氯吡格雷损伤胃黏膜上皮细胞机制及其靶向基因研究

With the use of clopidogrel to antiplatelet ，its side effects on the human gastrointestinal mucosa have become a common concern by clinicians, and its mechanism of damaging the gastrointestinal tract has not clear . Our previous study used gene chip demonstrate that clopidogrel through phosphorylate p38 / MAPK signaling pathway to induce endoplasmic reticulum stress proteins CHOP overexpression,gastric epithelial cell apoptosis, gastric epithelial tight junction structure destruct, gastric mucosal permeability increased, thus the mucosal barrier ultimately damaged.And this effect can be partly antagonized by p38 / MAPK inhibitor SB203580. Meanwhile Gene Chip prompted that after clopidogrel acting on the gastric mucosal cells , the MKP-5 kinase which could strongly inhibite the p38 / MAPK kinasehas been decreased.Foreign studies have confirmed that MKP-5, CHOP are closely related to gastrointestinal ulcers.Therefore, this study intends to construct the specific cDNA interference plasmid of MKP-5, and transfect into GES-1 cells, upregulate the expression of MKP-5 gene, thereby inhibiting p38 / MAPK phosphorylation induced by clopidogrel. To investigeta that if the MKP-5 cDNA interference plasmid can significantly antagonist clopidogrel injury gastric epithelial cells. From the genetic level to find the precise target genes, and thus provide a theoretical basis for the prevention and treatment of clopidogrel's side effects.

随着氯吡格雷的广泛应用，其对人胃粘膜的损伤作用引起关注，但具体机制尚未明确。我们前期通过基因芯片证实：氯吡格雷可通过磷酸化激活p38/MAPK信号通路使其下游内质网应激蛋白CHOP过表达，致胃黏膜上皮细胞凋亡，破坏胃黏膜上皮细胞紧密连接结构，使胃黏膜通透性增加、黏膜屏障受损。而此效应可被p38/MAPK阻断剂SB203580显著拮抗。同时芯片提示氯吡格雷作用于胃黏膜细胞后，对p38/MAPK激活有强烈抑制作用的MKP-5激酶表达量明显下降。国外已有研究证实MKP-5、CHOP均与消化道溃疡发生密切相关。因此本课题拟通过构建MKP-5的特异性cDNA的干扰质粒，转染入GES-1细胞，上调MKP-5基因的表达，从而抑制氯吡格雷引起的p38/MAPK的磷酸化，观察其是否可显著拮抗氯吡格雷对胃黏膜上皮细胞的损伤作用，从基因水平寻找氯吡格雷对胃黏膜损害的精确靶点，为其副作用的防治提供理论依据。

氯吡格雷作为抗血小板药物广泛运用于临床，但是随之而来的副作用如消化道出血和延迟胃溃疡愈合已引起了临床医生的广泛关注。我们之前的研究证实氯吡格雷可以激活p38/MAPK信号通路导致内质网应激，从而引起胃黏膜上皮细胞凋亡、破坏胃黏膜屏障。通过全基因表达谱分析，我们发现能够去磷酸化和失活p38/MAPK信号通路的MKP-5基因在氯吡格雷作用后表达明显下调，因此本研究旨在探讨MKP-5在氯吡格雷对胃黏膜损伤中的具体作用和机制。在临床上我们发现长期口服氯吡格雷达一年而出现消化道出血的患者，MKP-5 mRNA和蛋白表达水平显著降低。在体外研究中，我们发现氯吡格雷（1.0–2.0 mM）作用于胃黏膜上皮细胞后p38/MAPK磷酸化水平呈浓度依赖性增加，而 MKP-5则浓度依赖性下降；然而过表达MKP-5后减轻了氯吡格雷引起的胃粘膜凋亡和增殖抑制，并且抑制了内质网应激引起的转录因子CHOP和TRIB3的上调，从而使得胃粘膜紧密连接结构破坏程度减轻，以上对胃黏膜的保护效应都和上调MKP-5后抑制p38/MAPK磷酸化有关。因此本研究认为过表达MKP-5可以通过抑制磷酸化p38/MAPK信号通路而减轻氯吡格雷引起的胃黏膜损伤。MKP-5可能是防治氯吡格雷引起消化道出血的潜在靶基因。