【摘要】：[目的]:采用SD大鼠建立缺血性心力衰竭模型,術(shù)后4周存活大鼠予CSWT、PI3K/Akt信號通路阻斷劑LY294002處理,從整體動物水平驗證CSWT抑制心肌梗死后缺血性心力衰竭心肌細胞凋亡的作用,并探討其對PI3K/Akt信號通路的影響。[方法]:1、隨機入選60只成年雄性SD大鼠,體重220-250g,其中50只大鼠手術(shù)開胸,高位結(jié)扎左冠狀動脈前降支,建立SD大鼠缺血性心力衰竭模型。術(shù)前及術(shù)后采用高頻超聲心動圖評價大鼠心功能,評價建模成功率。2、所有實驗大鼠給予標準鼠糧及自來水自由飲食,術(shù)后4周存活大鼠隨機分為心衰組(IHF組9例)、心臟震波干預(yù)+心衰組(CSWT+IHF組9例)、LY294002+心衰組(LY294002+IHF組9例)、LY294002+心臟震波干預(yù)+心衰組(LY294002+CSWT+IHF組9例),另外10只正常大鼠作為對照組參與實驗。其中CSWT組實施心臟震波干預(yù),震波能量0.24MJ/mm2,每次200脈沖,頻率60次/min,每周三天,每天一次,共4周。3、CSWT干預(yù)4周后,采用高頻超聲評價大鼠心功能,利用TTC染色評估心肌梗死面積,應(yīng)用TUNEL凋亡檢測試劑盒測定心肌細胞凋亡,RT-PCR檢測心肌組織中Bcl-2、Bax、Casepase-3的mRNA表達水平,Western-blot檢測心肌組織中 Bcl-2、Bax、Casepase-3、AKT、p-AKT 的蛋白表達水平。[結(jié)果]:1、50只SD大鼠有47只完成急性心肌梗死模型制作,手術(shù)成功率84%。術(shù)后4周有36只心衰模型大鼠存活,存活率為76.6%。術(shù)后4周高頻超聲心動圖可見手術(shù)組大鼠左室擴大、左室壁變薄、室間隔運動幅度明顯減弱;LVESD(2.33±0.51 與 5.90±1.03,P0.001)和 LVEDD(4.92±0.73 與 7.54± 1.29,P0.001)較術(shù)前明顯擴大,且FS明顯減弱(51.14±6.25與20.16±2.92,P0.001),LVEF明顯降低(81.15±12.18 與 45.94±6.74,P0.001)。2、離體后利用TTC染色后評估心肌梗死面積占左室面積百分比;HF+CSWT組心肌梗死面積(17.10 ± 2.91%)均低于 HF 組(42.46 ± 5.39%),HF+LY 組(51.44±5.46%),HF+CSWT+LY 組(31.90±2.34%),且 P 均0.05。應(yīng)用 TUNEL凋亡檢測試劑盒測定心肌細胞凋亡比例證實HF+CSWT組心肌細胞凋亡比例(36.10±5.12)也明顯低于 HF 組(53.85±9.89%),HF+LY 組(68.01±4.74%),HF+CSWT+LY(47.08±0.25%),且 P 均0.05。3、Real timePCR檢測實驗報告示;抗凋亡基因Bcl-2在HF+CSWT組(0.81±0.07)中的 mRNA 量明顯高于 HF 組(0.36±0.03),HF+LY 組(0.25±0.04),HF+CSWT+LY(0.65 ±0.07)(P0.05)。而促凋亡基因 Bax 在 HF+CSWT 組的 mRNA(1.18±0.21 明顯低于 HF 組(1.65±0.16),HF+LY 組(2.38±0.22),HF+CSWT+LYC 1.42±0.10)(P 均0.05)。且促凋亡基因 Casepase-3 在 HF+CSWT組的 mRNA(1.04±0.12)也明顯低于 HF 組(1.81±0.25),HF+LY 組(2.42±0.33),HF+CSWT+LY(1.52±0.17)(P 均0.05)。4、Western-blot 檢測心肌組織中 Bcl-2、Bax、Casepase-3、AKT、p-AKT的蛋白表達水平示:抗凋亡蛋白Bcl-2在HF+CSWT組(0.73±0.10)中的相對表達量明顯高于 HF 組(0.35±0.03),HF+LY 組(0.17±0.08),HF+CSWT+LY(0.49±0.05)(P0.05)。同時在 HF+CSWT+LY組(0.49±0.05)中的相對表達量明顯高于HF+LY組(0.17±0.08)(P0.05)。而促凋亡蛋白BAX在HF+CSWT組(0.52±0.27)中的相對表達量較HF組(1.38±0.34)(P0.05)顯著下降。且在 HF+CSWT+LY 組(0.97±0.26)的相對表達量低于 HF+LY 組(2.35±0.40)(P0.05)。促凋亡蛋白 Caspase-3 的前體物質(zhì) pro-Caspase-3 在 HF+CSWT 組(0.92±0.04)中的相對表達量較HF組(0.46±0.09)(P0.05)顯著升高。且在 HF+CSWT+LY 組(0.66±0.16)的相對表達量高于 HF+LY 組(0.09±0.075)(P0.05)。同時發(fā)現(xiàn)磷酸化的Akt(P-Akt)在各實驗組的表現(xiàn)與抗凋亡蛋白Bcl-2中的趨勢相似。[結(jié)論]:1、開胸高位結(jié)扎左冠狀動脈前降支可以成功建立平行性良好的SD大鼠HF模型,可以為心血管領(lǐng)域提供理想的蛋白組學(xué)和基因組學(xué)動物模型。2、CSWT干預(yù)后的大鼠心肌梗死面積和心肌細胞凋亡比例較其他組明顯減少,提示CSWT治療可以抑制心力衰竭過程中的心肌細胞凋亡,減緩左室重構(gòu),從而改善HF大鼠的心功能。3、P-AKT蛋白與CSWT干預(yù)后的HF大鼠心功能改善、心室重塑緩解密切相關(guān),可能是CSWT治療IHF的關(guān)鍵因子,提示CSWT通過激活細胞內(nèi)PI3K/Akt信號通路而發(fā)揮抑制心肌細胞凋亡的作用,為今后進一步拓展CSWT治療IHF的分子機制研究奠定基礎(chǔ)。
[Abstract]:[Objective] To establish a model of ischemic heart failure in SD rats. The surviving rats were treated with CSWT and PI3K/Akt signaling pathway blocker LY294002 at 4 weeks after operation. The effects of CSWT on the apoptosis of myocardial cells in ischemic heart failure after myocardial infarction were validated at the whole animal level, and the effects of CSWT on PI3K/Akt signaling pathway were investigated. Sixty adult male SD rats weighing 220-250g were selected. Fifty of them underwent thoracotomy and high ligation of the left anterior descending coronary artery to establish the model of ischemic heart failure. The surviving rats were randomly divided into heart failure group (9 cases in IHF group), shock wave intervention + heart failure group (9 cases in CSWT + IHF group), LY294002 + heart failure group (9 cases in LY294002 + IHF group), LY294002 + shock wave intervention + heart failure group (9 cases in LY294002 + CSWT + IHF group), and 10 normal rats as control group. Shock energy 0.24MJ/mm2, 200 pulses per time, frequency 60 times/min, three days a week, once a day for 4 weeks. 3. After 4 weeks of CSWT intervention, the cardiac function of rats was assessed by high-frequency ultrasound, myocardial infarction area was assessed by TTC staining, myocardial apoptosis was detected by TUNEL apoptosis detection kit, and myocardial Bcl-2, Bax, Casepase-3 mRN was detected by RT-PCR. Western-blot was used to detect the expression of Bcl-2, Bax, Casepase-3, AKT and p-AKT in myocardial tissue. [Results]: 47 of the 1,50 SD rats completed the establishment of acute myocardial infarction model, the success rate of operation was 84%. 36 heart failure model rats survived 4 weeks after operation, the survival rate was 76.6%. Left ventricular enlargement, left ventricular wall thinning, and interventricular septal motion amplitude decreased significantly; LVESD (2.33 (+ 0.51) and 5.90 (+ 1.03, P 0.001) and LVEDD (4.92 (+ 0.73) and 7.54 (+ 1.29, P 0.001) were significantly enlarged, FS was significantly weakened (51.14 (+ 6.25) and 20.16 (+ 2.92), LVEF was significantly decreased (81.15 (+ 12.18) and 45.94 (+ 6.74), P 0.001). The percentage of myocardial infarction area to left ventricular area was assessed after staining. The myocardial infarction area of HF+CSWT group (17.10+2.91%) was lower than that of HF group (42.46+5.39%), HF+LY group (51.44+5.46%) and HF+CSWT+LY group (31.90+2.34%) respectively, and the percentage of myocardial cell apoptosis was all 0.05. The proportion of HF + LY group (68.01 + 4.74%), HF + CSWT + LY group (68.01 + 4.74%), HF + CSWT + LY group (47.08 + 0.25%), and P were all 0.05.3, Real time PCR test showed that the anti-apoapoapoptosis gene Bcl-2 in HF + CSWT group (0.81 + 0.07) was significantly higher than that in HF + CSWT group (0.81 + 0.07), HF + LY group (68.01 + LY + LY group (68.01 + 4.74%), HF + CSWT + LY + CSWT + LY group (47.08 + CSWT + LY + 47.08 + 0.08 + 0.25%), and P were all 0.05.07 (P 0.05) The mRNA of Bax in HF+CSWT group was significantly lower than that in HF+CSWT group (1.18 +0.21) in HF+CSWT group (1.18 +0.21 significantly lower than that in HF+CSWT group (1.18 +0.21) and HF+LY group (2.38 +0.22), HF+CSWT+LYC group (2.38 +0.22), HF+CSWT+LYC 1.42 +LYC 1.42 +0.10 (all P 0.05). The mRNAof Casepase-3 in HF+CSWT group (1.04 +0.12) in HF+CSWT group was also significantly lower than that in HF+CSWT group (1.81 +0.25 (P all 0. The expression of Bcl-2, Bax, Casepase-3, AKT and p-AKT in myocardium was detected by Western-blot. The relative expression of Bcl-2 in HF+CSWT group (0.73+0.10) was significantly higher than that in HF+CSWT group (0.35+0.03), HF+LY group (0.17+0.08), HF+CSWT+LY group (0.49+0.05) (P 0.05). The relative expression of BAX in HF+CSWT group was significantly lower than that in HF+CSWT group (1.38 +0.34) (P 0.05). The relative expression of BAX in HF+CSWT+LY group (0.97 +0.26) was significantly lower than that in HF+CSWT+LY group (2.35 +0.40) (P 0.05). The relative expression level in the + CSWT group was significantly higher than that in the HF group (0.92 + 0.04) (P 0.05). The relative expression level in the HF + CSWT + LY group (0.66 + 0.16) was higher than that in the HF + LY group (0.09 + 0.075) (P 0.05). The left anterior descending coronary artery can successfully establish HF model of SD rats with good parallelism and provide ideal animal models of proteomics and genomics for cardiovascular field. 2. The area of myocardial infarction and apoptosis rate of myocardial cells in rats after CSWT intervention were significantly reduced compared with other groups, suggesting that CSWT treatment can inhibit the process of heart failure. Cardiac myocyte apoptosis and left ventricular remodeling were slowed down to improve cardiac function in HF rats. 3. P-AKT protein was closely related to the improvement of cardiac function and the alleviation of ventricular remodeling in HF rats after CSWT intervention. It may be a key factor in the treatment of IHF by CSWT, suggesting that CSWT can inhibit cardiomyocyte apoptosis by activating PI3K/Akt signaling pathway. It will lay a foundation for further research on the molecular mechanism of CSWT in the treatment of IHF.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.6

【相似文獻】

相關(guān)期刊論文 前10條

1 ;某些因素對大鼠胃肌電的影響[J];基礎(chǔ)醫(yī)學(xué)與臨床;2001年S1期

2 陳同度,張昌穎;素食大鼠的貧血現(xiàn)象[J];營養(yǎng)學(xué)報;1957年04期

3 陳偉強;趙善廣;;自制注射用大鼠固定裝置[J];上海實驗動物科學(xué);1992年04期

4 肖柳英,林培英,馮昭明,張丹;不同周齡的SD大鼠生理、生化及體重的正常值測定[J];中藥新藥與臨床藥理;1996年03期

5 李淑云;簡易大鼠灌胃器的制作[J];錦州醫(yī)學(xué)院學(xué)報;2001年04期

6 楊明智,陳積圣;一種大鼠抓取與固定的新工具介紹[J];上海實驗動物科學(xué);2001年03期

7 戴英,陸群;復(fù)方H_(505)對Wistar大鼠外周血的血液流變學(xué)指標的影響[J];中國血液流變學(xué)雜志;2001年01期

8 韋應(yīng)波,孫喜慶,曹新生,姚永杰,馮岱雅,楊長斌;+Gz暴露時間對大鼠記憶功能和行為的影響[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2003年01期

9 呂學(xué)軍,郭俊生,李敏,周利梅,張永娟;暈船大鼠體內(nèi)鐵含量的變化[J];中國職業(yè)醫(yī)學(xué);2003年04期

10 湯仁仙,王迎偉,王慧,周峰;201A中藥合劑對大鼠抗腎小球基底膜腎炎病變的影響[J];徐州醫(yī)學(xué)院學(xué)報;2003年06期

相關(guān)會議論文 前10條

1 尹音;孫振宇;胡敏;李冬霞;;持續(xù)性高正加速度對大鼠顳頜關(guān)節(jié)損傷的作用[A];第八屆全國顳下頜關(guān)節(jié)病學(xué)及（牙合）學(xué)大會論文匯編[C];2011年

2 祝~=驤;iJ梊霞;洃克琴;崔素英;文允摪;，

本文編號：2208194