Survivin抑制缺氧性人肺动脉平滑肌细胞凋亡机制的初步研究
发布时间：2018-08-16 08:33
【摘要】：缺氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)是一种由多因素导致的疾病,目前发病机制尚不明确,早期缺乏特异性的临床症状,随动脉压力升高,可导致右心衰竭和死亡。HPH的主要始动因子是缺氧及缺氧性肺动脉收缩,其发生发展的核心是肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCS)凋亡受阻和过度增殖。肺动脉高压(pulmonary hypertension,PH)治疗的目的是使患者肺动脉压下降,心排血量增加,缓解症状,增强体质。理想的肺血管扩张药物是选择性的扩张肺血管,改善通气,提高PaO_2。但目前用于治疗PH的扩血管药物对肺循环无特异性且对体循环有较强的作用,因而影响动脉血压,甚至使PaO_2下降。肺动脉高压的主要病理改变是PASMCS增殖与凋亡的失衡,对已经发生肺血管重塑的肺动脉高压来说,扩血管药物作用相对较小,导致目前肺动脉高压治疗效果不佳,死亡率居高不下。因此从根本上抑制PASMCS增殖或促进其凋亡,从而减轻肺血管的重构,是目前治疗PH的关键及研究的热点。生存素(survivin)在凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族中凋亡作用最强,其具有双重功能:促进细胞增殖和抑制细胞凋亡。Survivin在绝大多数肿瘤中过度表达,而在正常组织中不表达或处于很低的水平。线粒体不仅是细胞内能量的产生场所,也是参与细胞凋亡的靶器官。在研究survivin抑制肿瘤细胞凋亡的机制中发现,survivin通过与线粒体凋亡途径中的Caspase-9结合,阻止凋亡小体的形成,进而阻断线粒体调控的凋亡通路。目前研究显示,线粒体与细胞凋亡以及细胞低氧感受器密切相关,并在PH发病机制中起到非常重要的作用。我们前期的研究表明:survivin在常氧(21%O_2)培养的人肺动脉平滑肌细胞(human PASMCs,HPASMCs)中不表达,而在缺氧(2.5%O_2)培养的HPASMCs中表达,并且促进HPASMCs增殖,抑制其凋亡。但survivin抑制缺氧HPAMSCs凋亡的机制目前仍不清楚。本实验以HPASMCs为研究对象,研究survivin抑制缺氧HPASMCs凋亡的机制,将HPASMCs分为6组:(1)N组:正常对照组;(2)H组:缺氧组;(3)NY组:常氧+YM155组;(4)HY1组:24h缺氧+YM155 1nmol/L;(5)HY10组:24h缺氧+YM155 10nmol/L;(6)HY100组:24h缺氧+YM155 100nmol/L。采用TMRE法检测HPASMCs线粒体膜电位,DCFH-DA法检测HPASMCs内活性氧(reactive oxygen species,ROS)含量,Western blot分析细胞色素C(cytochrome-C,Cyto-C)的分布与Caspase-9的蛋白表达。研究结果表明:(1)N组线粒体的膜电位为47.831±4.550,与H组膜电位(60.453±6.088)比较差异有统计学意义(q=5.199,P㩳0.05);各剂量YM155干预组线粒体的膜电位分别为49.183±1.007、37.180±1.047、17.568±5.836,与H组比较差异有统计学意义(q=4.642、9.585、17.663,P均㩳0.05),且在一定范围内呈浓度依赖性;(2)N组的细胞内ROS含量为26.160±3.500,与H组(17.662±3.116)比较差异有统计学意义(q=3.248,P㩳0.05)。各剂量YM155干预组的细胞内ROS含量为分别为34.825±3.225、52.225±5.794、71.744±7.050,与H组比较差异有统计学意义(q=6.561、13.212、20.674,P均㩳0.05),且在一定范围内呈浓度依赖性;(3)N组胞质Cyto-C/线粒体Cyto-C为0.733,与H组(0.173)比较差异有统计学意义(P㩳0.05)。HY1组、HY10组、HY100组中胞质Cyto-C/线粒体Cyto-C分别为0.846、1.908、3.258,与H组比较差异有统计学意义(P㩳0.05)。Caspase-9蛋白的表达:N组的表达量为0.531±0.102,与H组(0.124±0.113)比较,差异有统计学意义(q=4.284,P㩳0.05)。各剂量YM155的干预下,Caspase-9蛋白的表达量分别为1.016±0.225、1.732±0.156、1.732±0.156,与H组比较差异有统计学意义(q=9.389、14.400、29.042,P均㩳0.05),且在一定范围内呈浓度依赖性。上述结果表明:缺氧条件下HPAMSCs中survivin的表达,升高了HPAMSCs线粒体的膜电位,降低了细胞内ROS的含量,线粒体膜电位去极化受到抑制,进而抑制了Cyto-C从线粒体释放到细胞质及Caspase-9的活化,抑制了线粒体依赖的凋亡途径,因此通过抑制HPAMSCs中survivin的表达,促进线粒体依赖的凋亡通路,为HPH的治疗提供了新靶点。
[Abstract]:Hypoxic pulmonary hypertension (HPH) is a disease caused by many factors. At present, the pathogenesis of HPH is still unclear, and the early lack of specific clinical symptoms. With the increase of arterial pressure, it can lead to right heart failure and death. The core is pulmonary artery smooth muscle cells (PASMCS) apoptosis blocked and hyperplasia. The purpose of pulmonary hypertension (PH) treatment is to reduce pulmonary artery pressure, increase cardiac output, relieve symptoms and enhance physical fitness. The ideal pulmonary vasodilator is selective dilation of pulmonary blood. The main pathological changes of pulmonary hypertension are the unbalance of proliferation and apoptosis of PASMCS, which is the main pathological change of pulmonary hypertension. Vasodilators have relatively little effect on PH, which leads to poor therapeutic effect and high mortality rate of PASMCS. Therefore, inhibiting the proliferation or promoting apoptosis of PASMCS and alleviating the remodeling of pulmonary blood vessels is the key to PH treatment. Survivin is overexpressed in most tumors, but not expressed in normal tissues or at very low levels. Mitochondria are not only the site of energy production in cells, but also the target organs involved in apoptosis. Vitalin inhibits apoptosis of tumor cells by binding to caspase-9 in the mitochondrial apoptosis pathway, thus preventing the formation of apoptotic bodies and blocking the apoptotic pathway regulated by mitochondria. Our previous studies showed that survivin was not expressed in human PASMCs (HPASMCs) cultured in normoxia (21% O_2), but was expressed in HPASMCs cultured in hypoxia (2.5% O_2), and promoted the proliferation of HPASMCs and inhibited their apoptosis. HPASMCs were divided into six groups: (1) N group: normal control group; (2) H group: hypoxia group; (3) NY group: normoxia + YM155 group; (4) HY1 group: 24 h hypoxia + YM155 1 nmol / L; (5) HY10 group: 24 h hypoxia + YM15510 nmol / L; (6) HY100 group: 24 h hypoxia + YM155 100 nmol / L. ASTM / L was used to detect the apoptosis of HPASMCs by RE method. The contents of reactive oxygen species (ROS) in HPASMCs were detected by DCFH-DA, and the distribution of cytochrome-C (Cyto-C) and the expression of C aspase-9 protein were analyzed by Western blot. The results showed that: (1) The membrane potential of mitochondria in N group was 47.831 [4.550], which was significantly different from that in H group (60.453 [6.088]). Significance (q = 5.199, P? 0.05); the membrane potential of mitochondria in YM155 intervention group was 49.183 (+ 1.007), 37.180 (+ 1.047), 17.568 (+ 5.836) respectively, which was significantly different from that in H group (q = 4.642, 9.585, 17.663, P? 0.05) and was dose-dependent; (2) ROS content in N group was 26.160 (+ 3.500) compared with that in H group (17.662 (+ 3.116). There was significant difference (q = 3.248, P? 0.05). The intracellular ROS content of YM155 intervention group was 34.825 (+ 3.225), 52.225 (+ 5.794), 71.744 (+ 7.050), which was significantly different from that of H group (q = 6.561, 13.212, 20.674, P? 0.05), and in a certain range was dose-dependent; (3) Cyto-C / mitochondrial Cyto-C of N group was 0.733, 0.733, respectively. There was significant difference between group H (0.173) and group HY1 (P?0.05). The expression of Cyto-C/mitochondrial Cyto-C in group HY1, HY10 and HY100 was 0.846, 1.908 and 3.258, respectively. There was significant difference between group H and group HY1 (P?0.05). The expression of Caspase-9 protein in group N was 0.531 (+0.102) and group H (0.124 (+0.113)) with significant difference (q=4.284, P?0.05). The expression of Caspase-9 protein was 1.016 65507 Membrane potential of mitochondria decreased the content of ROS in cells, and depolarization of mitochondrial membrane potential was inhibited, which inhibited the release of Cyto-C from mitochondria to cytoplasm and the activation of Caspase-9, inhibited the mitochondrial-dependent apoptosis pathway. Therefore, the mitochondrial-dependent apoptosis pathway was promoted by inhibiting the expression of Survivin in HPAMSCs. Treatment provides a new target.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.1
本文编号：2185457
[Abstract]:Hypoxic pulmonary hypertension (HPH) is a disease caused by many factors. At present, the pathogenesis of HPH is still unclear, and the early lack of specific clinical symptoms. With the increase of arterial pressure, it can lead to right heart failure and death. The core is pulmonary artery smooth muscle cells (PASMCS) apoptosis blocked and hyperplasia. The purpose of pulmonary hypertension (PH) treatment is to reduce pulmonary artery pressure, increase cardiac output, relieve symptoms and enhance physical fitness. The ideal pulmonary vasodilator is selective dilation of pulmonary blood. The main pathological changes of pulmonary hypertension are the unbalance of proliferation and apoptosis of PASMCS, which is the main pathological change of pulmonary hypertension. Vasodilators have relatively little effect on PH, which leads to poor therapeutic effect and high mortality rate of PASMCS. Therefore, inhibiting the proliferation or promoting apoptosis of PASMCS and alleviating the remodeling of pulmonary blood vessels is the key to PH treatment. Survivin is overexpressed in most tumors, but not expressed in normal tissues or at very low levels. Mitochondria are not only the site of energy production in cells, but also the target organs involved in apoptosis. Vitalin inhibits apoptosis of tumor cells by binding to caspase-9 in the mitochondrial apoptosis pathway, thus preventing the formation of apoptotic bodies and blocking the apoptotic pathway regulated by mitochondria. Our previous studies showed that survivin was not expressed in human PASMCs (HPASMCs) cultured in normoxia (21% O_2), but was expressed in HPASMCs cultured in hypoxia (2.5% O_2), and promoted the proliferation of HPASMCs and inhibited their apoptosis. HPASMCs were divided into six groups: (1) N group: normal control group; (2) H group: hypoxia group; (3) NY group: normoxia + YM155 group; (4) HY1 group: 24 h hypoxia + YM155 1 nmol / L; (5) HY10 group: 24 h hypoxia + YM15510 nmol / L; (6) HY100 group: 24 h hypoxia + YM155 100 nmol / L. ASTM / L was used to detect the apoptosis of HPASMCs by RE method. The contents of reactive oxygen species (ROS) in HPASMCs were detected by DCFH-DA, and the distribution of cytochrome-C (Cyto-C) and the expression of C aspase-9 protein were analyzed by Western blot. The results showed that: (1) The membrane potential of mitochondria in N group was 47.831 [4.550], which was significantly different from that in H group (60.453 [6.088]). Significance (q = 5.199, P? 0.05); the membrane potential of mitochondria in YM155 intervention group was 49.183 (+ 1.007), 37.180 (+ 1.047), 17.568 (+ 5.836) respectively, which was significantly different from that in H group (q = 4.642, 9.585, 17.663, P? 0.05) and was dose-dependent; (2) ROS content in N group was 26.160 (+ 3.500) compared with that in H group (17.662 (+ 3.116). There was significant difference (q = 3.248, P? 0.05). The intracellular ROS content of YM155 intervention group was 34.825 (+ 3.225), 52.225 (+ 5.794), 71.744 (+ 7.050), which was significantly different from that of H group (q = 6.561, 13.212, 20.674, P? 0.05), and in a certain range was dose-dependent; (3) Cyto-C / mitochondrial Cyto-C of N group was 0.733, 0.733, respectively. There was significant difference between group H (0.173) and group HY1 (P?0.05). The expression of Cyto-C/mitochondrial Cyto-C in group HY1, HY10 and HY100 was 0.846, 1.908 and 3.258, respectively. There was significant difference between group H and group HY1 (P?0.05). The expression of Caspase-9 protein in group N was 0.531 (+0.102) and group H (0.124 (+0.113)) with significant difference (q=4.284, P?0.05). The expression of Caspase-9 protein was 1.016 65507 Membrane potential of mitochondria decreased the content of ROS in cells, and depolarization of mitochondrial membrane potential was inhibited, which inhibited the release of Cyto-C from mitochondria to cytoplasm and the activation of Caspase-9, inhibited the mitochondrial-dependent apoptosis pathway. Therefore, the mitochondrial-dependent apoptosis pathway was promoted by inhibiting the expression of Survivin in HPAMSCs. Treatment provides a new target.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R544.1
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