葡萄糖-6-磷酸脱氢酶缺陷对苯醌染毒K562细胞毒性的影响
Effects of Glucose-6-Phosphate Dehydrogenase Deficiency on Cytotoxicity of Benzoquinone on K562 Cells
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摘要:
目的 探讨葡萄糖-6-磷酸脱氢酶（G6PD）缺陷对苯醌（BQ）染毒人慢性髓性白血病（K562）细胞毒性的影响。
方法 用不同浓度的BQ（0、10、20、40 μmol/L）处理野生型K562细胞，应用MTT比色法检测BQ对受试细胞的增殖抑制作用，Western blot法检测G6PD蛋白表达的变化。构建
G6PD -set RNA干扰慢病毒，并感染野生型K562细胞株；以转染空载体的野生型K562细胞作为阴性对照细胞，荧光实时定量-聚合酶链反应（real-time PCR）法检测G6PD 基因的mRNA表达量。再用不同浓度的BQ处理G6PD 缺陷的K562-WT细胞（K562-G6PD△）和阴性对照细胞，检测细胞增殖抑制作用，比色法检测细胞中还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）水平。结果 MTT结果表明，与BQ浓度为0 μmol/L组相比，10、20、40 μmol/L的BQ作用24、48、72 h后，野生型K562细胞相对增殖率均明显降低（
P < 0.05）。Western blot结果显示，低剂量下（BQ浓度 < 40 μmol/L时）随着BQ浓度的增加，G6PD蛋白含量亦增加（r =0.809，P =0.008）；当BQ浓度升至40 μmol/L时，G6PD蛋白含量有所下降，但仍高于BQ浓度为0 μmol/L组（P < 0.05）。real-timePCR法检测结果显示，K562-G6PD△细胞的G6PD mRNA表达量较阴性对照降低了86.65%，表明K562-G6PD△细胞构建成功。干预G6PD后，MTT结果表明，与阴性对照细胞相比，K562-G6PD△细胞暴露于不同浓度BQ后，其相对增殖率均明显降低（P < 0.05）。比色法结果表明，当BQ浓度为20、40μmol/L时，K562-G6PD△细胞中GSH浓度明显降低，而在阴性对照细胞中，当BQ浓度为40μmol/L时GSH浓度明显降低；当BQ浓度为10μmol/L时，较阴性对照细胞相比，K562-G6PD△细胞中GSSG浓度显著升高（P < 0.05）。结论 野生型K562细胞暴露于BQ后细胞增殖受抑制，氧化产物增加的同时可能通过激活G6PD等抗氧化系统以抵抗机体所受的氧化损伤；而G6PD缺陷后，由于G6PD不能被激活，在暴露于较低剂量的BQ时即可使细胞内GSH耗竭导致GSSG在细胞内堆积而使细胞增殖抑制毒性增加。
Abstract:Objective To evaluate the effects of glucose-6-phosphate dehydrogenase (G6PD) deficiency on the cytotoxicity of benzoquinone (BQ) on K562 cells.
Methods After treatment with different concentrations of BQ (0, 10, 20, and 40 μmol/L), MTT assay was used to detect the relative growth rate of K562-WT cells, and Western blot assay was used to measure the protein expression level of G6PD. RNA interference lentivirus targeting
G6PD gene was constructed and transfected into K562-WT cells, while negative control group was transfected with empty vector. Quantitative real-time polymerase chain reaction (real-time PCR) was applied to measure the mRNA expression ofG6PD . MTT assay and colorimetric assay were used to detect the relative growth rate as well as reduced glutathione (GSH) and oxidized glutathione (GSSG) inG6PD defective K562-WT cells (K562-G6PD△) and negative control cells after treatment with different concentrations of BQ.Results The results of MTT assay indicated that the relative growth rates of K562-WT cells remarkably decreased with higher BQ concentrations compared with 0μmol/L BQ group (
P < 0.05) at 24, 48, and 72 h. The results of Western blot showed that the G6PD protein level was increased first (r =0.809,P =0.008) and then decreased when the cells were exposed to 40 μmol/L BQ, but was still higher than that of 0 μmol/L BQ group (P < 0.05). The results of real-time PCR showed theG6PD mRNA expression of K562-G6PD△ cells was decreased by 86.65% compared with the negative control cells. That suggested K562-G6PD△ cells were successfully constructed. The results of MTT assay indicated that the relative growth rate of K562-G6PD△ cells was remarkably decreased compared with the negative control cells at each concentration of BQ (P < 0.05). The results of colorimetric assay showed that the level of GSH in K562-G6PD△ cells was decreased when exposed to BQ concentrations of 20 and 40 μmol/L, while in the negative control cells the decrease occurred at 40 μmol/L. The level of GSSG in K562-G6PD△ cells increased significantly compared with the negative control cells when exposed to the BQ concentration of 10μmol/L (P < 0.05).Conclusion The results of this study suggested that the proliferation of K562-WT cells could be inhibited with exposure to BQ, while G6PD is activated to produce GSH to resist oxidative damage. So, G6PD couldn't be activated in G6PD defective cells, which might lead to GSH depletion and GSSG accumulation as well as increase the cytotoxicity when the cells are exposed to relatively low doses of BQ.
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