Effects of Leech Hirudo on Tumor Angiogenesis and Its Mechanisms
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摘要: 目的 探讨水蛭能否通过改善肿瘤细胞缺氧微环境来抑制肿瘤血管生成及其相关机制。方法以氯化钴模拟化学缺氧，采用MTT法观察水蛭含药血清对EA.hy926细胞的增殖抑制作用；采用RT-PCR法检测缺氧诱导因子-1α（HIF-1α）mRNA、血管内皮生长因子（VEGF）mRNA的表达；Western blot法检测HIF-1α蛋白水平；Matrigel基质胶模拟体外小管形成实验检测小管生成情况。结果在缺氧条件下，水蛭含药血清能抑制EA.hy926细胞的增殖，且呈时间和剂量依耐性。水蛭含药血清在低氧时可显著抑制HIF-1α mRNA、VEGF mRNA的表达和HIF-1α蛋白水平，与阴性对照组比较，差异有统计学意义（P<0.05）；水蛭高剂量组和阳性对照组HIF-1α mRNA表达及其蛋白表达水平相当（P>0.05），而VEGF mRNA在高剂量水蛭作用后的表达更少（P<0.05）。小管形成实验中，水蛭含药血清作用后小管数目与阴性对照组相比明显减少，差异有统计学意义（P<0.05），与阳性对照组相比，差异无统计学意义（P>0.05）。结论水蛭能通过改善肿瘤缺氧微环境抑制肿瘤血管生成来发挥抗肿瘤作用，其机制可能是通过降低HIF-1α蛋白水平和mRNA的表达，以及降低由HIF-1α所介导的靶基因VEGF mRNA的表达来实现的。Abstract: Objective To investigate whether Leech hirudo could inhibit tumor angiogenesis by improving the hypoxia microenvironment and its related anti-angiogenesis mechanisms. Methods Cobalt Chloride was added in the medium to simulate the chemical hypoxia environment.We used MTT method to observe the EA.Hy926 cells proliferation.The mRNA expressions of hypoxia inducible factor 1α(HIF-1α) and vascular endothelial growth factor(VEGF) were examined by real-time PCR,and the protein level of HIF-1α was detected by Western blot.The tube formation assay was employed to detect EA.Hy926 cells angiogenesis in vitro. Results The Leech hirudo medicated-serum inhibited EA.Hy926 cells proliferation obviously in time and dose-dependent manner in the hypoxic environment.The mRNA expressions of hirudo-medicated HIF-1α and VEGF,and the protein level of HIF-1α induced by Leech serum in hypoxia were remarkably inhibited,and significant differences were observed compared with the negative group (P<0.05).The mRNA and protein of HIF-1α in the high medicated group was equal to that of the positive group (P>0.05).However,the expression of VEGF mRNA of the high medicated group decreased significantly compared with the positive group (P<0.05).The tube number declined in both low and high hirudo medicated-serum dose groups,and significant differences were observed compared with negative group (P<0.05),but not in positive group(P>0.05). Conclusion Leech hirudo could inhibit tumor angiogenesis via improving tumor hypoxia microenvironment,which may be attributed to decrease the expression of mRNA and protein level of HIF-1α,and degrade the VEGF,the downstream gene of HIF-1α mRNA expression.
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