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目的:构建携带SAA3启动子的IκBα表达载体,观察其对NF-κB活性及脂多糖(LPS)诱导的小鼠炎症反应的影响,探讨脓毒症的治疗方法。方法:离体细胞实验:离体混合培养小鼠肝细胞和库普弗细胞,分为正常组、LPS处理组和LPS+基因转染组,LPS注射24 h后,测定各组细胞上清AST、ALT、LDH和TNF-α、IL-6水平。小鼠在体实验:(1)小鼠随机分为正常组、LPS处理组和LPS+基因转染组3组(n=10),小鼠腹腔注射250μg LPS或等量生理盐水,24 h后处死,取血清和肝组织测定TNF-α、IL-6水平。(2)小鼠随机分为LPS处理组和LPS+基因转染组(n=21),0、48 h二次注射150μg LPS,首次注射后不同时点(0、2、24、48、50、72、96 h)取肝组织测NF-κB和IκBα活性,以0 h测得值作为正常对照。(3)小鼠随机分为LPS处理组和LPS+基因转染组(n=20),腹腔注射350μg LPS后,继续饲养96 h,观察各时点(0、12、24、36、48、72、96 h)小鼠生存率。结果:与LPS组相比,LPS+基因转染组共培养细胞上清中AST、LDH和TNF-α、IL-6水平均降低,但仍高于正常组(P<0.05)。与LPS组相比,LPS+基因转染组肝组织、血清中TNF-α、IL-6水平降低(P<0.05);与LPS组相比,LPS+基因转染组2、24、50、72 h时NF-κB活性明显降低,但高于正常对照(P<0.05);LPS刺激72、96 h时LPS+基因转染组小鼠的存活率高于LPS组(P<0.05)。结论:以SAA3为启动子,IκBα靶基因能够在肝脏表达,并可有效动态抑制内毒素诱导的小鼠肝脏或全身炎症过激反应。
OBJECTIVE: To construct the IκBα expression vector carrying SAA3 promoter and observe its effect on the activity of NF-κB and the inflammatory response induced by lipopolysaccharide (LPS) in mice, and to explore the treatment of sepsis. Methods: In vitro experiment: The hepatocytes and Kupffer cells were cultured in vitro and divided into normal group, LPS treatment group and LPS + gene transfection group. After 24 h LPS injection, the levels of AST, ALT, LDH and TNF-α, IL-6 levels. Mice in vivo: (1) The mice were randomly divided into three groups (n = 10): normal group, LPS treatment group and LPS + gene transfection group. The mice were injected intraperitoneally with 250μg LPS or normal saline and sacrificed 24 hours later The levels of TNF-α and IL-6 in serum and liver tissues were determined. (2) The mice were randomly divided into LPS treatment group and LPS + gene transfection group (n = 21). The second injection of 150μg LPS at 0,48 h after injection for different time points (0,2,24,48,50, 72,96 h) to detect the activity of NF-κB and IκBα in the liver tissue, measured at 0 h as a normal control. (3) The mice were randomly divided into LPS treatment group and LPS + gene transfection group (n = 20). After intraperitoneal injection of 350μg LPS, mice were kept for 96 hours, and the mice were sacrificed at each time point (0,12,24,36,48,72 , 96 h) mouse survival rate. Results: Compared with LPS group, the levels of AST, LDH, TNF-α and IL-6 in the supernatants of LPS + gene transfection group were all lower than those of LPS group (P <0.05). Compared with LPS group, the levels of TNF-α and IL-6 in liver tissue and serum of LPS + gene transfected group decreased (P <0.05). Compared with LPS group, LPS + (P <0.05). The survival rate of LPS + gene transfection group was higher than that of LPS group (P <0.05) at 72 and 96 h after LPS stimulation. Conclusion: The target gene of IκBα can be expressed in the liver with SAA3 as a promoter, and can effectively inhibit endotoxin-induced liver or systemic inflammatory response in mice.