过表达TRPM7蛋白对HEK293细胞氧糖剥夺再氧合损伤后NF-κB活化的影响

发布时间：2018-02-10 16:43

本文关键词： TRPM Toll样受体 HEK细胞氧糖剥夺再氧合 NF-κB活化　出处：《华中科技大学学报(医学版)》2017年02期 　论文类型：期刊论文

【摘要】：目的探讨过表达瞬时受体电位通道M7(transient receptor potential melastatin type 7channel,TRPM7)蛋白对人胚肾293细胞(human embryonic kidney 293cells,HEK293)氧糖剥夺再氧合(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤后细胞核内核转录因子kappa B(nuclear factorκB,NF-κB)表达水平的影响,并研究上述变化是否通过Toll样受体4(toll-like receptor 4,TLR4)蛋白介导。方法运用重组DNA技术建立四环素调控稳定性表达TRPM7的HEK293细胞系(293-TRPM7/WT):加入四环素诱导稳定表达TRPM7的293-TRPM7/WT细胞称为Tet(+)细胞,未加入四环素的293-TRPM7/WT细胞称为Tet(-)细胞。通过免疫印迹技术鉴定上述细胞,予以氧糖剥夺1h/再氧合24h处理后,运用免疫印迹技术检测细胞核内NF-κB p65蛋白和TLR4蛋白的表达;在Tet(+)细胞OGD处理前1h加入TLR4特异性抑制剂TAK242或同体积二甲基亚砜(DMSO),使用免疫印迹技术检测细胞核内NF-κB p65蛋白表达的变化。结果 (1)Tet(-)细胞在经过OGD/R处理后,细胞核内NF-κB的表达增加(P0.05),而Tet(+)细胞在OGD/R后核内NF-κB的表达量与经过同样处理的Tet(-)细胞相比增加更为显著(P0.05);(2)Tet(-)细胞在OGD/R损伤后细胞内TLR4的表达升高(P0.05),与之相比,Tet(+)细胞在OGD/R处理后TLR4的表达量则更为升高(P0.05);(3)Tet(+)细胞经过OGD/R处理,并加入TLR4特异性抑制剂TAK242后,与未加药组或DMSO组相比,细胞核内NF-κB蛋白表达明显下降(均P0.05)。结论过表达TRPM7增加OGD/R后293-TRPM7/WT细胞内TLR4蛋白的表达,并通过TLR4介导加速OGD/R后NF-κB的活化。
[Abstract]:Objective to investigate the effect of M7 transient receptor potential melastatin type 7 channel (TRPM7) protein on the expression of nuclear transcription factor kappa B nuclear factor 魏 B (NF- 魏 B) in human embryonic kidney 293 cells (human embryonic kidney 293 cells) after oxygen-glucose deprivation and oxygen-glucose / reoxygenation / reoxygenation / OGDR / R injury, and to investigate the effects of M7 transient receptor potential melastatin type 7 channel (TRPM7) on the expression of nuclear transcription factor kappa Bnuclear factor 魏 B (NF- 魏 B) in human embryonic kidney 293 cells. To investigate whether these changes were mediated by the Toll like receptor 4toll-like receptor 4 (TLR4) protein. Methods the recombinant DNA technique was used to establish a tetracycline HEK293 cell line of stable expression of TRPM7: 293-TRPM7 / WTT cell line was induced by tetracycline to induce stable expression of TRPM7 in 293-TRPM7 / WT cell line called Tet() cells. The 293-TRPM7 / WT cells without tetracycline were identified by Western blotting technique, and the expression of NF- 魏 B p65 and TLR4 protein was detected by Western blotting after oxygen deprivation for 1 h / 24 h after reoxygenation. The expression of NF- 魏 B p65 protein in the cell nucleus was detected by immunoblotting with TLR4 specific inhibitor TAK242 or dimethyl sulfoxide dimethyl sulfoxide dimethyl sulfoxide (DMSO) at 1 hour before OGD treatment. Results after OGD/R treatment, the expression of NF- 魏 B p65 protein in the cell nucleus was detected. The expression of NF- 魏 B in the nucleus increased P0.05A, while the expression of NF- 魏 B in Tet-kappa cells after OGD/R was significantly higher than that in Tet-treated Tet-treated cells. The expression of TLR4 in the cells after OGD/R injury was significantly higher than that in Tet-treated cells. The expression of TLR4 in the cells treated with OGD/R was higher than that in the cells treated with OGD/R. After adding TAK242, a specific inhibitor of TLR4, the expression of NF- 魏 B in the nucleus decreased significantly compared with the control group or DMSO group (P 0.05). Conclusion overexpression of TRPM7 can increase the expression of TLR4 protein in 293-TRPM7 / WT cells after OGD/R, and accelerate the activation of NF- 魏 B after OGD/R by TLR4 mediated by TLR4.
【作者单位】：华中科技大学同济医学院附属普爱医院神经内科;
【基金】：国家自然科学基金资助项目(No.31300882) 武汉市卫生计生委临床医学科研项目(No.WX13C19)
【分类号】：R364.5

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