胃H,K-ATPase基因型影响雷贝拉唑抑制胃酸分泌功能的机制研究

Rabeprazole, one of the proton pump inhibitors (PPIs), treats peptic ulcer (PU) through binding to gastric H,K-ATPase to inhibit acid secretion. But in some patients, the therapeutic effects are not satisfactory, and the mechanism is still not understandable. Our preliminary test shows the result that there are 4 gene mutations (site 310, 796, 802 and 923) in α subunit of the gastric H,K-ATPase in PU patients; while in healthy volunteers, there are only 2 gene mutations (site 310 and 802).The incidence rates at site 310-gene mutation are 78.9% in PU patients and 34.8% in healthy volunteers.The amino acid converts from Ile (Hydrophobicity, Apolarity) to Met (Hydrophilicity, Polarity) at site 310. There was significant difference in gastric acid inhibition function between site 310 gene mutation group and control group in healthy volunteers, who were treated by single and successive intravenous administration of rabeprazole.(rabeprazole is less susceptible to the influence of genetic polymorphisms of either CYP2C19 or CYP3A4, both CYP2C19 and CYP3A4 contribute only a small fraction to the overall metabolism. 85% of rabeprazole is metabolismed by non-enzyme route in vivo.) The gastric acid inhibition funtion of the volunteers with site 310 gene mutation in α subunit of the gastric H,K-ATPase decreases significantly than that without mutation, and the mechanism deserves investigation. In order to explore the possible mechanism of action, we propose our hypothesis that the gene mutation in α subunit of gastric H,K-ATPase could lead to decrease of avidity and covalent bond stability of rabeprazoel (PPI) to gastric H,K-ATPase, resulting in the decline of the gastric acid inhibition function. In order to validate the hypothesis, Several experiments are in planning. First, we choose PU patients (with or without gene mutation at site 310) and healthy volunteers (without gene mutations) through gene sequencing, to analysis the difference in gastric acid inhibition function treated by single and successive administration of rabeprazole and famotidine (histamine-2 receptor antagonist). Then a series of techniques, such as site-directed mutagenesis, adenovirus vector transfection, immunofluorescence, immunoprecipitation, western blot, and isotope labeling, etc., will be used to construct rabit gastric parietal cells in vitro to evaluate the structure, activity and function of gastric H,K-ATPase. In the meanwhile, we will construct the site-directed mutagenesis mouse model to induce PU and to monitor the difference in gastric acid inhibition function treated by rabeprazole and famotidine. Thus we could identify the change of structure, activity and function of gastric H,K-ATPase with site 310 gene mutation in α subunit, and explain the partial reason why some of the patients with PU could not get satisfactory therapeutic effects from the treatment with rabeprazole or other PPIs. We will establish the rationale in gene diagnosis in peptic ulcer patients.

雷贝拉唑通过与胃H,K-ATP酶结合抑制胃酸分泌治疗消化性溃疡，但对部分患者疗效较差，其机制仍不清楚。预试验结果提示，消化性溃疡患者胃H,K-ATP酶α亚单位有4个位点碱基变异，健康志愿者有2个位点变异；其中雷贝拉唑对310位氨基酸变异的健康志愿者胃酸抑制功能显著下降，其机制还有待进一步探讨。为此，我们提出假说，胃H,K-ATP酶α亚单位碱基变异，致药物与其亲合力及共价键稳定性下降，胃酸抑制功能减弱。为了验证这一假说，我们拟通过基因测序筛选消化性溃疡α亚单位310位有/无变异的患者及无变异的健康者，统计分析雷贝拉唑和法莫替丁治疗前后胃酸分泌功能的差异。再利用定点诱变，腺病毒载体转染，免疫荧光，免疫沉淀，Western Blot及同位素标记等手段，构建定点突变体外家兔胃壁细胞及突变小鼠功能评价模型，明确该碱基变异影响酶的结构和功能。从而探明药效个体差异的来源，为溃疡患者基因诊断奠定理论基础。

胃H,K-ATP酶α亚单位可能存在不同基因亚型，影响质子泵抑制剂抑制胃酸分泌功能，国内外对此研究很重视。我们针对胃H,K-ATP酶α亚单位碱基变异进行了深入研究，结果发现：（1）消化性溃疡出血患者胃H,K-ATP酶α亚单位有6个碱基位点变异，氨基酸序号分别为：263、277、320、412、877和936位，消化性溃疡患者只有263位氨基酸碱基变异，而健康志愿者的变异氨基酸位点分别为：263和412位，这些位点变异都引起了相应的氨基酸结构改变。其中第263位氨基酸突变发生率在溃疡出血患者、溃疡患者和健康志愿者分别为：78.9、59.2和29.9%；（2）雷贝拉唑给药后胃酸抑制功能结果显示，雷贝拉唑对第263位氨基酸变异的健康志愿者与未变异者相比，胃酸抑制功能显著下降。因此初步可以确定胃H,K-ATPase α亚单位DNA序列呈多态性，可能存在2种甚至多种基因型，其中第263位氨基酸碱基变异可能对雷贝拉唑与之共价结合的亲合力、结合程度及稳定性下降，酶很快恢复活性，从而使雷贝拉唑胃酸抑制作用减弱，在临床表现为疗效降低或治疗失败。胃H,K-ATP酶多态性可能是质子泵抑制剂治疗消化性溃疡药效个体差异来源的主要因素之一。本课题研究(NSFC 81273593)共发表SCI收录论文3篇、国内核心期刊论文5篇，培养硕士研究生1名，在读硕士生1名。

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