本文選題：先天性心臟病 + 肺動(dòng)脈高壓�。� 參考：《第二軍醫(yī)大學(xué)》2013年博士論文

【摘要】：研究背景 先天性心臟�。–HD）是小兒時(shí)期最常見的心血管疾病，發(fā)病率約占全部活產(chǎn)嬰兒的7～8‰。據(jù)統(tǒng)計(jì)我國(guó)每年新出生的先心病患兒高達(dá)15萬(wàn)，其中5～10%左向右分流型先天性心臟病患兒因未及時(shí)治療而形成肺動(dòng)脈高壓（PAH）。若不加干預(yù)，患兒的肺動(dòng)脈壓力會(huì)不斷升高，進(jìn)而形成艾森曼格綜合征并最終導(dǎo)致右心衰竭而死亡。目前對(duì)PAH的治療主要包括藥物治療與手術(shù)治療。在藥物治療方面，幾種不同機(jī)制的抗肺動(dòng)脈高壓治療藥物已應(yīng)用于臨床，并改善了部分患者的生存質(zhì)量，但由于無法逆轉(zhuǎn)肺血管的重構(gòu)，最終病人仍將因不斷增高的肺動(dòng)脈壓力而導(dǎo)致右心衰竭及死亡。盡管手術(shù)治療可以徹底矯治先天性心內(nèi)缺損，且大部分在發(fā)生右向左分流前接受心內(nèi)缺損修補(bǔ)手術(shù)的患兒可獲得滿意的預(yù)后，但對(duì)發(fā)生雙向分流或以右向左分流為主的患兒，單純的手術(shù)治療并不能獲得滿意的療效，其原因在于該類患者的肺動(dòng)脈血管已經(jīng)發(fā)生了重構(gòu)；如果患者的肺動(dòng)脈血管重構(gòu)得不到有效的逆轉(zhuǎn)，那么手術(shù)治療反而會(huì)促使其死亡。由此可見，無論是藥物治療還是手術(shù)治療，肺動(dòng)脈血管重構(gòu)程度都是決定患兒預(yù)后的關(guān)鍵因素，因而研究CHD-PAH肺動(dòng)脈血管重構(gòu)的機(jī)制將有助于為減緩或逆轉(zhuǎn)肺動(dòng)脈重構(gòu)的治療提供新靶點(diǎn)。既往研究表明，BMPR2表達(dá)下降與肺動(dòng)脈重構(gòu)發(fā)生密切相關(guān)，文獻(xiàn)回顧及基因信息學(xué)分析表明miR-19a可能對(duì)BMPR2的表達(dá)具有調(diào)控作用。 研究目的 本課題希望通過臨床調(diào)查及相關(guān)的動(dòng)物實(shí)驗(yàn)明確miR-19a對(duì)BMPR2表達(dá)的調(diào)控作用及其促肺動(dòng)脈內(nèi)皮細(xì)胞增殖及肺動(dòng)脈血管重構(gòu)的調(diào)控機(jī)制,進(jìn)而為CHD-PAH手術(shù)治療提供依據(jù)（如相關(guān)miRNA的量化可以作為肺動(dòng)脈血管重構(gòu)可否逆轉(zhuǎn)的指標(biāo)）。此外，還可為PAHde藥物治療提供新靶點(diǎn)。 研究方法 1、以2011年10月-2012年10月在我院住院治療的重度肺動(dòng)脈高壓患者肺組織為實(shí)驗(yàn)組(n=10),自發(fā)性氣胸患者術(shù)中切除正常肺組織為對(duì)照組(n=10),進(jìn)行免疫組化及western-blotting檢測(cè)BMPR2表達(dá)水平差異；與此同時(shí)，在文獻(xiàn)回顧及基因信息學(xué)分析的基礎(chǔ)上，通過qRT-PCR方法檢測(cè)驗(yàn)證與CHD-PAH發(fā)生及BMPR2表達(dá)相關(guān)的miRNA。 2、建立左向右分流肺動(dòng)脈高壓大樹模型，以模型大鼠的肺組織為研究對(duì)象進(jìn)一步驗(yàn)證左向右分流血流動(dòng)力學(xué)改變對(duì)miR-19a及BMPR2表達(dá)水平的影響。方法如下：40只雄性SD大鼠隨機(jī)等分為4組：T1、T2、T3、T4； T1、T3為實(shí)驗(yàn)組，T2、T4為對(duì)照組。實(shí)驗(yàn)組腹正中切口開腹，穿刺腹主動(dòng)脈至下腔靜脈建立動(dòng)靜脈瘺，對(duì)照組腹正中切口開腹，但不穿刺血管。于術(shù)后第4周檢測(cè)T1、T2組的血流動(dòng)力學(xué)數(shù)據(jù)，第8周檢測(cè)T3、T4組的血流動(dòng)力學(xué)數(shù)據(jù)。并應(yīng)用免疫組化方法檢測(cè)BMPR2在肺動(dòng)脈表達(dá)改變；western-blotting及qRT-PCR分別檢測(cè)相關(guān)蛋白及miRNA表達(dá)。 3、將miR-19a及pcDNA-BMPR2通過脂質(zhì)體包裹的方法轉(zhuǎn)染大鼠肺動(dòng)阿密內(nèi)皮細(xì)胞，，繼而通過流式細(xì)胞學(xué)及AnnexinV/PI雙染色法觀察其對(duì)肺動(dòng)脈內(nèi)皮細(xì)胞增殖及調(diào)往的影響。 研究結(jié)果 1、與正常對(duì)照組相比，CHD-PAH患者肺組織中BMPR2蛋白表達(dá)水平顯著下降。通過文獻(xiàn)回顧及基因信息學(xué)網(wǎng)站預(yù)測(cè)，我們選定了與BMPR2相關(guān)性較強(qiáng)的miR-19a、miR-20a、miR-20b、miR-21、miR-130a作為目標(biāo)miRNA檢測(cè)其在實(shí)驗(yàn)組及對(duì)照組中的含量。實(shí)驗(yàn)結(jié)果顯示miR-19a在實(shí)驗(yàn)組肺組織中表達(dá)量明顯升高，其余miRNA表達(dá)水平無明顯差異。 2、模型組（左向右分流）較對(duì)照組肺動(dòng)脈壓力明顯升高（P＜0.05）。建模后4周組較正常對(duì)照組平均肺動(dòng)壓明顯升高(31.3±2.9mmHg vs15.5±1.08mmHg，p＜0.001）；建模后8周組平均肺動(dòng)脈壓較正常對(duì)照組明顯升高（40.8±2.6mmHgvs16.0±0.94mmHg，p＜0.001）；建模后4周組與建模后8周祖平均肺動(dòng)脈壓明顯升高（31.3±2.9mmHg vs40.8±2.6mmHg，p＜0.001）。模型組與對(duì)照組HE及P-VB染色比較，肺動(dòng)脈內(nèi)皮細(xì)胞及中層平滑肌細(xì)胞增生明顯，模型組較對(duì)照組中層膠原纖維增生明顯（P＜0.05）。免疫組化及western-boltting結(jié)果顯示模型組大鼠肺動(dòng)脈周圍BMPR2表達(dá)量較對(duì)照組明顯下降（P＜0.05）。qRT-PCR結(jié)果顯示模型組大鼠肺組織miR-19a表達(dá)明顯升高（P＜0.05）。 3、 SD大鼠內(nèi)皮細(xì)胞轉(zhuǎn)染miR-19a后24小時(shí)miR-19a增高約150倍；轉(zhuǎn)染miR-19a組western-blotting檢測(cè)BMPR2蛋白表達(dá)量明顯降低（P＜0.05）。轉(zhuǎn)染miR-19a+pcDNA-BMPR2組細(xì)胞增殖程度介于轉(zhuǎn)染miR-19a組與空白對(duì)照組之間，而轉(zhuǎn)染miR-19a組細(xì)胞增殖程度明顯高于空白對(duì)照組（P＜0.05）。轉(zhuǎn)染miR-19a組細(xì)胞凋亡率較其余兩組明顯下降（P＜0.05）。 研究結(jié)論 1、CHD-PAH患者肺組織中miR-19a表達(dá)量顯著升高，而BMPR2表達(dá)水平明顯下降。 2、左向右分流血流動(dòng)力學(xué)改變可導(dǎo)致大鼠肺組織miR-19a表達(dá)顯著升高，BMPR2表達(dá)水平顯著下降。 3、BMPR2是miR-19a的靶蛋白，過表達(dá)miR-19a可導(dǎo)致BMPR2的表達(dá)量顯著下降，進(jìn)而引發(fā)肺動(dòng)脈內(nèi)皮細(xì)胞的過度增殖。 綜上所述，miR-19a可通過下調(diào)BMPR2促肺動(dòng)脈內(nèi)皮細(xì)胞增殖，進(jìn)而在CHD-PAH血管重構(gòu)中發(fā)揮著重要的調(diào)控作用。
[Abstract]:Research background
Congenital heart disease (CHD) is the most common cardiovascular disease in children. The incidence of congenital heart disease is about 7~8 per thousand of all live births. According to statistics, 150 thousand of children born with congenital heart disease are born every year in China. Among them, 5 to 10% children with left to right shunt congenital heart disease form pulmonary hypertension (PAH) because they are not treated in time. The pressure of the pulmonary artery will continue to rise to form Eisen Mange's syndrome and eventually lead to the death of the right heart failure. The current treatment of PAH mainly includes drug treatment and surgical treatment. In the drug treatment, several different mechanisms of anti pulmonary hypertension have been applied to the bed, and the quality of life of some patients is improved, but the quality of life is improved in some patients. Due to the inability to reverse the remodeling of the pulmonary vessels, the patient will eventually lead to right heart failure and death due to increasing pulmonary arterial pressure. Although surgical treatment can completely correct congenital heart defects, and most of the children receiving the right to left shunt can receive a satisfactory prognosis, but there are two directions. In children with shunt or right to left shunt, simple surgical treatment can not achieve satisfactory results. The reason is that the pulmonary arteries of the patients have been reconstructed. If the patients' pulmonary artery reconfiguration is not effectively reversed, the surgical treatment will cause them to die. The degree of pulmonary vascular remodeling is the key factor in determining the prognosis of the children. Therefore, the study of the mechanism of CHD-PAH pulmonary artery remodeling will help to provide new targets for the treatment of the treatment of pulmonary remodeling. Previous studies have shown that the decline of BMPR2 expression is closely related to the occurrence of pulmonary artery remodeling. According to informatics analysis, miR-19a may play a regulatory role in the expression of BMPR2.
research objective
We hope to clarify the regulatory role of miR-19a on the expression of BMPR2 and the regulatory mechanism of the proliferation of pulmonary artery endothelial cells and pulmonary vascular remodeling through clinical investigation and related animal experiments, and then provide the basis for the treatment of CHD-PAH surgery (such as the quantification of related miRNA can be used as an indicator of the reversion of pulmonary vascular remodeling). In addition, it can also provide new targets for the treatment of PAHde.
research method
1, the lung tissue of patients with severe pulmonary hypertension hospitalized in our hospital in October -2012 October 2011 was the experimental group (n=10). The normal lung tissue was removed in the patients with spontaneous pneumothorax as the control group (n=10), and the differential expression of BMPR2 was detected by immunohistochemistry and Western-blotting. At the same time, the literature review and genetic information analysis were carried out. On the basis of the qRT-PCR method, we detected the miRNA. related to CHD-PAH occurrence and BMPR2 expression.
2, a left to right shunt pulmonary hypertension tree model was established. The effect of left to right shunt hemodynamic changes on miR-19a and BMPR2 expression levels was further verified by the model rats' lung tissue. Methods as follows: 40 male SD rats were randomly divided into 4 groups: T1, T2, T3, T4; T1, T3 as experimental group, T2, T4 as control group. The abdominal aorta was opened and the arteriovenous fistula was established in the abdominal aorta to the inferior vena cava. The control group was open to the abdomen in the middle incision, but no blood vessels were punctured. The hemodynamic data of group T1 and T2 were detected fourth weeks after the operation, and the hemodynamic data of group T3 and T4 were detected at eighth weeks, and the expression of BMPR2 in the pulmonary artery was detected by immunohistochemistry; Wester N-blotting and qRT-PCR were used to detect the expression of related proteins and miRNA respectively.
3, miR-19a and pcDNA-BMPR2 were transfected through liposomes to transfect rat pulmonary AMI endothelial cells, and then the effects on the proliferation and modulation of pulmonary artery endothelial cells were observed by flow cytometry and AnnexinV/PI double staining.
Research results
1, compared with the normal control group, the level of BMPR2 protein expression in the lung tissue of CHD-PAH patients decreased significantly. Through literature review and the prediction of the gene informatics website, we selected the miR-19a, miR-20a, miR-20b, miR-21, miR-130a as the target miRNA in the experimental group and the control group. The experimental results showed m. The results showed m. The expression of iR-19a in the lung tissue of the experimental group increased significantly, while the other miRNA levels showed no significant difference.
2, the model group (left to right shunt) was significantly higher than that in the control group (P < 0.05). The average pulmonary arterial pressure in the 4 weeks group after modeling was significantly higher than that in the normal control group (31.3 + 2.9mmHg vs15.5 + 1.08mmHg, P < 0.001). The average pulmonary arterial pressure in the 8 week group after modeling was significantly higher than that in the normal control group (40.8 + 2.6mmHgvs16.0 + 0.94mmHg, P < 0.001). The pulmonary arterial pressure in the 8 Zhou Zuping group after 4 weeks and after the modeling was significantly increased (31.3 + 2.9mmHg vs40.8 + 2.6mmHg, P < 0.001). Compared with the control group, HE and P-VB staining, the pulmonary artery endothelial cells and the middle layer smooth muscle cells proliferated obviously. The model group was significantly increased in the middle layer collagen fiber (P < 0.05) compared with the control group (P < 0.05). Immunohistochemistry and western-boltting junction were also found in the model group. The results showed that the expression of BMPR2 in the pulmonary artery in the model group was significantly lower than that in the control group (P < 0.05).QRT-PCR results showed that the expression of miR-19a in the lung tissue of the model group was significantly increased (P < 0.05).
3, the miR-19a of SD rat endothelial cells was increased about 150 times at 24 hours after transfection, and the expression of BMPR2 protein in the transfected miR-19a group was significantly decreased (P < 0.05). The proliferation degree of the transfected miR-19a+pcDNA-BMPR2 group was between the transfected miR-19a group and the blank control group, and the proliferation degree of the transfected miR-19a group was significantly higher than that of the empty cells. In the white control group (P < 0.05), the apoptotic rate in the transfected miR-19a group was significantly lower than that in the other two groups (P < 0.05).
research conclusion
1, the expression level of miR-19a in lung tissue of CHD-PAH patients increased significantly, while the expression level of BMPR2 decreased significantly.
2, hemodynamic changes in left to right shunt can lead to a significant increase in miR-19a expression and a significant decrease in BMPR2 expression in rat lung tissue.
3, BMPR2 is the target protein of miR-19a. Overexpression of miR-19a leads to a significant decrease in the expression of BMPR2, which leads to excessive proliferation of pulmonary artery endothelial cells.
In conclusion, miR-19a can promote the proliferation of pulmonary artery endothelial cells by downregulating BMPR2, and play an important regulatory role in CHD-PAH vascular remodeling.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R725.4

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相關(guān)期刊論文 前2條

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2 陳偉丹;聶宇;李守軍;;循環(huán)miR-19a作為先天性心臟病相關(guān)肺動(dòng)脈高壓的標(biāo)志物[J];中國(guó)分子心臟病學(xué)雜志;2010年05期

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