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目的探讨迷走神经递质P物质对肝癌细胞侵袭转移能力的影响及其相关分子机制。方法实时荧光定量PCR及琼脂糖凝胶电泳检测并分析两种人肝癌细胞株Hep G2和SMMC-7721中神经肽-1(NK-1)受体表达;Cell Titer Blue检测不同浓度P物质(0、100、250、500 nmol/L)在不同时间点(0、24、48、72、96、120 h)对肝癌细胞增殖能力的影响;肝癌细胞均经不同浓度的(0 nmol/L,对照组;100 nmol/L,实验组)P物质处理48 h,Transwell实验检测细胞迁移和侵袭功能,Western blot检测细胞中上皮-间质转化蛋白质标志表达变化,实时荧光定量PCR及Western blot检测NK-1受体表达变化。结果 Hep G2和SMMC-7721两种人肝癌细胞中均有NK-1受体表达;不同浓度P物质(0、100、250、500 nmol/L)在96 h内不影响两种细胞的增殖能力(P>0.05),而在120 h 250 nmol/L和500 nmol/L P物质可抑制两种细胞增殖(P<0.05);100 nmol/L P物质明显促进两种细胞迁移和侵袭能力(P<0.05);与对照组相比,实验组细胞NK-1受体表达明显上调(P<0.05),同时,细胞中上皮-间质转化蛋白质标志E-Cadherin表达下调,N-Cadherin和Slug表达上调(P<0.05)。结论迷走神经递质P物质与人肝癌细胞中表达的NK-1受体相互作用并使其表达上调,促进细胞发生上皮-间质转化,进而提高其侵袭转移能力。
Objective To investigate the effect of substance P of vagus nerve transmitter on the invasion and metastasis of hepatocellular carcinoma cells and its related molecular mechanisms. Methods The expression of neuropeptide-1 (NK-1) receptor in two human hepatocellular carcinoma cell lines Hep G2 and SMMC-7721 was detected by real-time fluorescence quantitative PCR and agarose gel electrophoresis. Cell Titer Blue was used to detect the expression of substance P , 100, 250, 500 nmol / L) at different time points (0,24,48,72,96,120 h) on the proliferation of hepatocellular carcinoma cells. The hepatoma cells were treated with different concentrations (0 nmol / L, Group, 100 nmol / L, experimental group) for 48 h. Transwell assay was used to detect cell migration and invasion. Western blot was used to detect the expression of epithelial-mesenchymal transition protein (EGF) 1 receptor expression changes. Results The expressions of NK-1 receptor in Hep G2 and SMMC-7721 cells were both higher than those in Hep G2 and SMMC-7721 cells. The concentrations of substance P (0, 100, 250 and 500 nmol / L) did not affect the proliferation of both cells (P <0.05). The proliferation of both cell lines was inhibited by 250 nmol / L and 500 nmol / L for 120 h (P <0.05), and 100 nmol / L promoted the migration and invasion ability of both cell lines ). Compared with the control group, the expression of NK-1 receptor in the experimental group was significantly increased (P <0.05), while the expression of E-Cadherin and N-Cadherin and Slug were up-regulated P <0.05). Conclusion The vagal neurotransmitter substance P interacts with the expressed NK-1 receptor in human hepatocellular carcinoma cells and up-regulates its expression and promotes the epithelial-mesenchymal transition of cells, thereby enhancing its invasion and metastasis.