本文選題：羅氟司特 + 膿毒血癥　； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：目的:膿毒血癥是指由感染所引起的全身炎癥反應綜合征,常伴隨多器官功能障礙,具有高發(fā)病率和高死亡率的特點。羅氟司特作為一種選擇性磷酸二酯酶抑制劑,具有強大的抗炎作用。于是,本課題采用盲腸結(jié)扎穿刺(cecal ligation and puncture,CLP)的方法建立膿毒血癥小鼠模型,考察羅氟司特對膿毒血癥小鼠的作用及可能涉及的作用機制。方法:1.采用CLP方法建立膿毒血癥小鼠模型。動物分組包括:假手術(shù)對照組、CLP模型組、CLP+羅氟司特低、中、高劑量組(0.3mg/kg、1.0mg/kg、3.0 mg/kg)。在造模前7天開始灌胃給藥處理,每天一次。造模后,每天觀察小鼠一次,連續(xù)觀察7天,記錄小鼠死亡時間,觀察存活率變化;按照評分標準對小鼠狀態(tài)進行評分,觀察臨床得分的變化。采用統(tǒng)計學方法檢驗存活率的組間差異、臨床觀察得分的組間差異是否具有統(tǒng)計學意義,考察羅氟司特對膿毒血癥小鼠是否具有保護作用。2.造模24 h后,收集血液、腹腔灌洗液、肝、肺、脾,將組織進行勻漿處理,采用平板表面涂布方法對樣品進行細菌計數(shù);將血液離心后收集血漿,組織進行勻漿處理,采用ELISA方法檢測五種樣品中TNF-α、IL-6的含量;檢測血漿中ALT、AST、LDH的含量,同時對肝、肺、脾組織切片進行HE染色,在光學顯微鏡下觀察組織病理變化。觀察這些指標在造模條件下的變化,羅氟司特對這些指標的影響,考察羅氟司特對膿毒血癥小鼠的保護作用可能涉及的途徑。3.造模24 h后,收集肝臟,將肝臟進行勻漿處理,采用ELISA方法檢測肝臟中cAMP的含量,采用Western blot方法檢測cAMP下游信號分子(p-CREB、CREB)、NF-κB通路相關(guān)蛋白(IκB-α、全蛋白NF-κB p65、胞漿蛋白NF-κB p65、胞核蛋白 NF-κB p65)、p38MAPK 通路相關(guān)蛋白(p-p38MAPK、p38MAPK)、STAT3 通路相關(guān)蛋白(p-JAK1、JAK1、p-JAK2、JAK2、p-STAT3、全蛋白 STAT3、胞漿蛋白STAT3、胞核蛋白STAT3)的蛋白表達。觀察這些蛋白在造模條件下的表達情況,以及羅氟司特對這些蛋白表達的影響,考察羅氟司特對膿毒血癥小鼠的保護作用可能涉及的信號通路。結(jié)果:1.羅氟司特對膿毒血癥小鼠具有保護作用。存活率實驗結(jié)果顯示,假手術(shù)組小鼠沒有出現(xiàn)死亡,造模后小鼠的存活率明顯下降,羅氟司特處理能夠顯著提高小鼠存活率。臨床觀察得分結(jié)果顯示,假手術(shù)組的分數(shù)是最低的,模型組小鼠的評分在造模后顯著升高,羅氟司特處理可以顯著地降低評分,將評分維持在較低水平。2.羅氟司特通過作用于膿毒血癥的多個環(huán)節(jié)介導對膿毒血癥小鼠的保護作用。細菌計數(shù)結(jié)果顯示,與假手術(shù)組相比,模型組各種樣本的細菌量均顯著增高。羅氟司特處理可以顯著降低腹腔灌洗液的細菌量,即局部細菌量,也可以明顯降低血液中的細菌量,即全身擴散的細菌量。此外,羅氟司特處理還可以有效降低肝、肺、脾中的細菌量。炎癥因子結(jié)果顯示,與假手術(shù)組相比,模型組小鼠各種樣本中IL-6、TNF-α的含量均顯著升高。羅氟司特處理能夠顯著降低血液、腹腔灌洗液、肝臟中的IL-6、TNF-α水平。這提示羅氟司特能夠抑制膿毒血癥小鼠全身炎癥反應、肝臟炎癥反應。此外,羅氟司特對肺和脾的炎癥因子升高也有一定抑制作用。酶活力結(jié)果顯示,與假手術(shù)組相比,模型組小鼠AST、ALT水平顯著上升,而羅氟司特處理顯著降低ALT、AST水平,這提示羅氟司特能夠緩解膿毒血癥小鼠肝組織損傷,HE切片結(jié)果進一步證實了羅氟司特的保護作用。此外,羅氟司特還可以一定程度緩解膿毒血癥肺損傷、脾損傷。3.羅氟司特對膿毒血癥小鼠的保護作用涉及多個信號通路。首先,結(jié)果顯示,與假手術(shù)組相比,造模后的cAMP含量和CREB磷酸化顯著下降,而羅氟司特能夠顯著抑制蛋白的下降,表明羅氟司特保護作用可能與cAMP/CREB的激活有關(guān)。此外,造模后IκB-α的降解、NF-κB p65核位移、p38 MAPK磷酸化均顯著增強,而羅氟司特處理可以顯著抑制這些蛋白的變化,表明而且羅氟司特的保護作用可能與NF-κB、p38MAPK的抑制有關(guān)。同時,造模后JAK1、JAK2、STAT3的磷酸化、STAT3核位移均顯著增強,而羅氟司特處理可以顯著抑制這些蛋白的變化,表明而且羅氟司特的保護作用可能與STAT3的抑制有關(guān)。結(jié)論:1.羅氟司特能夠提高膿毒血癥模型小鼠的存活率、緩解膿毒血癥癥狀,具有保護作用。2.羅氟司對特膿毒血癥模型小鼠的保護作用可能通過降低小鼠體內(nèi)的細菌量;降低炎癥因子IL-6、TNF-α水平,抑制炎癥:降低ALT、AST、LDH水平,減輕組織損傷。3.羅氟司特對膿毒血癥模型小鼠的保護作用可能通過激活cAMP/CREB信號通路,抑制NF-κκB、p38MAPK、STAT3信號通路。
[Abstract]:Objective: sepsis is a systemic inflammatory response syndrome caused by infection, often accompanied by multiple organ dysfunction, with high morbidity and high mortality. Roflurox is a selective phosphodiesterase inhibitor and has a strong anti-inflammatory effect. Therefore, cecal ligation and punct is used in this study. Ure, CLP) to establish a mouse model of sepsis to investigate the effect and possible mechanism of rolflurox on sepsis mice. Methods: 1. the mice model of sepsis was established by CLP method. The group of animals included the sham operation control group, the CLP model group, the low, middle, and high dose group (0.3mg/kg, 1.0mg/kg, and 3 mg/kg) of CLP+. After 7 days before the model, the gavage was given to the stomach. After the model was made, the mice were observed once a day for 7 days. The time of death and the change of survival rate were recorded. The state of the mice was evaluated according to the scoring standard and the changes of clinical scores were observed. The differences in the survival rate were examined by the statistical method and the clinical observation score was observed. Whether the difference between groups was statistically significant, to investigate whether rofluusate had protective effect on sepsis in mice with.2. 24 h, collect blood, peritoneal lavage fluid, liver, lung, spleen, homogenate the tissue, use the plate surface coating method to count the bacteria, collect blood after centrifugation, and homogenate the tissue. Treatment, ELISA method was used to detect the content of TNF- alpha and IL-6 in five samples; the content of ALT, AST, LDH in plasma was detected, and the liver, lung, and spleen tissue sections were stained with HE, and the pathological changes were observed under the optical microscope. The changes of these indexes under the model conditions and the effect of roflulesite on these indexes were observed and roflulein was examined. The protective effect of sepsis in mice may be involved in the pathway.3. 24 h, the liver is collected, the liver is homogenized, and the content of cAMP in the liver is detected by ELISA method. The Western blot method is used to detect the cAMP downstream signal molecules (p-CREB, CREB) and NF- kappa B pathway Guan Danbai. Nuclear protein NF- kappa B p65), p38MAPK pathway related proteins (p-p38MAPK, p38MAPK), STAT3 pathway related proteins (p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3, whole protein, Cytoplasmic Protein, cytoplasmic nucleoprotein). The protective effect of rolflurox on sepsis mice might be involved. Results: 1. rolllice had protective effect on sepsis in mice. The results of the survival rate test showed that the mice in the sham operation group had no death, the survival rate of the mice was significantly decreased, and the rolllethe treatment could significantly improve the survival rate of mice. The score of the sham operation group was the lowest, the score of the model group was significantly higher after the model, and the rolllethe treatment could significantly reduce the score, and the score was maintained at the lower level of.2. roflurox in the protective effect of multiple rings on sepsis in sepsis mice. The count results showed that the amount of bacteria in all the samples in the model group was significantly higher than that in the sham group. Roflurox treatment could significantly reduce the amount of bacteria in the peritoneal lavage fluid, that is, the amount of bacteria in the local bacteria and the amount of bacteria in the blood, that is, the amount of bacteria in the whole body. In addition, roflulein treatment can also effectively reduce the liver and lung, The amount of bacteria in the spleen. The results of inflammatory factors showed that the levels of IL-6 and TNF- a in all the samples of the model group were significantly higher than those in the sham group. The rolluset treatment could significantly reduce the blood, peritoneal lavage fluid, IL-6 and TNF- alpha levels in the liver. This suggests that rollluset can inhibit the systemic inflammatory response of mice with sepsis, liver, and liver. In addition, roflurox also inhibited the increase of inflammatory factors in the lungs and spleen. The results of enzyme activity showed that the AST, ALT level of the model mice increased significantly compared with the sham operation group, while rolllleat treatment significantly reduced ALT and AST levels, which suggested that rolllrex could relieve the liver tissue damage in mice with sepsis and HE section. The results further confirmed the protective effect of roflurolash. In addition, rolllrex could also relieve the pulmonary injury of sepsis to a certain extent. The protective effect of.3. rollrex on sepsis mice involved multiple signal pathways. First, the results showed that the content of cAMP and the phosphorylation of CREB after the model were significantly lower than those in the sham operation group. Roflurox could significantly inhibit the decrease of protein, indicating that the protection of rofluleat may be related to the activation of cAMP/CREB. In addition, the degradation of I kappa B- alpha, the nuclear shift of NF- kappa B p65, and the phosphorylation of p38 MAPK significantly increased, while roflurox treatment could significantly inhibit the changes of these proteins, indicating and the protective effect of rofluleat. It may be related to the inhibition of NF- kappa B and p38MAPK. At the same time, after the phosphorylation of JAK1, JAK2, STAT3, the nuclear displacement of STAT3 is significantly enhanced, and roflulethe treatment can significantly inhibit the changes of these proteins, indicating that the protective effect of rofluleat may be related to the inhibition of STAT3. Conclusion: 1. roflurox can improve the sepsis model small. The survival rate of rats, relieving the symptoms of sepsis, the protective effect of.2. rofluus on the mice with sepsis may be reduced by reducing the amount of bacteria in the mice; reducing the level of inflammatory factors IL-6, TNF- alpha, and inhibiting inflammation: lowering the level of ALT, AST, LDH, and reducing the tissue injury of the.3. rofluleus on the sepsis model mice. Protective effect may inhibit NF- kappa B, p38MAPK and STAT3 signaling pathway by activating cAMP/CREB signaling pathway.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R965

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相關(guān)期刊論文 前1條

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本文編號：2104455