低氧诱导因子-1α在心肌缺血预处理与后处理心肌保护中的作用

发布时间：2018-01-20 20:17

本文关键词： 低氧诱导因子-1α 心肌缺血/再灌注损伤缺血预处理缺血后处理　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 研究背景: 缺氧在心肌缺血引起的病理生理变化中起着关键的作用,它可引起心肌细胞丢失,导致收缩功能障碍。心肌缺血后尽早对缺血区进行再灌注是阻止心肌细胞死亡、恢复心肌功能的根本途径,然而,再灌注本身又会加重心肌细胞损伤,即发生缺血/再灌注损伤。缺血预处理与缺血后处理是目前公认的两种能减轻缺血/再灌注损伤的心肌保护措施,但其机制十分复杂,尚未充分阐明。近年发现的低氧诱导因子-1α(HIF-1α)是一种在氧平衡调节中起关键作用的转录因子,它通过调节多种靶基因的表达介导细胞缺氧反应,从而保护组织免受缺血损伤。一些临床及基础研究发现,HIF-1α可能是心肌缺血时首发的分子反应,提示它可能在缺血/再灌注损伤的内源性保护机制中起关键的始动作用;在缺血/再灌注以及缺氧/复氧的预处理过程中HIF-1α的蛋白表达量明显增加,但它在缺血后处理过程中表达变化如何,以及它在预处理,特别是后处理过程中是否起保护作用等问题尚未见文献报道。目的: 1.观察在心肌缺血/再灌注时给予缺血预处理及后处理后低氧诱导因子-1α的蛋白表达情况; 2.探讨低氧诱导因子-1α的表达变化与缺血预处理及后处理的心肌保护效应的关系。方法: 选取健康雄性Wistar大鼠40只,随机分为4组: ①伪手术组(sham,n=10):仅在左冠状动脉前降支(left anterior descending artery,LAD)下穿线,不结扎,持续225min; ②缺血/再灌注组(I/R,n=10):可逆性结扎LAD造成心肌缺血45min,再灌注3h; ③缺血预处理组(IP,n=10):给予3个循环的5min缺血/5min再灌注作为缺血预处理,随后给予可逆性结扎LAD造成心肌缺血45min,再灌注3h; ④缺氧后处理组(PC,n=10):可逆性结扎LAD造成心肌缺血45min,随即进行3个循环的再灌注10g/缺血10s的缺血后适应,再行再灌注3h。实验结束后用Even's blue和TTC双染,测定心肌危险区与梗塞区面积;利用免疫组织化学技术、Western蛋白免疫印迹检测心肌HIF-1α的表达:用DNA原位末端缺口标记法并配合caspase-3的活性检测对心肌细胞凋亡进行检测;制备血清,检测CK活性和MDA含量。结果: 1.缺血/再灌注模型的建立 1.1心肌缺血/再灌注损伤对心肌梗塞面积的影响危险区面积占左心室面积的百分比(AAR/LV比值):在伪手术组与缺血/再灌注组之间无显著性差异,各组动物模型的缺血程度大体一致,具有可比性。心肌梗塞面积占危险区面积百分比(AN/AAR比值):缺血/再灌注(I/R)组与伪手术(sham)组相比明显增高(44.17%±3.24%vs.3.36%±0.87%,P＜0.01)(表1,图2,图3)。 1.2心肌缺血/再灌注损伤对血清肌酸激酶(CK)活性的影响实验采用CK活性作为评价心肌损伤的指标。经CK试剂盒检测可见,缺血/再灌注(I/R)组与sham组相比,血清CK活性明显增高(10.17±2.36 U/ml vs.0.037±0.01U/ml,P＜0.01),(表1,图4)。 1.3心肌缺血/再灌注损伤对血清丙二醛(MDA)含量的影响 MDA含量可反应机体内脂质过氧化的程度,同时间接地反应出细胞损伤的程度。结果显示:缺血/再灌注(I/R)组血清MDA含量明显高于sham组(0.27±0.03 nmol/ml vs.0.04±0.01 nmol/ml,P＜0.01)(表1,图5)。 1.4心肌缺血/再灌注损伤对细胞凋亡的影响 DNA原位末端缺口标记法(TUNEL)心肌细胞凋亡检测结果显示:伪手术组(sham)大鼠心肌组织细胞凋亡指数为(1.45±0.41)%,缺血/再灌注组(I/R)心肌细胞凋亡指数明显增高达到(17.68±2.44)%,I/R与sham两组相比有统计学差异(P＜0.01)(图6,图7)。由于TUNEL法检测细胞凋亡具有敏感性高、但特异性较差的特点,因此我们配合检测了缺血/再灌注心肌组织的caspase-3比活性来共同说明心肌细胞的凋亡情况。caspase-3检测结果显示:与sham组(1.00±0.14)相比,缺血/再灌注组caspase-3比活性明显增高,达到2.22±0.39(P＜0.01)(图8)。以上结果均证明心肌缺血/再灌注模型建立成功。 2.缺血预处理和缺血后处理对心肌缺血/再灌注损伤的保护作用 2.1缺血预处理和缺血后处理对心肌梗塞面积的影响危险区面积占左心室面积的百分比(AAR/LV比值):在缺血/再灌注组、缺血预处理组和缺血后处理之间无显著性差异,各组动物模型的缺血程度大体一致,具有可比性(见表2,图2,图9a)。心肌梗塞面积占危险区面积百分比(AN/AAR比值):缺血预处理组(IP)的AN/AAR比值为(23.76±7.78)%,与缺血/再灌注组(44.17%±3.24%)相比明显降低(P＜0.01);缺血后处理(PC)的AN/AAR比值为(19.79±2.42)%,与I/R组相比也明显降低(P＜0.01);而IP与PC相比,则无统计学差异(表2,图2,图9b)。 2.2缺血预处理和缺血后处理对血清肌酸激酶(CK)活性的影响缺血/再灌注组(I/R)血清肌酸激酶活性为10.17±2.36 U/ml,缺血预处理组(IP)血清肌酸激酶活性为7.84±0.87U/ml,与I/R组相比明显降低(P＜0.05);缺血后处理(PC)组血清肌酸激酶活性(7.67±1.15U/ml)与I/R组相比也明显降低(P＜0.05);而IP组与PC组相比,则无统计学差异(表3,图10)。 2.3缺血预处理和缺血后处理对血清丙二醛(MDA)含量的影响血清MDA含量在缺血预处理组(IP)组为0.16±0.02 nmol/ml,缺血后处理组(PC)为0.16±0.02 nmol/ml。IP、PC组与缺血/再灌注组(0.27±0.03 nmol/ml)相比,均显著降低(P＜0.01);而IP组与PC组相比无统计学差异。(表3,图11)。 2.4缺血预处理和缺血后处理对细胞凋亡的影响 DNA原位末端缺口标记法(TUNEL)心肌细胞凋亡检测:缺血/再灌注组(I/R)心肌细胞凋亡指数为(17.68±2.44)%,预处理组(IP)及后处理组(PC)与I/R组相比心肌细胞的凋亡均显著减少,其细胞凋亡指数分别降至(13.79±1.29)%和(11.34±1.54)%,P值均小于0.01(表4,图12,图13a)。IP组与PC组相比,未见统计学差异。 caspase-3比活性分析:caspase-3比活性在IP组为1.53±0.25,PC组为1.23±0.26,与I/R组(2.22±0.39)相比均显著降低(P＜0.01)(表4,图13b)。IP组与PC组相比无统计学差异。检测结果提示,预处理及后处理均可抑制缺血/再灌注后心肌细胞的凋亡。其检测结果与TUNEL法基本一致。 3.心肌组织HIF-1α蛋白表达量检测 3.1心肌组织HIF-1α蛋白表达量的免疫组织化学检测伪手术(sham)组大鼠心肌组织仅有少量HIF-1α蛋白表达,缺血/再灌注组(I/R)与sham组相比,其HIF-1α免疫组化染色积分光密度(IOD)显著增强(8331.64±527.40 vs.2263.54±523.32,P＜0.01)。缺血预处理组(IP)和缺血后处理组(PC)与I/R组相比,它们的HIF-1αIOD均值明显增强,分别为9394.61±757.31和9847.20±940.66,均有统计学意义(P＜0.05和P＜0.01)(图14,图15)。免疫组化染色结果可见HIF-1α在细胞核与细胞浆内均有表达。 3.2心肌组织HIF-1α蛋白表达量Western-blot测定缺血/再灌注组(I/R)与sham组相比能明显诱导HIF-1α蛋白的表达(1.70±0.34 vs.1.0±0.20,P＜0.01),而缺血预处理.(IP)和缺血后处理(PC)均可显著增加I/R后心肌细胞的HIF-1α蛋白表达量,分别为(2.13±0.39)和(2.07±0.33),P值均小于0.05(图16,图17)。 4.HIF-1α蛋白表达量与反映心肌损伤各指标(心肌梗塞面积、CK活性、MDA含量及caspase-3比活性)之间的相关性分析采用SPSS 11.0软件对各组心肌梗塞面积、CK活性、MDA含量及caspase-3比活性分别与HIF-1α表达量进行相关分析,结果表明:心肌梗塞面积与HIF-1α蛋白表达量呈负相关,其相关系数为r=-0.842,P＜0.01(见图18);CK活性与HIF-1α蛋白表达量呈负相关,r=-0.796,P＜0.01(见图19);MDA含量与HIF-1α蛋白表达量呈负相关,r=-0.839(见图20);caspase-3比活性与HIF-1α蛋白表达量也呈负相关,r=-0.84,P＜0.01(见图21)。结论: (1).缺血预处理和后处理对心肌缺血/再灌注损伤均具有保护作用,能明显降低心梗面积、CK活性和MDA含量,并减少心肌细胞的凋亡。 (2).大鼠心肌在缺血/再灌注后,HIF-1α的蛋白表达量明显增加,提示该因子是心肌细胞缺血时的重要分子反应。 (3).心肌缺血预处理和后处理能使心肌缺血/再灌注后的HIF-1α蛋白表达量进一步明显增加,提示缺血预处理和后处理均有诱导心肌组织HIF-1α表达的效应。 (4).缺血预处理及后处理所引起的HIF-1α蛋白表达量的增加与反映心肌损伤各指标之间均有很高的负相关性,提示HIF-1α与缺血预处理以及后处理的心肌保护作用有密切关系。 (5).缺血预处理与后处理保护效应的始动机制可能是相同的。
[Abstract]:Research background:
Plays a key role in the pathophysiological changes caused by hypoxia in myocardial ischemia, it can cause myocardial cell loss, leading to systolic dysfunction after myocardial ischemia. Ischemia reperfusion as soon as possible to prevent the death of myocardial cells, the fundamental way, the recovery of myocardial function however, reperfusion itself may aggravate myocardial cell damage, which happened ischemia / reperfusion injury, ischemic preconditioning and ischemic postprocessing is currently recognized as the two kinds of protective measures can reduce ischemia / reperfusion injury of myocardium, but its mechanism is very complicated, have not yet been fully clarified. Hypoxia inducible factor found in recent years that -1 alpha (HIF-1 alpha) is a kind of play a key role in the regulation of oxygen balance the transcription factor, through the expression of multiple target genes mediated regulation of cell response to hypoxia, which protects tissues from ischemic injury. Some basic and clinical studies found that HIF-1 alpha may be myocardial ischemia When the first molecular reaction, suggesting that it may play a key role in the initiation of ischemia / reperfusion injury of the endogenous protective mechanisms in the pretreatment process; ischemia / reperfusion and hypoxia / reoxygenation in HIF-1 alpha protein expression was significantly increased, but its treatment after ischemia during expression, and it in the preprocessing, especially the postprocessing process whether the protection problem has not been reported.
Objective:
1. the protein expression of hypoxia inducible factor -1 alpha was observed after ischemic preconditioning and postconditioning in myocardial ischemia / reperfusion.
2. to investigate the relationship between the expression of hypoxia inducible factor -1 alpha and the myocardial protective effect of ischemic preconditioning and post-treatment.
Method:
40 healthy male Wistar rats were selected and divided into 4 groups randomly.
(1) the pseudo operation group (sham, n=10): only the left anterior descending branch of the coronary artery (left anterior descending artery, LAD) under the line, no ligation, continuous 225min;
(2) ischemia / reperfusion group (I/R, n=10): reversible ligation of LAD resulted in myocardial ischemia 45min, and reperfusion 3H;
(3) the ischemic preconditioning group (IP, n=10): 3 cycles of 5min ischemia /5min reperfusion were used as ischemic preconditioning, followed by reversible ligation of LAD, resulting in myocardial ischemia 45min and reperfusion 3H.
(4) hypoxic postconditioning group (PC, n=10): reversible ligation of LAD resulted in myocardial ischemia 45min, then 3 cycles of reperfusion, 10g/ ischemia, 10s ischemic postconditioning, and then reperfusion 3h..
After the end of the experiment by Even's blue and TTC double staining, the determination of the dangerous area and infarct size; using immunohistochemical technique, the expression of Western protein was detected by Western blot of myocardial HIF-1 alpha: DNA with TUNEL method and activity detection of Caspase-3 for detection of myocardial cell apoptosis; preparation of serum CK activity. And MDA were detected.
Result:
1. model of ischemia / reperfusion
The effect of 1.1 myocardial ischemia / reperfusion injury on the area of myocardial infarction
The percentage of the risk area occupied the ratio of the left ventricular area (AAR/LV ratio): there was no significant difference between the pseudo operative group and the ischemia-reperfusion group.
The area of myocardial infarction accounted for the percentage of the risk area (AN/AAR ratio): the ischemia / reperfusion (I/R) group was significantly higher than that of the pseudo operation (sham) group (44.17% + 3.24%vs.3.36% + 0.87%, P < 0.01) (Table 1, figure 2, figure 3).
The effect of 1.2 myocardial ischemia / reperfusion injury on the activity of serum creatine kinase (CK)
The activity of CK was used as an index to evaluate myocardial injury. CK kit test showed that the activity of serum CK in ischemia / reperfusion (I/R) group was significantly higher than that in sham group (10.17 + 2.36 U/ml vs.0.037 + 0.01U/ml, P < 0.01), (1, 4).
The effect of 1.3 myocardial ischemia / reperfusion injury on the content of serum malondialdehyde (MDA)
MDA content can reflect the extent of lipid peroxidation in the body, and indirectly reflect the extent of cell injury. The results showed that the serum MDA level in the ischemia / reperfusion (I/R) group was significantly higher than that in the sham group (0.27 + 0.03 nmol/ml vs.0.04 + 0.01 nmol/ml, P < 0.01) (table 1, figure 5).
The effect of 1.4 myocardial ischemia / reperfusion injury on cell apoptosis
DNA in situ nick end labeling (TUNEL) myocardial cell apoptosis test results show that: the sham operation group (sham) cell apoptosis index of myocardial tissue of rats was (1.45 + 0.41)%, ischemia / reperfusion group (I/R) myocardial apoptosis index was significantly increased to (17.68 + 2.44)%, I/R and sham two groups the difference was statistically significant (P < 0.01) (Figure 6, Figure 7).
The apoptotic cells were detected by TUNEL method has high sensitivity, but the specificity is low, so we detected with ischemia / reperfusion and myocardial perfusion caspase-3 activity showed together myocardial cells apoptosis and.Caspase-3 test results show that: the sham group (1 + 0.14) compared to the ischemia / reperfusion group caspase-3 activity significantly increased to 2.22 + 0.39 (P < 0.01) (Figure 8).
The above results all proved that the model of myocardial ischemia / reperfusion was established successfully.
2. protective effect of ischemic preconditioning and ischemic postconditioning on myocardial ischemia / reperfusion injury
2.1 the effect of ischemic preconditioning and ischemic postconditioning on the area of myocardial infarction
The percentage of the risk area occupied the percentage of the left ventricular area (AAR/LV ratio): there was no significant difference between the ischemic preconditioning group and the ischemic postconditioning group in the ischemia / reperfusion group. The degree of ischemia in each group was generally the same and comparable (see Table 2, Fig. 2, figure 9A).
Myocardial infarction area accounted for risk area percentage (AN/AAR ratio): ischemic preconditioning group (IP) AN/AAR ratio (23.76 + 7.78)%, and the ischemia / reperfusion group (44.17% + 3.24%) reduced significantly (P < 0.01); ischemic postprocessing (PC) AN/AAR ratio (19.79. 2.42)%, compared with the I/R group were significantly decreased (P < 0.01); and IP compared with PC, there was no significant difference (Table 2, figure 2, figure 9b).
2.2 effect of ischemic preconditioning and ischemic postconditioning on the activity of serum creatine kinase (CK)
Ischemia / reperfusion group (I/R) serum creatine kinase activity was 10.17 + 2.36 U/ml, ischemic preconditioning group (IP) and serum creatine kinase activity was 7.84 + 0.87U/ml, compared with the I/R group decreased significantly (P < 0.05); ischemic postprocessing (PC) serum creatine kinase activity (7.67 + 1.15U/ml) compared with the I/R group also decreased significantly (P < 0.05); and IP group compared with PC group, there was no significant difference (Table 3, figure 10).
2.3 effect of ischemic preconditioning and ischemic postconditioning on the content of serum malondialdehyde (MDA)
The serum MDA level in the ischemic preconditioning group (IP) group was 0.16 + 0.02 nmol/ml, and the ischemic postconditioning group (PC) was 0.16 + 0.02 nmol/ml.IP. Compared with the ischemia / reperfusion group (0.27 + 0.03 nmol/ml), the PC group decreased significantly (P < 0.01), while the IP group had no statistical difference compared with the PC group (tab 3, figure 11).
2.4 effect of ischemic preconditioning and ischemic postconditioning on cell apoptosis
DNA in situ nick end labeling (TUNEL) detection of myocardial cell apoptosis: the ischemia / reperfusion group (I/R) myocardial apoptosis index for (17.68 + 2.44)%, pretreatment group (IP) and postprocessing group (PC) compared with the I/R group myocardial apoptosis significantly decreased the apoptosis index respectively (13.79 + 1.29)% and (11.34 + 1.54)%, P values were less than 0.01 (Table 4, Figure 12, figure 13a) compared with.IP group and PC group, there was no statistical difference.
Analysis of specific activity of Caspase-3: caspase-3 activity in IP group was 1.53 + 0.25, 1.23 + 0.26 PC group, and I/R group (2.22 + 0.39) decreased significantly compared (P < 0.01) (Table 4, figure 13b).IP group and PC group compared with no significant difference. The results showed that pretreatment and postprocessing were inhibited after ischemia / reperfusion myocardial cells apoptosis. The results are consistent with the TUNEL method.
Detection of HIF-1 alpha protein expression in 3. myocardium
Immunohistochemical detection of the expression of HIF-1 alpha protein in 3.1 myocardium
Pseudo surgery (sham) in myocardial tissue of rats with only a small amount of HIF-1 protein expression, ischemia / reperfusion group (I/R) compared with sham group, the alpha HIF-1 immunohistochemical staining of the integral optical density (IOD) increased significantly (8331.64 + 527.40 vs.2263.54 + 523.32, P < 0.01). Ischemia preconditioning group (IP ischemia) and postprocessing group (PC) compared with I/R group, HIF-1 alpha IOD mean their obvious enhancement, were 9394.61 + 757.31 and 9847.20 + 940.66, there was statistically significant (P < 0.05 and P < 0.01) (Figure 14, Figure 15). The results of immunohistochemical staining showed HIF-1 expressed in alpha the nucleus and cytoplasm.
Determination of HIF-1 alpha protein expression by Western-blot in 3.2 myocardium
Ischemia / reperfusion group (I/R) was compared with the sham group could significantly induce HIF-1 alpha protein (1.70 + 0.34 vs.1.0 + 0.20, P < 0.01), and ischemic preconditioning. (IP) and ischemic postprocessing (PC) significantly increased HIF-1 protein expression in myocardial cells after I/R, respectively. (2.13 + 0.39) and (2.07 + 0.33), P values were less than 0.05 (Figure 16, Figure 17).
The correlation analysis between the expression of 4.HIF-1 alpha protein and the indexes of myocardial injury (myocardial infarction area, CK activity, MDA content and caspase-3 specific activity)
Using SPSS 11 software for each area of myocardial infarction, CK activity, MDA content and caspase-3 activity respectively with HIF-1 alpha expression by correlation analysis, the results show that the area of myocardial infarction and HIF-1 alpha protein expression was negatively correlated, the correlation coefficient is r=-0.842, P < 0.01 (see Figure 18); the activity of CK and HIF-1 alpha protein expression was negatively correlated with r=-0.796, P < 0.01 (see Figure 19); the content of MDA and HIF-1 protein expression was negatively correlated with r=-0.839 (see Figure 20); caspase-3 activity and HIF-1 protein expression was negatively correlated with r=-0.84, P < 0.01 (see Figure 21).
Conclusion:
(1) ischemic preconditioning and postconditioning have protective effects on myocardial ischemia / reperfusion injury, which can significantly reduce infarct size, CK activity and MDA content, and reduce cardiomyocyte apoptosis.
(2) after ischemia / reperfusion, the protein expression of HIF-1 alpha in the rat myocardium increased significantly, suggesting that the factor is an important molecular response to myocardial ischemia.
(3) myocardial ischemia preconditioning and postconditioning can further increase the expression of HIF-1 alpha protein after myocardial ischemia / reperfusion, suggesting that ischemic preconditioning and postconditioning can induce the expression of HIF-1 alpha in myocardium.
(4) the increase of HIF-1 alpha protein expression induced by ischemic preconditioning and postconditioning had a high negative correlation with all the indicators reflecting myocardial injury, suggesting that HIF-1 alpha is closely related to myocardial protection after ischemic preconditioning and postconditioning.
(5). The initial mechanism of ischemic preconditioning and postprocessing the protective effect may be the same.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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