HHSP70基因冠脈轉(zhuǎn)染對大鼠心肌缺血再灌注損傷的保護作用

1、文章來源 畢業(yè)論文網(wǎng) hhsp70基因冠脈轉(zhuǎn)染對大鼠心肌缺血再灌注損傷的保護作用文章來源 畢業(yè)論文網(wǎng) 畢業(yè)論文 【摘要】目的：研究hhsp70基因經(jīng)冠脈轉(zhuǎn)染對大鼠心肌抗缺血再灌注損傷的效應，并探討其可能機制。 方法：（1）構(gòu)建復制缺陷型重組腺病毒：采用分子克隆手段獲得攜帶增強型綠色熒光蛋白(egfp)基因的重組質(zhì)粒、攜帶人熱休克蛋白70的重組質(zhì)粒，以細胞內(nèi)同源重組法構(gòu)建復制缺陷型重組腺病毒adv-egfp、adv-hhsp70 ，在293細胞內(nèi)分別擴增，應用氯化銫密度梯度離心法純化病毒。經(jīng)氯化銫純化滴度分別為，adv-egfp：2.0109pfu/ml ； adv-hsp70: 2.51010

2、 pfu/ml 。 （2）選擇50只s-d大鼠隨機分成7組（n=10）。大鼠左側(cè)開胸，暴露心臟及升主動脈和肺動脈根部，用無損傷血管鉗鉗夾于主、肺動脈根部，阻斷循環(huán)10秒鐘，同時用27號針頭于左心尖部注射各組相應試劑0.1ml至左心室腔，使之在密閉的冠脈循環(huán)內(nèi)分布。第、組僅注射0.1ml生理鹽水；第、組分別注射含adv-egfp、adv-hhsp70（1109pfu)的生理鹽水；關(guān)胸蘇醒后，穩(wěn)定4天，使經(jīng)冠脈轉(zhuǎn)染的基因充分表達于心肌。 （3）冠脈轉(zhuǎn)染4天后，再次暴露胸腔，在動脈圓錐與左心耳根部間結(jié)扎冠狀動脈左前降支(lad)。結(jié)扎45min后,松開縫線使冠狀動脈再通180min。組只穿線不結(jié)扎。

3、組在lad結(jié)扎5min后開放5min，并重復3次，然后再行45min缺血、180min再灌注。 （4）采用evans blue和ttc雙染色法后利用image-pro plus5.0測定心肌梗死面積;光學顯微鏡下觀察心肌的炎癥改變；western blotting免疫印跡測定心肌基因表達的改變并利用bandscan 4.5進行灰度測量進而統(tǒng)計計算。 結(jié)果: （1）經(jīng)缺血預處理或hhsp70轉(zhuǎn)染后的心梗范圍(27.132.7%，27.885.3%，)較缺血再灌注組和腺病毒對照組(39.724.2%，32.737.0%)明顯縮小。hhsp70轉(zhuǎn)染組與缺血預處理組沒有統(tǒng)計學差別。 （2） hhsp7

4、0過量表達能夠增加bcl-2表達及減少caspase-3活化態(tài)的生成進。 （3） hhsp70過量表達能減少nf-b的活化。 （4） 心肌表達的蛋白發(fā)生相應的變化，hhsp70轉(zhuǎn)染組hhsp70過量表達，bcl-2表達增加，活化的caspase-3減少。 結(jié)論：（1）hhsp70轉(zhuǎn)染能通過hsp70的過表達，增加bcl-2表達，減少caspase-3的活化及其他機制來抑制凋亡。 （2）hhsp70轉(zhuǎn)染能通過抑制nf-b的表達及活化及其他機制起到抑制炎癥作用。 （3）在hsp70過表達的情況下對心肌梗死與缺血預處理組沒有統(tǒng)計學差別，可以起到與缺血預處理急性期同等程度的心肌的保護作用。 關(guān)鍵詞 缺

5、血再灌注損傷，腺病毒載體，hhsp70， 凋亡，炎癥 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 adenov

6、irus: 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 ultrac

7、entrifugation in cscl step gradient solutions. the concentration of recombinant adenovirus adv-egfp purified by ultracentrifugation in cscl step gradient solutions is 2.0109pfu/ml; that of adv-hsp70 is 2.51010pfu/ml. (2) 50 s-d rats were randomly divided into 5 groups (n=10). the left thoracotomy wa

8、s 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.

9、0109pfu) 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 group . in group adv-egfp was inj

10、ected. all of them were closed and recovered, and then stabilized without operative intervention for 4 day. (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 (e

11、thicon) 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 per

12、iod, the ligation was loosened and reperfusion was achieved for 180 min. group (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 were subjected to ischemic precond

13、itioning (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

14、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-b expression in heart were performed wit

15、h western blotting analysis. results: (1) the %aar, determined by the amount of negative staining region with evans blue after religation of the lad, showed no difference among lad ligated groups (goup). however, a significant reduction of the %infarct size was observed in the hhsp70 group (27.885.3

16、%), and ipc group (27.132.7%) compared with either saline control (39.724.2%) or adenoviral control (adv-egfp) (32.737.0%) 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-b active. (4)

17、 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-b downregulation. (2) transfection of hsp70 suppresses apoptosis through increas

18、ing 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,

19、 inflammation 目 錄 中文摘要 英文摘要 英文縮略4 前 言5 第1章 材料與方法9 1.1 重組腺病毒構(gòu)建9 1.2 動物15 1.3 手術(shù)器械和儀器試劑15 1.4 方法16 1.5 統(tǒng)計處理19 第2章 結(jié)果20 2.1 重組腺病毒構(gòu)建20 2.2 各組心肌hsp70表達量檢測22 2.3 心肌梗死范圍結(jié)果分析23 2.4 心肌缺血范圍結(jié)果方差分析23 2.5 hsp70轉(zhuǎn)染對心肌的炎癥作用24 2.5.1 心肌的炎癥光鏡表現(xiàn)24 2.5.2 nf-b結(jié)果分析 26 2.6 細胞凋亡的結(jié)果分析 26 2.6.1 hsp70對bcl-2表達影響26 2.6.2 hsp70對c

20、aspase-3激活的影響27 附圖 28 第3章 討論 29 第4章 結(jié)論32 第5章 參考文獻33 綜述36 致謝48 主要英文縮略詞表 ad adenovirus 腺病毒 adv adenoviral vector 腺病毒載體 adv-egfp adv encoding egfp 腺病毒介導的增強型綠色熒光蛋白基因 bcl-2 b-cell leukemia/lymphoma-2 b細胞淋巴瘤因子-2 egfp enhanced green fluorescence protein 增強型綠色熒光蛋白 cpb cardiopulmonary bypass 心肺轉(zhuǎn)流術(shù) ipc ischem

21、ic preconditioning 缺血預處理 sod superoxide dismutase 超氧化物歧化酶 hgf hepatocyte growth factor 肝細胞生長因子 hsp heat shock protein 熱休克蛋白 igf insulin-like growth factor 胰島素樣生長因子 il-10 interleukin 10 白細胞介素-10 i/r ischemia and reperfusion 缺血再灌注 luc luciferase 熒光素酶 mlc myosin light chain 肌球蛋白輕鏈 mrna messenger ribonucleic a