肺动脉高压大鼠肺动脉平滑肌细胞中转化生长因子β1调节胰岛素样生长因子结合蛋白的机制研究

发布时间：2018-07-09 20:33

  本文选题：肺动脉高压 + 野百合碱　； 参考：《南京医科大学》2015年硕士论文

【摘要】：目的：肺动脉高压(Pulmonary Arterial Hypertension, PAH)是WHO肺高压(pulmonary hypertension, PH)分类中的第一组,其特征是进行性右心衰竭从而导致死亡。过去多年开发出的多种靶向药物显著改善了PAH患者的预后,但是需要肺移植的患者数量依然很多。造成这一现象的主要原因是上述靶向药物常针对PAH的单一发病机制,而PAH的重要病理生理机制目前尚不完全清楚,已知的机制设计内皮细胞的炎性反应、平滑肌细胞的过度增殖和肺动脉(PA)收缩,进而导致PA病理性重构。促进PA重构的细胞因子众多,其中,胰岛素样生长因子(insulin-like growth factor, IGF)十分重要,它可以通过多种机制刺激血管平滑肌增殖。IGF对细胞的不同作用受到胰岛素样生长因子结合蛋白(insulin-like growth factor binding protein, IGFBP)家族复杂且严密的调控。转化生长因子β1(transforming growth factorβ1, TGF-β1)可调节IGFBPs的产生。然而,在PAH的肺动脉平滑肌细胞(pulmonary artery smooth muscle cells, PASMCs)中,TGF-β1调控IGFBPs的作用及机制尚不完全清楚。为此,本课题采用野百合碱(Monocrotaline, MCT)诱导建立PAH大鼠模型,分析PASMCs中TGF-β1对IGFBP3和IGFBP5的作用及可能的机制。方法：一次性腹腔注射野百合碱(1% MCT 60mg/kg)4周后诱导建立大鼠PAH模型。分离原代PASMCs,实验中选用第3-8代细胞,正常大鼠做为对照组。提取PASMCs中的总RNA,并逆转录成cDNA,应用R-TqPCR检测PASMCs中IGFBPs家族目的基因的表达变化。选取在两组间表达有统计学差异的基因,分别提取两组PASMCs全细胞蛋白,应用western blot方法检测上述差异表达基因的蛋白表达变化。为明确TGF-β1对IGFBP3和IGFBP5的调节作用,PASMCs无血清培养12h后加入重组TGF-β1或TGF-β1的中和抗体,应用western blot检测IGFBP3和IIGFBP5的蛋白表达的变化。为了解ERK及P13K对TGF-β1调节IGFBPs的作用,应用ERK特异性抑制剂PD98059或P13K特异性抑制剂LY294002预处理细胞后加入TGF-β1,再次检测IGFBP3、IGFBP5、P-Smad2及P-Smad3的表达变化。结果：1%MCT 60mg/kg-次性腹腔注射4周后,心脏超声结果显示：PAH组平均肺动脉压(mean pulmonary arterial pressure, mPAP:75.69±6.72 mmHg)、右室前壁厚度(right ventricular anterior wall,RVAW,0.79±0.02mm)、右室重量与左心室及室间隔的重量比[RV/(LV+ST)],47.0±1 0.0%]、及PA中膜厚度百分比(%MWT,24.0±1.10%)均显著高于对照组(34.58±2.49 mmHg,p0.001; 0.40±0.07mm,p=0.04; 20.0±9.50%, p=0.02,及17.0±3.0%,p=0.01) R-TqPCR结果示：PAH组PASMCs中IGFBP1、IGFBP2和IGFBP4的]mRNA表达水平与对照组间无统计学差异(p分别=0.17、p=0.18和p=0.90),而IGFBP3和IGFBP5的mRNA表达量显著高于对照组(p分别=0.04和=0.03)。为此,采用Western blot检测了IGFBP3和IGFBP5的蛋白水平,结果发现IGFBP3和IGFBP5的蛋白表达在PAH组也显著高于对照组(p分别=0.04和=0.02)。外源性TGF-β1可通过活化Smad2和Smad3而诱导PAH大鼠PASMCs中IGFBP3和IGFBP5的蛋白表达增加,这一作用可被TGF-β1的中和抗体及PD98059所抑制,而LY294002促进IGFBP5的表达,但却抑制IGFBP3的表达。结论：MCT诱导的PAH大鼠PASMCs中,IGFBP3及IGFBP5的mRNA和蛋白表达水平均显著高于对照组。TGF-β1通过活化Smad2和Smad3促进IGFBP3和IGFBP5的表达；P13K对TGF-β1调节IGFBP5和IGFBP3的表达,表现出差异性调节作用。ERK可通过活化Smad2和Smad3促进TGF-β1对IGFBP3和IGFBP5的调节作用。
[Abstract]:Objective: Pulmonary Arterial Hypertension (PAH) is the first group in the classification of WHO pulmonary hypertension (pulmonary hypertension, PH), characterized by progressive right heart failure and leading to death. The multiple targeting drugs developed over the years have significantly improved the prognosis of patients with PAH, but the number of patients requiring lung transplantation is still still in need. The main reason for this phenomenon is the single pathogenesis of PAH, and the important pathophysiological mechanism of PAH is not completely clear. The known mechanism is designed to design the inflammatory response of endothelial cells, the excessive proliferation of smooth muscle cells and the contraction of the pulmonary artery (PA), and then cause the pathological remodeling of PA, and promote the reconstruction of PA. There are many cytokines, among them, insulin-like growth factor (IGF) is very important. It can stimulate the proliferation of vascular smooth muscle through a variety of mechanisms and the different effects of.IGF on the cells are complicated and closely related to the insulin like growth factor binding protein (insulin-like growth factor binding protein, IGFBP) family. Regulation. Transforming growth factor beta 1 (transforming growth factor beta 1, TGF- beta 1) can regulate the production of IGFBPs. However, in the PAH pulmonary artery smooth muscle cells (pulmonary artery smooth muscle cells, PASMCs), the role and mechanism of regulating beta 1 are not completely clear. The PAH rat model was established to analyze the effect and possible mechanism of TGF- beta 1 on IGFBP3 and IGFBP5 in PASMCs. Methods: the rat PAH model was induced by intraperitoneal injection of monocrotaline (1% MCT 60mg/kg) for 4 weeks. The primary PASMCs was isolated and the 3-8 generation cells were selected in the experiment, and the normal rats were used as the control group. The total RNA in PASMCs was extracted and reverse transcriptase. CDNA, R-TqPCR was used to detect the expression changes of IGFBPs family target gene in PASMCs. The genes with statistical difference were selected between the two groups, two groups of PASMCs whole cell proteins were extracted, and the protein expression changes of the above expressed genes were detected by Western blot method. The regulation of TGF- beta 1 on IGFBP3 and IGFBP5, PASMC The neutralization antibody of recombinant TGF- beta 1 or TGF- beta 1 was added to the serum-free culture of S, and the changes in the protein expression of IGFBP3 and IIGFBP5 were detected by Western blot. In order to solve the effect of ERK and P13K on TGF- beta 1 regulating IGFBPs, the specific inhibitors or specific inhibitors were applied to the cells to be added to the beta 1. 3, IGFBP5, P-Smad2 and P-Smad3 expression changes. Results: after 4 weeks of intraperitoneal injection of 1%MCT 60mg/kg-, the echocardiographic results showed that the mean pulmonary arterial pressure in group PAH (mean pulmonary arterial pressure, mPAP:75.69 6.72 mmHg), right ventricle anterior wall thickness and left ventricle and left ventricle, The weight ratio of ventricular septum to [RV/ (LV+ST)], 47 + 1 0.0%], and the percentage of membrane thickness in PA (%MWT, 24 + 1.10%) were significantly higher than those of the control group (34.58 + 2.49 mmHg, p0.001; 0.40 + 0.07mm, p=0.04; 20 + 9.50%, p=0.02, 17 + 3%, p=0.01) R-TqPCR results showed no difference between the control group and the control group The statistical differences (P, =0.17, p=0.18 and p=0.90) were significantly higher than those of the control group (P =0.04 and =0.03). Therefore, Western blot was used to detect the protein levels of P and =0.03. - beta 1 can induce the increase in the expression of IGFBP3 and IGFBP5 in PAH rat PASMCs by activating Smad2 and Smad3. This effect can be suppressed by neutralizing antibodies and PD98059 of TGF- beta 1, while LY294002 promotes the expression of IGFBP5, but inhibits IGFBP3 expression. .TGF- beta 1 increased the expression of IGFBP3 and IGFBP5 by activating Smad2 and Smad3, and P13K showed a differential regulation of the expression of IGFBP5 and IGFBP3 by TGF- beta 1, which could regulate the regulating effect of.ERK by activating Smad2 and Smad3.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R544.1

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