B7-H3分子调控survivin表达在胰腺癌抵抗吉西他滨化疗中的作用机制

The resistance to chemotherapy of pancreatic cancer is one of the reasons of treatment failure. There is a previous study indicated that overexpression of new costimulatory molecule B7-H3 have something to do with resistance to gemcitabine, but the mechanism is not clear. We discovered that knocking down of B7-H3 expression in pancreatic cancer accompanied with gemcitabine treatment can downregulate anti-apoptosis molecule survivin expression, and increase sensitivity to chemotherapy. We also detect that there are several potential phosphorylation sites in the intra-cellular segment of B7-H3 by bioinformatics analysis, which suggest that B7-H3 is probably to transfer signal. Based on these researches, we suppose the scientific hypothesis that B7-H3 up-regulates survivin expression through transmit intra-cellular information, and then promotes resistance to chemotherapy. The study will investigate: ①Researching the relationship between B7-H3 expression and therapeutic effect of gemcitabine based on clinical materials; ②Detecting the mechanism of the resistance to gemcitabine leading by B7-H3 regulating survivin expression through experiments in vitro and in vivo by utilizing established different B7-H3 expression level cell lines; ③Confirming that there is molecular biological evidence that B7-H3 can transmit intra-cellular information, and investigate the molecular mechanism of the signal which regulates survivin expression. In all, the study will provide new clue for pancreatic cancer chemotherapy resistance.

胰腺癌的化疗抵抗是治疗失败的因素之一，既往研究证实胰腺癌高表达B7-H3与其抵抗吉西他滨化疗有关，但原因尚不清楚。我们课题组前期发现，敲减胰腺癌B7-H3表达增加了其对吉西他滨化疗的敏感性，进而发现这可能与下调了抗凋亡分子survivin的表达有关；生物信息学分析发现B7-H3胞内段存在多个潜在磷酸化位点，提示其具有传递信号的潜能。据此提出科学假说：胰腺癌中B7-H3通过调控survivin的表达在肿瘤化疗抵抗中发挥了重要作用。本项目拟进一步研究：①临床标本分析胰腺癌B7-H3表达、survivin表达及其与吉西他滨化疗疗效的相关性；②利用不同水平表达B7-H3的胰腺癌细胞株，探讨该分子调控survivin在化疗抵抗中的作用机制；③利用自主研发的激发型及阻断型B7-H3抗体，分析该分子的信号传导通路调控survivin的关键分子或途径。本项目旨在为揭示胰腺癌化疗抵抗机理提供新的线索。

胰腺癌的化疗抵抗是治疗失败的因素之一，既往研究证实胰腺癌高表达B7-H3与其抵抗吉西他滨化疗有关，但原因尚不清楚。我们课题组在前期发现的基础上，提出科学假说：胰腺癌中B7-H3通过调控survivin的表达在肿瘤化疗抵抗中发挥了重要作用。在研究期限内进行了如下工作：（1）应用ELISA试剂盒、qRT-PCR方法及免疫组化检测胰腺癌患者血清和组织样本标本中B7-H3的表达水平，分析其与肿瘤临床病理特征之间的关系。发现B7-H3在胰腺癌患者血清和癌组织中高表达，肿瘤组织中B7-H3的阳性表达与肿瘤的TNM分期、局部浸润、淋巴结转移等恶性进展指标呈显著正相关。肿瘤组织中B7-H3高表达的患者对吉西他滨化疗敏感性较低表达者差，预后较差，为制定合理化疗方案提供重要参考指标。临床成果在国内三甲三级医院应用和推广。（2）成功构建了特异性B7-H3基因的shRNA慢病毒载体。通过体外、体内实验，研究发现①在体外，B7-H3分子能够促进胰腺癌细胞粘附、水平迁移及侵袭转移，但不影响细胞生长曲线。在体内，B7-H3分子能够促进胰腺癌肿瘤生长，局部浸润和远处器官转移。（3）机制研究方面：①证实了B7-H3分子向胞内传导信号的分子生物学证据，用激发型抗体4H7刺激Patu8988细胞，可以使B7-H3胞内段发生磷酸化。②胰腺癌表达的B7-H3分子上调了肿瘤细胞EGFR的表达。③胰腺癌表达的B7-H3分子激活了胞内信号Erk 1/2以及NF-κB通路，但不激活Bcl-2, Bax, Bcl-xl通路。④胰腺癌表达的B7-H3分子抵抗吉西他滨诱导的凋亡。⑤胰腺癌表达的B7-H3分子通过调控抗凋亡分子survivin的上调表达，抑制肿瘤细胞的凋亡，而在拮抗吉西他滨的化疗中发挥着重要作用。总之，基本阐明了B7-H3在胰腺癌中促进肿瘤恶性进展的作用及其机制，阐明了B7-H3在胰腺癌抵抗吉西他滨化疗中的作用和机制，证实了B7-H3表达与胰腺癌细胞向肿瘤细胞内传递信号的分子生物学证据。为以B7-H3为靶点的肿瘤生物治疗提供理论基础，具有潜在临床应用价值。该研究获江苏省医学新技术引进奖二等奖。