miR-19a下调BMPR2促PAEC增殖在CHD-PAH血管重构中的作用机制研究

发布时间：2018-06-08 02:47

本文选题：先天性心脏病 + 肺动脉高压　；参考：《第二军医大学》2013年博士论文

【摘要】：研究背景先天性心脏病（CHD）是小儿时期最常见的心血管疾病，发病率约占全部活产婴儿的7～8‰。据统计我国每年新出生的先心病患儿高达15万，其中5～10%左向右分流型先天性心脏病患儿因未及时治疗而形成肺动脉高压（PAH）。若不加干预，患儿的肺动脉压力会不断升高，进而形成艾森曼格综合征并最终导致右心衰竭而死亡。目前对PAH的治疗主要包括药物治疗与手术治疗。在药物治疗方面，几种不同机制的抗肺动脉高压治疗药物已应用于临床，并改善了部分患者的生存质量，但由于无法逆转肺血管的重构，最终病人仍将因不断增高的肺动脉压力而导致右心衰竭及死亡。尽管手术治疗可以彻底矫治先天性心内缺损，且大部分在发生右向左分流前接受心内缺损修补手术的患儿可获得满意的预后，但对发生双向分流或以右向左分流为主的患儿，单纯的手术治疗并不能获得满意的疗效，其原因在于该类患者的肺动脉血管已经发生了重构；如果患者的肺动脉血管重构得不到有效的逆转，那么手术治疗反而会促使其死亡。由此可见，无论是药物治疗还是手术治疗，肺动脉血管重构程度都是决定患儿预后的关键因素，因而研究CHD-PAH肺动脉血管重构的机制将有助于为减缓或逆转肺动脉重构的治疗提供新靶点。既往研究表明，BMPR2表达下降与肺动脉重构发生密切相关，文献回顾及基因信息学分析表明miR-19a可能对BMPR2的表达具有调控作用。研究目的本课题希望通过临床调查及相关的动物实验明确miR-19a对BMPR2表达的调控作用及其促肺动脉内皮细胞增殖及肺动脉血管重构的调控机制,进而为CHD-PAH手术治疗提供依据（如相关miRNA的量化可以作为肺动脉血管重构可否逆转的指标）。此外，还可为PAHde药物治疗提供新靶点。研究方法 1、以2011年10月-2012年10月在我院住院治疗的重度肺动脉高压患者肺组织为实验组(n=10),自发性气胸患者术中切除正常肺组织为对照组(n=10),进行免疫组化及western-blotting检测BMPR2表达水平差异；与此同时，在文献回顾及基因信息学分析的基础上，通过qRT-PCR方法检测验证与CHD-PAH发生及BMPR2表达相关的miRNA。 2、建立左向右分流肺动脉高压大树模型，以模型大鼠的肺组织为研究对象进一步验证左向右分流血流动力学改变对miR-19a及BMPR2表达水平的影响。方法如下：40只雄性SD大鼠随机等分为4组：T1、T2、T3、T4； T1、T3为实验组，T2、T4为对照组。实验组腹正中切口开腹，穿刺腹主动脉至下腔静脉建立动静脉瘘，对照组腹正中切口开腹，但不穿刺血管。于术后第4周检测T1、T2组的血流动力学数据，第8周检测T3、T4组的血流动力学数据。并应用免疫组化方法检测BMPR2在肺动脉表达改变；western-blotting及qRT-PCR分别检测相关蛋白及miRNA表达。 3、将miR-19a及pcDNA-BMPR2通过脂质体包裹的方法转染大鼠肺动阿密内皮细胞，，继而通过流式细胞学及AnnexinV/PI双染色法观察其对肺动脉内皮细胞增殖及调往的影响。研究结果 1、与正常对照组相比，CHD-PAH患者肺组织中BMPR2蛋白表达水平显著下降。通过文献回顾及基因信息学网站预测，我们选定了与BMPR2相关性较强的miR-19a、miR-20a、miR-20b、miR-21、miR-130a作为目标miRNA检测其在实验组及对照组中的含量。实验结果显示miR-19a在实验组肺组织中表达量明显升高，其余miRNA表达水平无明显差异。 2、模型组（左向右分流）较对照组肺动脉压力明显升高（P＜0.05）。建模后4周组较正常对照组平均肺动压明显升高(31.3±2.9mmHg vs15.5±1.08mmHg，p＜0.001）；建模后8周组平均肺动脉压较正常对照组明显升高（40.8±2.6mmHgvs16.0±0.94mmHg，p＜0.001）；建模后4周组与建模后8周祖平均肺动脉压明显升高（31.3±2.9mmHg vs40.8±2.6mmHg，p＜0.001）。模型组与对照组HE及P-VB染色比较，肺动脉内皮细胞及中层平滑肌细胞增生明显，模型组较对照组中层胶原纤维增生明显（P＜0.05）。免疫组化及western-boltting结果显示模型组大鼠肺动脉周围BMPR2表达量较对照组明显下降（P＜0.05）。qRT-PCR结果显示模型组大鼠肺组织miR-19a表达明显升高（P＜0.05）。 3、 SD大鼠内皮细胞转染miR-19a后24小时miR-19a增高约150倍；转染miR-19a组western-blotting检测BMPR2蛋白表达量明显降低（P＜0.05）。转染miR-19a+pcDNA-BMPR2组细胞增殖程度介于转染miR-19a组与空白对照组之间，而转染miR-19a组细胞增殖程度明显高于空白对照组（P＜0.05）。转染miR-19a组细胞凋亡率较其余两组明显下降（P＜0.05）。研究结论 1、CHD-PAH患者肺组织中miR-19a表达量显著升高，而BMPR2表达水平明显下降。 2、左向右分流血流动力学改变可导致大鼠肺组织miR-19a表达显著升高，BMPR2表达水平显著下降。 3、BMPR2是miR-19a的靶蛋白，过表达miR-19a可导致BMPR2的表达量显著下降，进而引发肺动脉内皮细胞的过度增殖。综上所述，miR-19a可通过下调BMPR2促肺动脉内皮细胞增殖，进而在CHD-PAH血管重构中发挥着重要的调控作用。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4

【参考文献】