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目的观察低氧诱导人胰腺癌细胞系(SW1990)分泌高迁移率组蛋白B1(HMGB1)作用,并探讨胞外HMGB1在胰腺癌细胞凋亡抵抗及转移中的作用。方法取对数生长期SW1990细胞分别给予以下处理:(1)常氧+PBS;(2)低氧+PBS;(3)低氧+HMGB1(100μg/L);(4)低氧+HMGB1(100μg/L)+EP(胞外HMGB1特异性抑制剂)。细胞处理72 h后,ELISA检测(1)及(2)2组细胞培养上清中HMGB1表达的差异;Annexin V/PI流式细胞术检测(2)、(3)、(4)各组细胞的凋亡率;Transwell迁移实验检测(2)、(3)、(4)各组细胞迁移能力差异;Western-blotting检测(2)、(3)、(4)各组细胞凋亡相关蛋白(Bcl-2)及迁移相关蛋白(E-cadherin、MMP-9)表达的差异。结果 ELISA结果显示低氧条件下,细胞培养上清中HMGB1表达含量升高,是常氧条件下的5倍;Annexin V/PI流式细胞术检测以上3组细胞的凋亡率分别为:(23.47±2.20)%、(6.57±1.20)%、(20.03±1.89)%;低氧+HMGB1处理组细胞凋亡比率明显低于其他2组,差异有统计学意义(P<0.05);Transwell迁移实验中各组穿至下室的细胞数分别平均为(161.3±16.0)、(399.7±13.0)、(184.3±16.0)个,差异有统计学意义(P<0.05),提示低氧条件下HMGB1能明显促进人胰腺癌SW1990细胞的迁移;Western-blotting结果显示HMGB1处理组Bcl-2及MMP-9表达增高,E-cadherin表达降低,差异有统计学意义(P<0.05)。结论低氧可以促进肿瘤细胞释放HMGB1,从而促进人胰腺癌SW1990凋亡抵抗与迁移。
Objective To observe the effect of HMGB1 secreted by hypoxia-inducible human pancreatic cancer cell line (SW1990) and to explore the role of extracellular HMGB1 in apoptosis and metastasis of pancreatic cancer cells. Methods SW1990 cells in logarithmic growth phase were treated with (1) normoxia + PBS, (2) hypoxia and PBS, (3) hypoxia and HMGB1 (100 μg / L), hypoxia and HMGB1 100 μg / L) + EP (extracellular HMGB1 specific inhibitor). The cells were cultured for 72 hours, and the difference of HMGB1 expression in the cell culture supernatants of (1) and (2) 2 groups was detected by ELISA. The cells in each group were detected by Annexin V / PI flow cytometry (2), (3) and (2), (3) and (4) in each group were detected by Transwell migration assay; Western blotting was used to detect the expression of apoptosis related proteins (2), (3) and Bcl-2 and E-cadherin (MMP- Results The results of ELISA showed that under the condition of hypoxia, the content of HMGB1 in cell culture supernatant increased 5 times as that in normoxic condition. The apoptosis rates of the above three groups were detected by Annexin V / PI flow cytometry: 23.47 ± 2.20)%, (6.57 ± 1.20)% and (20.03 ± 1.89)%, respectively. The apoptosis rate in HUVEC + HMGB1 group was significantly lower than that in the other two groups (P <0.05) In the experiment, the average number of cells in the lower compartment reached (161.3 ± 16.0), (399.7 ± 13.0) and (184.3 ± 16.0) respectively, with statistical significance (P <0.05) The results of Western-blotting showed that the expression of Bcl-2 and MMP-9 increased and the expression of E-cadherin decreased in HMGB1-treated group. The difference was statistically significant (P <0.05). Conclusion Hypoxia can promote the release of HMGB1 by tumor cells and promote the apoptosis resistance and migration of human pancreatic cancer SW1990.