SHP-2酪氨酸磷酸酶在幽门螺杆菌诱发胃癌中的角色及隐丹参酮对其干预作用

Gastric cancer (GC) is the most common cancer of the digestive system, which occurrence is closely related to Helicobacter pylori (Hp) infection. Following attachment of Hp to the cell, CagA is translocated into the intracellular region of host cells and undergoes tyrosine phosphorylation by multiple members of the SRC family of kinases. Once phosphorylated, CagA specific binding and activation of oncoprotein SHP2. The availability of three-dimensional (3D) crystal structural information on SHP2 makes it possible to apply target-based computer-aided drug design (CADD) methods to find SHP2 inhibitors. In our previously study found that cryptotanshinone, which widely useded in clinical non-tumor treatment, may be a SHP2 inhibitor. Enzyme assay confirmed that this drug inhibited the SHP2-catalyzed hydrolysis of a phosphopeptide substrate with an IC50 of 22.50μM. It has a high selectivity for SHP2 and preferent inhibits the activated form of SHP2.After that the fluorescence quenching assay shows cryptotanshinone binds directly to SHP2. Further studies found that cryptotanshinone significantly inhibited Ba/F3 cell proliferation, and blocking the SHP2-mediated multiple cell signaling pathways. Our group first established a plasmid containing the CagA, intends to use cell and gene knock-in mouse studies the role of SHP2 in Hp-induced gastric epithelium lesions and gastric cancer,and confirm that similar mechanisms in human GC patients. Finally use cryptotanshinone intervention to investigate whether it is possible to suppress SHP2 protein so as to reduce or eliminate the Hp-induced gastric epithelial lesions. This research will provide a theoretical basis and experimental data for cryptotanshinone applied to Hp-induced gastric epithelial lesions and GC treatment.

胃癌是最常见的消化系统肿瘤，其发生与幽门螺杆菌(Hp)感染密切相关。Hp致病因素CagA进入宿主细胞内磷酸化后特异性结合并激活肿瘤蛋白SHP2。前期我们利用SHP2空间结构将其催化位点作为靶点通过CAAD筛选，发现在临床非肿瘤领域应用的隐丹参酮是一种SHP2抑制剂，酶动力学实验证实其对SHP2的IC50为22.5μM且对SHP2具有高选择性优先抑制活化的SHP2；同时荧光熄灭法证实它直接与SHP2蛋白相结合。进一步的实验发现其可显著抑制Ba/F3细胞的增殖并可阻断SHP2介导的多条细胞信号通路。本课题组首先建立含CagA质粒，拟用细胞及基因敲入鼠研究SHP2在幽门螺杆菌诱发胃上皮病变及胃癌中的作用并拟在临床确认类似机制，随后利用隐丹参酮干预，观察其是否可以抑制SHP2蛋白从而减轻或逆转Hp诱发的胃上皮病变。本课题研究将为隐丹参酮用于治疗Hp感染者胃上皮病变及胃癌提供理论依据和实验数据。

本研究以临床样本、胃癌细胞系及体内模型为研究对象，采用基因沉默、过表达及模型构建等实验手段，阐明了隐丹参酮对胃癌的体内外抑制作用以及拮抗幽门螺杆菌CagA-SHP2复合物诱发胃癌发生发展的机制。 主要研究结果：（1）51例胃癌患者癌组织中SHP2磷酸化水平与肿瘤长径、肿瘤浸润深度、淋巴结转移、临床分期阳性淋巴结数相关，且与患者血清中CagA阳性率相关。（2）隐丹参酮可抑制胃癌细胞增殖，促进胃癌细胞发生凋亡，减弱其迁移能力。（3）异隐丹参酮可通过靶向抑制STAT3信号通路，从而抑制胃癌增殖，促进其发生细胞周期阻滞和细胞凋亡，同时也可抑制体内瘤体的生长。（4）隐丹参酮可以拮抗胃癌细胞内CagA结合并活化SHP2的过程，从而减弱CagA诱导的胃癌增殖转移行为。（5） C57BL/6小鼠胃内幽门螺杆菌（SS1、PMSS1 野生型 和 PMSS1 CagA-）定植模型建立。综上研究证实SHP-2 在幽门螺杆菌诱发的胃癌发展进程中的关键价值，隐丹参酮在体内外具有抗胃癌作用，且可以通过破坏 CagA-SHP2 复合物，拮抗CagA诱导的胃癌细胞的增殖转移作用。本研究结果为今后隐丹参酮治疗Hp感染者胃上皮病变及胃癌提供理论依据和实验数据。