缺氧对肝癌血管生成拟态的影响及其机制

Hypoxia promotes vasculogenic mimicry formation in hepatocellular carcinoma

目的:研究缺氧对肝癌血管生成拟态(vasculogenic mimicry,VM)的影响及其可能机制.方法:建立基质胶Matrigel三维培养体系,观察高转移人肝癌细胞系MHCC97-H在常氧(常氧组)和缺氧(缺氧组)条件下形成VM的差异;应用RT-PCR和Western blot技术检测两组缺氧诱导因子-1α(HIF-1α)、基质金属蛋白酶(matrix metalloproteinase,MMP)-2和MMP-9的表达.选择151例肝癌组织标本,免疫组织化学方法检测VM是否存在及HIF-1α、MMP-2和MMP-9的表达.结果:三维培养48h,缺氧组能形成基质型VM或管形VM,而常氧组不形成基质型VM或形成较短的管形VM.缺氧组HIF-1α、MMP-2和MMP-9 mRNA和蛋白表达水平明显较常氧组高(mRNA:0.70±0.05vs0.51±0.10,P=0.03;0.90±0.09vs0.67±0.09,P=0.013;0.64±0.10vs0.42±0.07,P=0.01;蛋白:0.52±0.09vs0.41±0.09,P=0.035;0.63±0.11vs0.50±0.11,P=0.023;0.50±0.14vs0.38±0.09,P=0.008).151例肝癌组织标本中31例存在VM,存在VM的标本HIF-1α、MMP-2和MMP-9免疫组织化学染色综合得分较不存在VM的标本高(4.5±1.6vs3.4±1.7,P=0.001;4.5±1.6vs3.4±1.7,P=0.030;3.9±1.9vs2.9±1.8,P=0.007).结论:缺氧能促进肝癌VM的形成,诱导HIF-1α、MMP-2和MMP-9表达可能是其作用机制之一.

AIM：To investigate the effect of hypoxia on vasculogenic mimicry（VM） formation in hepatocellular carcinoma（HCC） and to explore possible mechanisms involved.METHODS：A three-dimensional cell culture system for human HCC cell line MHCC97-H was established to observe VM formation under normoxic and hypoxic conditions.Semi-quantitative reverse transcription-polymerase chain reaction（RT-PCR） and Western blot were used to detect the effect of hypoxia on the mRNA and protein expression of hypoxic inducible factor1α（HIF-1α）,matrix metalloproteinase（MMP）-2 and MMP-9,respectively.Immunohistochemistry was used to identify patterned matrix VM and investigate the expression of HIF-1α,MMP-2 and MMP-9 in 151 HCC samples.RESULTS：Patterned matrix VM or tubular VM was produced under hypoxic conditions,while cells cultured under normoxic conditions for 48 h failed to form patterned matrix VM or formed shorter tubular VM.After cells were cultured for 48 h,the mRNA and protein expression levels of HIF-1α,MMP-2 and MMP-9 were significantly higher under hypoxia than under normoxia（mRNA：0.70 ± 0.05 vs 0.51 ± 0.10,0.90 ± 0.09 vs 0.67 ± 0.09,0.64 ± 0.10 vs 0.42 ± 0.07,P = 0.03,0.013,0.01;protein;0.52 ± 0.09 vs 0.41 ± 0.09,0.63 ± 0.11 vs 0.50 ± 0.11,0.50 ± 0.14 vs 0.38 ± 0.09,P = 0.035,0.023,0.008）.Thirty-one of 151 HCC samples exhibited evidence of patterned matrix VM.The staining intensity of HIF-1α,MMP-2 and MMP-9 expression was signifi cantly higher in the VM group than in the non-VM group（4.5 ± 1.6 vs 3.4 ± 1.7,4.5 ± 1.6 vs 3.4 ± 1.7,3.9 ± 1.9 vs 2.9 ± 1.8,P = 0.001,0.030,0.007）.CONCLUSION：Hypoxia can induce VM formation in HCC by regulating the expression of HIF-1α,MMP-2 and MMP-9.