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目的探讨微缺氧与人结肠癌细胞HT-29侵袭能力的关系及其机制。方法建立微缺氧模型,噻唑蓝(MTT)比色试验测定HT-29细胞的异质性黏附能力,transwell侵袭试验判定其侵袭游走能力,内皮细胞小管形成实验检测促进新生血管形成的能力;将HT-29细胞微缺氧处理0、6、12、24、48 h,明胶酶谱分析检测分泌的MMP-2/9活性变化,逆转录-聚合酶链反应(RT-PCR)和Western blot检测骨桥蛋白(OPN)的mRNA和蛋白表达,Western blot检测胞核内核转录因子(NF-κB/p65)蛋白表达。结果微缺氧诱导24 h后,HT-29细胞的异质性黏附能力、侵袭游走能力显著增加,明显促进人脐静脉内皮细胞(HUVECs)形成管状结构。微缺氧下6 h,MMP-2/9活性无明显变化,尔后随着微缺氧时间的延长,其活性逐渐显著上调,24 h达顶峰,48与24 h差异无统计学意义(P>0.05)。HT-29细胞在微缺氧的诱导下,其OPN的mRNA和蛋白以及胞核内NF-κB/p65蛋白的表达趋势与MMP-2/9活性变化趋势类同。结论微缺氧能诱导HT-29细胞异质性黏附能力、侵袭游走能力显著增加,MMP-2/9活性上调,促进新生血管形成而促使其向恶性表型转化。OPN- NF-κB可能是微缺氧下继HIF-I之外的另一重要调节途径,该途径与微缺氧诱导的恶性表型密切相关。
Objective To investigate the relationship between micro-hypoxia and invasiveness of human colon cancer cell line HT-29 and its mechanism. Methods Hypoxia model was established. The heterogeneous adhesion of HT-29 cells was determined by MTT colorimetric assay. The invasion and migration of HT-29 cells were determined by transwell invasion assay. The ability of endothelial cell tubule formation assay to detect neovascularization was determined. HT-29 cells were subjected to hypoxia for 0, 6, 12, 24, and 48 h. Gelatin zymography was used to detect the changes of MMP-2/9 activity. Reverse transcription polymerase chain reaction The expression of osteopontin (OPN) mRNA and protein was detected by Western blot. The expression of NF-κB / p65 protein was detected by Western blot. Results After 24 hours of micro-hypoxia induction, the heterogeneous adhesion and migration of HT-29 cells significantly increased, and promoted the formation of tubular structure of human umbilical vein endothelial cells (HUVECs). After 6 hours of micro-anoxia, the activity of MMP-2/9 did not change significantly. Afterwards, the activity of MMP-2/9 increased gradually with the prolongation of hypoxia-hypoxia, reached the peak at 24 hours and no significant difference at 48 and 24 hours (P> 0.05). Under micro-hypoxia induction, the expression of OPN mRNA and protein and the expression of NF-κB / p65 protein in HT-29 cells were similar to that of MMP-2/9. Conclusion Hypoxia can induce the heterogeneous adhesion of HT-29 cells, and significantly increase the invasiveness and migration of MMP-2/9 cells, and up-regulate the activity of MMP-2/9 and promote the formation of neovascularization. OPN-NF-κB may be another important regulatory pathway besides HIF-I under micro-hypoxia, which is closely related to the hypoxic-induced malignant phenotype.