生存素对缺氧人肺动脉平滑肌细胞凋亡与增殖的影响

发布时间：2018-06-12 04:34

  本文选题：生存素 + 肺动脉平滑肌细胞　； 参考：《河北北方学院》2015年硕士论文

【摘要】：缺氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)是呼吸系统常见疾病,可导致右心衰竭,甚至死亡。迄今为止,HPH发病机制尚未完全阐释清楚,肺血管重塑被认为是HPH的主要病理特征,而肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)过度增殖和凋亡抑制是肺血管重塑的重要机制。当前大多数治疗肺动脉高压(pulmonary hypertension,PH)的药物主要集中在扩张肺血管,然而对已经发生肺血管重塑的患者来说,降低肺动脉压的程度有限,且这种扩血管作用对肺循环无特异性。因此,如何抑制PASMCs增殖促进其凋亡是目前PH治疗的热点课题。目前关于肿瘤细胞的过度增殖和凋亡受阻已经有了较为详尽的研究,值得借鉴。生存素(survivin)是凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族的新成员,是目前发现的最强的凋亡抑制蛋白,在人类多种恶性肿瘤细胞中过度表达,具有抑制细胞凋亡和促进细胞增殖的双重功能。近些年研究结果显示,survivin参与PASMCs增殖凋亡失衡形成的肺血管重塑。我们前期的研究结果表明,survivin在慢性缺氧肺动脉高压大鼠肺组织中高表达,且主要位于肺内小动脉的中膜。但survivin在缺氧人PASMCs(human PASMCs,HPASMCs)中是否表达国内外尚未见报道。本研究选用HPASMCs为研究对象,研究suvivin在缺氧HPASMCs中的表达情况及survivin的小分子抑制剂YM155对HPASMCs增殖与凋亡的影响。将HPASMCs进行常氧或缺氧24 h培养并将其分为6组:①常氧对照组(N组);②24 h缺氧组(H组);③常氧+YM155 10nM组(NY组);④24 h缺氧+YM155 1nM组(HY1组);⑤24 h缺氧+YM155 10n M组(HY10组);⑥24 h缺氧+YM155 100nM组(HY100组)。采用Western Blot的方法检测HPASMCs survivin蛋白表达,RT-PCR的方法检测HPASMCs survivin mRNA表达。采用活细胞计数试剂盒(CCK-8)法检测HPASMCs增殖,原位末端标记(TUNEL)法检测HPASMCs凋亡。研究发现N组未见survivin蛋白(0.016±0.006)和mRNA(1)表达,H组可见survivin蛋白(0.837±0.027)、survivin mRNA(17086±1044)表达。各剂量(1n M、10nM、100nM)YM155干预组survivin蛋白、survivin mRNA含量分别为0.382±0.041、0.281±0.025、0.021±0.002、8074±2135、5614±709、1382±347,较H组显著降低(q值分别为20.26、24.77、36.36、8.59、11.14、15,53,均P0.05),且在一定范围内呈现浓度依赖性。N组细胞增殖活性(以CCK-8法检测出的A值表示)和细胞凋亡率分别为0.988±0.071、2.683±1.360,与H组(1.438±0.121、0.612±0.500)比较差异有统计学意义(q值分别为6.484、3.532,均P0.05)。各剂量(1nM、10nM、100nM)YM155干预组细胞增殖活性和细胞凋亡率分别为1.318±0.067、1.168±0.071、0.845±0.129、5.517±1.711、6.658±1.487、7.972±1.934,与H组比较差异有统计学意义(q值分别为2.581、3.980、8.733、6.014、7、413、9.023,均P0.05),且在一定范围内呈现浓度依赖性。上述结果证实survivin在缺氧(2.5%O2)HPASMCs中表达,而在常氧(21%O2)HPASMCs中不表达。Survivin的小分子抑制剂YM155可以在基因和蛋白水平下调缺氧HPAMCs中表达的survivin,且呈现浓度依赖性。YM155抑制缺氧性HPASMCs增殖,促进其凋亡,并在一定范围内随YM155浓度的增加,作用增强。Survivin的表达异常在HPH的发生发展中发挥重要作用,可能成为治疗HPH的新靶点。
[Abstract]:Hypoxic pulmonary hypertension (HPH) is a common disease of the respiratory system, which can lead to right heart failure and even death. So far, the pathogenesis of HPH has not been fully elucidated. Pulmonary vascular remodeling is considered to be the main pathological feature of HPH, while pulmonary arteria smooth muscle cells (pulmonary arterial smooth muscle cells, PASM) Cs) excessive proliferation and inhibition of apoptosis is an important mechanism for pulmonary vascular remodeling. Most of the current drugs for the treatment of pulmonary hypertension (pulmonary hypertension, PH) are mainly concentrated in the dilation of pulmonary vessels. However, for patients who have undergone pulmonary vascular remodeling, the reduction of pulmonary arterial pressure is limited and this vasodilator is not specific to the pulmonary circulation. Therefore, how to inhibit the proliferation of PASMCs and promote its apoptosis is a hot topic of PH therapy at present. There has been a more detailed study on the overproliferation and apoptosis of tumor cells, which is worthy of reference. Survivin (survivin) is a new member of the inhibitor of apoptosis proteins (IAP) family, which is currently found. The strongest apoptosis suppressor protein, overexpressed in a variety of human malignant tumor cells, has the dual function of inhibiting cell apoptosis and promoting cell proliferation. In recent years, the results showed that survivin was involved in pulmonary vascular remodeling in the imbalance of PASMCs proliferation and apoptosis. Our previous research results showed that survivin was in chronic hypoxic pulmonary artery high. High expression in rat lung tissue and mainly located in the middle membrane of the pulmonary arterioles, but the expression of Survivin in PASMCs (human PASMCs, HPASMCs) has not been reported at home and abroad. This study selected HPASMCs as a study object to study the expression of suvivin in anoxic HPASMCs and a small molecule inhibitor YM155 for survivin YM155 to HPASMCs. The influence of colonization and apoptosis. HPASMCs was cultured and divided into 6 groups: normoxic or anoxic 24 h groups: (1) normal oxygen control group (group N), 24 h hypoxia group (group H); (group NY); (4) 24 h hypoxia +YM155 1nM group (HY1 group); 5. 24 The expression of HPASMCs survivin protein was detected by the method, and the expression of HPASMCs survivin mRNA was detected by RT-PCR. The proliferation of HPASMCs was detected by the live cell count Kit (CCK-8), and the apoptosis of HPASMCs was detected by the in situ terminal labeling (TUNEL) method. The survivin protein (0.016 + 0.006) and the expression of 1 were found in N group. ), survivin mRNA (17086 + 1044) expressed survivin protein in each dose (1n M, 10nM, 100nM) YM155 intervention group, survivin mRNA content was 0.382 + 0.041,0.281 + 0.0028074 + 21355614 + 7091382 + 347, respectively. The cell proliferation activity of the dependent.N group (the A value detected by CCK-8) and the apoptosis rate were 0.988 + 0.071,2.683 + 1.360, respectively, and compared with the H group (1.438 + 0.121,0.612 + 0.500), the difference was statistically significant (Q value was 6.484,3.532, all P0.05). The cell proliferation activity and the cell apoptosis rate of each dose (1nM, 10nM, 100nM) The difference between 1.318 + 0.067,1.168 + 0.071,0.845 + 0.129,5.517 + 1.711,6.658 + 1.487,7.972 + 1.934 and H group was statistically significant (Q value was 2.581,3.980,8.733,6.014,7413,9.023, all P0.05), and showed a concentration dependence in a certain range. The above results confirmed that survivin was expressed in the hypoxia (2.5%O2) HPASMCs, while in the normal oxygen (21). %O2) small molecule inhibitor YM155, which does not express.Survivin in HPASMCs, can downregulate the survivin expressed in anoxic HPAMCs at the gene and protein level, and present a concentration dependent.YM155 inhibiting the proliferation of anoxic HPASMCs, promoting its apoptosis and increasing the concentration of YM155 in a certain range, and the abnormal expression of the action enhancement.Survivin is in HPH. Playing an important role in development may become a new target for the treatment of HPH.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R544.1

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