基于浸润性膀胱移行上皮癌-癌间质代谢偶联的双靶向代谢干预实验研究

Muscle-invasive bladder cancer is one of the serious diseases threatening the health of humans. Till now, the prognosis of bladder cancer is still poor out of the unclear molecular mechanism. 30% of the patients demonstrate microscopic evidence of metastasis at the time of diagnosis and 90% of the patients died of metastasis at last. Studies have shown that the invasion and metastasis of muscle-invasive bladder cancer intimately related to metabolic pathways. Our previous proteomic studies had discovered that both cancer cells themselves and corresponding stromal cells exist significantly metabolic changes. Namely, significant tendencey of oxidative phosphorylation in cancer cells and significant tendencey of glycolysis in stromal cells. Based on our findings, we deduce that the stromal cells undergo aerobic glycolysis and produce the energy-rich metabolites.Meanwhile,these metabolites transferred to cancer cells, where they enter oxidative phosphorylation. Thus a metabolic-coupling is constructed. The present research item is just focus on the metabolic changes in muscle-invaisve bladder cancer, and furthe to observe the deriveing changes in cancer cells and stromal cells in a prerequisite of co-culture. All the scientistic contents are under the circumstance of offer evidences to metabolic-coupling and mainly include oxidative stress, DNA damage, autophagy, glycolysis, mitochondrial function, etc. Furthermore, combined target small interfering technique is performed to block energy transfer and thus to produce artificial metabolic-uncoupling. The successor of combined target treatment is investigated by in vitro and in vivo experiments. Above all things, present research item will help to explain the mechanism of bladder cancer and lay the foundation to novel targeted antitumor therapy.

浸润性膀胱癌是危及人类健康的重大疾病，发病机制不详，预后差。30%的浸润性膀胱癌患者确诊时出现镜下转移，超过90%的患者最终死于肿瘤转移。有研究表明，浸润性膀胱癌的侵袭、转移与代谢通路改变密切相关。我们前期蛋白质组学研究中亦发现浸润性膀胱癌及其间质中均发生了显著代谢改变，即膀胱癌细胞中的氧化磷酸化改变与癌间质细胞中的有氧酵解改变。我们推测膀胱癌间质成分通过有氧酵解以及营养素定向转运饲育膀胱癌细胞氧化磷酸化，从而形成代谢偶联。因此本课题正是着眼于膀胱癌代谢改变，进一步通过体内外实验观察共生环境下膀胱癌细胞及间质细胞的衍生变化，从氧化应激、DNA损伤、自体吞噬、糖酵解、线粒体功能等多方面为代谢偶联提供佐证，并利用联合靶向性小干扰RNA技术阻断定向转运通路导致代谢脱偶联，探讨其体内外抗增殖及侵袭作用，为阐明膀胱癌的分子发病机制及其靶向治疗夯实理论基础。

浸润性膀胱癌是危及人类健康的重大疾病，发病机制不详，预后差。有研究表明，浸润性膀胱癌的侵袭、转移与代谢通路改变密切相关。我们前期蛋白质组学研究中发现浸润性膀胱癌及其间质中均发生了显著代谢改变，即膀胱癌细胞中的氧化磷酸化改变与癌间质细胞中的有氧酵解改变。我们推测膀胱癌间质成分通过有氧酵解以及营养素定向转运饲育膀胱癌细胞氧化磷酸化，从而形成代谢偶联。因此本课题着眼于膀胱癌代谢改变，进一步通过体内外实验观察共生环境下膀胱癌细胞及间质细胞的衍生变化，从氧化应激、DNA 损伤、自体吞噬、糖酵解、线粒体功能等多方面为代谢偶联提供佐证，并利用联合靶向性小干扰RNA技术阻断定向转运通路导致代谢偶联，探讨其体内外抗增殖及侵袭作用，为阐明膀胱癌的分子发病机制及其靶向治疗提供理论基础。结果如下：1. 三维共培养环境下膀胱癌细胞可以诱导成纤维细胞具有CAF表型，且CAF可导致膀胱癌细胞MCT1上调表达，成纤维细胞MCT4上调表达，这两种乳酸定向转运体的过表达标志着CAF与肿瘤代谢、发展及血管生成有着密切的关系；2. 共培养条件下膀胱癌细胞线粒体相关蛋白的表达较成纤维细胞明显增高，为“肿瘤-间质代谢偶联”提供了进一步的证据；3. 共培养体中MCTs 介导的CAFs-T24 乳酸定向转运机制研究表明siRNA干扰之后培养基中乳酸浓度明显降低；4. 构建含有RNA干扰序列的重组质粒载体pSUPER-MCT1，pSUPER-MCT4，联合靶向阻断MCT1，MCT4可以有效抑制膀胱癌细胞增殖。本课题着重于探讨膀胱癌与间质细胞的代谢偶联机制，为联合多位点分子靶向治疗浸润性膀胱癌提供理论依据。