KCNJ5基因148dupT突变对H295R细胞醛固酮分泌的影响

Influence of 148dupT mutation of KCNJ5 gene on the aldosterone secretion of H295R cells

目的:研究KCNJ5基因148dupT突变对H295R细胞醛固酮分泌的影响及相关机制。方法:构建KCNJ5基因148dupT突变和非突变型质粒,选取H295R肾上腺腺瘤细胞系和293T人胚肾细胞系,应用jetPRIME转染试剂将突变和非突变以及对照质粒转染H295R细胞系以及293T细胞系,使用DiSBAC2染料检测293T细胞膜电位的变化、放射免疫分析法检测H295R细胞醛固酮的合成以及实时定量PCR检测CYP11B1和CYP11B2基因的表达。结果:148dupT突变型KCNJ5基因转入293T细胞可以导致膜电位去极化;转入H295R细胞可以导致醛固酮分泌增加,和CYP11B1和CYP11B2基因的表达增加。结论:148dupT突变型KCNJ5基因可能通过细胞膜电位去极化使得醛固酮合酶基因表达增加,进而导致H295R细胞醛固酮分泌增多和醛固酮瘤的发生发展。

Objective： To investigate the effect and mechanisms of 148dupT mutation of KCNJ5 gene on the al- dosterone secretion of H295R cells. Method： The plasmid with mutant and wildtype KCNJ5 sequence were constructed. The two plasmids were then transfected into the H295R cells and 293T cells using jetPRIME transfec- tion reagents. DiSBAC2 was used to detect the membrane voltage of 293T cells. Radio immunoassay was used to detect the synthesis of aldosterone. Real-time PCR was used to determine the expression of CYPllB1 and CYP11B2. Result： Transfection of 148dupT mutation of KCNJ5 gene into 293T cells could depolarize the mere brane voltage. Transfection of 148dupT mutation of KCNJ5 gene into H295R cells could increase the aldosterone secretion and the expression of CYPllB1 and CYPllB2. Conclusion： The mutant type of 148dupT of KCNJ5 gene may increase the expression of genes related to the synthesis of aldosterone by depolarizing the membrane voltage, which lead to increased aldosterone synthesis and the development of aldosteronoma.