缺氧性肺动脉高压新生大鼠体内热休克蛋白70对HIF-1α的阻断作用研究

Hypoxic pulmonary hypertension (HPH) is one of the intractable diseases in neonates that do harm to their health, which pulmonary vasoconstriction and remodeling is the main change of the disease. Preliminary study in our group found that the increased expression of hypoxia-inducible factor-1α (HIF-1α) activated the expression of two downstream target genes, endothelin-1 (ET-1) and inducible nitric oxide synthase (iNOS), and then caused pulmonary vasoconstriction and remodeling. Currently treatments were limited among these intervention of downstream target genes of HIF-1α, but usage was limited because of its limitations and side effects in the newborn. If the role of HIF-1α could be blocked, then inhibiting the activity of its downstream gene effectively, the process of HPH may be delayed. Studies found that Heat Shock Protein 70 (HSP70) promote the degradation of HIF-1α by the ubiquitin pathway and thus blocking the expression of downstream target genes, but it is still unclear whether HSP70 suppress the occurrence of neonatal HPH development through this pathway. Based on the preliminary studies, this study is aimed to using HSP70 gene transfection in the HPH newborn rat lung tissue to degradate HIF-1α and to investigate whether HSP70 could block the role of HIF-1α to delay the development of HPH in the neonatal rats, and provide new ideas of prevention HPH in newborns.

新生儿缺氧性肺动脉高压（HPH）是危害其健康的难治性疾病之一，肺血管收缩、重塑是该病的主要环节。本课题组前期研究发现，在HPH新生大鼠体内缺氧诱导因子-1α（HIF-1α）表达增加可激活下游靶基因内皮素-1（ET-1）、诱导型一氧化氮合酶（iNOS），使肺血管收缩、重塑，促进该病发生。目前治疗多限于对其下游靶基因干预，但因其局限性及毒副作用等问题在新生儿使用受限。如能直接阻断HIF-1α的作用，可能会抑制其下游基因的活性，从而延缓HPH的进程。研究发现热休克蛋白70（HSP70）可通过泛素化途径促进HIF-1α降解进而阻断下游靶基因表达，但有关HSP70能否通过此途径抑制新生儿HPH的发生发展国内外未见报道。本课题拟在前期研究基础上，通过基因转染技术探讨HSP70在HPH新生大鼠肺组织中能否阻断HIF-1α的作用，从而延缓新生大鼠HPH的发生发展，为防治新生儿HPH提供理论依据。

新生儿缺氧性肺动脉高压（HPH）是危害其健康的难治性疾病之一，肺血管收缩、重塑是该病的主要环节。本课题组前期研究发现，在HPH新生大鼠体内缺氧诱导因子-1α（HIF-1α）表达增加可激活下游靶基因内皮素-1（ET-1）、诱导型一氧化氮合酶（iNOS），使肺血管收缩、重塑，促进该病发生。目前治疗多限于对其下游靶基因干预，但在新生儿使用受限。如能直接阻断HIF-1α的作用，可能会抑制其下游基因的活性，从而延缓HPH的进程。研究发现热休克蛋白70（HSP70）可通过泛素化途径促进HIF-1α降解进而阻断下游靶基因表达，但相关研究在新生儿HPH未见报道。本研究在前期建立新生大鼠HPH模型基础上，成功构建携带外源性基因HSP70 的表达载体pHBAd-MCMV-GFP-HSP70，与腺病毒骨架质粒pHBAd-BHG 共同转染进入人胚肾HEK293 细胞中，包装出重组腺病毒Ad-HSP7（含有增强型绿色荧光信号标记蛋白），经酶切电泳和基因测序证实扩增产物与Genebank 中的HSP70 基因序列相同，将腺病毒介导的HSP70通过尾静脉注射转染到HPH新生儿大鼠肺组织，在荧光显微镜下观察到转染肺组织中绿色荧光信号标记蛋白，用病理、免疫组化、RT-PCR、Western blot方法研究不同实验组HPH新生大鼠肺动脉压力的变化、HPH新生大鼠肺血管重塑指标的变化，发现HPH新生大鼠肺组织HSP70高效表达后，下调HIF-1α的表达，进而阻止其下游靶基因ET-1、iNOS的表达，降低肺动脉压力，减轻肺血管重塑，延缓或减轻了新生大鼠HPH的发生发展，为HSP70从基因水平治疗新生儿HPH奠定了一定的理论基础。