250 and 300g, were randomly divided into control(A) and brain-death(B) groups (n=10). Brain-death model was established in group B. The group A were placed Foley balloon catheter in intracalvarium only; no brain-death model established. Blood sample were collected at the time point of anesthesia 15 min (TO) and 0 h (T1),2 h (T2),6h (T3) and 12 h (T4) after brain-death model established. The samples of lung tissues were harvested at T4 time point.1.2 Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. Histological alterations in lung tissues were examined by HE staining. And at the same time lung index and lung water content were measured. The levels of CGRP in lung tissues were examined by RT-PCR and Western blot.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. P< 0.05 was considered statistically significant.2 Results2.1 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. MAP of group B were significantly lower at T1 and after T1 time points than that of TO time point, HR of group B were significantly faster at T1 and after T1 time points than that of TO time point (P<0.05). At the peak time point MAP and HR of group B were significant increase (P<0.05). Comparison of MAP at T1 and after T1 time points, MAP of group B, were significant lower than that of group A, comparison of HR at T1 and after T1 time points, HR of group B, were significant faster than that of group A (P<0.05).2.2 In group A, the levels of CGRP in serum was higher at T1 than that at T0, and lower at T2 than that at T1, and the difference was significant (P<0.05). In group B, the levels of CGRP was higher at than that at TO, and lower at T2-T4 than that at T0, and the difference was significant (P<0.05). The levels of CGRP in group B were higher than that in group A at T1 time points, but lower than that in group A at other time points, and the difference was significant (P<0.05).2.3 The levels of ET-1 was higher at other time points except T4 than that at TO in group A, and the difference were statistically significant (P<0.05). The levels of ET-1 was higher at other time points than that at TO in group B, and the difference was significant (P<0.05). The levels of ET-1 in group A was higher at same time points than that in group B, and the difference were statistically significant (P<0.05).2.4 The levels of mRNA of CGRP in lung tissues were significantly up-regulation in group B compared with that in group A and protein of CGRP in lung tissues were significantly down-regulation in group B compared with that in group A (P<0.05).2.5 Histological alterations:The lung injury was confirmed by HE examination in groupB. The lung Histological examination in group A was normal.2.6 In contrast to group A, lung index and lung water content were increased significantly in group B (P<0.05).Part two Effect of CGRP on the lung injury in brain death rats and its mechanisms1 Methods1.1 Seventy-five adult Wistar rats, weighted between 250 and 300g, and were randomly divided into control group (A), brain-death group (B) and CGRP treatment group (n=25). Brain-death model was established in group B and group C. The group A were placed Foley balloon catheter in intracalvarium only; no brain-death model established. In group C, CGRP 3μg/kg was injected intravenously immediately after establishment of brain-death model, followed by CGRP 6μg/kg continously injected for 12h. Blood sample and lung tissues of five rats in each group were collected at the time point of anesthesia 15 min (TO) and 0 h (T1),2 h (T2),6 h (T3) and 12 h (T4) after brain-death model established.1.2 The levels of TNF-α, IL-1βand IL-6 in the serum were measured by Enzyme-linked immunosorbent assay (ELISA). Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. The MPO and SOD activity were evaluated in the lung tissues at T4. The lung index and lung water content were measured. Histological alterations in lung tissues were examined by HE staining. The levels ofγ-GCS、AQP-1、CGRP were measured by RT-PCR and Western blot at T4.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. P value of<0.05 was considered statistically significant.2 Results2.1 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B and C, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. MAP of group B and C were significantly lower at T1 and after T1 time points than that of TO time point, HR of group B and C were significantly faster at T1 and after T1 time points than that of TO time point (P<0.05). At the peak time point MAP and HR of group B and C were significant increase (P<0.05). Comparison of MAP at T1 and after T1 time points, MAP of group B and C, were significant lower than that of group A, comparison of HR at T1 and after T1 time points, HR of group B and C, were significant faster than that of group A (P<0.05)。2.2 TNF-α:There were no significant difference for the levels of TNF-a among time points in group A. The levels of TNF-a were higher at other time points than that at T2 in group B and C, and the difference was significant (P<0.05). From T2, the level of TNF-αwas higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05)2.3 IL-1β:There were no significant difference for the levels of IL-lβamong time points in group A. The levels of IL-1βwere higher at other time points than that at T2 in group B and C (P<0.05). From T2, the level of IL-1 (3 was higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05)2.4 IL-6:The levels of IL-6 were higher at T2, T3 and T4 than that at TO and T1 in group A (P<0.05). The levels of IL-6 were higher at other time points than that at T2 in group B and C(P<0.05).From T2, the level of IL-6 was higher in group C than that in group A, and higher in group B than that in group A and C (P<0.05) 2.5 CGRP:The levels of CGRP in serum were higher at T1 than that of TO in group A, and lower at T2 than that at T1 (P<0.05).In group B, the levels of CGRP were higher at T1 than that at T0, but lower at other time points than that at TO (P<0.05). The levels of CGRP were higher at all the time points than that at TO in group C (P<0.05). The level of CGRP was higher in group B than that in group A at T1, but lower at other time points. The level of CGRP was higher in group C than that in group A from T1, and higher than that in group B from T2 (P<0.05)2.6 ET-1:The levels of ET-1 in serum were higher at T1 and T2 than that of TO in group A (P<0.05). The levels of ET-1 were higher at other time points than that of TO in group B (P<0.05). The levels of ET-1 were higher at other time points than that of TO in group C (P<0.05). The levels of ET-1 were higher in group B and C than that in group A from T1. The levels of ET-1 were lower in group C than that in group B from T2 (P<0.05)。2.7 MDA and SOD activity:MDA activity of lung tissues in group B was higher than that in group A and C, and group C was higher than that in group A (P<0.05) SOD activity of lung tissues in group B was lower than that in group A and C (P<0.05)2.8 The mRNAs levels ofγ-GCS in group B and group C were higher than that in group A, and group C were higher than that in group B (P<0.05). The mRNAs levels of AQP-1 in group B were lower than that in group A, and group C were higher than that in group B (P<0.05). The mRNAs levels of CGRP in group B were higher than that in group A, and the mRNAs levels of CGRP in group C were lower than that in group A (P<0.05).The proteins levels of AQP-1、γ-GCS、CGRP in group B were lower than that in group A, and group C were higher than that in group A and B, and the differences were statistically significant (P<0.05).2.9 Histological alterations:The lung injury was confirmed by HE examination in group B and C, while less pathologic alterations were shown in group C. The lung Histological examination in group A was normal.2.10 In contrast to group A, lung index and lung water content were increased significantly in group B and C. But in contrast to group B, lung index and lung water content were decreased significantly in group C (P<0.05).Part three Effect of CGRP gene on lung injury in brain death rats1 Methods1.1 Fifty adult Wistar rats, weighted between 250 and 300g, and were randomly divided into control group (A), brain-death group (B), CGRP treatment group(C), empty vector group (D) and CGRP gene transfer group (E). Brain-dead model was established in group B, C, D and E. The group A were placed Foley balloon catheter in intracalvarium only; no brain-dead model established. In group C, CGRP was injected via vein immediately after establishment of brain-dead model. In group D, empty vector was transferred after establishment of brain-dead model. In group E, CGRP with enhanced green fluorescent protein gene mediated by recombinant adenovirus (Ad) vector was transinfected via trachea after establishment of brain-dead model. Blood sample and lung tissues in each group were collected at the time point of anesthesia (TO) and 12 h (T1) after brain-dead model established.1.2 The levels of TNF-α, IL-1βand IL-6 were measured by ELISA. Radioimmunoassay was used to examine the level of CGRP and ET-1 in the serum. The gene transfect was accessed by fluorescence microscope. The MPO and SOD activity were evaluated in the lung tissues at T1. The lung index and lung water content were measured. Histological alterations in lung tissues were examined by HE staining. The expression of y-GCS and AQP-1 was analyzed by immunohistochemistry. The levels of y-GCS, AQP-1, CGRP were measured by RT-PCR and Western blot at T1.1.3 Statistical analysis:Data are expressed as means±SEM. Statistical analysis was performed using the SPSS software version 16.0. All the data were analyzed by ANOVA. a level was set at 0.05.2 Results2.1 There was no green fluorescence in group A, B and C. Enhanced green fluorescence were seen in group D and E.2.2 Hemodynamic changes:BP and HR of group A during the whole experiment had no significant changes (P>0.05). For group B, C, D and E, BP increased significantly after increasing intracranial pressure and HR first decreased slowly then increased. Than that of TO time point, MAP of group B, C, D and E were significantly lower at the time point of brain-death model established and after. Than that of TO time point HR of group B, C, D and E were significantly faster at the time point of brain-death model established and after (P<0.05). At the peak time point MAP and HR of group B, C, D and E were significant increase (P<0.05). Comparison of MAP at the time point of brain-death model established and after, MAP of group B, C, D and E, were significant lower than that of group A, comparison of HR at the time point of brain-death model established and after, HR of group B, C, D and E, were significant faster than that of group A (P<0.05)。2.3 TNF-α, IL-1βand IL-6:The levels of TNF-α, IL-1βand IL-6 in group B and D were higher than those in group E, C and A, and group E and C were higher than those in group A, group C were higher than those in group E (P<0.05)。2.4 CGRP:There were no significant differences among groups at TO. The levels of CGRP were higher in group E than those in group D, C, B and A at T1. The levels of CGRP were higher in group C than those in group D, B and A. The levels of CGRP were higher in group A than those in group D and B (P<0.05)2.5 ET-1:There were no significant differences among groups at T0. The levels of ET-1 were lower in group E than those in group D, C, B and A at T1. The levels of ET-1 were lower in group C than those in group D, B and A. The levels of ET-1 were lower in group A than those in group D and B (P<0.05)2.6 MDA and SOD activity:In the lung tissues, the MDA activity in group B and D was higher than that in group A, C and E, while group C was higher than that in group A and E, and group E was higher than that in group A (P<0.05).The SOD activity in group C and E was higher than that in group A, B and D, while group E was higher than that in group C (P<0.05)2.7 The mRNAs levels of y-GCS in group B and group C and group D and group E were higher than that in group A, and group C were higher than that in group B and D, while group E were higher than that in group C (P<0.05). The mRNAs levels of AQP-1 in group B and group D were lower than that in group A, The mRNAs levels of AQP-1 were higher in group C and E than that in group A, while group E were higher than that in group C (P<0.05). The mRNAs levels of CGRP in group B and D were higher than that in group A, and group C were lower than that in group A, while group E were higher than those in group B and D, and the differences were statistically significant (P<0.05) The protein levels of y-GCS, AQP-1 and CGRP were lower in group B and in group D than those in group A. The protein levels of y-GCS, AQP-1 and CGRP were higher in group C and E than those in group A, while group E were higher than those in group C, and the differences were statistically significant (P<0.05)2.8γ-GCS and AQP-1 immunohistochemistry:The levels ofγ-GCS and AQP-1 in group B and D were lower than those in group E, C and A, while group E and C were higher than those in group B and D, and group E were higher than those in group C (P<0.05)2.9 Histological alterations:The lung injury was confirmed by HE examination in group B and D, while less pathologic alterations were shown in group C and E. The lung Histological examination in group A was normal.2.10 Lung index and lung water:Lung index and lung water content in group B and D was higher than that in group E, C and A, while group E and C was higher than that in group A, and group E was lower than that in group C (P<0.05)Conclusion1. The CGRP level changes after brain death in rats. Lung injury after brain death has a relationship with the decreased level of CGRP, the release of TNF-a, IL-1βand IL-6, the enhanced of oxidative stress and the expression down-regulated of AQP-1.2. Exogenous CGRP has protective effect on lung injury after brain death in rats. 3. CGRP gene transfection can up-regulated the expression of CGRP in lung tissues after brain death in rats. The levels of TNF-α, IL-1βand IL-6 in serum are decreased after CGRP gene transfection in brain death rats, while the expression ofy-GCS and AQP-1 in lung tissues are increased.4. CGRP gene transfection attenuates lung injury and lung water content after brain death in rats. The mechanism may have relation with its effect of anti-inflammation, anti-oxidative stress and AQP-1 up-regulation.更多还原