Survivin抑制缺氧性人肺動脈平滑肌細(xì)胞凋亡機制的初步研究
發(fā)布時間：2018-08-16 08:33
【摘要】：缺氧性肺動脈高壓(hypoxic pulmonary hypertension,HPH)是一種由多因素導(dǎo)致的疾病,目前發(fā)病機制尚不明確,早期缺乏特異性的臨床癥狀,隨動脈壓力升高,可導(dǎo)致右心衰竭和死亡。HPH的主要始動因子是缺氧及缺氧性肺動脈收縮,其發(fā)生發(fā)展的核心是肺動脈平滑肌細(xì)胞(pulmonary arterial smooth muscle cells,PASMCS)凋亡受阻和過度增殖。肺動脈高壓(pulmonary hypertension,PH)治療的目的是使患者肺動脈壓下降,心排血量增加,緩解癥狀,增強體質(zhì)。理想的肺血管擴張藥物是選擇性的擴張肺血管,改善通氣,提高PaO_2。但目前用于治療PH的擴血管藥物對肺循環(huán)無特異性且對體循環(huán)有較強的作用,因而影響動脈血壓,甚至使PaO_2下降。肺動脈高壓的主要病理改變是PASMCS增殖與凋亡的失衡,對已經(jīng)發(fā)生肺血管重塑的肺動脈高壓來說,擴血管藥物作用相對較小,導(dǎo)致目前肺動脈高壓治療效果不佳,死亡率居高不下。因此從根本上抑制PASMCS增殖或促進其凋亡,從而減輕肺血管的重構(gòu),是目前治療PH的關(guān)鍵及研究的熱點。生存素(survivin)在凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族中凋亡作用最強,其具有雙重功能:促進細(xì)胞增殖和抑制細(xì)胞凋亡。Survivin在絕大多數(shù)腫瘤中過度表達(dá),而在正常組織中不表達(dá)或處于很低的水平。線粒體不僅是細(xì)胞內(nèi)能量的產(chǎn)生場所,也是參與細(xì)胞凋亡的靶器官。在研究survivin抑制腫瘤細(xì)胞凋亡的機制中發(fā)現(xiàn),survivin通過與線粒體凋亡途徑中的Caspase-9結(jié)合,阻止凋亡小體的形成,進而阻斷線粒體調(diào)控的凋亡通路。目前研究顯示,線粒體與細(xì)胞凋亡以及細(xì)胞低氧感受器密切相關(guān),并在PH發(fā)病機制中起到非常重要的作用。我們前期的研究表明:survivin在常氧(21%O_2)培養(yǎng)的人肺動脈平滑肌細(xì)胞(human PASMCs,HPASMCs)中不表達(dá),而在缺氧(2.5%O_2)培養(yǎng)的HPASMCs中表達(dá),并且促進HPASMCs增殖,抑制其凋亡。但survivin抑制缺氧HPAMSCs凋亡的機制目前仍不清楚。本實驗以HPASMCs為研究對象,研究survivin抑制缺氧HPASMCs凋亡的機制,將HPASMCs分為6組:(1)N組:正常對照組;(2)H組:缺氧組;(3)NY組:常氧+YM155組;(4)HY1組:24h缺氧+YM155 1nmol/L;(5)HY10組:24h缺氧+YM155 10nmol/L;(6)HY100組:24h缺氧+YM155 100nmol/L。采用TMRE法檢測HPASMCs線粒體膜電位,DCFH-DA法檢測HPASMCs內(nèi)活性氧(reactive oxygen species,ROS)含量,Western blot分析細(xì)胞色素C(cytochrome-C,Cyto-C)的分布與Caspase-9的蛋白表達(dá)。研究結(jié)果表明:(1)N組線粒體的膜電位為47.831±4.550,與H組膜電位(60.453±6.088)比較差異有統(tǒng)計學(xué)意義(q=5.199,P㩳0.05);各劑量YM155干預(yù)組線粒體的膜電位分別為49.183±1.007、37.180±1.047、17.568±5.836,與H組比較差異有統(tǒng)計學(xué)意義(q=4.642、9.585、17.663,P均㩳0.05),且在一定范圍內(nèi)呈濃度依賴性;(2)N組的細(xì)胞內(nèi)ROS含量為26.160±3.500,與H組(17.662±3.116)比較差異有統(tǒng)計學(xué)意義(q=3.248,P㩳0.05)。各劑量YM155干預(yù)組的細(xì)胞內(nèi)ROS含量為分別為34.825±3.225、52.225±5.794、71.744±7.050,與H組比較差異有統(tǒng)計學(xué)意義(q=6.561、13.212、20.674,P均㩳0.05),且在一定范圍內(nèi)呈濃度依賴性;(3)N組胞質(zhì)Cyto-C/線粒體Cyto-C為0.733,與H組(0.173)比較差異有統(tǒng)計學(xué)意義(P㩳0.05)。HY1組、HY10組、HY100組中胞質(zhì)Cyto-C/線粒體Cyto-C分別為0.846、1.908、3.258,與H組比較差異有統(tǒng)計學(xué)意義(P㩳0.05)。Caspase-9蛋白的表達(dá):N組的表達(dá)量為0.531±0.102,與H組(0.124±0.113)比較,差異有統(tǒng)計學(xué)意義(q=4.284,P㩳0.05)。各劑量YM155的干預(yù)下,Caspase-9蛋白的表達(dá)量分別為1.016±0.225、1.732±0.156、1.732±0.156,與H組比較差異有統(tǒng)計學(xué)意義(q=9.389、14.400、29.042,P均㩳0.05),且在一定范圍內(nèi)呈濃度依賴性。上述結(jié)果表明:缺氧條件下HPAMSCs中survivin的表達(dá),升高了HPAMSCs線粒體的膜電位,降低了細(xì)胞內(nèi)ROS的含量,線粒體膜電位去極化受到抑制,進而抑制了Cyto-C從線粒體釋放到細(xì)胞質(zhì)及Caspase-9的活化,抑制了線粒體依賴的凋亡途徑,因此通過抑制HPAMSCs中survivin的表達(dá),促進線粒體依賴的凋亡通路,為HPH的治療提供了新靶點。
[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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R544.1
本文編號：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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R544.1
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