胰腺癌细胞通过高表达核因子E2相关因子2促进巨噬细胞血管内皮生长因子表达的实验研究

目的探讨胰腺癌细胞对巨噬细胞血管内皮生长因子(VEGF)表达的影响及核因子E2相关因子2(Nrf2)在其中的作用。方法检测单一人胰腺癌细胞(PANC-1)培养组、单一巨噬细胞培养组、肿瘤细胞和巨噬细胞共培养组中各细胞VEGF表达。利用野生组、Nrf2过表达组、Nrf2敲减组中PANC-1的条件培养基刺激巨噬细胞,检测巨噬细胞VEGF的表达。将Nrf2过表达PANC-1条件培养基分为乳酸正常组,乳酸减低组和乳酸减低恢复组利用各组条件培养基刺激巨噬细胞,探究乳酸在抑制巨噬细胞VEGF表达中的作用,并且检测乳酸可能激活的巨噬细胞内的信号通路。两组间相关性采用费舍尔检验,两组间比较采用独立样本t检验,多组间比较采用单因素方差分析。结果肿瘤细胞与巨噬细胞共培养组中巨噬细胞VEGF表达高于其他各组中各细胞,差异有统计学意义(F=99.836,P<0.05)。Nrf2过表达的PANC-1条件培养基刺激组中巨噬细胞VEGF相对表达量为2.875±0.446,分泌水平为(326.744±27.507)ng/L,显著高于其他组,差异有统计学意义(F=26.952、48.996,P<0.05)。Nrf2过表达组中PANC-1乳酸分泌高于对照组,差异有统计学意义(t=5.974,P<0.05)。调节Nrf2过表达PANC-1条件培养基中乳酸浓度,利用条件培养基刺激巨噬细胞,乳酸减少组中巨噬细胞内活性氧物质(ROS)生成显著低于其他组,差异有统计学意义(F=26.952,P<0.05)。抑制巨噬细胞内ROS,抑制组VEGF表达(0.582±0.063)和分泌量(156.560±23.200)ng/L]显著减少,差异有统计学意义(t=4.791、7.333,P<0.05)。结论Nrf2通过增加胰腺癌细胞乳酸分泌诱导巨噬细胞内ROS生成,从而促进巨噬细胞VEGF表达。

Nuclear factor E2-related factor 2 overexpressed pancreatic cancer cells induced vascular endothelial growth factor expression of macrophages

Objective To investigate the effect of pancreatic cancer cells on the expression of vascular endothelial growth factor(VEGF)of macrophages and the role of nuclear factor E2-related factor 2(Nrf2)during pancreatic cancer cells and macrophages interaction.Methods The expressions of VEGF of cells in human pancreatic cancer cell(PANC-1)mono-culture group,macrophage mono-culture group,tumor cell and macrophage co-culture group were detected.Macrophages were stimulated with conditioned medium of PANC-1 from wild-type group,Nrf2-overexpression group and Nrf2-knockdown group and then the expressions of VEGF were analyzed.The conditioned medium of Nrf2-overexpressed PANC-1 was divided into normal group,lactate-decreased group and lactate-rescued group.Macrophages were stimulated by conditioned medium of each group to explore the role of lactate in the expression of VEGF in macrophages,and the related pathway was detected.Fisher test was used for correlation between the two groups,independent sample t test was used for comparison between the two groups,and one-way analysis of variance was used for multi group comparison.Results VEGF expression of macrophages in co-culture group was 10.258±1.622,which was remarkably higher than that in other groups(F=99.836,P<0.05).The expression and secretion of VEGF in macrophages stimulated by conditioned medium of Nrf2 overexpressed PANC-1 were 2.875±0.446 and(326.744±27.507)ng/L respectively,which were significantly higher than those in other groups(F=26.952,P<0.05;F=48.996,P<0.05).The lactate secretion of Nrf2-overespressed PANC-1 group was(10.387±0.686)ng/μl and significantly higher than that in control group(t=5.974,P<0.05).The production of reactive oxygen species(ROS)in macrophages in lactic acid reduced group was significantly lower than that in other groups(F=26.952,P<0.05).The expression and secretion of VEGF were 0.582±0.063 and(156.560±23.200)ng/L respectively,which were significantly decreased in the ROS inhibition group as compared with those in the control group(t=4.791,P<0.05;t=7.333,P<0.05).Conclusion Nrf2 overexpressed pancreatic cancer cells could induce ROS production in macrophages by increasing lactate secretion,thereby promoting VEGF expression of macrophages.