成纤维生长因子受体4在胃癌中作用的功能研究及其抑制剂联合5-Fu作用机制的研究

Currently, targeted drugs have become a hotspot in the treatment field of gastric cancer. However, most targeted drugs have not achieved breakthrough except heceptin. Recently, more and more researches concerning Fibroblast growth factor receptor (FGFR) family molecules involved in different tumors. Some investigators considered that FGFR family was an emerging field in terms of the diagnosis and treatment of malignant tumors and had a wide application prospects. However, seldom researches regarding FGFR4 were found in the field of gastric cancer. Based on our previous studies, we plan to decrease and upregulate the expression of FGFR4 by means of constructing lentiviral vector mediated gastric cancer cell lines of FGFR4 RNAi and FGFR4 overexpression. Then gastric cancer model of nude mouse was established in order to perform a series of experiments in vitro and in vivo, including detecting the expression of biological features associated proteins in gastric cancer cell lines and tissues. Thus, the influence of FGFR4 in the tumorigenesis and development of gastric cancer was much more comprehensively clarified, which might provide a novel prognostic factor. Furthermore, the single and combination of 5-Fu and PD173074 (FGFR inhibitor) were used to intervene lentivirus treated gastric cancer cell lines and gastric cancer model of nude mouse, including a series of experiments in vitro and in vivo. Then we could explore the impact and mechanism of two drugs' combination concerning the biological characteristic of gastric cancer cells, which might provide some evidences that PD173074, as heceptin, is applied in the treatment of patients with gastric cancer.

目前靶向药物在胃癌治疗领域的研究成为一大热点。然而除赫赛汀外，多数靶向药物在胃癌研究中尚未取得突破。近来，关于FGFR家族分子在不同肿瘤中的报道越来越多。有学者认为，FGFR家族在恶性肿瘤诊治方面是一个新兴的领域，有广阔的应用前景，但FGFR4在胃癌中的研究甚少。本课题在前期研究的基础上，通过慢病毒构建FGFR4 RNAi稳转株和FGFR4过表达株以下调和上调FGFR4的表达，继而用于裸鼠胃癌模型的建立，进行一系列体外和体内实验，并检测胃癌生物学特性相关蛋白表达的变化，更全面地研究FGFR4在胃癌发生发展中的作用，可能为胃癌的预后提供新的评价指标。此外，5-Fu和FGFR4抑制剂PD173074单药或联合，干预以上慢病毒处理过的细胞株及裸鼠胃癌模型，进行一系列体外和体内实验，探讨两药联合对胃癌细胞及组织生物学行为的影响及机制，为PD173074像赫赛汀一样应用于胃癌的临床治疗提供一些依据。

我们前期的研究显示, FGFR4可能是胃癌治疗新的靶点。该课题首先探讨PD173074(PD)和5-Fu对胃癌细胞生物学行为的影响及其机制。将FGF19、PD和5-Fu单药或联合干预MKN45胃癌细胞株。结果显示，随着PD及5-Fu浓度的增加，细胞存活率逐渐降低；5-Fu参与的处理组FGFR4表达明显降低。PD显著增加MKN45细胞的凋亡率，而5-Fu和PD联合组比单药组效果更显著。此外，PD降低p-ERK 和 Bcl-xl的表达而增加Caspase-3的表达。抑制FGFR4的活性可能是PD疗效的主要机制，而5-Fu主要通过降低FGFR4表达起作用。5-Fu和PD两药联合可能是治疗胃癌的有效方法。.经慢病毒过表达载体处理后的MGC803和BGC823细胞株（FGFR4-LV5）的侵袭能力更强、凋亡率降低。5-Fu和PD单药或联合用药处理后的胃癌细胞的增殖能力均降低，而对照组较FGFR4-LV5组细胞增殖率降低更显著（P<0.05）。与对照组相比，5-Fu和PD单药或联合用药处理后的胃癌细胞的凋亡率显著增加；而FGFR4-LV5组的凋亡率低于对照组（P<0.05）。5-Fu和PD单药或联合用药处理后的胃癌细胞PCNA和Bcl-xl的表达明显降低，而Caspase-3表达显著升高。体内实验结果显示，FGFR4-LV5组裸鼠成瘤体积明显小于对照组。FGFR4过表达无论体内或体外均可提高胃癌细胞的增殖能力。FGFR4过表达可能抑制两药的效果。.另外，我们构建慢病毒载体以稳定下调胃癌细胞株FGFR4的表达。无论体内或体外实验结果均显示，shRNA组HGC27和MKN45胃癌细胞株的增殖能力、侵袭能力显著降低,而凋亡率明显升高(P<0.05)。shRNA组Bcl-xl, FLIP, PCNA的表达明显降低，而Caspase-3和E-cadherin 表达显著增强。shRNA组经5-Fu和BGJ398处理后的胃癌细胞株的增殖能力明显降低。与对照相比，5-Fu和BGJ398单药组和联合组均可增加胃癌细胞的凋亡率，尤其是联合组。5-Fu和BGJ398单药组和联合组减弱PCNA, Bcl-xl和FLIP的表达，增加Caspase-3的表达，而shRNA组的作用尤其显著。5-Fu和BGJ398联合在抑制胃癌细胞增殖能力、增加其凋亡率方面具有协同作用，提示其可能是胃癌治疗新的靶向药物。