目的：以小干扰RNA沉默人眼脉络膜黑色素瘤细胞中的低氧诱导因子-1(hypoxia inducible factor-1，HIF-1)基因，在缺氧状态下观察其对人眼脉络膜黑色素瘤(human uveal melanoma cell，OCM-1)细胞中基质金属蛋白酶-2(matrix metalloproteinase-2，MMP-2)基因表达的影响。

方法：将脉络膜黑色素瘤细胞分为正常组与缺氧组，其中将缺氧组再分成单纯缺氧组、干扰组、脂质体对照组、阳性对照组和阴性对照组； 对正常组细胞用含有浓度为10%的胎牛血清和1%的双抗(青-链霉素混合液)的DMEM高糖培养基进行培养。在缺氧组人眼OCM-1细胞的培养瓶中加入100μmol/L CoCl₂以构建肿瘤细胞内部的缺氧微环境。干扰组转染HIF-1α siRNA，阴性对照组转染无义siRNA，阳性对照组转染β-actin siRNA，脂质体对照组转染空脂质体。RT-PCR检测细胞中HIF-1α和MMP-2 mRNA的表达，行Western-blot检测HIF-1α和MMP-2蛋白的表达。

结果：与正常组相比，单纯缺氧组HIF-1α蛋白的表达增高(P<0.05)，而其mRNA表达量无明显变化(P>0.05)； MMP-2 mRNA及蛋白的表达升高(P<0.05)。缺氧培养的各组之间相比，阳性对照组β-actin mRNA表达下降(P<0.05)，证实转染成功； 干扰组HIF-1αmRNA和MMP-2 mRNA和蛋白表达均下降(P<0.05)，阴性对照组、脂质体对照组各检测因素均无明显差别(P>0.05)。

结论：缺氧条件下可以提高人眼脉络膜黑色素瘤细胞中HIF-1α蛋白的表达，而且HIF-1α蛋白的表达水平可以影响MMP-2的转录与翻译，从而可能进一步影响人眼脉络膜瘤细胞的外周浸润和远处转移能力。

AIM: To silent hypoxia inducible factor-1α(HIF-1α)gene in malignant melanoma of the choroid cell by small interference RNA(siRNA)and investigate its effect on the expression of matrix metalloproteinase-2(MMP-2)in the choroid cell line human uveal melanoma cell(OCM-1)in hypoxia environment.

METHODS: OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO₂ incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl₂ was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA(siRNA+negative control+positive control), the target sequences of the HIF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HIF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM-1 cell through Lipofectamine2000. The expression of HIF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot.

RESULTS: Compared with the normal group, the expression of HIF-1α mRNA was not obviously changed(P>0.05), but the expression of HIF-1α protein and MMP-2 mRNA protein was significantly higher(P<0.05). Compared with the other hypoxia groups,β-actin mRNA expression of positive control group decreased(P<0.05), which proved successful transfection. The expression of HIF-1α mRNA and the expression of its protein and both MMP-2 mRNA and its protein was significantly lower(P<0.05). The negative control group, liposome control group had no significant difference in the detection of factors(P>0.05).

CONCLUSION: Hypoxia status may upregulate the HIF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HIF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HIF-1α siRNA.