本文選題：肺動(dòng)脈高壓 + 野百合堿��； 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：肺動(dòng)脈高壓(Pulmonary Arterial Hypertension, PAH)是WHO肺高壓(pulmonary hypertension, PH)分類中的第一組,其特征是進(jìn)行性右心衰竭從而導(dǎo)致死亡。過去多年開發(fā)出的多種靶向藥物顯著改善了PAH患者的預(yù)后,但是需要肺移植的患者數(shù)量依然很多。造成這一現(xiàn)象的主要原因是上述靶向藥物常針對(duì)PAH的單一發(fā)病機(jī)制,而PAH的重要病理生理機(jī)制目前尚不完全清楚,已知的機(jī)制設(shè)計(jì)內(nèi)皮細(xì)胞的炎性反應(yīng)、平滑肌細(xì)胞的過度增殖和肺動(dòng)脈(PA)收縮,進(jìn)而導(dǎo)致PA病理性重構(gòu)。促進(jìn)PA重構(gòu)的細(xì)胞因子眾多,其中,胰島素樣生長(zhǎng)因子(insulin-like growth factor, IGF)十分重要,它可以通過多種機(jī)制刺激血管平滑肌增殖。IGF對(duì)細(xì)胞的不同作用受到胰島素樣生長(zhǎng)因子結(jié)合蛋白(insulin-like growth factor binding protein, IGFBP)家族復(fù)雜且嚴(yán)密的調(diào)控。轉(zhuǎn)化生長(zhǎng)因子β1(transforming growth factorβ1, TGF-β1)可調(diào)節(jié)IGFBPs的產(chǎn)生。然而,在PAH的肺動(dòng)脈平滑肌細(xì)胞(pulmonary artery smooth muscle cells, PASMCs)中,TGF-β1調(diào)控IGFBPs的作用及機(jī)制尚不完全清楚。為此,本課題采用野百合堿(Monocrotaline, MCT)誘導(dǎo)建立PAH大鼠模型,分析PASMCs中TGF-β1對(duì)IGFBP3和IGFBP5的作用及可能的機(jī)制。方法：一次性腹腔注射野百合堿(1% MCT 60mg/kg)4周后誘導(dǎo)建立大鼠PAH模型。分離原代PASMCs,實(shí)驗(yàn)中選用第3-8代細(xì)胞,正常大鼠做為對(duì)照組。提取PASMCs中的總RNA,并逆轉(zhuǎn)錄成cDNA,應(yīng)用R-TqPCR檢測(cè)PASMCs中IGFBPs家族目的基因的表達(dá)變化。選取在兩組間表達(dá)有統(tǒng)計(jì)學(xué)差異的基因,分別提取兩組PASMCs全細(xì)胞蛋白,應(yīng)用western blot方法檢測(cè)上述差異表達(dá)基因的蛋白表達(dá)變化。為明確TGF-β1對(duì)IGFBP3和IGFBP5的調(diào)節(jié)作用,PASMCs無(wú)血清培養(yǎng)12h后加入重組TGF-β1或TGF-β1的中和抗體,應(yīng)用western blot檢測(cè)IGFBP3和IIGFBP5的蛋白表達(dá)的變化。為了解ERK及P13K對(duì)TGF-β1調(diào)節(jié)IGFBPs的作用,應(yīng)用ERK特異性抑制劑PD98059或P13K特異性抑制劑LY294002預(yù)處理細(xì)胞后加入TGF-β1,再次檢測(cè)IGFBP3、IGFBP5、P-Smad2及P-Smad3的表達(dá)變化。結(jié)果：1%MCT 60mg/kg-次性腹腔注射4周后,心臟超聲結(jié)果顯示：PAH組平均肺動(dòng)脈壓(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)、右室重量與左心室及室間隔的重量比[RV/(LV+ST)],47.0±1 0.0%]、及PA中膜厚度百分比(%MWT,24.0±1.10%)均顯著高于對(duì)照組(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結(jié)果示：PAH組PASMCs中IGFBP1、IGFBP2和IGFBP4的]mRNA表達(dá)水平與對(duì)照組間無(wú)統(tǒng)計(jì)學(xué)差異(p分別=0.17、p=0.18和p=0.90),而IGFBP3和IGFBP5的mRNA表達(dá)量顯著高于對(duì)照組(p分別=0.04和=0.03)。為此,采用Western blot檢測(cè)了IGFBP3和IGFBP5的蛋白水平,結(jié)果發(fā)現(xiàn)IGFBP3和IGFBP5的蛋白表達(dá)在PAH組也顯著高于對(duì)照組(p分別=0.04和=0.02)。外源性TGF-β1可通過活化Smad2和Smad3而誘導(dǎo)PAH大鼠PASMCs中IGFBP3和IGFBP5的蛋白表達(dá)增加,這一作用可被TGF-β1的中和抗體及PD98059所抑制,而LY294002促進(jìn)IGFBP5的表達(dá),但卻抑制IGFBP3的表達(dá)。結(jié)論：MCT誘導(dǎo)的PAH大鼠PASMCs中,IGFBP3及IGFBP5的mRNA和蛋白表達(dá)水平均顯著高于對(duì)照組。TGF-β1通過活化Smad2和Smad3促進(jìn)IGFBP3和IGFBP5的表達(dá)；P13K對(duì)TGF-β1調(diào)節(jié)IGFBP5和IGFBP3的表達(dá),表現(xiàn)出差異性調(diào)節(jié)作用。ERK可通過活化Smad2和Smad3促進(jìn)TGF-β1對(duì)IGFBP3和IGFBP5的調(diào)節(jié)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R544.1

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