Hedgehog信号通路对新疆维、汉民族胰腺癌干细胞生物学行为的调控及其分子机制研究

Pancreatic cancer stem cells are considered to be the core of research field about pancreatic cancer. In the preliminary study, we successfully separated side population cells from pancreatic cancer tissue and identified them, confirmed that they were the cell subsets which were rich in pancreatic cancer stem cells. For Hedgehog (Hh) signal path, which plays a key role in many kinds of cancer stem cells, our results indicated that the key members of it (Shh, Smo, Ptch1, Gli1, Bcl-2, HHIP-1, JAG-2, MYCN) revealed differential expression between pancreatic cancer and para-carcinoma tissue or Uyghur and Han nationality, However, the researches about these differences within pancreatic cancer stem cells and Uyghur or Han nationality in Xinjiang have not been seen at home and abroad. Therefore, this project is proposed to screen out the key members of Hh signal path, which are closely to pancreatic cancer stem cells from Uyghur and Han nationality in Xinjiang, by using gene expression difference detection between SP cells and NSP cells or Uyghur and Han nationality , analysing the change about characteristics of pancreatic cancer stem cells after RNAi. Through using fluorescence report enzyme and gene mutation method, the control mechanism of Hh signal path and the effect to properties of pancreatic cancer stem cells can be clarified in this project, so as to lay a good foundation for further revealing the molecular mechanism of malignant biological behavior of pancreatic cancer.

胰腺癌干细胞是目前胰腺癌研究领域的核心。前期研究中，我们成功分离了胰腺癌SP细胞并鉴定其为富集胰腺癌干细胞的细胞亚群。针对在多种肿瘤干细胞中被证实有关键作用的Hedgehog信号通路，我们的研究结果及相关文献报道提示，其关键成员（Shh、Smo、Ptch1、Gli1、Bcl-2、HHIP-1 、JAG-2、MYCN）在胰腺癌与癌旁组织间或新疆维吾尔族与汉族胰腺癌组织间存在一定的表达差异，但这些差异在新疆维汉民族胰腺癌SP细胞中是否同样存在国内外并未见相关报道。故本项目拟通过胰腺癌SP细胞/非SP细胞及新疆维汉民族间上述基因的表达差异检测与靶向干扰后胰腺癌干细胞特性变化分析，筛选出新疆维汉民族胰腺癌干细胞相关的Hh信号通路关键成员；通过Hh通路调控因子靶向干扰、荧光报告酶与基因突变法检测靶基因表达，从而阐明Hh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奠定良好的基础。

目前的观点认为肿瘤干细胞是肿瘤发生发展过程的核心，其生物学行为受多条细胞信号通路的共同调节，其中以Hedgehog信号通路、Notch信号通路及WNT信号通路尤为关键。为阐明Hedgehog信号通路与Notch信号通路在胰腺癌干细胞中的表达及调控机制，本研究采用Affymetrix基因表达谱芯片对10例新疆维吾尔族及汉族胰腺癌组织及其癌旁组织标本进行了分析，结果显示Hedgehog信号通路中的SHH、SMO、PTCH1、GLI1、GLI2、SUFU及靶基因BCL2，Notch信号通路中的NOTCH1、NOTCH2、NOTCH3、JAG2、DLL1、DLL3及靶基因HES1均存在显著差异表达，经胰腺癌原代细胞实时定量PCR及Western blot验证，证实上述通路中的SMO、GLI1、SUFU、NOTCH1、NOTCH2、JAG2及靶基因BCL2、HES1在胰腺癌组织及胰腺癌原代细胞中的表达均显著高于癌旁组织或正常胰腺导管细胞；汉族胰腺癌细胞来源CD24+CD44+ESA+胰腺癌干细胞中干细胞特性相关基因OCT4、Nanog，上述信号通路成员SMO、GLI1、SUFU、NOTCH1及NOTCH2 mRNA与蛋白的表达均显著上调升，维吾尔族胰腺癌细胞来源CD24+CD44+ESA+胰腺癌干细胞中OCT4、Nanog、SMO、GLI1、BCL2、NOTCH2、JAG2及HES1 mRNA与蛋白的表达均显著上调，证实这两条信号通路在胰腺癌组织、原代细胞及CD24+CD44+ESA+干细胞亚群中均存在异常激活。通过转染构建的携带SMO siRNA慢病毒表达载体后，胰腺癌原代细胞中Hedgehog信号通路靶基因BCL2的表达显著下降，且细胞增殖及侵袭转移能力显著被抑制，分选出的CD24+CD44+ESA+干细胞亚群培养过程中增殖缓慢，证实Hedgehog信号通路可能与胰腺癌增殖及侵袭转移过程相关；转染携带JAG2 siRNA慢病毒表达载体后，胰腺癌原代细胞增殖及侵袭转移能力显著被抑制，但细胞凋亡显著增多，分选出的CD24+CD44+ESA+干细胞亚群培养过程中同样增殖缓慢，证实NOTCH信号通路可能与胰腺癌增殖、凋亡及侵袭转移过程相关。本研究为进一步实现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，并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。