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1、上海交通大学硕士学位论文原发性醛固酮增多症时醛固酮合成与调节相关基因的变化姓名：田秀丽申请学位级别：硕士专业：内分泌与代谢病学指导教师：汤正义20090401上海交通大学医学院硕士毕业论文 原发性醛固酮增多症时醛固酮合成与调节相关基因的变化 摘 要 【目的及意义】原发性醛固酮增多症中导致醛固酮高分泌的机制仍不明确；本研究利用基因表达谱芯片检测原醛症肾上腺组织中全基因组基因的表达变化， 结合醛固酮合成路径相关酶与调节因子基因的表达改变，探讨原醛症中醛固酮高分泌的可能机制。 【研究方法】1.所取 10 例醛固酮瘤、7 例结节性肾上腺增生及 7 例正常肾上腺组织，均经临床表现和术后病理证实，采用 H
2、umanRef-6 v2 芯片，分别检测比较三组标本全基因组基因的 mRNA 表达水平，并用real-time RT-PCR 技术对醛固酮合成路径相关酶与调节因子基因的表达进行验证。2.采用 Matlab 和 Graphviz 软件对所得数据进行生物信息学分析，包括差异基因的筛选，基因功能的显著性分析，信号传导通路的显著性分析，构建基因的功能关联网络。 【研究结果】1.醛固酮合成路径的五个酶中，醛固酮瘤组和肾上腺结节性增生组 CYP11B2 的表达分别为正常组的 70.29 倍和 38.57 倍，CYP11A1、HSD3B2、CYP21A2 和 CYP11B1 的表达在原醛组和正常组间无统计学
3、差异；与醛固酮合成酶系有关的调节因子中，醛固酮瘤组RENBP 和 NR1H2 的表达上调，CYB5A、CYP17A1、DUSP1、HMGCR、上海交通大学医学院硕士毕业论文 HSD11B2、JUN、RGS2、STAR 表达下调，CYP17/HSD3B2 的表达比值降低；肾上腺结节性增生组 RENBP 和 NR1H2 的表达上调，HMGCR、HSD11B2、JUN 表达下调，CYP17/HSD3B2 的表达比值降低但无统计学意义。2.醛固酮瘤组上调基因主要涉及到的显著性功能是：经 G 蛋白偶联受体信号通路，加快蛋白质的去磷酸化，蛋白质的转运，机体对刺激作出响应，集中加强细胞粘附，加大胞吞作用以及
4、加强机体的免疫反应。而下调基因涉及到的显著性功能主要是： 减弱氧的运输， 调节 NF-kappaB的向核转移，调控细胞周期及细胞凋亡，抑制细胞分化。肾上腺结节性增生组上调基因主要涉及到葡萄糖代谢，离子调控，通过 JNK 通路和Wnt 信号通路，加强免疫反应（与细胞抗病毒感染的保护有关） ；而下调基因与半胱氨酸、丝氨酸的代谢，脂肪的代谢，激素的分泌，细胞增殖，粒细胞的趋化性有关。醛固酮瘤和单侧肾上腺结节性增生的发病机制均与 MAPK、PPAR、Apoptosis、ErbB、Bladder cancer 等通路异常有关；而醛固酮瘤还可能与 JAK-STAT 等通路的异常有关，结节性增生组还可能与
5、p53 等通路的异常有关。 【结论】参与醛固酮合成的酶中，CYP11B2 基因表达升高；生理情况下大多数调节醛固酮合成的因子均下调，仅 RENBP 和 NR1H2 上调，提示肿瘤有独特的促进醛固酮合成路径； 醛固酮瘤组中 CYP17/HSD3B2 比值降低可能促进醛固酮高分泌；筛选得到的众多差异基因提示：醛固酮瘤和单侧肾上腺结节性增生均存在肿瘤特性。 上海交通大学医学院硕士毕业论文 关键词：原发性醛固酮增多症，醛固酮合成酶，微阵列分析，基因调节，实时定量 PCR 上海交通大学医学院硕士毕业论文 EXPRESSION OF ALDOSTERONE SYNTHESIS RELATED ENZYME
6、 AND ASSOCIATED REGULATORY FACTOR GENES IN PRIMARY ALDOSTERONISM ABSTRACT Objectives The pathogenesis of aldosterone over-secretion in primary aldosteronism(PA) is still not clear. The whole genomic expression including aldosterone synthesis related enzyme and associated regulatory factor genes in a
7、drenal glands with PA was investigated with microarray to explore the possible mechanism of hyperaldosterone. Methods 1. 10 aldosterone-producing adenoma, 7 unilateral nodular adrenal hyperplasia and 7 normal adrenal tissues were studied, which were confirmed by clinical and pathologic diagnosis. Ch
8、ips Illumina HumanRef-6 v2 were applied to analyze these gene expressions of the three groups. Real-time RT-PCR was used to confirm the mRNA level of the changed expressions among aldosterone synthesis related enzyme and associated 上海交通大学医学院硕士毕业论文 regulatory factor genes. 2. Matlab and Graphviz soft
9、ware was used to do data analysis including TwoClassDif, GO-Analysis, Path-Finder and GeneFunNet. Results 1. Among the aldosterone synthesis related enzymes, CYP11B2 transcript levels showed 70.29- and 38.57-fold higher in APA and UNAH over those in NA. No significant difference was observed about t
10、he levels of CYP11A1, HSD3B2, CYP21A2 and CYP11B1 mRNA between primary aldosteronism and normal adrenals. Among the aldosterone synthesis associated regulatory factor genes, RENBP and NR1H2 were up regulated, CYB5A, CYP17A1, DUSP1, HMGCR, HSD11B2, JUN, RGS2 and STAR were down regulated in APA. RENBP
11、 and NR1H2 were up regulated, HMGCR, HSD11B2 and JUN were down regulated in UNAH. Ratio of CYP17/HSD3B2 mRNA was lower in APA and not significantly changed in UNAH than in NA.2. According to significant GO category, APA main activate positive regulation of protein amino acid dephosphorylation, focal
12、 adhesion formation, regulation of exocytosis and inhibit regulation of NF-kappaB import into nucleus, defense response to protozoan, negative regulation of myeloid cell differentiation. UNAH main activate regulation of JNK cascade, embryonic forelimb morphogenesis, ER overload response and inhibit
13、defense response to protozoan, long-chain fatty acid metabolic process, regulation of translational termination. APA and UNAH both with the 上海交通大学医学院硕士毕业论文 downregulation in pathways of MAPK, PPAR, Apoptosis, ErbB and Bladder cancer may have partly same pathogenesis. In addition, APA may also correl
14、ate with the abnormal of JAK-STAT pathway, and UNAH may also correlate with the abnormal of p53 pathway. Conclusion CYP11B2 was over-expressed in aldosterone synthesis related enzymes. Most of the regulators in aldosterone synthesis down regulated but RENBP and NR1H2 suggested that a special aldoste
15、rone synthesis stimulating pathway may exist. The lower ratio of CYP17/HSD3B2 mRNA may be a promoter in hyperaldosteronism. According to the changes of main gene expression pathways, both APA and UNAH have the characteristics of tumor. KEY WORDS: Primary aldosteronism, aldosterone synthase, Microarr
16、ay analysis, gene regulation, real-time RT-PCR 上海交通大学医学院硕士毕业论文 英文缩略词表 英文全称,缩写 中文译名 aldosterone-producing adenoma, APA 产醛固酮腺瘤 unilateral nodular adrenal hyperplasia, UNAH 单侧肾上腺结节性增生 idiopathic hyperaldosteronism, IHA 特发性醛固酮增多症 adrenal vein samlpe, AVS 肾上腺静脉导管术采血 cholesterol side-chain cleavage, CYP11A1 胆固醇侧链裂解酶 3-hydroxysteroid dehydrogenase, 3-HSD 3-羟基类
