【作者】 钟久昌；

【导师】 黄东阳；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 高血压病（essential hypertension,EH）被普遍认为是一种环境和遗传因素共同作用的多基因疾病。肾素血管紧张素系统（RAS）是机体内调控血压稳定的最重要机制之一,其中血管紧张素转换酶（ACE）及其新近发现的同源酶ACE2起关键性作用。ACE2可直接对抗ACE,它不仅能竞争性地作用于ACE的底物血管紧张素Ⅰ（angiotensinⅠ,AngⅠ）,还能清除ACE的催化产物Ang Ⅱ,使之代谢生成舒血管物质Ang-（1-7）。由于ACE2、apelin以及APJ三个新成员的加入,RAS体系较既往变得更为复杂。新的RAS体系ACE2/ACE参与血压调控可能依赖于两条路径:其中一条为ACE与AngⅡ,起升压效应;另一条为ACE2与Ang-（1-7）,通过对抗前一路径,引起血压下降。另外,RAS新体系中还可能存在一条独立于ACE2/Ang-（1-7）之外的降压路径,被命名为apelinAPJ信号系统。apelin-APJ系统可能通过直接拮抗AngⅡ-AT₁信号途径以及促进一氧化氮（NO）生成与释放来实现其降压效应。APJ是最早被克隆的一种与AT₁相关的孤儿受体。尽管APJ“孤儿”了多年,最近已分离出其内源性配体并命名为apelin,后者与AngⅡ高度同源。ACE2,apelin和APJ基因在维持心、肾功能以及血压稳定中起重要作用,其表达异常可能促成EH的发生、发展。 血压分子镶嵌理论是长期以来较为认可的EH致病机制模型,认为EH的致病涉及多个环节,各环节彼此相互作用形成一个血压调控网络,而基因是该网络的中心。与作用于表面的传统降压药物相比,转录调节剂通过在基因水平（镶嵌模型的中心）直接调整高血压相关基因的转录、表达失控,可以从根本上达到EH防治目的。近年来有关转录因子及核激素受体的研究进展给转录调节降压剂提供了新的视点,维生素A的活性代谢产物全反式维甲酸（atRA）即是其中的一种。己证实atRA通过核内维甲酸受体（RAR）和维甲类X受体（RXR）的激活而起作用。AtRA可激活异二聚体RAR/RXR,后者通过识别和结合维甲酸受体反应元件（RARE）,进而引起靶基因的转录激活或转录抑制。研究表明atRA能够调节RAS体系多种成分的基因表达,如肾素、ACE、AngⅡ、AT₁等。鉴于ACE2、apelin、APJ分别为ACE、AngⅡ和AT₁的同源类似物,无论在基因序列还是组织表达分更多还原

【Abstract】 Essential hypertension is universally acknowledged as a multifactorial quantitative trait controlled by both genetic and environmental factors. The renin-angiotensin system （RAS） is currently considered a central regulator of blood pressure, in which angiotensin-converting enzyme （ACE） and its newly identified homologue ACE2 play a key role. ACE2 might counteract ACE by competing with ACE for the substrate angiotensin I （Ang I） or by directly metabolizing the Ang II to generate a vasodilator Ang-（l-7）. The RAS has been shown to be a far more complex system than initially thought with the discoveries of ACE2, apelin and APJ. The novel RAS participates in the regulation of blood pressure depending likely on two pathways: ACE/Ang II, evoking pressor effect; and ACE2/Ang-（l-7）, inducing depressor effect by directly antagonizing the former. In addition, there is another hypotensive pathway independent of ACE2/Ang-（l-7） termed apelin-APJ signaling pathway, which modulates the blood pressure through dampening Ang II-induced AT₁ signaling and potentiating the synthesis and release of nitric oxide （NO）. The APJ is among the earliest cloned orphan receptor. Although "orphan" for many years, its natural ligand was recently isolated and named apelin, a homologue of Ang II. ACE2, apelin and APJ emerges as important regulators of cardiorenal function and blood pressure homeostasis, in which abnormalities may be critical contributors to the initiation and maintenance of hypertension.The Molecular Mosaic Theory is generally accepted as the pathogenesis of hypertension, in which multiple regulatory systems controlled by multiple genetic factors interact to promote essential hypertension. In contrast to conventional antihypertensive agents that treat surface facets of the mosaic, transcription-modulating drugs can be used to modify gene expression and attack essential hypertension at the core of the mosaic. Research advances on the key transcription factors and nuclear hormone receptors provide fresh insight into thedevelopment of transcription-modulating antihypertensive drugs, among which is all-trans retinoic acid （atRA）, a biologically active metabolite of vitamin A. atRA exerts its effects by binding the retinoic acid receptor （RAR） and retinoid X receptor （RXR）. Initially, atRA activates a heterodimer RAR/RXR, which further recognizes and binds to retinoic acid receptor response element （RARE） consensus sequence, thereby activating or repressing target gene transcription. Experimental documents have demonstrated that atRA influences the gene expression of components of the RAS including renin, ACE, Ang II and ATi receptor. In view of the sequence and distribution similarities between ACE2 and ACE （as well as apelin and Ang II; APJ and ATi）, we predicted that atRA might affect gene expression and actions of ACE2, apelin and APJ, and therefore elicit some beneficial effects in hypertension. In the present work, we evaluate above-mentioned possibilities of atRA by examining changes of mRNA and protein of ACE2, apelin and APJ receptor and levels of serum NO in spontaneously hypertensive rats （SHR） after chronic atRA treatment.Methods: （1） Animals: SHR and WKY rats were randomly assigned to five treatment groups: WKY-C （WKY control）, WKY-R （WKY treated with atRA）, SHR-C （SHR control）, SHR-L and SHR-H （SHR treated with low-dose or high-dose atRA）. Rats received daily intraperitoneal injection of atRA （10²0 mg ? kg’1 ? day"1 ） or vehicle for a month. Systolic blood pressure （SBP） of rats was measured by the tail-cuff method. （2） Isolation of RNA: Total RNA was isolated from heart, aorta and kidney tissues using Trizol Reagent. cDNA were synthesized by standard techniques. （3） Real-time Quantitative PCR: Primers and TaqMan probes were designed and synthesized. The positive standards of rat ACE2, apelin, APJ and ATi were successfully cloned through plasmid recombination and DNA sequencing, then used as standard templates to construct the calibration curves for sample array. Rat GAPDH mRNA was measured as an internal control. （4） Northern Blotting: A 32P-labeled rat ACE2 cDNA probe was prepared with the Random Primes DNA Labeling System. Rat (3-actin probe was used as an internal control. 30 ug of RNA was resolved on a 1.2% FA gel and electrophoresed, then blotted to nylon membrane and fixed by the ultraviolet cross-linking. Blots were prehybridized and hybridized with the probes. With washes and exposure, the blots were then detected and analyzed in a Scanner. （5） Western Blotting: Protein samples were loaded onto precast denaturing gels, followed by separation andtransblotting to nitrocellulose filter. After blocking, the primary and AP-labeled secondary antibody incubation, the positive bands representing target protein were developed and quantified with an Automated Imaging System. Rat P-actin protein was used as an internal control. （6） Measurement of Serum NO Levels: Serum NO concentrations were determined at 550 nm with the Greiss reagent after reduction with nitrate reductase. The positive and negative controls were established in the meantime. （7） Transmission Electron Microscope （TEM） Analysis: Samples of rat hearts were prefixed by 2.5% Glutaral and washed, followed by postfixation and stainning, and dehydration in a graded series of ethanol. After infiltration and polymerization, ultrathin sections were cut, and observed by TEM.Results: （1） A marked rise of SBP was shown in SHR compared with WKY rats （PO.01）. SBP did not differ among SHR groups before and 1, 2 weeks after treatment. However, a significant reduction of SBP was shown in SHR-L and SHR-H 3, 4 weeks after atRA treatment （PO.05, respectively）. （2） In placebo-treated SHR, a significant reduction of ACE2, APJ and apelin mRNA expression and an obvious rise of ATj mRNA expression were shown in heart, aorta and kidney, compared with WKY rats （PO.01, respectively）. Supplementation with atRA led to upregulated mRNA expression of ACE2, APJ and apelin and downregulated mRNA expression of ATi in atRA-treated SHR （PO.05, respectively）. （3） In placebo-treated SHR, protein expressions of ACE2, APJ and apelin were markedly depressed, whereas ATi protein expression was significantly increased when compared with WKY rats （PO.01, respectively）. By contrast, in atRA-treated SHR, ACE2, APJ and apelin protein expression were significantly enhanced, whereas ATi protein expression was significantly decreased （P<0.05, respectively）. （4） Serum NO was greatly reduced in SHR compared with WKY rats （P<0.01）, but significantly elevated in atRA-treated SHR （PO.05）. Serum NO levels did not differ among WKY rats. （5） Severe myocardial damage of the left ventricle was represented in SHR from TEM compared with WKY rats. However, in atRA-treated SHR, the myocardial damage of left ventricle was obviously attenuated.Conclusion: （1） Compared with conventional PCR, real-time quantitative PCR is a simple, convenient and accurate method, which can combine high sensitivity with reliable specificity and be available in detecting quantitatively and studying the genes relevant to the更多还原