内毒素耐受小鼠Toll样受体2、4及其下游分子Tollip蛋白表达变化的研究

发布时间：2018-05-14 00:01

本文选题：内毒素耐受 + Toll样受体2　；参考：《东南大学》2005年硕士论文

【摘要】： 目的:建立内毒素耐受小鼠模型,确定建立模型合适的LPS剂量和LPS刺激持续时间,并观察TLR4表达与内毒素耐受的关系。方法:SPF级C57BL/6J小鼠随机分组。1)LPS剂量组:分为腹腔注射LPS 0.1、0.5、1.0、1.5、2.0mg/kg组,每日一次持续一周;对照组,腹腔注射同体积生理盐水;共6小组,每组3只。2)LPS刺激时间组:0.5mg/kg LPS连续每日腹腔注射一周、二周、三周,共3小组,每组3只动物。观察各组小鼠的一般情况。Western免疫印迹法测定小鼠脾脏TLR4表达变化。结果:各组小鼠在注射不同剂量的内毒素后,均出现不同程度的嗜睡、少动等感染征象,0.1mg/kg、0.5mg/kg组在第五天症状好转;剂量越大,小鼠对LPS反应越明显。0.5mg/kg组延长LPS刺激时间对小鼠日常行为无明显影响。脾脏TLR4蛋白定量结果:与对照组比较,各LPS剂量组脾脏TLR4表达均下调(P0.05)。LPS剂量组间比较,0.1mg/kg组TLR4表达高于其它各组(P0.05)。0.5mg/kg/天LPS刺激小鼠一周、二周、三周,脾脏TLR4表达量无差异。结论: 0.5mg/kg/天LPS、连续刺激二周,是较理想的内毒素耐受模型。内毒素耐受与脾脏TLR4下调有关。目的:研究内毒素对内毒素耐受小鼠Toll样受体2、4及其下游信号分子Tollip在不同器官表达的影响方法:SPF级C57BL/6J小鼠随机分成两大组:内毒素耐受组(ET组),每日腹腔注射LPS 0.5mg/kg,连续二周;正常对照组(NC组),每日腹腔注射同体积生理盐水,连续二周。第十五天,两组动物尾静脉注射LPS 10mg/kg,分别于0h(注射大剂量LPS前)、1h、6h、20h处死小鼠。共8小组,每组6只。Western免疫印迹法测定小鼠脾、肝、心组织TLR4、TLR2、Tollip表达。免疫组织化学法鉴定TLR4、TLR2、Tollip组织上定位。结果: (1)脾脏:与NC 0h组比较,ET 0h组TLR4显著下调(P0.05);大剂量LPS攻击后,ET各组TLR4无明显变化, NC组TLR4在6h上调,20h达到高峰。与NC 0h组比较,ET 0h组TLR2显著下调(P0.05);大剂量LPS攻击后,ET各组TLR2无明显变化, NC组TLR2在1h上调,20h达到高峰。与NC 0h组比较,ET 0h组Tollip显著下调(P0.05);大剂量LPS攻击后,ET组、NC组Tollip在1h继续下调至几乎不表达,之后有上调趋势,但仍低于各自0h水平。免疫组化结果证实,TLR2、TLR4、Tollip阳性表达均分布于脾脏髓质区。 (2)肝脏:与NC 0h组比较,ET 0h组TLR2下调不明显;大剂量LPS攻击后,ET各组TLR2无明显变化, NC组TLR2在1h迅速达到高峰,在20h有所下降但仍高于其0h水平。与NC 0h组比较,ET 0h组Tollip无显著下调;大剂量LPS攻击后,ET组、NC组Tollip在1h均明显上调,但ET组变化幅度略小。各组小鼠肝脏均不表达TLR4。免疫组化结果证实:Tollip阳性表达位于肝窦内。 (3)心脏:与NC 0h组比较,ET 0h组TLR4显著下调(P0.05);大剂量LPS攻击后,ET各组TLR4无明显变化,NC组TLR4在6h达到高峰。与NC 0h组比较,ET 0h组Tollip无显著下调;大剂量LPS攻击后,ET组、NC组Tollip在1h均明显上调,6h达到高峰,但ET组变化幅度略小,持续时间短。各组小鼠心脏均不表达TLR2。免疫组化结果证实:TLR4阳性表达位于心肌细胞膜上,Tollip阳性表达位于心肌细胞浆内。结论:内毒素耐受状态与各器官TLR2、4表达下调有关。再次受LPS攻击后,内毒素耐受动物TLR2、4表达量无变化,Tollip表达量仍有所增加,提示内毒素耐受降低机体对LPS反应性与TLR2、4不敏感有关。
[Abstract]:Objective: to establish an endotoxin tolerance mouse model, to determine the appropriate dose of LPS and the duration of LPS stimulation, and to observe the relationship between TLR4 expression and endotoxin tolerance.
Methods: SPF class C57BL/6J mice were randomly divided into.1) LPS dose group, divided into group LPS 0.1,0.5,1.0,1.5,2.0mg/kg with LPS 0.1,0.5,1.0,1.5,2.0mg/kg daily for one week; control group, intraperitoneal injection of same volume of physiological saline, 6 groups, each group of 3.2) LPS stimulation time group: 0.5mg/kg LPS consecutive daily intraperitoneal injection of one week, two weeks, three weeks, a total of 3 groups, 3 animals in each group, 3 animals in each group. Each group was treated with a total of 3 groups. The general condition of mice in each group was observed. The expression of TLR4 in spleen of mice was detected by.Western blotting.
Results: the mice in each group were injected with different doses of endotoxin. The symptoms of somnolence and less movement were observed in different degrees. The symptoms of 0.1mg/kg and 0.5mg/kg were improved on the fifth day. The greater the dose, the more obvious the LPS reaction in the mice was the prolongation of the LPS stimulation time in the.0.5mg/kg group. The quantitative results of the spleen TLR4 protein were as follows: Compared with group LPS, the expression of spleen TLR4 in each LPS dose group was down down (P0.05).LPS dose group, TLR4 expression in 0.1mg/kg group was higher than that of other groups (P0.05).0.5mg/kg/ days, LPS stimulation mice for one week, two weeks, three weeks, the spleen TLR4 expression was no difference.
Conclusion: 0.5mg/kg/ days LPS, continuous stimulation for two weeks, is an ideal endotoxin tolerance model. Endotoxin tolerance is related to downregulation of spleen TLR4.
Objective: To study the effect of endotoxin on the expression of Toll like receptor 2,4 and its downstream signaling molecule Tollip in different organs of endotoxin tolerant mice.
Methods: SPF C57BL/6J mice were randomly divided into two groups: endotoxin tolerance group (group ET), LPS 0.5mg/kg was injected daily for two weeks, and normal control group (group NC) was injected with the same volume of normal saline daily for two weeks for two weeks. Two groups of animals were injected with LPS 10mg/kg in the tail vein, respectively, 0h (injection of large dose LPS), 1H, 6h, and death. Mice. A total of 8 groups, each group of 6.Western immunoblotting methods were used to determine the spleen, liver, and cardiac tissue of TLR4, TLR2, and Tollip. Immunohistochemical staining was used to identify TLR4, TLR2, and Tollip tissues.
Result:
(1) spleen: compared with group NC 0h, TLR4 in group ET 0h was significantly down (P0.05). After large dose of LPS attack, TLR4 had no obvious changes in ET groups, TLR4 in NC group was up and reached the peak. In group ET 0h, Tollip was significantly down (P0.05); after large dose of LPS attack, ET group, NC group Tollip continued to decrease to almost non expression in 1H, and then up trend, but still lower than the level of each 0h. Immunohistochemistry results confirmed that TLR2, TLR4, and positive expression were distributed in the medullary region of the spleen.
(2) liver: compared with the NC 0h group, the decrease of TLR2 in the ET 0h group was not obvious. After the large dose of LPS attack, there was no obvious change in TLR2 in each group of ET, and the TLR2 in the NC group reached the peak rapidly in 1H, but decreased in 20h, but still higher than that of the ET.
Compared with the NC 0h group, there was no significant reduction in Tollip in group ET 0h. After large dose of LPS attack, ET group and NC group Tollip increased obviously in 1H, but the amplitude of ET group was slightly smaller. All mice in each group did not express TLR4..
Immunohistochemistry showed that the positive expression of Tollip was located in the hepatic sinusoid.
(3) heart: compared with group NC 0h, TLR4 in ET 0h group was significantly down regulated (P0.05). After high-dose LPS attack, TLR4 did not change significantly in ET group, and NC reached peak in NC group.
Compared with the NC 0h group, there was no significant reduction in Tollip in group ET 0h. After large dose of LPS attack, ET group and NC group Tollip increased obviously in 1H, 6h reached the peak, but the amplitude of ET group was slightly smaller and the duration of NC was short.
The heart of the mice in each group did not express TLR2.
Immunohistochemical results showed that the positive expression of TLR4 was located in the myocardial cell membrane, and Tollip positive expression was located in the cytoplasm of the myocardium.
Conclusion: the state of endotoxin tolerance is related to the downregulation of TLR2,4 expression in various organs. After LPS attack, the expression of TLR2,4 in endotoxin tolerant animals is not changed, and the expression of Tollip is still increased, suggesting that endotoxin tolerance reduces the responsiveness of the body to the insensitivity of TLR2,4 to the insensitivity to LPS.

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

【参考文献】