中文摘要
KCNJ5基因编码G蛋白偶联的内向整流型钾离子通道亚型4（GIRK4），其突变可导致醛固酮分泌瘤的发生。新疆高血压研究所对醛固酮分泌瘤组织进行该基因测序发现了新的突变率高达100%的X2位点，而该突变位点引起的通道功能改变，及其是否参与肾上腺球状带醛固酮分泌和细胞增殖的发生机制尚需进一步研究。本课题拟利用分子克隆技术构建含KCNJ5 X2突变型重组体并稳定转染至NCI-H295R细胞，通过细胞外应用不同类型通道阻断剂并运用RT-RCR技术、MTT法、EdU法等推测突变引起的通道功能改变；利用磷酸化标签（Phos-tag）检测突变型GIRK4的磷酸化水平，从而验证该突变是否参与促进醛固酮分泌和细胞增殖。
英文摘要
KCNJ5 gene encode GIRK4, the G-protein-coupled inwardly rectifying potassium channels, its somatic mutations have been implicated as a cause of aldosterone-producing adenomas.Researchers in hypertension institute of xinjiang sequenced KCNJ5 gene in adenomas tissues and found a novel missense mutation X2 which the ratio of it was 100%. However, the mechanisms of channel function changed by this mutation of KCNJ5 as well as whether to participate in the adrenocortical cell aldosterone overproduction and proliferation were remainly unknown.To test the function of mutant KCNJ5 in an “adrenal-like” system,we expressed the KCNJ5 X2 mutation in the adrenocortical carcinoma cell line NCI H295R by using molecular cloning technology. Various kinds of inhibitors of ion channels will be used extracellularly and using RT-PCR technique, MTT assay and Edu assay to speculate the functional change of channel caused by the mutation, then using phos-tag western blot to detect phosphorylation of GIRK4 harbouring the mutation, and thereby to verify whether this mutation involved in promoting aldosterone secretion and cell proliferation.
结题摘要
KCNJ5基因编码G蛋白偶联的内向整流型钾离子通道亚型4（GIRK4），研究已证实其突变可导致醛固酮分泌瘤的发生，然而突变引起的通道功能的改变及其参与调控肾上腺球状带细胞增殖的机制尚未阐明。本课题通过制备KCNJ5 L102Q突变的293T细胞株，利用MTT法、EdU法观察细胞增殖情况，同时应用不同种类的通道阻断剂观察其对细胞增殖的影响，最后运用western blot 技术检测三种细胞中Akt、pAkt （Thr308和Ser473），其下游靶蛋白GSK-3β、pGSK-3β的表达, 从而推测突变所引起的通道功能改变。我们发现（1）经过12小时、24小时孵育后KCNJ5突变型细胞的增殖率明显高于野生型和空白质粒型细胞，进一步EDU实验也证实转染KCNJ5突变可使细胞增殖率显著增加；（2）细胞外利用不同浓度的氯化钡、布比卡因和维拉帕米等离子通道阻断剂，进行24小时孵育后，布比卡因可显著抑制KCNJ5突变型细胞的增殖，这可能与突变引起的GIRK4通道离子选择性缺失，允许Na+通过的通道改变有关。（3）与在野生型和空载质粒细胞相比，pAkt （Thr308和Ser473）、pGSK-3β等蛋白在KCNJ5 突变型细胞中的表达未见明显差异，提示该突变独立于PI3K/Akt通路的磷酸化参与醛固酮瘤的发病。本课题从增殖的角度进一步验证该突变的功能学意义，推测突变所引起的通道功能改变，并为今后的药物治疗提供理论基础。