经胸震波治疗缺血性心力衰竭的机制研究

发布时间：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,术后4周存活大鼠予CSWT、PI3K/Akt信号通路阻断剂LY294002处理,从整体动物水平验证CSWT抑制心肌梗死后缺血性心力衰竭心肌细胞凋亡的作用,并探讨其对PI3K/Akt信号通路的影响。[方法]:1、随机入选60只成年雄性SD大鼠,体重220-250g,其中50只大鼠手术开胸,高位结扎左冠状动脉前降支,建立SD大鼠缺血性心力衰竭模型。术前及术后采用高频超声心动图评价大鼠心功能,评价建模成功率。2、所有实验大鼠给予标准鼠粮及自来水自由饮食,术后4周存活大鼠随机分为心衰组(IHF组9例)、心脏震波干预+心衰组(CSWT+IHF组9例)、LY294002+心衰组(LY294002+IHF组9例)、LY294002+心脏震波干预+心衰组(LY294002+CSWT+IHF组9例),另外10只正常大鼠作为对照组参与实验。其中CSWT组实施心脏震波干预,震波能量0.24MJ/mm2,每次200脉冲,频率60次/min,每周三天,每天一次,共4周。3、CSWT干预4周后,采用高频超声评价大鼠心功能,利用TTC染色评估心肌梗死面积,应用TUNEL凋亡检测试剂盒测定心肌细胞凋亡,RT-PCR检测心肌组织中Bcl-2、Bax、Casepase-3的mRNA表达水平,Western-blot检测心肌组织中 Bcl-2、Bax、Casepase-3、AKT、p-AKT 的蛋白表达水平。[结果]:1、50只SD大鼠有47只完成急性心肌梗死模型制作,手术成功率84%。术后4周有36只心衰模型大鼠存活,存活率为76.6%。术后4周高频超声心动图可见手术组大鼠左室扩大、左室壁变薄、室间隔运动幅度明显减弱;LVESD(2.33±0.51 与 5.90±1.03,P0.001)和 LVEDD(4.92±0.73 与 7.54± 1.29,P0.001)较术前明显扩大,且FS明显减弱(51.14±6.25与20.16±2.92,P0.001),LVEF明显降低(81.15±12.18 与 45.94±6.74,P0.001)。2、离体后利用TTC染色后评估心肌梗死面积占左室面积百分比;HF+CSWT组心肌梗死面积(17.10 ± 2.91%)均低于 HF 组(42.46 ± 5.39%),HF+LY 组(51.44±5.46%),HF+CSWT+LY 组(31.90±2.34%),且 P 均0.05。应用 TUNEL凋亡检测试剂盒测定心肌细胞凋亡比例证实HF+CSWT组心肌细胞凋亡比例(36.10±5.12)也明显低于 HF 组(53.85±9.89%),HF+LY 组(68.01±4.74%),HF+CSWT+LY(47.08±0.25%),且 P 均0.05。3、Real timePCR检测实验报告示;抗凋亡基因Bcl-2在HF+CSWT组(0.81±0.07)中的 mRNA 量明显高于 HF 组(0.36±0.03),HF+LY 组(0.25±0.04),HF+CSWT+LY(0.65 ±0.07)(P0.05)。而促凋亡基因 Bax 在 HF+CSWT 组的 mRNA(1.18±0.21 明显低于 HF 组(1.65±0.16),HF+LY 组(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组的 mRNA(1.04±0.12)也明显低于 HF 组(1.81±0.25),HF+LY 组(2.42±0.33),HF+CSWT+LY(1.52±0.17)(P 均0.05)。4、Western-blot 检测心肌组织中 Bcl-2、Bax、Casepase-3、AKT、p-AKT的蛋白表达水平示:抗凋亡蛋白Bcl-2在HF+CSWT组(0.73±0.10)中的相对表达量明显高于 HF 组(0.35±0.03),HF+LY 组(0.17±0.08),HF+CSWT+LY(0.49±0.05)(P0.05)。同时在 HF+CSWT+LY组(0.49±0.05)中的相对表达量明显高于HF+LY组(0.17±0.08)(P0.05)。而促凋亡蛋白BAX在HF+CSWT组(0.52±0.27)中的相对表达量较HF组(1.38±0.34)(P0.05)显著下降。且在 HF+CSWT+LY 组(0.97±0.26)的相对表达量低于 HF+LY 组(2.35±0.40)(P0.05)。促凋亡蛋白 Caspase-3 的前体物质 pro-Caspase-3 在 HF+CSWT 组(0.92±0.04)中的相对表达量较HF组(0.46±0.09)(P0.05)显著升高。且在 HF+CSWT+LY 组(0.66±0.16)的相对表达量高于 HF+LY 组(0.09±0.075)(P0.05)。同时发现磷酸化的Akt(P-Akt)在各实验组的表现与抗凋亡蛋白Bcl-2中的趋势相似。[结论]:1、开胸高位结扎左冠状动脉前降支可以成功建立平行性良好的SD大鼠HF模型,可以为心血管领域提供理想的蛋白组学和基因组学动物模型。2、CSWT干预后的大鼠心肌梗死面积和心肌细胞凋亡比例较其他组明显减少,提示CSWT治疗可以抑制心力衰竭过程中的心肌细胞凋亡,减缓左室重构,从而改善HF大鼠的心功能。3、P-AKT蛋白与CSWT干预后的HF大鼠心功能改善、心室重塑缓解密切相关,可能是CSWT治疗IHF的关键因子,提示CSWT通过激活细胞内PI3K/Akt信号通路而发挥抑制心肌细胞凋亡的作用,为今后进一步拓展CSWT治疗IHF的分子机制研究奠定基础。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.6

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