先心病室间隔缺损合并不同程度肺高压患儿外周血单核细胞中转录因子T-bet和GATA-3mRNA的表达

发布时间：2018-05-17 01:07

  本文选题：先心病室间隔缺损合并肺高压 + T-bet　； 参考：《河北医科大学》2012年硕士论文

【摘要】：目的：先心病室间隔缺损(ventricular septal defect, VSD)简称室缺，是最常见的先心病之一，约占23～30%。患儿出生后由于肺循环打开，使肺动脉压力骤减，右室压力也随之减小，使左右室压差显著。当室间隔出现缺损时，则左室血液将有一部分经缺损流入右心室，长期左向右分流，使肺小动脉中层增生，内膜增厚，肺血管阻力进一步增加，形成肺动脉高压，如果没有及时治疗，肺小动脉病变进行性加剧，形成不可逆性肺血管阻塞性病变，肺血管阻力甚至超过体循环的阻力，导致右向左分流，患儿出现紫绀，即艾森曼格综合症。 肺动脉高压(pulmonary arterial hypertension, PAH)是先心病室间隔缺损(VSD)最常见的并发症。另外，VSD患儿在围手术期可出现PAH危象，是围手术期死亡的重要原因之一。国内对于低月龄、低体重的VSD合并PAH患儿的外科治疗，与国际先进水平尚有相当大的差距，是当今我国小儿心外科的主要挑战之一。 有研究报道先心病患儿存在免疫功能低下和免疫功能紊乱，即存在Thl→Th2漂移，并且肺充血型先心病较肺缺血型先心病患儿免疫功能紊乱更为明显。Th1、Th2及其相互之间的平衡在免疫应答中起着关键作用。在T细胞亚群分化过程中，转录因子具有重要的调节作用。转录因子T-bet和GATA-3分别特异性地表达于Th1和Th2，是决定Th0细胞向Thl细胞或Th2细胞分化的两个关键且特异性转录因子。 实时荧光定量PCR是一种基于传统PCR检测方法上的新技术，它有效地解决了传统定量只能终点检测的局限，具有高特异性和可靠性，实验结果稳定，重复性好等优点。 本实验拟通过检测小年龄、低体重室间隔缺损合并不同程度肺高压患儿围手术期外周血单核细胞(Peripheral blood mononuclear cells, PBMCs)中转录因子T-bet mRNA和GATA-3mRNA的表达水平，从调控转录水平探讨Th1和Th2细胞在先天性室间隔缺损合并不同程度肺高压围术期免疫功能紊乱中的作用及意义。 方法：2011年3月至2011年12月在本院心脏外科住院接受先心病手术治疗并符合以下条件的VSD合并不同程度PAH患者，入选标准：①所有病例均经体检、辅助检查及彩色心脏超声检查确诊为先天性心脏病室间隔缺损患者，年龄1岁以内，体重＜10Kg；②严格按照纳入标准选VSD合并轻度PAH (肺动脉压/主动脉压在0.3～0.45)、中度PAH (0.45～0.75)、重度PAH (＞0.75)以及未合并PAH者各10例，所有入选患儿均在体外循环开始前直接测压，确定肺高压程度；③所有患儿其他脏器无严重疾病史，无其它缺氧相关疾病史，无严重感染和自身免疫性疾病史；④排除原发性肺动脉高压；⑤本实验经河北省儿童医院伦理委员会批准，患儿家属签署知情同意书。 研究对象抽取外周静脉血，采用SYBR GreenⅠ实时荧光定量RT-PCR技术检测PBMCs中转录因子T-bet mRNA和GATA-3mRNA的表达水平。所有数据应用SPSS13.0统计软件分析，，多组间比较用单因素方差分析。P0.05有统计学意义。 结果： 1先心病室间隔缺损合并不同程度PAH患儿(共4组)之间的统计比较结果如下： 1.1T-bet基因的统计结果：无PAH组、轻度PAH组、中度PAH组、重度PAH组ΔCt值分别为5.74±2.15、6.93±1.45、7.51±1.96、7.43±1.31。先天性室间隔缺损合并中度PAH组T-bet基因的表达较未合并PAH组低(P=0.024＜0.05)；先天性室间隔缺损合并重度PAH组T-bet基因的表达较未合并PAH组低(P=0.033＜0.05)；其余各组之间无差别(P＞0.05)。 1.2GATA-3基因的统计结果：无PAH组、轻度PAH组、中度PAH组、重度PAH组ΔCt值分别为3.79±2.10、5.43±1.29、5.36±1.36、5.35±1.20。先天性室间隔缺损合并轻度PAH组GATA-3基因的表达较未合并PAH组低(P=0.025＜0.05)；先天性室间隔缺损合并中度PAH组GATA-3基因的表达较未合并PAH组低(P=0.018＜0.05)；先天性室间隔缺损合并重度PAH组GATA-3基因的表达较未合并PAH组低(P=0.020＜0.05)；其余各组之间无差别(P＞0.05)。 2先心病室间隔缺损在围手术期(术前、术后1d、术后3d、术后7d)，各组之间的统计比较结果如下： 2.1T-bet基因的统计结果：先心病室间隔缺损在围手术期术前、术后1d、术后3d、术后7dΔCt值分别为5.97±1.49、6.74±2.29、7.30±1.22、7.17±1.27。术前患者T-bet基因的表达较术后3d患者低(P=0.045＜0.05)；术前患者T-bet基因的表达较术后7d患者低(P=0.002＜0.05)；术后1d患者T-bet基因的表达较术后7d患者低(P=0.038＜0.05)；其余各组之间无差别(P＞0.05)。 2.2GATA-3基因的统计结果：各组之间均无统计学差别（P值均＞0.05）。 结论：本研究从转录水平上提示先天性室间隔缺损合并不同程度PAH患儿的在围手术期存在免疫功能紊乱。先天性室间隔缺损合并中、重度PAH组T-bet基因的表达较未合并PAH组低，即先天性室间隔缺损合并中、重度PAH组患儿Th1细胞数量减少的可能性大，可能存在细胞免疫下降；先天性室间隔缺损合并轻、中、重度PAH组GATA-3基因的表达较未合并PAH组低，即先天性室间隔缺损合并轻、中、重度PAH组患儿存在Th2细胞数量减少的可能性大，可能存在体液免疫也降低；先心病室间隔缺损在围手术期(术前、术后1d、术后3d、术后7d)，各组之间的统计比较结果提示患儿在术后第1d、3d、7d时T-bet基因的表达均降低，即术后均存在Th1细胞数量减少，细胞免疫下降，第3d最低，7d后开始恢复，但并未达到正常水平；术前及术后体液免疫未见明显变化。
[Abstract]:Objective: ventricular septal defect (VSD) is one of the most common congenital heart disease, which accounts for about 23 to 30%. of children after the birth of the pulmonary circulation, the pulmonary artery pressure is reduced, the right ventricular pressure decreases, and the left and right ventricular pressure is significant. When the ventricular septal defect occurs, there will be a part of the left ventricle blood. Through the defect into the right ventricle, the long-term left to right shunt, the middle layer of pulmonary artery hyperplasia, the thickening of the intima, the pulmonary vascular resistance to further increase, the formation of pulmonary hypertension, if not timely treatment, the pulmonary arteriopathy is aggravated, the formation of irreversible pulmonary vascular obstruction venereal disease, pulmonary vascular resistance even more than body circulation resistance, leading to right Left to shunt, children with cyanosis, that is, Eisen Mange's syndrome.
Pulmonary arterial hypertension (PAH) is the most common complication of ventricular septal defect (VSD). In addition, the PAH crisis can occur in children in the perioperative period of VSD, which is one of the important causes of perioperative death. The surgical treatment of low month, low weight VSD with PAH in children and the international advanced level of Shang Youxiang The big gap is one of the main challenges of pediatric cardiac surgery in China.
Some studies have reported that children with congenital heart disease have low immune function and immune function disorder, that is, there is Thl to Th2 drift, and the immune function disorder of the pulmonary blood type congenital heart disease is more obvious than that of the pulmonary ischemic congenital heart disease in children with.Th1. Th2 and the balance between them play a key role in the immune response. In the process of the differentiation of T cell subsets, the transcription is transcribed. Factor T-bet and GATA-3 are specifically expressed in Th1 and Th2, respectively, which are two key and specific transcription factors that determine the differentiation of Th0 cells into Thl or Th2 cells.
Real time fluorescence quantitative PCR is a new technology based on traditional PCR detection method. It effectively solves the limitation of traditional quantitative detection only. It has the advantages of high specificity and reliability, stable experimental results and good repeatability.
This experiment is to detect the expression of T-bet mRNA and GATA-3mRNA in peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) in children with small age and low weight ventricular septal defect combined with different degrees of pulmonary hypertension, and to discuss the combination of Th1 and Th2 cells in congenital ventricular septal defect with the regulation of transcription. The role and significance of perioperative pulmonary function disorder in the same degree of pulmonary hypertension.
Methods: from March 2011 to December 2011, the patients receiving congenital heart disease in our hospital heart surgery were treated with VSD and the following conditions were combined with different degrees of PAH. The criteria were selected: (1) all cases were diagnosed as congenital heart disease ventricular septal defect by physical examination, auxiliary examination and color echocardiography, within 1 years of age and body weight. < 10Kg; (2) strictly according to the inclusion criteria, VSD combined with mild PAH (pulmonary arterial pressure / aortic pressure from 0.3 to 0.45), moderate PAH (0.45 ~ 0.75), severe PAH (> 0.75) and 10 cases without PAH, all the selected children were directly measured before cardiopulmonary bypass to determine the degree of pulmonary hypertension; third, there were no serious diseases in other organs of all children. History, no other history of hypoxia related diseases, no serious infection and the history of autoimmune diseases; 4. Elimination of primary pulmonary hypertension; 5. This experiment was approved by the ethics committee of Hebei children's Hospital, and the families of the children signed the informed consent.
The study subjects extracted peripheral venous blood and detected the expression level of T-bet mRNA and GATA-3mRNA in PBMCs by SYBR Green I real-time fluorescence quantitative RT-PCR. All data were analyzed with SPSS13.0 statistical software, and the single factor variance analysis of.P0.05 was statistically significant in multiple groups.
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