Wnt信号通路主要成员在新疆维吾尔族与汉族胰腺癌干细胞中的表达及其调控机制研究

Recent view considered that the pancreatic cancer stem cells are the core of the malignant biological behavior of pancreatic cancer. The study about the gene control of cancer stem cells also has become a new hot spot of pancreatic cancer research. Wnt signaling pathway is one of which were known to have the closest relationship with the cancer stem cells. The recently study reported that the principle members of it has abnormal expression in pancreatic cancer tissue. However, the study of dependency between Wnt signal pathway and pancreatic cancer is still in its infancy, there is only a little coverage about it. Our preliminary research show that, in pancreatic cancer tissue, there is a visible difference of the expression of Wnt signaling pathway members between Uyghur and Han patients in Xinjiang. While, does such difference also appear in pancreatic cancer stem cells? How about its regulatory mechanism? To answer the above questions, this project intends to analysis the difference of expression profile between Uyghur and Han nationality, detect the expression of mRNA and protein of Wnt signaling pathway members, and Interference with Wnt signaling pathway members' gene expression. By these methods, we will screen out the Wnt signaling pathway members which are correlation with pancreatic cancer stem cells. Then, this project also intends to illuminate the molecular biochemistry mechanism of this signal pathway by using dual-luciferase reporter assey system and gene mutation analysis. All of above research results should be conducive to revealing the molecular biochemistry mechanism of the malignant biological behavior of pancreatic cancer and finding Gene targets for early diagnosis and treatment.

最近的观点认为胰腺癌干细胞是胰腺癌恶性生物学行为产生与维持的核心，针对肿瘤干细胞的调控研究也已成为胰腺癌研究领域新的热点。Wnt信号通路是已知与肿瘤干细胞调控关系最为密切的信号通路之一，且最近的研究也报道了其主要成员在胰腺癌组织中的异常表达，然而国内关于胰腺癌干细胞中Wnt信号通路调控作用的研究尚处于起步阶段，相关报道较少。我们前期研究的结果显示，新疆维吾尔族与汉族胰腺癌组织中Wnt通路部分成员的表达存在差异，这种差异是否同样存在与不同民族的胰腺癌干细胞中？其调控机制如何？为回答上述问题，本项目拟通过不同民族胰腺癌干细胞表达谱差异分析、mRNA与蛋白定量检测及基因过表达与表达阻遏等方法，筛选出胰腺癌干细胞相关的Wnt信号通路关键成员，并通过荧光报告酶与基因突变法阐明该信号通路的调控机制，以期为进一步揭示胰腺癌恶性生物学行为的分子机制及早期诊断与靶向治疗位点的确定奠定良好的基础。

胰腺癌干细胞是胰腺癌恶性生物学特性的源泉，其发生发展可能与多条调控胚胎发育的细胞信号通路组成的调控网络异常激活密切相关，经典WNT通路即WNT/β-catenin信号通路是该调控网络最核心的成员之一。本研究旨在阐明WNT信号通路与胰腺癌干细胞的关联及其可能存在的调控机制。研究组采用10例新疆维吾尔族与汉族患者胰腺癌组织及其癌旁组织Affymetrix基因表达谱芯片差异表达基因分析及胰腺癌原代细胞实时定量PCR验证的方法从WNT通路中筛选出存在显著差异表达的成员β-catenin、LEF1、MMP7、SOX4及WNT5A；采用流式细胞仪从胰腺癌原代细胞中分选出CD24+CD44+ESA+胰腺癌干细胞并通过干细胞特征性基因OCT4及Nanog检测与裸鼠成瘤实验加以验证，经实时定量PCR分析上述差异表达基因在胰腺癌干细胞中的表达均显著高于胰腺癌原代细胞；结合文献报道，研究组选择通路核心调控因子β-catenin构建过表达慢病毒载体并通过转染胰腺癌原代细胞构建稳定过表β-catenin的胰腺癌原代细胞，经流式细胞仪分析，该原代细胞中CD24+CD44+ESA+胰腺癌干细胞的比率明显高于未转染组原代细胞；干细胞生物学特性研究结果显示，稳定过表β-catenin的胰腺癌原代细胞分选出的胰腺癌干细胞成球能力、克隆形成能力、细胞活性（增殖能力）及侵袭迁移能力均显著高于未转染组，且该组胰腺癌干细胞处于S期的细胞比率也明显高于未转染组，但两组胰腺癌干细胞细胞凋亡率则未见显著差异；上述差异表达基因及干细胞特征性基因中，LEF1、SOX4、OCT4及Nanog 在过表达组胰腺癌干细胞中的表达均显著高于为转染组，而WNT5A与MMP7 的表达在两组细胞间则无显著差异。研究证实，胰腺癌干细胞恶性生物学特性的产生与维持可能与WNT信号通路的异常激活密切相关，但该通路具体调控机制尚不明确。本研究为阐明经典WNT信号通路在胰腺癌发生发展中的重要作用及机制提供了真实可靠的研究数据，为后续深入分析研究新疆地区胰腺癌早期诊断及个体化治疗奠定了扎实的实验基础，对于拟采用胰腺癌原代细胞行肿瘤干细胞研究的学者具有一定的参考价值。