經(jīng)胸震波治療缺血性心力衰竭的機(jī)制研究
發(fā)布時(shí)間：2018-08-27 19:17
【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(hào)通路阻斷劑LY294002處理,從整體動(dòng)物水平驗(yàn)證CSWT抑制心肌梗死后缺血性心力衰竭心肌細(xì)胞凋亡的作用,并探討其對(duì)PI3K/Akt信號(hào)通路的影響。[方法]:1、隨機(jī)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(dòng)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(dòng)圖評(píng)價(jià)大鼠心功能,評(píng)價(jià)建模成功率。2、所有實(shí)驗(yàn)大鼠給予標(biāo)準(zhǔn)鼠糧及自來水自由飲食,術(shù)后4周存活大鼠隨機(jī)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(duì)照組參與實(shí)驗(yàn)。其中CSWT組實(shí)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(píng)價(jià)大鼠心功能,利用TTC染色評(píng)估心肌梗死面積,應(yīng)用TUNEL凋亡檢測(cè)試劑盒測(cè)定心肌細(xì)胞凋亡,RT-PCR檢測(cè)心肌組織中Bcl-2、Bax、Casepase-3的mRNA表達(dá)水平,Western-blot檢測(cè)心肌組織中 Bcl-2、Bax、Casepase-3、AKT、p-AKT 的蛋白表達(dá)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(dòng)圖可見手術(shù)組大鼠左室擴(kuò)大、左室壁變薄、室間隔運(yùn)動(dòng)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(kuò)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(píng)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(cè)試劑盒測(cè)定心肌細(xì)胞凋亡比例證實(shí)HF+CSWT組心肌細(xì)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(cè)實(shí)驗(yàn)報(bào)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(cè)心肌組織中 Bcl-2、Bax、Casepase-3、AKT、p-AKT的蛋白表達(dá)水平示:抗凋亡蛋白Bcl-2在HF+CSWT組(0.73±0.10)中的相對(duì)表達(dá)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(shí)在 HF+CSWT+LY組(0.49±0.05)中的相對(duì)表達(dá)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(duì)表達(dá)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(duì)表達(dá)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(duì)表達(dá)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(duì)表達(dá)量高于 HF+LY 組(0.09±0.075)(P0.05)。同時(shí)發(fā)現(xiàn)磷酸化的Akt(P-Akt)在各實(shí)驗(yàn)組的表現(xiàn)與抗凋亡蛋白Bcl-2中的趨勢(shì)相似。[結(jié)論]:1、開胸高位結(jié)扎左冠狀動(dòng)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(dòng)物模型。2、CSWT干預(yù)后的大鼠心肌梗死面積和心肌細(xì)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(xì)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(xì)胞內(nèi)PI3K/Akt信號(hào)通路而發(fā)揮抑制心肌細(xì)胞凋亡的作用,為今后進(jìn)一步拓展CSWT治療IHF的分子機(jī)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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R541.6
[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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R541.6
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