本文選題：鐵死亡 + 膿毒癥�。� 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：目的:本課題的研究目的為通過盲腸結(jié)扎穿孔法(CLP法)建立小鼠膿毒癥模型,檢測T淋巴細(xì)胞內(nèi)鐵死亡重要指標(biāo)隨膿毒癥嚴(yán)重程度的變化情況,研究T淋巴細(xì)胞鐵死亡與膿毒癥的關(guān)系及Nrf2在膿毒癥T淋巴細(xì)胞鐵死亡中的作用。方法:雄性C57BL/6j小鼠共計(jì)51只,分為:生存分析組、假手術(shù)(對照)組、輕度膿毒癥組、重度膿毒癥組。生存分析組繼續(xù)分為假手術(shù)亞組、輕度膿毒癥亞組、重度膿毒癥亞組,觀察各時(shí)間點(diǎn)小鼠的表現(xiàn)及生存情況。假手術(shù)組小鼠在中下腹部腹中線做切口,暴露腹腔,找到盲腸后不做處理立即將盲腸回納腹腔,關(guān)腹縫合。輕度膿毒癥組、重度膿毒癥組分別在找到盲腸后在距離盲腸遠(yuǎn)端1/3和2/3交接點(diǎn)處結(jié)扎盲腸和部分血管,再用27G針頭貫穿扎孔3次后回納腹腔,關(guān)腹縫合。48h后留取小鼠的全血、腹腔灌洗液、脾臟、肺泡灌洗液、肺組織等標(biāo)本。分別用比色法和考馬斯亮藍(lán)法測定肺灌洗液中白蛋白和總蛋白含量,二者相比計(jì)算肺通透指數(shù)(LPI),觀察小鼠有無急性肺損傷發(fā)生,并測定肺組織中Nrf2的表達(dá)水平。檢測小鼠腹腔灌洗液和血清中鐵離子、轉(zhuǎn)鐵蛋白、鐵蛋白的表達(dá)水平。檢測小鼠外周血及腹腔灌洗液中T淋巴細(xì)胞數(shù)目;RT-PCR檢測各組小鼠T淋巴細(xì)胞中caspase 3、caspase 4、caspase 8、caspase 9、caspase 12、Bcl-1、Bax、CHOP mRNA的表達(dá)情況;測定各組小鼠T淋巴細(xì)胞中ROS、GSH含量;RT-PCR和Western blot法檢測各組小鼠T淋巴細(xì)胞中Nrf2的表達(dá)情況并對比分析;流式細(xì)胞儀測定各T淋巴細(xì)胞亞群凋亡比例。結(jié)果:(1)根據(jù)各亞組小鼠的生存情況用SPSS軟件進(jìn)行生存分析,結(jié)果可見小鼠死亡率隨著膿毒癥加重而明顯增加。(2)測定肺通透指數(shù)(LPI),結(jié)果示輕度膿毒癥組肺通透指數(shù)與假手術(shù)組相比無顯著差異,但重度膿毒癥時(shí)肺通透指數(shù)升高,出現(xiàn)急性肺損傷。說明膿毒癥模型構(gòu)建成功。(3)測定肺組織中Nrf2的表達(dá)水平:WB檢測肺組織中Nrf2蛋白及其磷酸化水平表達(dá)情況,結(jié)果示輕度膿毒癥組中的表達(dá)量較假手術(shù)組略有降低,而重度膿毒癥組中的表達(dá)量較假手術(shù)組明顯升高。磷酸化的Nrf2在各動物間表達(dá)差異較大,但重度膿毒癥組中的表達(dá)量較假手術(shù)組還是有所升高。RT-PCR檢測Nrf2在mRNA水平表達(dá)情況,結(jié)果示Nrf2的mRNA表達(dá)水平在輕度膿毒癥組小鼠中略有升高,且在重度膿毒癥組小鼠中顯著升高。(4)檢測小鼠腹腔灌洗液中鐵離子、轉(zhuǎn)鐵蛋白、鐵蛋白含量,結(jié)果顯示:重度膿毒癥組小鼠腹腔灌洗液中的鐵離子、鐵蛋白、轉(zhuǎn)鐵蛋白含量較假手術(shù)組均有所升高。(5)檢測小鼠血清中鐵離子、轉(zhuǎn)鐵蛋白、鐵蛋白含量,結(jié)果顯示:重度膿毒癥組小鼠血清中鐵離子的含量較假手術(shù)組顯著降低,而血清中鐵蛋白和轉(zhuǎn)鐵蛋白的含量較假手術(shù)組均有顯著升高。(6)檢測CLP建模后小鼠脾臟中各T淋巴細(xì)胞亞群數(shù)目變化,結(jié)果顯示:重度膿毒癥組小鼠脾臟中CD3、CD4、CD8的陽性率較假手術(shù)組均有顯著下降。(7)驗(yàn)證T淋巴細(xì)胞死亡是否為鐵死亡:RT-PCR檢測各組小鼠T淋巴細(xì)胞中caspase 3、caspase 4、caspase 8、caspase 9、caspase 12、Bcl-1、Bax、CHOP mRNA的表達(dá),結(jié)果可見各基因在輕度膿毒癥組中的表達(dá)均略有升高,而在重度膿毒癥組中除caspase 12外,其余各基因的表達(dá)均與假手術(shù)組和輕度膿毒癥組相比升高顯著,caspase 12雖也有升高,但各組間差異較大。輕度膿毒癥組小鼠T淋巴細(xì)胞中的ROS含量較假手術(shù)組略有升高,重度膿毒癥組小鼠T淋巴細(xì)胞中的ROS含量較假手術(shù)組顯著升高。輕度膿毒癥組和重度膿毒癥組小鼠T淋巴細(xì)胞中的GSH含量較假手術(shù)組均顯著降低。Western blot檢測各組小鼠T淋巴細(xì)胞中Nrf2的表達(dá)情況時(shí),Nrf2和p-Nrf2無法得到合適條帶。RT-PCR檢測結(jié)果示:輕度膿毒癥組小鼠T淋巴細(xì)胞中Nrf2的表達(dá)量較假手術(shù)組略有升高,重度膿毒癥組小鼠Nrf2表達(dá)量較假手術(shù)組顯著升高。用流式細(xì)胞術(shù)檢測各組小鼠T淋巴細(xì)胞凋亡比例,結(jié)果顯示:輕度膿毒癥組小鼠與假手術(shù)組相比,T淋巴細(xì)胞的凋亡比例明顯升高,且隨著膿毒癥加重,凋亡比例進(jìn)一步增加。結(jié)論:通過本課題研究,證明嚴(yán)重膿毒癥小鼠T淋巴細(xì)胞發(fā)生鐵死亡,并明確Nrf2在嚴(yán)重膿毒癥T淋巴細(xì)胞鐵死亡中具有重要作用。
[Abstract]:Objective: the purpose of this study was to establish a mouse sepsis model by cecal ligation and perforation (CLP), to detect the change of the important index of iron death in T lymphocytes with the severity of sepsis, and to study the relationship between T lymphocyte iron death and sepsis and the role of Nrf2 in the death of T lymphocytes in sepsis. Methods: male C57 The total of 51 BL/6j mice were divided into the survival analysis group, the sham operation group, the mild sepsis group and the severe sepsis group. The survival analysis group continued to divide into the sham operation subgroup, the mild sepsis subgroup and the severe sepsis subgroup, and observed the performance and survival of the mice at each time point. The sham operation group was exposed to the midline abdominal midline incision and exposure. After finding the cecum, the cecum was immediately returned to the abdominal cavity and closed to the abdomen. The mild sepsis group and the severe sepsis group ligated the cecum and part of the blood vessels at the 1/3 and 2/3 junction points of the cecum after finding the cecum, then reused the 27G needle through the ligation hole for 3 times and then returned to the abdominal cavity, and the abdomen was sutured to keep the whole blood of the mice after the closure of the abdominal suture, and the abdomen was left to keep the whole blood of mice, abdomen and abdomen. The contents of albumin and total protein in lung lavage fluid were measured by colorimetric method and Coomassie brilliant blue method respectively. The two groups compared the lung permeability index (LPI), observed the incidence of acute lung injury in mice, and measured the expression level of Nrf2 in the lung fabric. The peritoneal lavage fluid and serum of mice were detected. The expression level of iron ion, transferrin and ferritin. The number of T lymphocytes in the peripheral blood and peritoneal lavage liquid of mice was detected. The expression of caspase 3, caspase 4, caspase 8, caspase 9, caspase 12, Bcl-1, Bax, CHOP mRNA in the T lymphocytes of mice were detected by RT-PCR. Tern blot method was used to detect and compare the expression of Nrf2 in T lymphocytes of mice in each group. Flow cytometry was used to determine the percentage of T lymphocyte subsets apoptosis. Results: (1) survival analysis was carried out by SPSS software based on the survival of each subgroup of mice. The results showed that the mortality of mice increased significantly with the aggravation of sepsis. (2) detection of lung permeability. The index (LPI) showed that the lung permeability index in the mild sepsis group had no significant difference compared with the sham group, but the lung permeability index increased in the severe sepsis and the acute lung injury. (3) the expression level of Nrf2 in the lung tissue: WB detection of the expression of Nrf2 protein and its phosphorylation level in lung tissue, The expression in the mild sepsis group was slightly lower than that in the sham operation group, but the expression in the severe sepsis group was significantly higher than that in the sham operation group. The expression of phosphorylated Nrf2 was significantly different among the animals, but the expression of severe sepsis in the severe sepsis group was higher than that in the sham operation group and the expression of Nrf2 at the level of mRNA was higher than that in the sham operation group. The result was the result of the expression of mRNA. The expression level of mRNA in Nrf2 was slightly elevated in the mice of mild sepsis group and significantly increased in the severe sepsis mice. (4) the iron ions, transferrin and ferritin content in the peritoneal lavage fluid of mice were detected. The results showed that the iron, ferritin, and transferrin content in the abdominal lavage fluid of severe sepsis mice was more than that of the sham operation group. The content of iron ion, transferrin and ferritin in serum of mice was detected (5). The results showed that the content of iron ion in the serum of severe sepsis mice was significantly lower than that of sham operation group, while the content of ferritin and transferrin in serum was significantly higher than that of sham operation group (6) the detection of CLP model mice spleen in the spleen of T lymphatic thin. The changes in the number of cell subgroups showed that the positive rates of CD3, CD4 and CD8 in the spleen of the severe sepsis mice were significantly lower than those in the sham operation group. (7) whether the death of T lymphocyte was iron death: RT-PCR detection of caspase 3, caspase 4, caspase 8, caspase 9, caspase 12 in the T lymphocytes of each group. There was a slight increase in the expression of each gene in the mild sepsis group, while in the severe sepsis group, the expression of the other genes was significantly higher than that in the sham operation group and the mild sepsis group, and the caspase 12 was also elevated, but the difference was larger among the groups. The ROS content in the T lymphocyte of the mild sepsis mice was less than that of the mild sepsis group. The ROS content in T lymphocyte in the severe sepsis group was significantly higher than that of the sham operation group. The GSH content in the T lymphocyte in the mild sepsis group and the severe sepsis group was significantly lower than that in the sham group. The Nrf2 and p-Nrf2 could not be obtained when the Nrf2 expression in the T lymphocytes of each group was detected by.Western blot. The results of.RT-PCR detection in the appropriate strip showed that the expression of Nrf2 in the T lymphocyte of mice with mild sepsis was slightly higher than that in the sham operation group, and the expression of Nrf2 in the severe sepsis group was significantly higher than that in the sham operation group. The percentage of T lymphocyte apoptosis in each group was detected by flow cytometry. The results showed that the mice in the mild sepsis group and the Pseudomonas aeruginosa were in the sham operation. Compared with the group, the proportion of T lymphocyte apoptosis increased significantly, and with the aggravation of sepsis, the proportion of apoptosis was further increased. Conclusion: through this study, it is proved that the T lymphocytes of severe sepsis mice have iron death, and that Nrf2 plays an important role in the iron death of severe sepsis T lymphocyte.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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