【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。
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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。
（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 myo免费论文网 【http://www.51lunwen.net】cardial 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 with 本文来自：免费论文网 [http://www.51lunwen.net]