Nrf2在脓毒症T淋巴细胞铁死亡中的作用研究

发布时间：2018-05-08 12:22

本文选题：铁死亡 + 脓毒症　；参考：《第二军医大学》2017年硕士论文

【摘要】：目的:本课题的研究目的为通过盲肠结扎穿孔法(CLP法)建立小鼠脓毒症模型,检测T淋巴细胞内铁死亡重要指标随脓毒症严重程度的变化情况,研究T淋巴细胞铁死亡与脓毒症的关系及Nrf2在脓毒症T淋巴细胞铁死亡中的作用。方法:雄性C57BL/6j小鼠共计51只,分为:生存分析组、假手术(对照)组、轻度脓毒症组、重度脓毒症组。生存分析组继续分为假手术亚组、轻度脓毒症亚组、重度脓毒症亚组,观察各时间点小鼠的表现及生存情况。假手术组小鼠在中下腹部腹中线做切口,暴露腹腔,找到盲肠后不做处理立即将盲肠回纳腹腔,关腹缝合。轻度脓毒症组、重度脓毒症组分别在找到盲肠后在距离盲肠远端1/3和2/3交接点处结扎盲肠和部分血管,再用27G针头贯穿扎孔3次后回纳腹腔,关腹缝合。48h后留取小鼠的全血、腹腔灌洗液、脾脏、肺泡灌洗液、肺组织等标本。分别用比色法和考马斯亮蓝法测定肺灌洗液中白蛋白和总蛋白含量,二者相比计算肺通透指数(LPI),观察小鼠有无急性肺损伤发生,并测定肺组织中Nrf2的表达水平。检测小鼠腹腔灌洗液和血清中铁离子、转铁蛋白、铁蛋白的表达水平。检测小鼠外周血及腹腔灌洗液中T淋巴细胞数目;RT-PCR检测各组小鼠T淋巴细胞中caspase 3、caspase 4、caspase 8、caspase 9、caspase 12、Bcl-1、Bax、CHOP mRNA的表达情况;测定各组小鼠T淋巴细胞中ROS、GSH含量;RT-PCR和Western blot法检测各组小鼠T淋巴细胞中Nrf2的表达情况并对比分析;流式细胞仪测定各T淋巴细胞亚群凋亡比例。结果:(1)根据各亚组小鼠的生存情况用SPSS软件进行生存分析,结果可见小鼠死亡率随着脓毒症加重而明显增加。(2)测定肺通透指数(LPI),结果示轻度脓毒症组肺通透指数与假手术组相比无显著差异,但重度脓毒症时肺通透指数升高,出现急性肺损伤。说明脓毒症模型构建成功。(3)测定肺组织中Nrf2的表达水平:WB检测肺组织中Nrf2蛋白及其磷酸化水平表达情况,结果示轻度脓毒症组中的表达量较假手术组略有降低,而重度脓毒症组中的表达量较假手术组明显升高。磷酸化的Nrf2在各动物间表达差异较大,但重度脓毒症组中的表达量较假手术组还是有所升高。RT-PCR检测Nrf2在mRNA水平表达情况,结果示Nrf2的mRNA表达水平在轻度脓毒症组小鼠中略有升高,且在重度脓毒症组小鼠中显著升高。(4)检测小鼠腹腔灌洗液中铁离子、转铁蛋白、铁蛋白含量,结果显示:重度脓毒症组小鼠腹腔灌洗液中的铁离子、铁蛋白、转铁蛋白含量较假手术组均有所升高。(5)检测小鼠血清中铁离子、转铁蛋白、铁蛋白含量,结果显示:重度脓毒症组小鼠血清中铁离子的含量较假手术组显著降低,而血清中铁蛋白和转铁蛋白的含量较假手术组均有显著升高。(6)检测CLP建模后小鼠脾脏中各T淋巴细胞亚群数目变化,结果显示:重度脓毒症组小鼠脾脏中CD3、CD4、CD8的阳性率较假手术组均有显著下降。(7)验证T淋巴细胞死亡是否为铁死亡:RT-PCR检测各组小鼠T淋巴细胞中caspase 3、caspase 4、caspase 8、caspase 9、caspase 12、Bcl-1、Bax、CHOP mRNA的表达,结果可见各基因在轻度脓毒症组中的表达均略有升高,而在重度脓毒症组中除caspase 12外,其余各基因的表达均与假手术组和轻度脓毒症组相比升高显著,caspase 12虽也有升高,但各组间差异较大。轻度脓毒症组小鼠T淋巴细胞中的ROS含量较假手术组略有升高,重度脓毒症组小鼠T淋巴细胞中的ROS含量较假手术组显著升高。轻度脓毒症组和重度脓毒症组小鼠T淋巴细胞中的GSH含量较假手术组均显著降低。Western blot检测各组小鼠T淋巴细胞中Nrf2的表达情况时,Nrf2和p-Nrf2无法得到合适条带。RT-PCR检测结果示:轻度脓毒症组小鼠T淋巴细胞中Nrf2的表达量较假手术组略有升高,重度脓毒症组小鼠Nrf2表达量较假手术组显著升高。用流式细胞术检测各组小鼠T淋巴细胞凋亡比例,结果显示:轻度脓毒症组小鼠与假手术组相比,T淋巴细胞的凋亡比例明显升高,且随着脓毒症加重,凋亡比例进一步增加。结论:通过本课题研究,证明严重脓毒症小鼠T淋巴细胞发生铁死亡,并明确Nrf2在严重脓毒症T淋巴细胞铁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.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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