低氧环境对黑素瘤细胞增殖及LDH表达的影响

Influence of Low Oxygen Environment on Melanoma Cells Proliferation and Expression of LDH

目的探讨低氧对黑素瘤细胞增殖、乳酸脱氢酶(lactate dehydrogenase,LDH)同工酶亚基(LDH-A、LDH-*/B)蛋白表达及LDH同工酶谱的影响。方法使用氯化钴(CoCl_2)构建黑素瘤A375细胞株低氧模型。用MTT法观察CoCl_2诱导低氧下对A375细胞的增殖,探索CoCl_2诱导A375细胞增殖的适宜浓度和时间。Western blot技术检测低氧诱导因子(hypoxia-inducible factor,HIF-1α)蛋白表达水平以验证低氧模型成功;Western blot技术检测低氧对黑素瘤细胞LDH-A、LDH-B蛋白表达的影响;琼脂糖凝胶电泳技术检测低氧下LDH同工酶谱活性的变化情况。结果MTT法显示CoCl_2诱导的低氧使MM细胞的增殖能力在一定范围内随着CoCl_2作用浓度的递增和作用时间的延长而逐渐下降。其中,CoCl_2的作用浓度50、100、150、200、250μmol/L时细胞增殖无明显抑制(P>0.05)。CoCl_2对A375细胞作用的最佳浓度、时间分别是150μmol、48h。Western blot技术结果显示CoCl_2可上调A375细胞中HIF-1α、LDH-A蛋白的表达,下调LDH-B蛋白表达(P<0.05),并呈现出一定的浓度及时间范围依赖性关系;琼脂糖凝胶电泳技术检测结果显示随着低氧浓度的递增,LDH同工酶谱中LDH3、LDH4和LDH5活性逐渐增高,LDH1和LDH2下降(P<0.05)。结论适量CoCl_2可成功诱导A375细胞低氧;低氧具有促肿瘤细胞增殖效应,其可能的机制与HIF-1α、LDH-A过表达,LDH-B表达下调及LDH同工酶谱变化有关。

Objective To explore the influence of hypoxia on proliferation and the expression of the lactate dehydrogenase（LDH）in human melanoma A375 cell line. Methods The hypoxia model of melanoma cells was induced by CoCl2,then MTT assay was used to observe the effects of hypoxic on the proliferation of human melanoma cells; HIF-1α,LDH-A and LDH-B protein expression was detected by Western blotting to make sure that the model was successful; The activity of LDH isozyme was examined by agarose gel electrophoresis. Results MTT showed that the proliferation of human melanoma cells was significantly promoted by CoCl2 -induced hypoxia,in a concentration- and time-dependent way. For instance,CoCl2 could not inhibit the proliferation of A375 melanoma cells at 50,100,150,100,250 μmol/μL（P〉 0.05）. The optimal concentration and time for cell proliferation is 150μmol/L,48h. Western blotting showed that HIF-1α,LDH-A expression were up-regulated,and LDH-B protein expression was down-regulated（P〈 0.05）,both of them were dependent on concentration an incubation time.Within more hypoxic environment,the activity of LDH3,LDH4,LDH5 increased and LDH1 and LDH2 decreased（P〈0.05）.Conclusion A hypoxia model of human melanoma A375 cell line could be induced by a moderate amount of CoCl2; Hypoxia could promote tumor cell proliferation. It might be related to the overexpression of HIF-1α and LDH-A,down regulation of LDH-B expression and the changes of LDH isozyme profile.