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hHSP70基因冠脉转染对大鼠心肌缺血再灌注损伤的

2014-07-11 01:05 毕业

【摘要】目的：研究hHSP70基因经冠脉转染对大鼠心肌抗缺血再灌注损伤的效应，并探讨其可能机制。
方法：（1）构建复制缺陷型重组腺病毒：采用分子克隆手段获得携带增强型绿色荧光蛋白(EGFP)基因的重组质粒、携带人热休克蛋白70的重组质粒，以细胞内同源重组法构建复制缺陷型重组腺病毒Adv-EGFP、Adv-hHsp70 ，在293细胞内分别扩增，应用氯化铯密度梯度离心法纯化病毒。经氯化铯纯化滴度分别为，Adv-EGFP：2.0×109Pfu/ml ； Adv-Hsp70: 2.5×1010 Pfu/ml 。
（2）选择50只S-D大鼠随机分成7组（n=10）。大鼠左侧开胸，暴露心脏及升主动脉和肺动脉根部，用无损伤血管钳钳夹于主、肺动脉根部，阻断循环10秒钟，同时用27号针头于左心尖部注射各组相应试剂0.1ml至左心室腔，使之在密闭的冠脉循环内分布。第Ⅰ、Ⅱ、Ⅲ组仅注射0.1ml生理盐水；第Ⅳ、Ⅴ组分别注射含Adv-EGFP、adv-Hhsp70（1×109pfu)的生理盐水；关胸苏醒后，稳定4天，使经冠脉转染的基因充分表达于心肌。
（3）冠脉转染4天后，再次暴露胸腔，在动脉圆锥与左心耳根部间结扎冠状动脉左前降支(LAD)。结扎45min后,松开缝线使冠状动脉再通180min。Ⅰ组只穿线不结扎。Ⅲ组在LAD结扎5min后开放5min，并重复3次，然后再行45min缺血、180min再灌注。
（4）采用Evan's Blue和TTC双染色法后利用image-pro plus5.0测定心肌梗死面积;光学显微镜下观察心肌的炎症改变；Western Blotting免疫印迹测定心肌基因表达的改变并利用bandscan 4.5进行灰度测量进而统计计算。
结果: （1）经缺血预处理或hHSP70转染后的心梗范围(27.13±2.7%，27.88±5.3%，)较缺血再灌注组和腺病毒对照组(39.72±4.2%，32.73±7.0%)明显缩小。hHSP70转染组与缺血预处理组没有统计学差别。 （科教范文网http://fw.nseac.com）
（2） hHSP70过量表达能够增加Bcl-2表达及减少caspase-3活化态的生成进。
（3） hHSP70过量表达能减少NF-κB的活化。
（4） 心肌表达的蛋白发生相应的变化，hHSP70转染组hHSP70过量表达，，Bcl-2表达增加，活化的caspase-3减少。
结论：（1）hHSP70转染能通过HSP70的过表达，增加Bcl-2表达，减少caspase-3的活化及其他机制来抑制凋亡。
（2）hHSP70转染能通过抑制NF-κB的表达及活化及其他机制起到抑制炎症作用。
（3）在HSP70过表达的情况下对心肌梗死与缺血预处理组没有统计学差别，可以起到与缺血预处理急性期同等程度的心肌的保护作用。

关键词 缺血再灌注损伤，腺病毒载体，hHSP70， 凋亡，炎症

ABSTRACT

Objectives：To observe the protective effect against myocardial ischemia reperfusion injury after transfer of human HSP70 gene via coronary artery and to investigate the potential mechanisms.
Methods：
(1) Construction of the replication-deficient recombinant adenovirus: Recombinant plasmids PDC-EGFP (containing enhanced green fluorescence protein EGFP gene), PDC-hsp70 (containing human HSP gene). Recombinant adenovirus Adv-EGFP, Adv-hsp70 were produced by homologous recombinant in 293 cells, amplified also in 293 cells on a large scale, and purified by ultracentrifugation in CsCl step gradient solutions. The concentration of recombinant adenovirus Adv-EGFP purified by ultracentrifugation in CsCl step gradient solutions is 2.0×109Pfu/ml; that of Adv-hsp70 is 2.5×1010Pfu/ml.
(2) 50 S-D rats were randomly divided into 5 groups (n=10). The left thoracotomy was performed and the pericardium opened with the exposure of the heart and the base of pulmonary artery and ascending aorta. Pulmonary artery and ascending aorta were occluded by applying a nontraumatic vascular clamp to achieve complete cessation of outflow, at the same time adenovirus particles (1.0×109pfu) in 0.1 ml of 0.9% saline solution were injected into the left ventricle cavity using a 1-ml syringe with a 27-gauge needle. After 10 seconds of complete outflow occlusion, the clamp was removed. 0.1ml saline solution alone was injected into the rat heart in GroupⅠ Ⅱ Ⅲ. In Group Ⅳ Adv-EGFP was injected. All of them were closed and recovered, and then stabilized without operative intervention for 4 day. （转载自http://zw.ＮＳＥaＣ.com科教作文网）
(3) After 4 days, a repeat thoracotomy was performed to expose the anterior surface of the heart. The proximal left anterior descending coronary artery (LAD) was identified and a 6/0 suture (Ethicon) was placed around the artery below the left atrial appendage and the surrouding myocardium. Regional left ventricular ischemia was established for 45 min by ligation of LAD. Ischemia was confirmed by discoloration of myocardium and by changes in cardiac rhythm. At the end of the ischemia period, the ligation was loosened and reperfusion was achieved for 180 min. GroupⅠ (sham-operated group) served as surgical controls and were subjected to the same surgical procedures as the the experimental group, with the exception that the LAD was not ligated. Group Ⅲ were subjected to ischemic preconditioning (IPC) before I/R by repeated ischemia and reperfusion through 3 cycles of induced LAD ischemia for 5 min followed by 5 min of reperfusion.
(4) To evaluate the effect of 45min ischemia followed by180min reperfusion of the LAD, the area at risk (AAR) and infarct region were determined by Evan’s blue dye perfusion and triphenyl tetrazolium chloride (TTC) staining. Aliquots of paraffin section were hematoxylin and eosin stained and examined under the light microscope to measure cardiac inflammation. Determination of Bcl-2， active caspase-3，active NF-κB expression in heart were performed with Western Blotting analysis.

Results: (1) The %AAR, determined by the amount of negative staining region with Evan’s blue after religation of the LAD, showed no difference among LAD ligated groups (GoupⅡ~Ⅴ). However, a significant reduction of the %infarct size was observed in the Hhsp70 group (27.88±5.3%), and IPC group (27.13±2.7%) compared with either saline control (39.72±4.2%) or adenoviral control (adv-EGFP) (32.73±7.0%) by TTC staining in the regional I/R model.
(2) Gene hHSP70 over expression can raise Bcl-2 production、restrain caspase-3 activated.
(3) Gene hHSP70 can restrain NF-κB active. （科教范文网http://fw.nseac.com）
(4) The expression of protein in rat heart altered. In Adv-HSP70 group the he expression of HSP70 and Bcl-2 were raised, activated caspase-3 decreased.

Conclusions：(1) Transfection of HSP70 inhibits inflammation through NF-κB downregulation.
(2) Transfection of HSP70 suppresses apoptosis through increasing Bcl-2 production and decreasing Caspase-3 activation.
(3) Transfection of HSP70 demonstrates synergistic effects of myocardial tolerance to I/R injury, which can analogue the protective effect of IPC to a larger degree.

KEY WORDS：ischemia reperfusion injury, adenovirus vector, hHSP70, apoptosis, inflammation


目 录

中文摘要……………………………………………………………………………Ⅰ
英文摘要………………………………………………………………………Ⅲ
英文缩略……………………………………………………………………………4
前 言………………………………………………………………………………5
第1章 材料与方法………………………………………………………………9
1.1 重组腺病毒构建…………………………………………………………9
1.2 动物………………………………………………………………………15
1.3 手术器械和仪器试剂……………………………………………………15
1.4 方法………………………………………………………………………16
1.5 统计处理…………………………………………………………………19
第2章 结果………………………………………………………………………20
2.1 重组腺病毒构建…………………………………………………………20
2.2 各组心肌HSP70表达量检测……………………………………………22
2.3 心肌梗死范围结果分析…………………………………………………23 （科教作文网http://zw.ΝsΕAc.com发布）
2.4 心肌缺血范围结果方差分析……………………………………………23
2.5 HSP70转染对心肌的炎症作用……………………………………………24
2.5.1 心肌的炎症光镜表现…………………………………………………24
2.5.2 NF-κB结果分析 ………………………………………………………26
2.6 细胞凋亡的结果分析 ……………………………………………………26
2.6.1 HSP70对Bcl-2表达影响………………………………………………26
2.6.2 HSP70对Caspase-3激活的影响………………………………………27
附图………………………………………………………………………………… 28
第3章 讨论……………………………………………………………………… 29
第4章 结论………………………………………………………………………32
第5章 参考文献…………………………………………………………………33
综述…………………………………………………………………………………36
致谢…………………………………………………………………………………48
主要英文缩略词表

Ad Adenovirus 腺病毒
Adv Adenoviral vector 腺病毒载体
Adv-EGFP Adv encoding EGFP 腺病毒介导的增强型绿色荧光蛋白基因
Bcl-2 B-cell leukemia/lymphoma-2 B细胞淋巴瘤因子-2
EGFP enhanced green fluorescence protein 增强型绿色荧光蛋白
CPB cardiopulmonary bypass 心肺转流术
IPC Ischemic Preconditioning 缺血预处理
SOD superoxide dismutase 超氧化物歧化酶
HGF hepatocyte growth factor 肝细胞生长因子
HSP heat shock protein 热休克蛋白
IGF insulin-like growth factor 胰岛素样生长因子
IL-10 interleukin 10 白细胞介素-10
I/R Ischemia and reperfusion 缺血再灌注
LUC luciferase 荧光素酶
MLC myosin light chain 肌球蛋白轻链

mRNA messenger ribonucleic acid 信息核糖核酸
OFR Oxygen free radical 氧自由基
AIF Apoptosis inducing factor 凋亡诱导因子
Pfu plaque forming unit 斑块形成单位
FGF Fibroblast growth factor 成纤维细胞生长因子
TNF Tumor necrosis factor 肿瘤坏死因子
TUNEL TdT-mediated dUTP digoxigenin nick end labeling 原位缺口末端
标记技术
VEGF vascular endothelial growth factor 血管内皮细胞
生长因子


前 言

缺血/再灌注损伤是指器官、组织在缺血的基础上恢复血液灌注时,细胞代谢功能障碍及结构破坏进1步加重的现象。 随着心脏骤停复苏成功率提高，动脉搭桥术、PTCA、心脏体外循环术的广泛开展，心肌的缺血/再灌注损伤的预防和治疗成为1个待进1步解决的课题。防治心肌I/R损伤的有效办法除了尽快恢复心肌血流的再灌注外，控制炎症和抑制凋亡，就成为防治心肌I/R损伤的主要途径。
HSP70是应激条件下产生的主要应激蛋白。HSP70对心肌再灌注损伤的保护作用是多方面的：（1）抗炎症作用。（2）抗凋亡作用。（3）分子伴侣作用。
HSP70与炎症反应
HSP70的抗炎机制(1)与其抑制核因子NF-κB的活性有关。HSP70通过抑制NF-κB的核转运来调节促炎介质的表达，具体是通过抑制IκB kinase (IKK)起作用的(2)。

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