【作者】 吴益和；

【导师】 胡盛寿；

【作者基本信息】 北京协和医学院 ， 外科学， 2012， 博士

【摘要】 背景：完全性先天性大动脉转位(Transposition of great arteries, TGA)患儿左心室与肺动脉相连,随着出生后肺血流阻力的下降,左心室压力后负荷也逐渐下降。长期的左室后负荷低压状态,将导致左室心肌发生功能性退化。在肺动脉放置环扎带的左室训练术通过增加左室压力后负荷,使左室在较高的压力负荷刺激后再度发育,心肌细胞出现肥大反应,这也为我们研究压力负荷下未成熟心肌肥厚、发育的分子机制提供了独特而宝贵的人类疾病模型。识别并鉴定TGA患儿左室训练术后的血浆中出现的特异性生物标记物,一方面可以为研究人未成熟心肌的肥厚、发育的分子机制提供新的线索；另一方面,将这些生物标志物作为新的潜在的分子靶点,为开发新的心肌肥厚干预药物带来新的希望。方法：用差异凝胶电泳(Differential gel electrophoresis, DIGE)蛋白质组学技术筛选并鉴定TGA病人行左室训练术后48hrs血浆中的差异表达蛋白。然后应用ELISA方法检测差异表达蛋白血浆铜蓝蛋白(Ceruloplasmin, CP),α1-抗胰蛋白酶(Alpha-1-antitrypsin, SERPINA1),转铁蛋白(Transferrin, TF),和小清蛋白(Parvalbumin, PVALB)在TGA病人左室训练术前、术后血浆,非体外循环心脏手术病人术前、术后血浆,以及非先心病手术病人术前血浆中的表达情况。进一步用TF和促肥厚因子内皮素-1(Endothelin-1,ET-1)处理体外培养乳鼠心肌细胞,观察心肌细胞形态大小变化,心肌肥厚标志物利钠肽前体A (Natriuretic peptide precursor A, Nppa).利钠肽前体B (Natriuretic peptide precursor B, Nppb) mRNA的表达变化,以及PVALB的表达变化。结果：采用DIGE蛋白质组学技术,成功鉴定出TGA病人行左室训练术后血浆差异表达蛋白25种,其中19种蛋白与心脏发育、铁离子稳态、蛋白质生物合成与折叠、物质代谢、信号转导等方面相关;ELISA分析结果显示：CP、TF和PVALB3种差异蛋白在TGA病人行左室训练术后48hrs血浆中的表达水平较其术前水平显著升高,而在非体外循环心脏手术病人中则无类似的显著改变。TF和ET-1处理乳鼠心肌细胞48hrs后,细胞面积显著增大,肥厚标志物Nppa. Nppb mRNA表达显著增强,PVALB mRNA表达水平、PVALB蛋白阳性百分率显著升高；去铁转铁蛋白(apotransferrin, apo-TF)和铁饱和转铁蛋白(holotransferrin, holo-TF)均能诱导心肌细胞肥大和增强肥厚标志物Nppa, Nppb mRNA的表达。结论：铁离子稳态相关蛋白CP、TF和心脏发育相关蛋白PVALB是TGA患儿左室训练术后的血浆特异性蛋白生物标志物,是与人未成熟心肌肥厚、发育相关的蛋白生物标志物；TF诱导乳鼠心肌细胞肥大的体外实验研究证实TF是有效的未成熟心肌细胞促肥厚因子,且同剂量的apo-TF和holo-TF的促肥厚效应类似；体外细胞水平研究证实PVALB是未成熟心肌细胞肥大的生物标志物。背景：先天性心脏病(Congenital heart disease, CHD)是主要的出生缺陷之一。缺氧和肥厚能够诱导Ca2+调节蛋白的表达改变,抑制心肌收缩力,是CHD主要病理生理改变。Ca2+调节蛋白调节细胞内游离Ca2+浓度,维持细胞内Ca2+稳态,因此在兴奋-收缩偶联过程中发挥核心作用,其表达变化可以影响心肌细胞的收缩能力。当前较少有关于人右心室在肥厚和／或缺氧条件下Ca2+调节蛋白变化特点的研究。我们期望通过研究缺氧和肥厚对人右心室Ca2+调节蛋白表达的影响,加深我们对CHD患儿右心室肥厚和缺氧的分子机制的认识,进而为此类疾病的治疗提供新的思路。方法：25例不伴缺氧的肺动脉瓣狭窄患儿作为单纯肥厚组(Hypertrophy group, H group),25例法乐四联症患儿作为缺氧肥厚组(Hypoxia and hypertrophy group, HH group),25例不伴缺氧和肥厚的限制性小室间隔缺损患儿作为对照组(Control group, C group),在行手术矫治时收集右室流出道心肌组织标本。心肌组织的石蜡切片用细胞膜绿色荧光探针DiO进行染色后用于心肌细胞形态大小比较。用Real-time PCR、Western Blot或免疫荧光技术检测Ca2+调节蛋白肌浆网Ca2+-ATPase (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a)、兰尼碱受体2(Ryanodine receptor2,RyR2)、钠钙交换体(Sodiumcalcium exchanger, NCX)、肌脂蛋白(Sarcolipin,SLN)、受磷蛋白(Phospholamban, PLN)的表达水平。另外,用Western Blot检测RyR和PLN的磷酸化水平以及检测蛋白磷酸酶1(protein phosphatase1, PP1)的表达水平。结果：通过细胞形态大小比较,证实了肥厚组(+31%,P<0.001vs对照组)和缺氧肥厚组(+22%,P<0.001vs对照组)病人右室心肌细胞存在轻微的肥大改变。与对照组相比,缺氧肥厚组SERCA2a mRNA水平显著降低(P<0.01),单纯肥厚组SERCA2a mRNA水平也有下降趋势,但无统计学意义。与对照组相比,无论是单纯肥厚组还是缺氧肥厚组,右心室心肌细胞RyR2、NCX、SLN和PLN的mRNA和蛋白表达均无变化。与对照组相比,缺氧肥厚组的PLN-Ser16表达下调(P<0.01),PP1表达增强(P<0.01)。结论：SERCA2a mRNA表达水平的下调是同时存在缺氧和右室肥厚的先天性心脏病早期阶段病理过程的生物标志物；缺氧和肥厚的共同作用诱导了PLN-Ser16脱磷酸化这一不利效应,对同时存在缺氧和右室肥厚的先天性心脏病患儿进行早期的外科手术矫治,可能可以加速PLN-Ser16磷酸化水平的恢复,进而改善心肌收缩能力。增强的PP1表达使PLN-Ser16水平下降,抑制PP1的表达是儿科病人心功能不全的潜在的治疗靶点。更多还原

【Abstract】 Background:The left ventricle of complete congenital transposition of great arteries (TGA) children was connected with pulmonary artery. With the decline in pulmonary vascular resistance after birth, the left ventricular pressure load decreased gradually. The long-term low left ventricular pressure load will lead the functional degradation of left ventricle. Increased left ventricular pressure load induced by left ventricle training with pulmonary artery banding surgery would stimulate left ventricular development and cardiomyocyte hypertrophy, which provides an unique and valuable human disease model to study pressure overload hypertrophy and development of immature myocardium. Identification of the plasma biomarkers involved in transposition of great arteries children undergoing left ventricular training surgery will provide new clues for studying the molecular mechanisms of hypertrophy and development of immature myocardium. Additionally, using these biomarkers as a new potential molecular target can bring hope for developing new myocardial hypertrophy intervention drugs.Methods:Differential gel electrophoresis (DIGE) proteomics was used to screen and identify the differential proteins from the plasma of the anterior and posterior left ventricular training surgery in TGA children. Then, enzyme linked immunosorbent assay (ELISA) was used to detect the expression level of the differential proteins including ceruloplasmin (CP), alpha-1-antitrypsin (SERPINA1), transferrin (TF), and parvalbumin (PVALB) in the preoperative and postoperative plasma of TGA children undergoing left ventricular training surgery, the preoperative and postoperative plasma of children undergoing off-pump cardiac surgery, and the preoperative plasma of children undergoing non-congenital heart disease surgery. Furthermore, the in vitro cultured neonatal rat cardiomyocytes was treated with TF or Endothelin-1(ET-1) to observe changes in the size of cells, in the mRNA expression of myocardial hypertrophy biomarkers including natriuretic peptide precursor A (Nppa) and natriuretic peptide precursor B (Nppb), as well as in the PVALB expression.Results:By using DIGE proteomics,25differential proteins were successfully identified in postoperative plasma of TGA children undergoing left ventricular training surgery.19of them were involved in heart development, iron ion homeostasis, protein biosynthesis and protein folding, metabolism, signal transduction and others. ELISA analysis showed that compared with the levels in their preoperative plasma,3differential proteins including CP, TF and PVALB levels rose significantly in postoperative plasma of TGA children undergoing left ventricular training surgery, while there was no significant change in children undergoing off-pump cardiac surgery. The in vitro study showed that after48hours’incubation with TF or ET-1, the size of cardiomyocytes increased significantly, the expression of hypertrophic biomarkers including Nppa and Nppb was significantly enhanced, as well as the PVALB mRNA expression level and PVALB positive protein percentage rose significantly. Both the apotransferrin (apo-TF) and holotransferrin (holo-TF) could induce cardiomyocyte hypertrophy and enhanced expression of Nppa and Nppb mRNA.Conclusions:Iron homeostasis related proteins CP and TF and heart development-associated protein PVALB were plasma biomarkers of posterior left ventricular training surgery in TGA children, so were the biomarkers associated with hypertrophy and development of human immature myocardium. The in vitro study confirmed that TF was a factor which can induce immature cardiomyocyte hypertrophy effectively, and the same dose of apo-TF or holo-TF has similar effect on cardiomyocyte hypertrophy. The in vitro study also confirmed that PVALB was a biomarker of the immature cardiomyocyte hypertrophy. Background:Congenital heart disease (CHD) is a major birth defect around the world. Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Ca2+-regulatory proteins regulate intracellular free Ca2+concentrations and maintain intracellular Ca2+homeostasis so they are very important for excitation-contraction coupling and for myocyte contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Research about the alteration of Ca2+-regulatory proteins in right ventricular hypertrophy (RVH) with or without hypoxia will help to understand the cellular and molecular bases of RVH and hypoxia in the populations of children with CHD.Methods:Right ventricle tissues were collected from children with pulmonary stenosis [n=25, hypertrophy group (H group)], tetralogy of Fallot [n=25, hypoxia and hypertrophy group (HH group)], or small isolated ventricular septal defect [n=25, control group (C group)] during open-heart surgery. Paraffin sections of tissues were stained with3,3’-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), ryanodine receptor2(RyR2), sodiumcalcium exchanger (NCX), sarcolipin (SLN) and phospholamban (PLN)] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase1(PP1) were evaluated using western blot.Results:Mild cardiomyocyte hypertrophy of the right ventricle in H (+31%, P<0.001vs C group) and HH groups (+22%, P<0.001vs C group) was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P<0.01) was observed in the HH group compared with the C group, and a similar trend of decrease was detected in the H group, but there was not a statistically significant difference. No change in the mRNA or protein expression of RyR2, NCX, SLN and PLN in H group and HH group when compared with the C group. The level of Ser16-phosphorylated PLN was down-regulated (P<0.01) and PP1was increased (P<0.01) in the HH group compared to that in the C group.Conclusions:The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser’6dephosphorylation, and early surgical repair might accelerate the recovery of the phosphorylated state of PLN and thereby contribute to improved cardiac contractility in cyanotic CHD with right ventricular hypertrophy. Increased PP1could result in the decreased PLN-Ser16and inhibition of PP1is a potential therapeutic target for heart dysfunction in pediatrics.更多还原