本文選題：砷 + 肺��； 參考：《石河子大學》2017年碩士論文

【摘要】：目的:原花青素(PC)具有較強抗炎性損傷作用,探討葡萄籽原花青素(GSPE)拮抗砷誘導的肺臟炎性損傷機制及NF-κB信號通路在其中的作用,為砷中毒防治提供參考。方法:本研究以人支氣管上皮細胞(Human bronchial epithelial cells,BEAS-2B)細胞為對象,根據As_2O_3(0、5、10和20μmol/L)、GSPE(0、25和50mg/L)和BAY 11-7082(NF-κB抑制劑,抑制刺激物所誘導的IκB-α磷酸化,0和5μmol/L)的劑量分為48組。采用MTT法測量細胞的存活率,用流式細胞術測定細胞的凋亡率;依據ELISA法測定BEAS-2B細胞勻漿中IL-1β、IL-6、IL-10、TNF-α和CRP的表達水平;q RT-PCR測定BEAS-2B細胞勻漿中IκB-α、IKKα、IKKβ、NF-κBp65、NF-κBp50的m RNA水平;Western-Blot測定BEAS-2B細胞勻漿中P-IκB-α、IκB-α、IKKα/β、NF-κBp65、NF-κBp50的蛋白表達水平。多組之間的比較采用方差分析,兩兩之間的比較使用Bonferroni法。結果:1.隨著As_2O_3的染毒劑量增加,暴露砷24h時BEAS-2B的存活率下降(100%至65.26%,P均0.05),凋亡率升高(1.7%至30.4%,P均0.05);于48h時呈現出類似的趨勢。在施加BAY 11-7082后,細胞存活率升高,凋亡率下降;當使用GSPE拮抗砷時,細胞存活率升高(65.26%至83.38%,P均0.05),凋亡率下降(30.4%至13%,P均0.05)。2.與空白對照組相比,砷染毒于BEAS-2B24h時使促炎因子(IL-1β、IL-6、TNF-α和CRP),ROS和LPO的表達水平增加(P均0.05),抑炎因子(IL-10)的表達水平減少(P0.05);且隨著砷的染毒劑量增加,促炎因子,ROS和LPO的表達水平升高(P均0.05),抑炎因子下降(P0.05)。相較于砷染毒組,在施加BAY 11-7082之后發(fā)現,促炎因子,ROS和LPO的表達量明顯下降(P均0.05),而抑炎因子上升(P0.05);使用GSPE干預后,促炎因子,ROS和LPO的表達水平下降(P均0.05),而抑炎因子的表達量增加(P0.05)。3.qRT-PCR結果表明,相較于空白對照組,砷促進IKKα、IKKβ、NF-κBp65、NF-κBp50 m RNA的水平升高(P均0.05),降低IκB-αm RNA的水平(P0.05)。與砷染毒組相比較,在施加BAY 11-7082或GSPE后發(fā)現,IKKα、IKKβ、NF-κBp65、NF-κBp50 m RNA的水平降低(P均0.05),IκB-αm RNA的水平增加(P0.05)。4.Western-Blot結果顯示,同空白對照組相比較,砷可以增加P-IκB-α、IKKα/β、NF-κBp65、NF-κBp50與β-actin的比值(P均0.05),減少IκB-α與β-actin的比值(P0.05)。相較于砷染毒組,在施加BAY 11-7082或GSPE后,P-IκB-α、IKKα/β、NF-κBp65、NF-κBp50與β-actin的比值(P均0.05)減少,IκB-α與β-actin的比值增加(P0.05)。結論:砷暴露會增加BEAS-2B的凋亡率,降低存活率,可能是激活了NF-κB信號通路,誘導促炎性因子的表達水平增加,降低抑炎因子的表達量,導致炎性損傷;而GSPE可以抑制砷所致的NF-κB信號通路的激活,減少促炎性因子的表達,增加了抑炎因子的表達,表明了GSPE可以通過抑制砷所致NF-κB信號通路的激活,從而調節(jié)下游炎性因子的表達量,拮抗了砷所致的炎性損傷作用。
[Abstract]:Objective: proanthocyanidin (PC) has a strong anti-inflammatory injury effect. To explore the mechanism of GSPE antagonizing arsenic induced pulmonary inflammatory injury and the role of NF- 魏 B signaling pathway in preventing and treating arsenic poisoning. Methods: human bronchial epithelial cells BEAS-2B) cells were divided into 48 groups according to the doses of As2O3 / L (10 and 20 渭 mol / L) and BAY11-7082 (NF- 魏 B inhibitor). Cell viability was measured by MTT assay and apoptosis rate was measured by flow cytometry. The expression of IL-10 TNF- 偽 and CRP in BEAS-2B cell homogenate was determined by Elisa. The protein expression of P-I 魏 B- 偽 -taik 魏 Bp65, NF- 魏 Bp50 in BEAS-2B cell homogenate was determined by Western-Blot. The expression level of P-I 魏 B- 偽 -taik- 偽 -kapp65 NF- 魏 Bp50 protein in BEAS-2B cell homogenate was determined by Western Blot, and the expression of I 魏 B- 偽 / 尾 -NF- 魏 Bp65 NF- 魏 Bp50 protein in BEAS-2B cell homogenate was determined by Western-Blot. The analysis of variance was used for the comparison between the two groups and the Bonferroni method was used for the comparison between the two groups. The result is 1: 1. The survival rate of BEAS-2B decreased by 100% to 65.26% and the apoptosis rate increased by 1.7% to 30.4% after exposure to arsenic for 24 h, and a similar trend was observed at 48 h. After application of BAY11-7082, the cell survival rate increased and the apoptosis rate decreased, and the cell survival rate increased by 65.26% to 83.38%, and the apoptosis rate decreased by 30.4% to 0.05%, respectively, when GSPE was used to antagonize arsenic. Compared with the control group, arsenic exposed to BEAS-2B for 24 hours increased the expression levels of IL-6TNF- 偽, CRPG-Ros and LPO, and decreased the expression level of anti-inflammatory factor IL-10 (P0.05N), and with the increase of arsenic exposure dose, The expression levels of Ros and LPO were both increased (P 0.05), and those of anti-inflammatory factor were decreased (P 0.05). Compared with arsenic exposure group, after applying BAY11-7082, it was found that the expression of Ros and LPO decreased significantly, while the anti-inflammatory factor increased P0.050.After GSPE treatment, the expression of Ros and LPO decreased significantly. The expression level of Ros and LPO decreased (P 0.05), while the expression of anti-inflammatory factor increased (P 0.05). The results of qRT-PCR showed that compared with the control group, arsenic increased the level of NF- 魏 Bp65, NF- 魏 Bp50 mRNA and decreased the level of I 魏 B- 偽 mRNA. Compared with the arsenic exposed group, it was found that the level of Ike 偽 -IKK 尾 -NF- 魏 Bp65, NF- 魏 Bp50 mRNA decreased, the level of I 魏 B- 偽 mRNA increased by P0.05. 4. Western-Blot results showed that compared with the control group, arsenic could increase the ratio of P-I B- 偽 -kek 偽 / 尾 -NF- 魏 Bp65 to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 偽 to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 偽 and 尾 -actin to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 偽 and 尾 -actin to 尾 -actin (P 0.05), compared with the control group (P 0.05. 4) Western-Blot showed that arsenic could increase the ratio of P-I B- 偽 to 尾 -kek 偽 / 尾 -kappa Bp65, and decrease the ratio of I 魏 B- 偽 to 尾 -actin. Compared with arsenic-exposed group, the ratio of Ike 偽 / 尾 -NF- 魏 Bp65, NF- 魏 Bp50 to 尾 -actin was decreased after BAY11-7082 or GSPE (P 0.05) and the ratio of I 魏 B- 偽 to 尾 -actin increased by P0.05. Conclusion: arsenic exposure can increase the apoptosis rate of BEAS-2B and decrease the survival rate. It may be that arsenic exposure activates NF- 魏 B signaling pathway, induces the increase of pro-inflammatory factor expression, decreases the expression of anti-inflammatory factor, and leads to inflammatory injury. GSPE can inhibit the activation of arsenic induced NF- 魏 B signaling pathway, reduce the expression of pro-inflammatory factors, and increase the expression of anti-inflammatory factor, suggesting that GSPE can inhibit the activation of arsenic induced NF- 魏 B signaling pathway. Thus regulate the expression of downstream inflammatory factors and antagonize the inflammatory injury induced by arsenic.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R595

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本文編號：2014329