Role of 14-3-3σgene in the regulation of endotoxin/lipopolysaccharide-induced inflammatory responses in human pulmonary epithelial cells
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摘要: 目的 探讨14-3-3σ基因在调控内毒素/脂多糖(LPS)引发人肺上皮细胞炎症反应中的机制。 方法 (1)取培养的对数生长期人正常肺上皮细胞BEAS-2B,按照随机数字表法分为对照组、PCMV6-14-3-3σ组,每组3孔。对照组细胞转染空载质粒,PCMV6-14-3-3σ组细胞转染PCMV6-14-3-3σ质粒。转染48 h,蛋白质印迹法检测细胞内14-3-3σ蛋白表达。(2)取培养的对数生长期人正常肺上皮细胞BEAS-2B,按照随机数字表法分为对照组、PCMV6-14-3-3σ组、PCMV6-14-3-3σ+LPS组、LPS组,每组3孔。对照组细胞转染空载质粒孵育42 h,PCMV6-14-3-3σ组细胞转染PCMV6-14-3-3σ质粒孵育42 h,PCMV6-14-3-3σ+LPS组细胞转染PCMV6-14-3-3σ质粒42 h后加入LPS(终质量浓度1 μg/mL,下同)刺激6 h,LPS组细胞仅加入LPS刺激6 h。蛋白质印迹法检测Bax、B淋巴细胞瘤-2基因(Bcl-2)的蛋白表达,计算两者比值;流式细胞仪检测细胞凋亡率;实时荧光定量反转录PCR技术检测细胞内肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)mRNA表达量;酶联免疫吸附测定法检测细胞上清液中TNF-α、IL-1β含量。对数据行
t 检验、单因素方差分析、LSD检验。 结果 (1)与对照组(0.78±0.04)比较,转染48 h PCMV6-14-3-3σ组细胞14-3-3σ蛋白表达水平(1.05±0.03)明显升高(t =5.41,P <0.01)。(2)与对照组比较,PCMV6-14-3-3σ组细胞Bax/Bcl-2比值、细胞凋亡率、TNF-α mRNA及IL-1β mRNA表达量、细胞上清液中TNF-α及IL-1β含量均无明显变化(P >0.05),LPS组细胞上述指标均明显升高或增加(P <0.01)。与LPS组比较,PCMV6-14-3-3σ+LPS组细胞上述指标明显下降或减少(P <0.01)。 结论 14-3-3σ是调控细胞凋亡的重要因子,通过调节凋亡调控因子Bax/Bcl-2比值,抑制人肺上皮细胞凋亡,从而减轻LPS诱导的炎症反应。Abstract: Objective To explore the mechanism of 14-3-3σgene in regulating inflammatory response of human pulmonary epithelial cells induced by endotoxin/lipopolysaccharide (LPS). Methods (1) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group and PCMV6-14-3-3σgroup using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid, and cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid. The protein expression of 14-3-3σin cell was detected by Western blotting at 48 hours after transfection. (2) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group, PCMV6-14-3-3σgroup, PCMV6-14-3-3σ+ LPS group, and LPS group using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid for 42 hours. Cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in PCMV6-14-3-3σ+ LPS group were stimulated with 1 μg/mL LPS (the same final mass concentration below) for 6 hours after being transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in LPS group were stimulated by LPS for 6 hours. The protein expressions of Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting, and the ratio of Bax to Bcl-2 was calculated. Apoptotic rate was detected by flow cytometry. The mRNA expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1β) in cells were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction technique. Content of TNF-α and IL-1β in cell culture supernatant was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed witht test, one-way analysis of variance, and least significant difference test. Results (1) At 48 hours after transfection, the protein expression of 14-3-3σin cells of PCMV6-14-3-3σgroup (1.05±0.03) was significantly higher than that in control group (0.78±0.04,t =5.41,P <0.01). (2) Compared with those in control group, the ratio of Bax to Bcl-2, apoptotic rate, mRNA expressions of TNF-α and IL-1β, and content of TNF-α and IL-1β in cell supernatant in PCMV6-14-3-3σgroup showed no significant difference (P >0.05); the above-mentioned indexes of cells in LPS group were significantly higher or increased (P <0.01). Compared with those in LPS group, the above-mentioned indexes of cells in PCMV6-14-3-3σ+ LPS group were significantly lower or decreased (P <0.01). Conclusions 14-3-3σis a key factor in regulating apoptosis. It can alleviate the LPS-induced inflammatory responses by regulating the ratio of apoptotic regulators Bax to Bcl-2 and inhibiting apoptosis of human pulmonary epithelial cells.-
Key words:
- Endotoxins /
- Lipopolysaccharides /
- Inflammation /
- 14-3-3σ /
- Bax/B-cell lymphoma-2 /
- Pulmonary epithelial cell
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