本文選題：生存素 + 肺動脈平滑肌細胞�。� 參考：《河北北方學(xué)院》2015年碩士論文

【摘要】：缺氧性肺動脈高壓(hypoxic pulmonary hypertension,HPH)是呼吸系統(tǒng)常見疾病,可導(dǎo)致右心衰竭,甚至死亡。迄今為止,HPH發(fā)病機制尚未完全闡釋清楚,肺血管重塑被認為是HPH的主要病理特征,而肺動脈平滑肌細胞(pulmonary arterial smooth muscle cells,PASMCs)過度增殖和凋亡抑制是肺血管重塑的重要機制。當(dāng)前大多數(shù)治療肺動脈高壓(pulmonary hypertension,PH)的藥物主要集中在擴張肺血管,然而對已經(jīng)發(fā)生肺血管重塑的患者來說,降低肺動脈壓的程度有限,且這種擴血管作用對肺循環(huán)無特異性。因此,如何抑制PASMCs增殖促進其凋亡是目前PH治療的熱點課題。目前關(guān)于腫瘤細胞的過度增殖和凋亡受阻已經(jīng)有了較為詳盡的研究,值得借鑒。生存素(survivin)是凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族的新成員,是目前發(fā)現(xiàn)的最強的凋亡抑制蛋白,在人類多種惡性腫瘤細胞中過度表達,具有抑制細胞凋亡和促進細胞增殖的雙重功能。近些年研究結(jié)果顯示,survivin參與PASMCs增殖凋亡失衡形成的肺血管重塑。我們前期的研究結(jié)果表明,survivin在慢性缺氧肺動脈高壓大鼠肺組織中高表達,且主要位于肺內(nèi)小動脈的中膜。但survivin在缺氧人PASMCs(human PASMCs,HPASMCs)中是否表達國內(nèi)外尚未見報道。本研究選用HPASMCs為研究對象,研究suvivin在缺氧HPASMCs中的表達情況及survivin的小分子抑制劑YM155對HPASMCs增殖與凋亡的影響。將HPASMCs進行常氧或缺氧24 h培養(yǎng)并將其分為6組:①常氧對照組(N組);②24 h缺氧組(H組);③常氧+YM155 10nM組(NY組);④24 h缺氧+YM155 1nM組(HY1組);⑤24 h缺氧+YM155 10n M組(HY10組);⑥24 h缺氧+YM155 100nM組(HY100組)。采用Western Blot的方法檢測HPASMCs survivin蛋白表達,RT-PCR的方法檢測HPASMCs survivin mRNA表達。采用活細胞計數(shù)試劑盒(CCK-8)法檢測HPASMCs增殖,原位末端標記(TUNEL)法檢測HPASMCs凋亡。研究發(fā)現(xiàn)N組未見survivin蛋白(0.016±0.006)和mRNA(1)表達,H組可見survivin蛋白(0.837±0.027)、survivin mRNA(17086±1044)表達。各劑量(1n M、10nM、100nM)YM155干預(yù)組survivin蛋白、survivin mRNA含量分別為0.382±0.041、0.281±0.025、0.021±0.002、8074±2135、5614±709、1382±347,較H組顯著降低(q值分別為20.26、24.77、36.36、8.59、11.14、15,53,均P0.05),且在一定范圍內(nèi)呈現(xiàn)濃度依賴性。N組細胞增殖活性(以CCK-8法檢測出的A值表示)和細胞凋亡率分別為0.988±0.071、2.683±1.360,與H組(1.438±0.121、0.612±0.500)比較差異有統(tǒng)計學(xué)意義(q值分別為6.484、3.532,均P0.05)。各劑量(1nM、10nM、100nM)YM155干預(yù)組細胞增殖活性和細胞凋亡率分別為1.318±0.067、1.168±0.071、0.845±0.129、5.517±1.711、6.658±1.487、7.972±1.934,與H組比較差異有統(tǒng)計學(xué)意義(q值分別為2.581、3.980、8.733、6.014、7、413、9.023,均P0.05),且在一定范圍內(nèi)呈現(xiàn)濃度依賴性。上述結(jié)果證實survivin在缺氧(2.5%O2)HPASMCs中表達,而在常氧(21%O2)HPASMCs中不表達。Survivin的小分子抑制劑YM155可以在基因和蛋白水平下調(diào)缺氧HPAMCs中表達的survivin,且呈現(xiàn)濃度依賴性。YM155抑制缺氧性HPASMCs增殖,促進其凋亡,并在一定范圍內(nèi)隨YM155濃度的增加,作用增強。Survivin的表達異常在HPH的發(fā)生發(fā)展中發(fā)揮重要作用,可能成為治療HPH的新靶點。
[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.
【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】：2015
【分類號】：R544.1

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