死亡相关蛋白3 DAP3促进胃癌新辅助化疗诱导肿瘤细胞凋亡的分子生物学基础

The R0 resection rate of gastric cancer remains low due to the diagnosis at late stage, leading to the poor prognosis. The combined therapy including preoperative chemotherapy、D2 radical resection and postoperative chemotherapy had become the main stream therapeutic model. The efficacy of neoadjuvant chemotherapy correlate well with patients’ outcome who receive the combined therapy, thus prediction and evaluation of chemotherapy response become the focus.Inducing apoptosis is the main mechanism of most chemotherapeutics for gastric cancer, our preliminary results also indicated that apoptosis level correlate well with response to neoajduvant chemotherapy and prognosis of gastric cancer patient following preoperative chemotherapy. Thus we choose the Death associated protein 3 (DAP3) as the apoptosis associated marker, and found its expression level is related to patients survival following preoperative chemotherapy, and the trend it correlated with chemotherapy efficacy could be observed. The cell assay and flow cytometry with DAP3 expression knocked down cell lines indicate that DAP3 expression knocked down could inhibit the chemotherapy induced tumor cell apoptosis...In this study, we plan to use immunohistochemical staining to prove whether the relationship between DAP3 expression and patients survival following neoadjuvant chemotherapy lie in the DAP3 prognostic role in gastric cancer patients without preoperative chemotherapy; we also plan to confirm DAP3’s role in promoting chemotherapy induced apoptosis using cell lines over expresse DAP3 ; and explore the downstream effect factors, and further the apoptosis pathway that DAP3 may be involved due to the changed tumor cell microenviroment after the preoperative chemotherapy. And finally find several candidate molecules for joint detection aimed at predicting the preoperative chemotherapy efficacy, so as to further enhance the overall efficacy of gastric cancer combined therapy.

我国胃癌多为进展期，R0切除率低，围手术期化疗+胃癌D2根治术的综合治疗已成为趋势，其中术前化疗疗效与预后显著相关，故疗效的预测和评价成为焦点。诱导凋亡是化疗主要机理之一，本组前期结果提示新辅助化疗胃癌患者术后病理切片凋亡指数与疗效、预后密切相关；故进一步选择死亡相关蛋白3（DAP3）作为凋亡相关标志物，发现其mRNA，蛋白表达水平均与接受新辅助化疗胃癌患者预后相关，且与化疗疗效具有相关趋势。一系列细胞实验以及流式细胞仪凋亡检测均提示DAP3表达敲除可抑制化疗诱导的肿瘤细胞凋亡。.本研究拟通过免疫组化明确DAP3是否因为是胃癌预后因子进而与新辅助化疗患者预后相关；还拟通过DAP3过表达进一步验证其在化疗诱导凋亡过程中的调节作用，并探索其下游作用因子，及其在新辅助化疗肿瘤微环境改变后可能参与调节凋亡的途径；从而为新辅助化疗疗效预测的联合检测提供备选因子，从而进一步提高胃癌综合治疗的整体疗效。

我国胃癌多为进展期，R0切除率低，围手术期化疗+胃癌D2根治术的综合治疗已成为趋势，其中术前化疗疗效与预后显著相关，故疗效的预测和评价成为焦点。诱导凋亡是化疗主要机理之一，本组前期结果提示新辅助化疗胃癌患者术后病理切片凋亡指数与疗效、预后密切相关；故进一步选择死亡相关蛋白3（DAP3）作为凋亡相关标志物，发现其mRNA，蛋白表达水平均与接受新辅助化疗胃癌患者预后相关，且与化疗疗效具有相关趋势。一系列细胞实验以及流式细胞仪凋亡检测均提示DAP3表达敲除可抑制化疗诱导的肿瘤细胞凋亡。.本研究拟通过免疫组化明确DAP3是否因为是胃癌预后因子进而与新辅助化疗患者预后相关；还拟通过DAP3过表达进一步验证其在化疗诱导凋亡过程中的调节作用，并探索其下游作用因子，及其在新辅助化疗肿瘤微环境改变后可能参与调节凋亡的途径；从而为新辅助化疗疗效预测的联合检测提供备选因子，从而进一步提高胃癌综合治疗的整体疗效。.我们选取了120例患者的肿瘤与癌旁组织的冰冻新鲜组织提取RNA，以及89 例未接受新辅助化疗胃癌患者术后病理切片，行DAP3免疫组化染色，分析 DAP3 表达与临床病理参数以及预后的相关性。总的样本分析结果显示免疫组化和RNA的QPCR结果在很多方面具有一致性，包括在肿瘤组织，癌旁组织中的差异表达；Lauren病理分型；T，N，M分期等方面。DAP3与预后相关性方面，蛋白水平未显示出显著的差别，RNA水平可以看出DAP3高表达与更好预后相关的趋势。.在多种胃癌细胞系检测中发现DAP3表达水平接近管家基因的表达水平，总体表达水平较高，构建DAP3过表达肿瘤细胞系的难度较大，因此我们后续继续采用基因敲除的方法去挖掘DAP3参与调整化疗疗效的机制。后续结果显示DAP3通过B-catenin/LGR5/Bcl-2调控轴来调节肿瘤细胞对化疗的敏感性。