醛固酮合酶和11-β羟化酶基因单体型影响醛固酮瘤发生的分子机制研究

To date, the exact mechanisms of aldosterone-producing adenoma (APA) have not been completely elucidated. CYP11B2 and CYP11B1 genes have been recognized to participate regulating the synthesis of aldosterone. We have firstly found that the polymorphisms of CYP11B1 gene are associated with APA and that the genotypes and haplotypes of CYP11B2/CYP11B1 are significantly associated with the risk and the phenotype of APA. However, the exact mechanism of the polymorphisms in CYP11B2/CYP11B1 on APA is not clear. In the previous study, we found that the polymorphisms in CYP11B2/CYP11B1 were associated with the expression of β-catenin, a key protein of Wnt signaling pathway. Therefore, we hypothesize that the polymorphisms in CYP11B2/CYP11B1 are possiblly affect the incidence of APA via the Wnt signaling pathway. In order to verify the hypothesis, we will go on detecting the other single nucleotide polymorphisms (SNPs) of CYP11B2/CYP11B1 and trying to find the special haplotypes associated with the risk and phenotype of APA. We will start with Wnt pathway to elucidate the molecular mechanism of the special CYP11B2/CYP11B1 haplotypes on APA by use of the molecular cloning and transgenic techniques so as to provide some new idea for the treatment of APA.

醛固酮瘤（APA）的发病机制尚不完全清楚。已知醛固酮合酶基因(CYP11B2)和11-β羟化酶基因(CYP11B1)参与醛固酮合成调控。本项目组首次发现CYP11B1基因多态性与APA相关，并发现 CYP11B2/CYP11B1基因型和单体型与APA的发病风险和表型显著相关。但其影响APA发生的确切机制尚不清楚。在前期研究中，我们发现CYP11B2/CYP11B1基因多态性与Wnt信号通路重要的信号因子β-catenin的表达相关。我们推测，CYP11B2/CYP11B1基因多态性可能通过Wnt信号通路影响APA的发生。为验证这一假说，我们继续检测CYP11B2/CYP11B1基因的其它单核苷酸多态性（SNPs）；分析其与APA发病风险和表型相关联的特定单体型；采用分子克隆和转基因等技术，从Wnt信号通路入手阐明特定单体型影响APA发生的分子机制，为APA临床治疗提供新的线索和思路。

资料表明，CYP11B2/CYP11B1基因多态性与醛固酮瘤（APA）的发病风险相关。但其影响APA发生的确切机制尚不清楚。本项目将继续检测CYP11B2/CYP11B1基因的其它单核苷酸多态性（SNPs），探讨其与APA的相关性及分子机制。.项目组选择了醛固酮瘤（APA）患者80例为APA组，101名健康体检者或住院良性疾病患者为对照组。选择CYP11B2和CYP11B1基因中SNP位点共7个（rs3802228、rs3802230、rs4545、rs4546、rs6433、rs5301和rs6471570），采用TaqMan-MGB 探针法进行检测。结果显示，APA组中rs5301位点的A等位基因、rs3802228位点的A等位基因、rs6433位点的G等位基因频率均高于对照组；与对照组相比，APA组中rs4545位点AG基因型显著低于正常对照。单体型GAACAAC、GCGAAGT频率显著低于对照组。.采用Real-time RT-PCR法检测两组CYP11B2和CYP11B1 mRNA表达。结果显示，实验组CYP11B2 mRNA的相对表达量高于对照组。CYP11B2 mRNA的相对表达量与醛固酮（PAC）水平呈正相关，与血钾水平呈负相关。CYP11B1 mRNA的相对表达量与术前最高收缩压呈负相关。.采用Western blot法检测两组CYP11B2和CYP11B1蛋白表达。结果显示，CYP11B1蛋白在实验组的平均相对含量低于对照组。APA中CYP11B1蛋白的表达与年龄呈负相关。实验组CYP11B2蛋白平均相对含量高于对照组。CYP11B2蛋白表达水平与CYP11B1 mRNA表达水平呈负相关。.按照特定单体型成功构建CYP11B1和CYP11B2基因突变载体，转染HAdCC细胞。结果表明特定单体型可以促进HADCC细胞增殖，抑制HADCC细胞凋亡；这种作用可能通过Wnt信号通路实现。.总之，CYP11B2/CYP11B1基因多态性与APA显著相关，其作用可能通过Wnt信号通路实现。本项目研究可为APA的临床治疗和预防以及其发病机制的研究提供新的线索和思路。

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