紫绀型先天性心脏病体外循环致急性肺损伤中miR-204的作用机制研究

Infants with cyanotic congenital heart disease are more likely suffered from acute lung injury(ALI) than non-cyanotic congenital heart disease infants after cardiopulmonary bypass(CPB), which results in ICU staying and hospital mortality increased, however, lacking of basic experiments related to such lung injury and randomized controlled clinical study. Our previous study found that miR-204 expression of non-cyanotic piglet lung tissue declined after ALI induced by CPB, further research also found that predicted target gene cyclooxygenase-2 (Cox-2) mRNA and its protein expression increased, which can aggravate damaged parts with synthesis and accumulation of inflammatory PGs which would worsen lung damage. Whether the existence of the change can be found in cyanotic congenital heart disease ALI after CPB? This project intends to prepare cyanotic infant rat model by using the hypoxic animals incubator, and induce ALI model by perform CPB surgery, and mimic ischemia-reperfusion model by using pulmonary vascular endothelial cells to study regulation of Cox-2 expression by miR-204 and the protective role in vivo and vitro. By building reports vectors, cell transfection, applications such as real-time PCR technology to validate Cox-2 is a target gene of miR-204 . Due to the current lack of effective treatment, exploring the regulatory mechanism of miR-204 in-depth understanding the pathogenesis and pathological processes of ALI induced by CPB in cyanotic congenital heart disease infants will make attributes to the development of drugs and treatment, thereby provide theoretical and experimental evidence to reducing the symptom of ALI, improving the patient’s outcomes.

紫绀型先心病较非紫绀型先心病体外循环（CPB）后更易发生急性肺损伤（ALI），致ICU停留时间及住院病死率显著升高，目前尚无相关实验及临床研究。我们前期研究发现，非紫绀乳猪CPB后ALI后肺组织中miR-204表达降低，预测靶基因Cox-2表达升高，后者可加重受损部位炎性PGs等的合成和聚积。紫绀型先心病CPB后ALI是否也存在相应变化？本实验拟采用低氧动物培养箱制备紫绀型幼年大鼠模型后再行CPB术致ALI，同时构建肺血管内皮细胞缺血再灌注模型，体内及离体研究ALI时miR-204的作用及对Cox-2表达调节；制备病毒载体，应用细胞转染、实时PCR等技术验证Cox-2是否为miR-204的靶基因。由于目前缺乏有效治疗手段，探索miR-204的调控机制为深入了解紫绀型先心病CPB后ALI发病机制和病理过程，减轻ALI、改善紫绀先心病预后提供理论和实验依据。

右室功能保护是目前国际研究热点，但在紫绀型先心病中相关研究还很少。法洛四联症是最常见的紫绀型先心病，其根治术后可能发生肺动脉瓣反流，使右室容量负荷增加，对右心室功能产生慢性持续性的损害，诱导右心室发生心室重构。外科手术进行肺动脉瓣置换可以有效终止肺动脉瓣反流，但即使进行换瓣手术后，很多患者的右室功能无法恢复正常。导致这一情况的机制尚不清楚，该领域研究几乎空白。本研究通过利用该疾病的临床标本，比较肺动脉瓣置换术后右室功能转归不同患者的右室心肌在蛋白水平的差异，初步发现肌动蛋白解聚因子（DSTN）可能是在右室重构中起重要作用的蛋白，其表达异常调控的F-actin积聚导致心肌收缩-舒张功能障碍，是右室功能障碍的重要原因。但DSTN在心肌细胞发生作用的主要通路机制还尚不明确，因此本研究建立新型的低氧肺动脉瓣反流SD大鼠模型，旨在对其通路进行探索和验证。希望本研究初步探索的结果，可以为该疾病的研究提供新的思路，为未来临床上确定该类患者的手术时机或延缓右室功能损害提供帮助。