微缺氧对人结肠癌细胞HT-29血管生成能力及分泌基质金属蛋白酶2/9活性的影响

Moderate hypoxia up-regulated the expression of osteopontin and augmented angiogenesis and MMP-2/9 activities in colon carcinoma cell line HT-29

目的观察HT-29细胞在微缺氧下的血管生成能力,检测OPN的表达和MMP-2/MMP-9的活性变化,以探讨其向恶性表型转化的潜在机制。方法参照体内肿瘤的氧分压,建立微缺氧模型。内皮细胞小管形成试验检测体外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和48 h,明胶酶谱分析检测分泌的MMP-2/MMP-9活性变化,RT-PCR和Western blot检测OPN的mRNA和蛋白表达。结果微缺氧组内皮细胞形成的管状结构数为(28.5±3.6),明显多于对照组(12.4±2.8),其差异具有显著性(P<0.01)。微缺氧6 h,MMP-2/MMP-9活性无明显变化,随着微缺氧时间的延长,其活性逐渐上调,24 h达顶峰,48 h与24 h差异无显著性。HT-29细胞在微缺氧的诱导下,其OPN mRNA和蛋白的表达趋势与MMP-2/9活性变化趋势类同。结论微缺氧能诱导HT-29细胞促进新生血管形成,上调MMP-2/MMP-9活性而促进其向恶性表型转化。该实验检测到在微缺氧下OPN和MMP-2/MMP-9普遍上调,且时限同步。因此,笔者推测:OPN可能是微缺氧下继HIF-1α之外的另一重要调控因子,该调控因子与微缺氧诱导的恶性表型密切相关。

【objective】 To investigate correlation between moderate hypoxia and tumor angiogenesis in colon carcinoma cell line HT-29,detect the expression of osteopontin and activities of MMP-2/9 so as to explore its latent mechanism of maliglant transformation.【Methods】 The moderate hypoxia model was established in accordance with the PO2 of tumor in vivo.Tube formation of human umbilical vein endothelial cells（HUVECs） was adopted to evaluate the ability of tumor angiogenesis in vitro.After 0,6,12,24 or 48 h of moderate hypoxia,MMP-2/9 activities secreted by HT-29 cells were assessed by gelatin zymography,and osteopontin mRNA and protein levels detected by RT-PCR and Western blot respectively.【Result】 The numbers of tubes formed by HUVECs were： control group with（12.4±2.8）,moderate hypoxia group with（28.5±3.6） and showed significant difference.MMP-2/9 activities under moderate hypoxia at 6h showed no significant change as compared with that under normoxia,then gradually up-regulated and reached the peak at 24 h,there was no significant difference between hypoxia for 24 h and 48 h.The expression of osteopontin mRNA,protein were similar to that of MMP-2/9 under moderate hypoxia.【Conclusion】 Moderate hypoxia could induce malignant transformation in HT-29 cells through up-regulating tumor angiogenesis and MMP-2/9 activities.There was a general and synchronous up-regulation of osteopontin and MMP-2/9 in a time-dependent manner.OPN may be an important modulator besides HIF-1α under hypoxic condition,which involves malignant phenotype induced by moderate hypoxia.