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目的探讨ＮＦＫＢ以及抗氧化剂在大鼠肺微血管内皮细胞中对ＴＮＦα加 ＬＰＳ诱导的 ｉＮＯＳ基因表达的作用及其机制。方法通过免疫组化染色ＡＢＣ法用抗内皮细胞抗体ＣＤ３１对培养的大鼠肺微血管内皮细胞进行鉴定、Ｇｎｅｓｓ法测定细胞培养上清液中的亚硝酸盐浓度以反映 ＮＯ的生成。 Ｎｏｒｔｈｅｒｎ ｂｌｏｔ结合ＲＴ－ＰＣＲ分析ｉＮＯＳｍＲＮＡ水平。ＥＭＳＡ法测定细胞核内ＮＦ。Ｂ的结合活性、结果 ＴＮＦ。（１００μ／ｍｌ）加 ＬＰＳ（１μｇ／ｍｌ）处理内皮细胞２小时后ｉＮＯＳｍＲＮＡ水平明显上升（Ｐ＜０．０５），２４小时后能明显增加 ＮＯ的生成（Ｐ＜０．０１）、用放线菌酮（１０ｍｇ／ｍｌ）或抗氧化剂毗咯烷二硫代氨基甲酸酯（ｐｙｒｒｏｌｉｄｉｎｅｄｉｔｈｉｏｃａｒｂａｍａｔｅ，ＰＤＴＣ，０．１ｍｍｏｌ／Ｌ）或氯乙酸基半胱氨酸（Ｎ－ａｃｅｔｙｌｃｙｓｔｅｉｎｅ， ＮＡＣ， ２０ｍｍｏｌ／Ｌ）处理内皮细胞有显著降低ＴＮＦ。和ＬＰＳ诱导的亚硝酸盐（ＮＯ）生成和ｉＮＯＳｍＲＮＡ表达（Ｐ＜０．０５）。抗氧化剂ＰＤＴＣ和ＮＡＣ对ＮＯ诱导生成的抑制作用呈现剂量－效应关系。 ＴＮＦα（１００／ｍｌ）加 ＬＰＳ（１＼μｍ／ｍｌ）能刺激内皮细胞中ＮＦＫＢ?
Objective To investigate the effects and mechanisms of NFKB and antioxidants on TNFα and LPS-induced iNOS gene expression in rat pulmonary microvascular endothelial cells. Methods Immunohistochemical ABC method was used to identify the cultured rat pulmonary microvascular endothelial cells with anti-endothelial cell antibody CD31. The nitrite concentration in the cell culture supernatant was measured by Gness method to reflect the production of NO. Northern blot combined with RT-PCR analysis of iNOS mRNA levels. EMSA assay of nuclear NF. B binding activity, resulting in TNF. The level of iNOS mRNA increased significantly (P <0.05) after treated with LPS (100μg / ml) and LPS (1μg / ml) for 2 hours, and significantly increased the production of NO at 24 hours (10 mg / ml) or pyrrolidinedithiocarbamate (PDTC, 0.1 mmol / L) or N-acetylcysteine (NAC, 20 mmol / L) Treatment of endothelial cells significantly reduced TNF. And LPS-induced nitrite (NO) production and iNOS mRNA expression (P <0.05). The anti-oxidants PDTC and NAC showed a dose-response relationship with the inhibition of NO induction. TNFα (100 / ml) plus LPS (1μm / ml) can stimulate NFKB in endothelial cells?