本文關(guān)鍵詞： TRPM Toll樣受體 HEK細(xì)胞 氧糖剝奪再氧合 NF-κB活化　出處：《華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年02期 　論文類型：期刊論文

【摘要】：目的探討過表達(dá)瞬時(shí)受體電位通道M7(transient receptor potential melastatin type 7channel,TRPM7)蛋白對人胚腎293細(xì)胞(human embryonic kidney 293cells,HEK293)氧糖剝奪再氧合(oxygen-glucose deprivation/reoxygenation,OGD/R)損傷后細(xì)胞核內(nèi)核轉(zhuǎn)錄因子kappa B(nuclear factorκB,NF-κB)表達(dá)水平的影響,并研究上述變化是否通過Toll樣受體4(toll-like receptor 4,TLR4)蛋白介導(dǎo)。方法運(yùn)用重組DNA技術(shù)建立四環(huán)素調(diào)控穩(wěn)定性表達(dá)TRPM7的HEK293細(xì)胞系(293-TRPM7/WT):加入四環(huán)素誘導(dǎo)穩(wěn)定表達(dá)TRPM7的293-TRPM7/WT細(xì)胞稱為Tet(+)細(xì)胞,未加入四環(huán)素的293-TRPM7/WT細(xì)胞稱為Tet(-)細(xì)胞。通過免疫印跡技術(shù)鑒定上述細(xì)胞,予以氧糖剝奪1h/再氧合24h處理后,運(yùn)用免疫印跡技術(shù)檢測細(xì)胞核內(nèi)NF-κB p65蛋白和TLR4蛋白的表達(dá);在Tet(+)細(xì)胞OGD處理前1h加入TLR4特異性抑制劑TAK242或同體積二甲基亞砜(DMSO),使用免疫印跡技術(shù)檢測細(xì)胞核內(nèi)NF-κB p65蛋白表達(dá)的變化。結(jié)果 (1)Tet(-)細(xì)胞在經(jīng)過OGD/R處理后,細(xì)胞核內(nèi)NF-κB的表達(dá)增加(P0.05),而Tet(+)細(xì)胞在OGD/R后核內(nèi)NF-κB的表達(dá)量與經(jīng)過同樣處理的Tet(-)細(xì)胞相比增加更為顯著(P0.05);(2)Tet(-)細(xì)胞在OGD/R損傷后細(xì)胞內(nèi)TLR4的表達(dá)升高(P0.05),與之相比,Tet(+)細(xì)胞在OGD/R處理后TLR4的表達(dá)量則更為升高(P0.05);(3)Tet(+)細(xì)胞經(jīng)過OGD/R處理,并加入TLR4特異性抑制劑TAK242后,與未加藥組或DMSO組相比,細(xì)胞核內(nèi)NF-κB蛋白表達(dá)明顯下降(均P0.05)。結(jié)論過表達(dá)TRPM7增加OGD/R后293-TRPM7/WT細(xì)胞內(nèi)TLR4蛋白的表達(dá),并通過TLR4介導(dǎo)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.
【作者單位】： 華中科技大學(xué)同濟(jì)醫(yī)學(xué)院附屬普愛醫(yī)院神經(jīng)內(nèi)科;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(No.31300882) 武漢市衛(wèi)生計(jì)生委臨床醫(yī)學(xué)科研項(xiàng)目(No.WX13C19)
【分類號】：R364.5

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