脓毒症大鼠肝脏细胞TLR4和TNF-α表达及细胞凋亡研究

摘要

目的 观察脓毒症时期大鼠肝脏Toll样受体4(toll-like receptor 4,tLR4)及肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)表达及肝脏细胞凋亡情况.方法 (1)建立动物模型及分组:72只SPF级SD大鼠随机分为正常对照组、脓毒症组及脓毒性休克组,脓毒症组腹腔注射10 mg/kg内毒素(lipopolysaccharide,LPS)、脓毒性休克组腹腔注射12 mg/kg内毒素建立大鼠模型;正常对照组腹腔注射12 mg/kg生理盐水.(2)各组大鼠在注射后1h、6h、24 h处死,每时点每组各8只,采集肝脏组织标本;标本制作石蜡切片,并设计原位杂交探针序列,进行mRNA原位杂交检测;TUNEL(原位末端1转移酶标记技术)法测定细胞凋亡;结果进行定量分析,并做数据统计.结果 (1) mRNA原位杂交检测TLR4在脓毒症及脓毒症休克组大鼠肝脏表达在LPS攻击后1h、6h、24 h随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组均显著高于对照组,脓毒性休克组TLR4表达显著高于脓毒症组(P＜0.05).(2)TNF-α在LPS攻击大鼠后1h、6h、24 h随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组均显著高于对照组,脓毒性休克组均显著高于脓毒症组(P＜0.05).(3)脓毒症及脓毒性休克大鼠肝脏组织细胞凋亡情况随时间呈上升趋势,且每个时段脓毒症组、脓毒性休克组显著高于对照组,脓毒性休克组细胞凋亡显著高于脓毒症组(P＜0.05).结论 (1)TLR4及TNF-α在肝脏的高表达,尤其在脓毒性休克大鼠肝脏的高表达,进一步证实由于LPS导致机体通过TLR4介导产生TNF-α等炎症因子的过度释放,产生炎症因子的瀑布反应,引起重要脏器组织细胞凋亡,对机体造成严重影响.(2)通过阻断TLR4的表达,可能抑制脓毒症大鼠炎症因子的大量释放,对减轻机体各重要脏器的损害可能有益.%Objective To observe the period sepsis in the rat liver toll-like receptor 4 (TLR4) and Tumor necrosis factor-α(TNF-α) expression and liver cell apoptosis.Methods 72 SPF SD rats were randomly divided into three groups.Sepsis group rats were injected by intraperitoneal 10 mg/kg endotoxin lipopolysaccharide,LPS,and septic shock group rats were injected by intraperitoneal 12 mg/kg endotoxin to establish the rat model.Normal control group rats were injected by intraperitoneal 12 mg/kg saline solution.After injection 1 h,6 h,24 h,each group rats to be put to death,all the points in each group 8,and liver tissue samples was collected.Make specimens of paraffin section,and design in situ hybridization probe sequence,was used to detect the mRNA in situ hybridization.Apoptosis was detected by TUNEL in situ terminal (transferase markers) and results were analyzed.Results mRNA in situ hy bridization detection of TLR4 in sepsis and sepsis shock group expression of rat liver in LPS attack after 1 h,6 h,24 h range upward trend over time,and each time sepsis and septic shock group were significantly higher than that of control group,septic shock TLR4 expression was significantly higher than sepsis groups,(P＜0.05).TNF-α in LPS attack rats after 1 h,6 h,24 h range upward trend over time,and each time sepsis and septic shock group were significantly higher than that of control group,septic shock group were significantly higher than that of sepsis group,(P＜0.05).Sepsis and septic shock in the rat liver tissue apoptosis situation was on the rise over time,and each time sepsis and septic shock group was significantly higher than that of control group,and there were statistical significantly differences between septic shock group and sepsis group (P＜0.05).Conclusion TLR4 and TNF-α high expression in the liver,may cause serious influence to the body.By blocking the expression of TLR4,may inhibit sepsis in the rat inflammatory factor of a large number of release,to reduce the damage to the body each important viscera may be beneficial.