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目的本研究旨在阐述CXCLl2/CXCR4轴对胰腺癌神经相互作用的影响,探讨CXCLl2/CXCR4在胰腺癌嗜神经过程中的相关分子机制。方法体外培养胰腺癌Panc-1、Miapaca-2细胞,实验分为空白对照组、外源性CXCLl2组及CXCR4受体特异性阻断剂AMD3100组。采用RT-PCR检测胰腺癌细胞CXCR4及CXCL12mRNA的表达,检测基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)及人尿激酶型纤溶酶原激活物(human urokinase plasminogen activator,uPA)mRNA的表达;采用Transwell侵袭实验观察各实验组中细胞侵袭性的变化;提取大乳鼠脊髓背根神经节,建立胰腺癌细胞Miapaca-2与脊髓背根神经节(DRG)共培养模型,倒置显微镜成像系统动态观察不同实验组中神经轴突生长差异。结果胰腺癌细胞Panc-1、Miapaca-2细胞存在CXCR4mRNA的表达,而CXCL12mRNA无表达;两种胰腺癌细胞之MMP-2、MMP-9及uPAmRNA在AMD3100组、对照组、CXCLl2组中的表达差异具有统计学意义(P<0.01);Transwell细胞侵袭性实验中,外源性CXCLl2明显增强Panc-1、Miapaca-2细胞的侵袭性,而AMD3100有效抑制肿瘤细胞的侵袭能力,组间存在显著差异(P<0.05)。胰腺癌细胞与脊髓背根神经节共培养模型中,外源性CXCLl2明显增强神经轴突的生长,促使胰腺癌细胞与神经轴突接触,而AMD3100有效抑制神经轴突生长及与肿瘤细胞接触;细胞共培养144h后各组神经生长轴突数差异有统计学意义(P<0.01)。结论 CXCLl2/CXCR4一方面增强胰腺癌细胞的侵袭能力,促进胰腺癌细胞神经浸润;另一方面,神经元与施万细胞等构成神经纤维分泌CXCL12并通过化学趋化作用,诱导表达CXCR4的胰腺癌细胞向神经迁移,导致神经浸润的发生。
Objective This study was designed to investigate the effects of CXCL12 / CXCR4 axis on the neural interaction of pancreatic cancer and to explore the molecular mechanism of CXCL12 / CXCR4 in the process of neuropathic process of pancreatic cancer. Methods Panc-1 and Miapaca-2 cells were cultured in vitro. The experiment was divided into blank control group, exogenous CXCL12 group and CXCR4 receptor-specific blocker AMD3100 group. The expression of CXCR4 and CXCL12 mRNA in pancreatic cancer cells was detected by RT-PCR. The expressions of MMP-2, MMP-9 and human urokinase Transwell invasion assay was used to observe the changes of cell invasiveness in each experimental group. The dorsal root ganglion (DRG) of neonatal rat spinal cord was extracted to establish the pancreatic cancer cell line Miapaca- 2 and dorsal root ganglion (DRG) were co-cultured in vitro. Inverted microscope imaging system was used to dynamically observe the difference of axon growth in different experimental groups. Results The expression of CXCR4 mRNA was detected in Panc-1 and Miapaca-2 cells but not CXCL12 mRNA in pancreatic cancer cells. The expressions of MMP-2, MMP-9 and uPA mRNA in the two pancreatic cancer cell lines were different between AMD3100 group, control group and CXCL12 group (P <0.01) .Expression of CXCL12 in transwell invasion assay significantly enhanced the invasiveness of Panc-1 and Miapaca-2 cells. AMD3100 effectively inhibited the invasiveness of tumor cells with significant difference (P <0.05). In pancreatic cancer cells and spinal cord dorsal root ganglion co-culture model, exogenous CXCL12 significantly enhances the growth of axons and promotes the contact of pancreatic cancer cells with axons, while AMD3100 effectively inhibits axonal growth and contacts with tumor cells. After co-cultured for 24 hours, the number of neurite outgrowth in each group was significantly different (P <0.01). Conclusions CXCL12 / CXCR4 can enhance the invasiveness of pancreatic cancer cells and promote the invasion of pancreatic cancer cells. On the other hand, CXCL12 is secreted by nerve fibers and Schwann cells and induces CXCR4-expressing pancreatic cancer through chemoattractant Cells migrate to the nerve, resulting in the occurrence of nerve infiltration.