發(fā)布時(shí)間：2018-05-14 00:01

  本文選題：內(nèi)毒素耐受 + Toll樣受體2　； 參考：《東南大學(xué)》2005年碩士論文

【摘要】： 目的:建立內(nèi)毒素耐受小鼠模型,確定建立模型合適的LPS劑量和LPS刺激持續(xù)時(shí)間,并觀察TLR4表達(dá)與內(nèi)毒素耐受的關(guān)系。 方法:SPF級(jí)C57BL/6J小鼠隨機(jī)分組。1)LPS劑量組:分為腹腔注射LPS 0.1、0.5、1.0、1.5、2.0mg/kg組,每日一次持續(xù)一周;對(duì)照組,腹腔注射同體積生理鹽水;共6小組,每組3只。2)LPS刺激時(shí)間組:0.5mg/kg LPS連續(xù)每日腹腔注射一周、二周、三周,共3小組,每組3只動(dòng)物。觀察各組小鼠的一般情況。Western免疫印跡法測(cè)定小鼠脾臟TLR4表達(dá)變化。 結(jié)果:各組小鼠在注射不同劑量的內(nèi)毒素后,均出現(xiàn)不同程度的嗜睡、少動(dòng)等感染征象,0.1mg/kg、0.5mg/kg組在第五天癥狀好轉(zhuǎn);劑量越大,小鼠對(duì)LPS反應(yīng)越明顯。0.5mg/kg組延長(zhǎng)LPS刺激時(shí)間對(duì)小鼠日常行為無明顯影響。脾臟TLR4蛋白定量結(jié)果:與對(duì)照組比較,各LPS劑量組脾臟TLR4表達(dá)均下調(diào)(P0.05)。LPS劑量組間比較,0.1mg/kg組TLR4表達(dá)高于其它各組(P0.05)。0.5mg/kg/天LPS刺激小鼠一周、二周、三周,脾臟TLR4表達(dá)量無差異。 結(jié)論: 0.5mg/kg/天LPS、連續(xù)刺激二周,是較理想的內(nèi)毒素耐受模型。內(nèi)毒素耐受與脾臟TLR4下調(diào)有關(guān)。 目的:研究?jī)?nèi)毒素對(duì)內(nèi)毒素耐受小鼠Toll樣受體2、4及其下游信號(hào)分子Tollip在不同器官表達(dá)的影響 方法:SPF級(jí)C57BL/6J小鼠隨機(jī)分成兩大組:內(nèi)毒素耐受組(ET組),每日腹腔注射LPS 0.5mg/kg,連續(xù)二周;正常對(duì)照組(NC組),每日腹腔注射同體積生理鹽水,連續(xù)二周。第十五天,兩組動(dòng)物尾靜脈注射LPS 10mg/kg,分別于0h(注射大劑量LPS前)、1h、6h、20h處死小鼠。共8小組,每組6只。Western免疫印跡法測(cè)定小鼠脾、肝、心組織TLR4、TLR2、Tollip表達(dá)。免疫組織化學(xué)法鑒定TLR4、TLR2、Tollip組織上定位。 結(jié)果: (1)脾臟:與NC 0h組比較,ET 0h組TLR4顯著下調(diào)(P0.05);大劑量LPS攻擊后,ET各組TLR4無明顯變化, NC組TLR4在6h上調(diào),20h達(dá)到高峰。與NC 0h組比較,ET 0h組TLR2顯著下調(diào)(P0.05);大劑量LPS攻擊后,ET各組TLR2無明顯變化, NC組TLR2在1h上調(diào),20h達(dá)到高峰。與NC 0h組比較,ET 0h組Tollip顯著下調(diào)(P0.05);大劑量LPS攻擊后,ET組、NC組Tollip在1h繼續(xù)下調(diào)至幾乎不表達(dá),之后有上調(diào)趨勢(shì),但仍低于各自0h水平。免疫組化結(jié)果證實(shí),TLR2、TLR4、Tollip陽性表達(dá)均分布于脾臟髓質(zhì)區(qū)。 (2)肝臟:與NC 0h組比較,ET 0h組TLR2下調(diào)不明顯;大劑量LPS攻擊后,ET各組TLR2無明顯變化, NC組TLR2在1h迅速達(dá)到高峰,在20h有所下降但仍高于其0h水平。 與NC 0h組比較,ET 0h組Tollip無顯著下調(diào);大劑量LPS攻擊后,ET組、NC組Tollip在1h均明顯上調(diào),但ET組變化幅度略小。各組小鼠肝臟均不表達(dá)TLR4。 免疫組化結(jié)果證實(shí):Tollip陽性表達(dá)位于肝竇內(nèi)。 (3)心臟:與NC 0h組比較,ET 0h組TLR4顯著下調(diào)(P0.05);大劑量LPS攻擊后,ET各組TLR4無明顯變化,NC組TLR4在6h達(dá)到高峰。 與NC 0h組比較,ET 0h組Tollip無顯著下調(diào);大劑量LPS攻擊后,ET組、NC組Tollip在1h均明顯上調(diào),6h達(dá)到高峰,但ET組變化幅度略小,持續(xù)時(shí)間短。 各組小鼠心臟均不表達(dá)TLR2。 免疫組化結(jié)果證實(shí):TLR4陽性表達(dá)位于心肌細(xì)胞膜上,Tollip陽性表達(dá)位于心肌細(xì)胞漿內(nèi)。 結(jié)論:內(nèi)毒素耐受狀態(tài)與各器官TLR2、4表達(dá)下調(diào)有關(guān)。再次受LPS攻擊后,內(nèi)毒素耐受動(dòng)物TLR2、4表達(dá)量無變化,Tollip表達(dá)量仍有所增加,提示內(nèi)毒素耐受降低機(jī)體對(duì)LPS反應(yīng)性與TLR2、4不敏感有關(guān)。
[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.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R363

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