重症联合免疫缺陷小鼠内毒素血症巨噬细胞的生物学特性

发布时间：2018-07-05 00:32

本文选题：内毒素血症 + 脂多糖　；参考：《重庆医科大学》2011年博士论文

【摘要】：背景与目的脓毒症是严重感染、创伤、烧伤、外科大手术患者常见的并发症,进一步发展可导致脓毒症休克和多器官功能障碍综合征(MODS),是临床危重患者死亡的最主要原因之一。全球脓毒症患者总病例数约为1800万/年,美国患病人数为75万/年,欧洲为13.5万/年。全世界死亡人数超过1.4万/天,美国21.5万/年,并成为美国非心脏ICU死亡的主因。目前对脓毒症的认识及治疗措施有所改进,但脓毒症的病死率仍然居高不下,已成为现代危重病医学面临的突出难题。根本原因源于脓毒症的根本发病环节及作用机制尚未充分阐明,故而缺乏早期有效的预防与治疗措施。失控的全身炎症反应是脓毒症预后不良的重要原因之一。固有免疫细胞是炎症反应的重要参与者,其异常活化可导致失控炎症反应的发生。感染相关的噬血细胞综合征(IAHS)及重症急性呼吸道综合征(SARS)、重症H1N1等高致死性流行病毒感染性疾病,其病理生理特点亦为失控的全身炎症反应综合征。以上疾病存在着固有免疫细胞的异常活化,是疾病发生的重要原因之一,但对于何种原因导致固有免疫细胞的异常活化需要进一步研究。近年的研究显示了固有免疫细胞与适应性免疫细胞在炎症反应中相互作用的复杂性。我们的前期研究显示T细胞缺陷裸鼠感染呼吸道合胞病毒(RSV)后,炎症反应重于野生型小鼠,提示T细胞可能抑制炎症反应发生。Kim的研究显示炎症早期,淋巴细胞可抑制固有免疫细胞活化。由此,我们推测,脓毒症,IAHS及SARS等的发生可能与机体体内淋巴细胞不同程度功能缺陷导致固有免疫细胞异常活化相关。若能对淋巴细胞缺陷动物脓毒症中固有免疫细胞活化及功能特征进行研究,也许能为以上疾病的发病环节及作用机制研究提供新的思路。内毒素在革兰阴性细菌感染所致脓毒症的发病机制中扮演重要作用,甚至细菌血培养阴性时,也存在内毒素血症,提示内毒素血症可是脓毒血症的单独致病因素。内毒素具有极广泛而又复杂的生物学效应,脓毒症病理过程中出现的失控炎症反应、免疫机能紊乱、高代谢状态及多脏器功能损害均可由内毒素直接或间接触发。淋巴细胞缺陷的重症联合免疫缺陷(SCID)小鼠革兰阴性细菌感染模型的病理性免疫反应(炎症反应)取决于细菌的持续繁殖扩散和宿主的免疫反应。这一模型给研究单一免疫反应带来困难。为避免此类情况的发生,我们拟采用脂多糖(LPS)腹腔注射诱导T、B细胞缺陷重症联合免疫缺陷小鼠内毒素血症,通过观察SCID小鼠巨噬细胞生物学特性,从而揭示淋巴细胞缺陷对炎症反应的影响以及对巨噬细胞活化的调控作用及其作用可能的分子机制,为脓毒症的防治提供新的方向和理论依据。目的在LPS诱导的BALB/c小鼠和SCID小鼠内毒素血症模型基础上,比较两种小鼠炎症反应的差异,观察淋巴细胞缺陷对固有免疫应答及炎症反应的影响。方法LPS腹腔注射BALB/c小鼠和SCID小鼠后,观察小鼠的一般状况及存活率。将32只雄性BALB/c小鼠和32只雄性SCID小鼠随机分为正常对照组,LPS注射后3 h,6 h和12 h组。取小鼠的血清,肝脏及肺脏组织;用全自动生化分析仪检测两种小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)及血尿素氮(BUN)水平;用H.E染色,双盲法评估肝脏,肺脏的炎症病理损伤;用流式细胞术微球阵列法(CBA)检测两种小鼠血清TNF-α, IFN-γ, IL-10, IL-6及MCP-1的水平;用ELISA检测两种小鼠血清IL-12, IL-4及IL-17的水平及肝组织匀浆TNF-α及IL-10的水平。结果LPS诱导内毒素血症后,SCID小鼠于12～24 h均死亡(8/8),BALB/c小鼠仅1只死亡(1/8);LPS注射后12 h,SCID小鼠血清ALT、AST均高于BALB/c小鼠,两种小鼠BUN无显著差异;SCID小鼠肝脏、肺脏病理评分均高于BALB/c小鼠;LPS注射后,两种小鼠血清TNF-α,IFN-γ,IL-10,IL-6,MCP-1及IL-4的水平均显著升高,且SCID小鼠以上细胞因子水平明显高于BALB/c小鼠;LPS注射后3 h,SCID小鼠肝组织匀浆TNF-α水平高于BALB/c小鼠,注射后6 h,IL-10水平高于BALB/c小鼠。结论LPS诱导小鼠内毒素血症后,与野生型BALB/c小鼠比较,SCID小鼠分泌过量的炎性及抗炎细胞因子,出现失控的炎症反应,导致严重的脏器损伤,是SCID小鼠死亡的重要原因。固有免疫应答在缺乏淋巴细胞的状态下异常增强,可能是发生危及生命的重症全身炎症反应综合征的重要原因。第二部分脂多糖诱导的BALB/c小鼠与SCID小鼠内毒素血症腹腔巨噬细胞活化及功能的比较目的在LPS诱导的BALB/c小鼠和SCID小鼠内毒素血症模型基础上,比较BALB/c小鼠及SCID小鼠腹腔巨噬细胞活化及功能的差异。观察淋巴细胞缺陷对巨噬细胞活化及功能的影响。方法将40只雄性BALB/c小鼠和40只雄性SCID小鼠各自随机分为正常对照组,LPS注射后1 h,3 h,6 h和12 h组。取小鼠的腹腔灌洗液,腹腔巨噬细胞和小鼠脾脏NK细胞;用ELISA检测腹腔灌洗液TNF-α和IL-10的水平;用实时荧光定量PCR检测腹腔巨噬细胞TNF-α和IL-10 mRNA表达;流式细胞术检测BALB/c小鼠及SCID小鼠正常对照组及LPS注射后3 h组腹腔巨噬细胞TLR4,MHC-II,CD80,CD86及CD40的表达及LPS注射后12 h脾脏NK细胞胞内细胞因子IFN-γ水平;鸡红细胞吞噬实验检测两种小鼠正常对照组腹腔巨噬细胞吞噬功能;并提取两种小鼠正常对照组腹腔巨噬细胞体外LPS刺激培养,测定上清中TNF-α, IL-6, IL-10, IL-17及IFN-γ的水平。结果LPS注射后1 h,SCID小鼠腹腔灌洗液TNF-α水平高于BALB/c小鼠,注射后3 h,IL-10水平明显低于BALB/c小鼠;正常对照组及LPS注射后3 h,6 h和12 h组,SCID小鼠腹腔巨噬细胞TNF-αmRNA表达高于BALB/c小鼠;正常对照组及LPS注射后各时间点,SCID小鼠腹腔巨噬细胞IL-10 mRNA表达均低于BALB/c小鼠。BALB/c小鼠腹腔巨噬细胞吞噬百分率及吞噬指数均高于SCID小鼠腹腔巨噬细胞;LPS注射后,BALB/c小鼠腹腔巨噬细胞TLR4, MHC-II, CD80, CD86表达均明显升高,SCID小鼠腹腔巨噬细胞TLR4, MHC-II, CD86表达无明显变化,BALB/c小鼠腹腔巨噬细胞CD40及SCID小鼠腹腔巨噬细胞CD80,CD40均明显下降;SCID小鼠NK细胞胞内IFN-γ平均荧光值高于BALB/c小鼠NK细胞。体外实验LPS刺激20h后,SCID小鼠腹腔巨噬细胞较BALB/c小鼠腹腔巨噬细胞分泌更多的TNF-α,IL-6,但IL-10的分泌明显偏少。结论与野生型BALB/c小鼠比较,SCID小鼠腹腔巨噬细胞共刺激分子CD80、CD86的表达自发性增高,吞噬功能明显下降。经LPS刺激后,腹腔巨噬细胞TLR4和MHC-II和CD80、CD86分子表达无增高,但分泌的炎性细胞因子显著增加,且NK细胞胞内IFN-γ水平增高。以上表现是SCID小鼠内毒素血症炎症反应失控的主要特点。第三部分MKP-1在淋巴细胞对巨噬细胞活化调控中的作用目的在LPS诱导的BALB/c小鼠和SCID小鼠内毒素血症模型基础上,筛选出可能参与淋巴细胞对巨噬细胞活化调控的信号分子。于体外建立单独腹腔巨噬细胞组及腹腔巨噬细胞+pan-T细胞混合培养组模型,采用LPS刺激后,明确所筛选出分子的表达及作用。方法将40只雄性BALB/c小鼠和40只雄性SCID小鼠各自随机分为正常对照组,LPS注射后1 h,3 h,6 h和12 h组。提取腹腔巨噬细胞;采用实时荧光定量PCR检测腹腔巨噬细胞SOCS1, SOCS3及MKP-1的mRNA表达,免疫荧光检测腹腔巨噬细胞MKP-1蛋白表达。提取BALB/c小鼠腹腔巨噬细胞及脾脏pan-T细胞建立单独腹腔巨噬细胞组及腹腔巨噬细胞+pan-T细胞混合培养组模型;LPS刺激后,提取贴壁腹腔巨噬细胞及细胞培养上清;实时荧光定量PCR检测MKP-1 mRNA的表达;Western Blot检测腹腔巨噬细胞MKP-1蛋白表达水平;用ELISA检测上清中TNF-α、IL-6及IL-10的水平。结果在LPS注射后3 h及6 h,SCID小鼠腹腔巨噬细胞SOCS1 mRNA表达明显高于BALB/c小鼠;在LPS注射后1 h及12 h,BALB/c小鼠SOCS3表达明显高于SCID小鼠。正常对照组及LPS注射后, BALB/c小鼠腹腔巨噬细胞MKP-1 mRNA的表达均高于SCID小鼠腹腔巨噬细胞。体外实验,LPS刺激后,腹腔巨噬细胞与pan-T细胞共培养细胞中腹腔巨噬细胞MKP-1 mRNA及蛋白表达高于单独培养腹腔巨噬细胞;上清中TNF-α、IL-6水平明显低于单独培养巨噬细胞,IL-10水平无显著差异。结论内毒素血症小鼠淋巴细胞并非通过SOCS1和SOCS3信号分子调控巨噬细胞活化。Pan-T细胞可抑制LPS刺激下巨噬细胞炎性细胞因子释放。在LPS诱导炎症反应小鼠模型中淋巴细胞可能通过促进巨噬细胞MKP-1表达,抑制其炎性细胞因子的产生。
[Abstract]:Background and objective sepsis is a common complication of severe infection, trauma, burns, and major surgical operations. Further development can lead to septic shock and multiple organ dysfunction syndrome (MODS), which is one of the most important causes of death in critically ill patients. The total number of cases of sepsis in the world is about 18 million / year, and the number of patients in the United States is 75. Ten million / a year, Europe is 135 thousand / a year. The number of deaths in the world is more than 14 thousand / day, and the United States is the main cause of non cardiac ICU death in the United States. The current understanding and treatment of sepsis have improved, but the mortality of sepsis remains high and has become a prominent problem facing modern critical medicine. The root cause is the pus. The basic pathogenesis and mechanism of toxosis have not been fully elucidated. Therefore, early effective prevention and treatment measures are lacking.
The uncontrolled systemic inflammatory response is one of the important reasons for the poor prognosis of sepsis. The innate immune cells are important participants in the inflammatory response, and their abnormal activation can lead to the occurrence of uncontrolled inflammatory reactions. Infection related hemophagocytic syndrome (IAHS) and severe acute respiratory syndrome (SARS), severe H1N1, and so on. The pathological and pathophysiological characteristics of dyed diseases are also out of control systemic inflammatory response syndrome. The abnormal activation of innate immune cells in the above diseases is one of the important causes of the disease. But it is necessary to further study the causes of the abnormal activation of the innate immune cells. The complexity of the interaction of the immune cells in the inflammatory response. Our previous study showed that after the infection of the respiratory syncytial virus (RSV) in the T cell deficiency nude mice, the inflammatory response was heavier than the wild type mice, suggesting that the T cells may inhibit the.Kim in the inflammatory response. Therefore, we speculate that the occurrence of sepsis, IAHS and SARS may be related to the abnormal activation of innate immune cells in the body of lymphocytes in different degrees in the body. The study of the activation and function of the innate immune cells in the lymphocytic sepsis may be the link and effect of the disease. The mechanism research provides a new way of thinking.
Endotoxin plays an important role in the pathogenesis of sepsis caused by gram-negative bacterial infection. Even when the bacterial blood culture is negative, endotoxemia is also present, suggesting that endotoxemia is a separate pathogenic factor of sepsis. Endotoxin has a very extensive and complex biological effect. It is out of control in the pathological process of sepsis. Inflammatory reaction, immune dysfunction, high metabolic state and multiple organ dysfunction can be triggered directly or indirectly by endotoxin. The pathological immune response (inflammatory reaction) of the gram negative bacterial infection model of the severe combined immunodeficiency (SCID) mice depends on the continuous propagation of bacteria and the immune response of the host. This model is difficult to study the single immune response. In order to avoid this kind of situation, we intend to use LPS to induce T, B cell deficiency and severe combined immunodeficiency mice endotoxemia. By observing the biological characteristics of macrophages in SCID mice, the effects of lymphocyte defects on the inflammatory response are revealed. It also provides a new direction and theoretical basis for the prevention and treatment of sepsis.
Objective on the basis of LPS induced BALB/c mice and SCID mice endotoxemia model, the difference of inflammatory response in two mice was compared and the effects of lymphocyte defects on the inherent immune response and inflammatory response were observed.
Methods the general condition and survival rate of LPS mice were observed by intraperitoneal injection of BALB/c and SCID mice. 32 male BALB/c mice and 32 male SCID mice were randomly divided into normal control group, 3 h, 6 h and 12 h groups after LPS injection. The serum, liver and lung tissues of the mice were taken. The serum cereal rotation of two mice was detected by automatic biochemical analyzer. The levels of ammonia enzyme (ALT), glutamic grass transaminase (AST) and blood urea nitrogen (BUN) were measured by H.E staining and double blind method was used to evaluate the pathological damage of the liver and lung. The level of TNF- alpha, IFN- gamma, IL-10, IL-6 and MCP-1 in two kinds of mice was detected by flow cytometry microsphere array (CBA), and the level of serum IL-12, the level and liver of the two kinds of mice were detected by ELISA. Weave the level of TNF- alpha and IL-10 in the homogenate.
Results after LPS induced endotoxemia, SCID mice died at 12~24 H (8/8), and only 1 of BALB/c mice died (1/8); LPS injected 12 h, SCID mice serum ALT, AST were higher than that of BALB/c mice, two mice had no significant difference. The level of -10, IL-6, MCP-1 and IL-4 increased significantly, and the level of cytokines above SCID mice was significantly higher than that of BALB/c mice, and the level of TNF- a in the liver homogenate of SCID mice was higher than that of BALB/c mice after LPS injection, and the level of 6 h was higher than that of the mice after the injection.
Conclusion after LPS induced endotoxemia in mice, compared with the wild type BALB/c mice, SCID mice secreted excessive inflammatory and anti-inflammatory cytokines, resulting in uncontrolled inflammatory reaction and serious organ damage, which was an important cause of death in SCID mice. And the important cause of severe systemic inflammatory response syndrome.
The second part is the comparison of activation and function of lipopolysaccharide induced peritoneal macrophages in BALB/c mice and SCID mice with endotoxemia.
Objective to compare the activation and function of peritoneal macrophages in BALB/c mice and SCID mice on the basis of LPS induced endotoxemia model in BALB/c mice and SCID mice, and to observe the effect of lymphocyte defects on the activation and function of macrophages.
Methods 40 male BALB/c mice and 40 male SCID mice were randomly divided into normal control group, 1 h, 3 h, 6 h and 12 h groups after LPS injection. The peritoneal lavage fluid, peritoneal macrophage and spleen NK cells of mice were taken, and TNF- alpha and IL-10 of peritoneal lavage liquid were detected by ELISA; real-time fluorescent quantitative PCR was used to detect peritoneal macrophages. Expression of alpha and IL-10 mRNA; flow cytometry was used to detect the expression of TLR4, MHC-II, CD80, CD86 and CD40 of peritoneal macrophages in 3 h groups of BALB/c mice and SCID mice after LPS injection and the level of intracellular cytokines in the 12 spleen cells after LPS injection; and two mice of normal control group of peritoneal macrophages were tested by phagocytosis. Phagocytosis; two normal mice were extracted from the normal control group and cultured in vitro stimulated by LPS. The levels of TNF-, IL-6, IL-10, IL-17 and IFN- gamma in the supernatant were measured.
Results the level of TNF- alpha in peritoneal lavage fluid of SCID mice was higher than that of BALB/c mice after 1 h injection, and the level of IL-10 in SCID mice was significantly lower than that of BALB/c mice after injection, and the level of IL-10 was significantly lower than that of BALB/c mice after injection. The normal control group and 3 h, 6 h and 12 h groups after LPS injection were higher than those of the mice; the normal control group and the time points after the injection were in the abdominal cavity. The expression of IL-10 mRNA in macrophages was lower than that of BALB/c mouse.BALB/c mice. The phagocytic percentage and phagocytic index of peritoneal macrophages in.BALB/c mice were higher than that of peritoneal macrophages in SCID mice. TLR4, MHC-II, CD80, CD86 expression of peritoneal macrophages in BALB/c mice were significantly increased after LPS injection, and there was no obvious expression in the peritoneal macrophages in SCID mice. BALB/c mice peritoneal macrophages CD40 and SCID mice peritoneal macrophages CD80, CD40 significantly decreased, SCID mice NK cell IFN- gamma average fluorescence value is higher than the BALB/c mouse NK cells. There is less obvious secretion.
Conclusion compared with the wild type BALB/c mice, the co stimulatory molecule CD80 of peritoneal macrophages of SCID mice increased spontaneously and the phagocytic function decreased obviously. After LPS stimulation, the expression of TLR4 and MHC-II and CD80 in peritoneal macrophages was not increased, but the secreted inflammatory cell factor increased significantly, and the IFN- gamma level in the cell cell of NK cells increased. The above features are the main features of the inflammatory reaction of SCID mice with endotoxemia.
The third part is the role of MKP-1 in regulating lymphocyte activation.
Objective on the basis of the endotoxemia model of LPS induced BALB/c mice and SCID mice, the signal molecules that may participate in the regulation of macrophage activation were screened. The model of a mixed culture group of single peritoneal macrophage and peritoneal macrophage +pan-T cells was established in vitro. After LPS stimulation, the molecular table was clearly screened. Reach and function.
Methods 40 male BALB/c mice and 40 male SCID mice were randomly divided into normal control group. After LPS injection, 1 h, 3 h, 6 h and 12 h were used to extract peritoneal macrophages. The expression of SOCS1, SOCS3 and MKP-1 mRNA in peritoneal macrophages was detected by real-time fluorescent quantitative PCR, and the expression of peritoneal macrophage was detected by immunofluorescence. C mice peritoneal macrophages and spleen pan-T cells were established by the mixed culture group of peritoneal macrophages and +pan-T cells in peritoneal macrophages. After LPS stimulation, the peritoneal macrophages and cell culture supernatants were extracted. The expression of MKP-1 mRNA was detected by real-time fluorescent quantitative PCR; Western Blot was used to detect the expression of MKP-1 protein in peritoneal macrophages. Levels of TNF-, IL-6 and IL-10 in supernatant were detected by ELISA.
Results after LPS injection of 3 h and 6 h, the expression of SOCS1 mRNA in peritoneal macrophages of SCID mice was significantly higher than that of BALB/c mice; 1 h and 12 h after LPS injection, BALB/c mice SOCS3 expression was significantly higher than that of the mice. After LPS stimulation, the expression of MKP-1 mRNA and protein in peritoneal macrophages co cultured cells of peritoneal macrophages and pan-T cells was higher than that of peritoneal macrophages alone. The level of TNF- alpha and IL-6 in the supernatant was significantly lower than that of macrophages alone, and there was no significant difference in the level of IL-10.
Conclusion the lymphocyte of mice with endotoxemia is not mediated by SOCS1 and SOCS3 signaling molecules to regulate macrophage activation of.Pan-T cells to inhibit the release of inflammatory cytokines in macrophages under LPS stimulation. In the mouse model induced by LPS, the lymphocyte may inhibit the production of inflammatory cytokines by promoting the expression of MKP-1 in macrophages.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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