肾上腺腺瘤、增生患者GIRK4基因突变及相关分子机制研究

GIRK4 is the forth subunit of G-protein coupled inwardly rectifying K current. At present, the studies on the association between GIRK4 genetic variants and adrenal aldosterone-producing adenoma (APA) have made a rapid progress. Our previous studies have shown two SNPs (rs2624204 and rs4937391) in GIRK4 are associated with primary aldosteronism (PA). In the study, 70 cases of adrenal APA and 30 cases of hyperplasia and normal tissues as control are collected for operation specimens and DNA extraction. PCR is used to amplify the three extrons and promoter of GIRK4, following, the PCR products is sequenced to examine the reported variants and novel variants in GIRK4 gene. Real time PCR and Western-blot method are used to assess the expression of GIRK4 in patients with APA and hyperplasia. The whole cell patch clamp technique and Edu method and ELISA are applied to detect the function of studied variants. PKC and Akt may play roles in aldosterone synthesis as regulator of GIRK4, the expression of different subunits of PKC will be detected by Western-blot method or immunohistochemical staining. The signal molecular- - Akt and NF-κB which are related to cell proliferation and apoptosis, are determined by Western-blot method. The study aims to elucidate the relationship among the several signal molecular and PA, and to explore the etiology of APA and hyperplasia.

GIRK4为G蛋白偶联的内向整流型钾离子通道亚型4，其基因突变与肾上腺腺瘤的相关研究目前进展迅速。新疆高血压研究所研究发现GIRK4基因两个SNP位点与原发性醛固酮增多症相关。本课题提取肾上腺腺瘤70例、增生患者30例的手术标本DNA，PCR扩增GIRK4外显子及启动子区序列并测序，以验证这些患者是否存在GIRK4已报道的突变，并明确是否存在新的突变。利用实时荧光定量PCR法和Western-blot法检测GIRK4在腺瘤、增生组织及正常肾上腺组织中的表达。运用全细胞膜片钳技术、Edu法、Elisa等方法验证突变位点的功能。本课题研究肾上腺腺瘤、增生组织中PKC、Akt等信号分子的信号分子，试图从增殖、凋亡角度阐明疾病发生机制，从而为揭示肾上腺腺瘤、增生的发病机制奠定基础。

GIRK4 为G 蛋白偶联的内向整流型钾离子通道亚型4，其基因突变与肾上腺腺瘤的相关研究目前进展迅速。新疆高血压研究所研究发现GIRK4 基因两个SNP 位点与原发性醛固酮增多症相关。本课题提取肾上腺腺瘤75 例、增生患者18例的手术标本DNA，PCR 扩增GIRK4 外显子及启动子区序列并测序，测验结果显示，肾上腺瘤组织中存在4种错义突变，3种为已报道的突变类型，其中一种为新发现的错义突变；在肾上腺增生组织中检测出3种错义突变。运用实时定量荧光PCR Array法对经基因测序明确的肾上腺瘤GIRK4突变型、野生型及瘤旁正常对照组织进行了主要的离子通道相关基因进行检测，发现肾上腺腺瘤中钾离子通道、钙离子通道及钠离子通道基因均有异常表达。进一步运用全细胞膜片钳技术、Edu 法、Elisa 等方法验证了新发现的突变位点的功能。此外，还本课题还从肾上腺腺瘤、增生组织中PKC、Akt 等信号分子的研究， 从增殖、凋亡角度阐明疾病发生机制，从而揭示了肾上腺腺瘤、增生的发病机制。

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