【作者】 刘伟；

【导师】 华树成；

【作者基本信息】 吉林大学 ， 内科学， 2016， 博士

【摘要】 背景:重症社区获得性肺炎(severe community-acquired pneumonia,SCAP)病死率高、预后差,导致病人死亡的重要原因是感染,及感染后引起的全身炎症反应,继而引起脓毒症(sepsis)、严重脓毒症(severe sepsis)、脓毒症休克(sepsis shock),引发多器官功能不全。免疫系统多参与参与其中,所以有关重症肺炎免疫学机制的研究已引起了国内外研究者的极大关注。树突状细胞(dendritic cells,DC)是人体免功系统的重要组成部分。作为抗原呈递细胞,它们启动并控制针对呼吸道病原体的适应性免疫应答。此外,它们在先天免疫系统的各种功能,缩小了先天和适应性免疫之间的间隙。然而,重症社区获得性肺炎患者的外周血树突细胞亚群及其表型的变化及其作用,目前仍不十分明确。目的:通过观察研究重症社区获得性肺炎患者外周血树突细胞表型及其相关功能分子的变化,分析其与重症肺炎患者临床指标间的相关性。此外,通过观察不同时间点重症肺炎模型小鼠脾脏、肺脏树突细胞的变化,探讨树突状细胞(DCs)亚群及表型在重症社区获得性肺炎早期免疫亢进及晚期免疫抑制阶段中的变化及其作用。方法:采用流式细胞术检测SCAP患者及健康对照外周血中m DC和p DC的频数及细胞表面共刺激分子CD80、CD86和共抑制分子PD-L1的表达水平,并分析DC频数与SCAP患者氧合指数(Pa O2/Fi O2)、血乳酸(Lac)的相关性。将72只雄性SPF昆明小鼠随机被分成重症肺炎组(n=36)和健康对照组(n=36),每组再随机分成6组(4h组、12h组、1d组、3d组、5d组、7d组),重症肺炎组、健康对照组每小组均有6只小鼠。通过向气管内注射肺炎克雷伯杆菌的方法,制作重症肺炎小鼠模型。以等量生理盐水替代肺炎克雷伯杆菌液按上述时间注射。按不同时间点分批处死每组小鼠,摘取小鼠的双肺和脾脏,按不同组别分别进行福尔马林固定、超低温冻存、酶消化或匀浆处理。HE染色法检测重症肺炎组不同时间点小鼠脾脏病理变化,免疫组织化学法观察小鼠脾脏组织中CD205、CD86树突细胞的分布,流式细胞术检测测定CDllc/CD86标记阳性细胞的表达率和外周血中T细胞亚群的变化,应用末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术(TUNEL)对不同时间点的重症肺炎组小鼠脾脏树突细胞进行凋亡原位检测。通过ELISA方法测定小鼠们血清中和肺组织匀浆中的TNF-α浓度。采用光镜观察小鼠肺组织的大体病理变化,并对其进行CD86、CD205免疫组化,CDllc免疫荧光技术标记小鼠肺组织中的DCs。密度梯度离心法分离小鼠肺组织中单个核细胞,运用MACS磁珠分选小鼠肺组织中单个核细胞中的DCs,通过流式细胞学技术进行DCs的鉴定和DCs表面细胞表型的鉴定。采用ELISA方法测定DCs孵育过的上清液中细胞因子IL-10的浓度。结果:(1)SCAP患者外周血中树突细胞亚群m DC和p DC相对健康对照组其表达频数明显下降(患者组分别为0.37±0.19和0.19±0 12,对照组为0.51±0.18和0.29±0.13,P<0.05)。(2)重症肺炎患者外周血m DC频数与氧合指数(Pa O2/Fi O2)、血乳酸(Lac)水平呈负相关(r=-0.5878,P<0.0001;r=-0.4628,P=0.003)。(3)SCAP患者外周血中树突细胞表面的共刺激分子CD80、CD86相对健康对照组其表达频数下降。相关共抑制分子PDL1相对健康对照组表达频数升高。(4)正常小鼠脾脏DC含量较少,主要分布在脾脏边缘区。实验4h重症肺炎小鼠脾脏DC细胞组显著增生且功能活跃,CD86+/CDIlc+DC占脾脏单个核细胞的比例明显增加,与正常组比较有极显著差异(P<0.01);CD205细胞显著增加,尤以边缘区显著,呈片状分布,正常对照组比较差异有显著性(P<0.01);CD86细胞含量显著增加,以边缘最丰富,弥漫分布,可见DC s棕黄色阳性染色的胞膜呈树枝状向周围延伸,与正常对照组比较差异有显著性(P<0.01);白髓和红髓中有散在分布小片状TUNEL阳性细胞数目明显增多,呈大片、灶状或巢状分布;CD4+T细胞含量明显下降,而CD8+T细胞含量明显增加,CD4+/CD8+比值降低,与正常对照组比较差异具有显著性(P均<0.05)。(5)重症肺炎模型小鼠12~24h组CD86+/CDIlc+DC比例略有减少,与正常组比较仍有显著差异(P<0.05);CD205细胞较4 h组略减少,分布较稀疏,但仍明显多于正常对照组(P<0.05);CD4+T细胞含量继续下降至最低点,而CD8+T细胞含量也减少,CD4+/CD8+比值降低,与正常对照组比较差异具有显著性(P均<0.01)。3~5d组CD86+/CDllc+DC表达比例接近正常组;CD205细数接近正常对照组。(6)重症肺炎模型小鼠3~5d组脾脏凋亡细胞较12~24h减少,略多于正常对照组;3-5d CD4+T细胞含量增加,CD4+/CD8+比值回升,但仍低于正常对照组。(7)重症肺炎模型小鼠5~7d组脾脏CD86+/CDIlc+DC表达比例明显下降,与正常组比较有极显著差异(P<0.01);CD205细胞含量再次明显增加,呈片状弥漫分布,与正常对照组和实验组3~5d比较差异具有显著性(P<0.05);CD86胞膜和部分胞浆呈深棕色,未见树枝状胞膜突起,阳性细胞含量较3~5d组略增加,但远不如4h增加明显,CD+86细胞数与正常对照组和实验组3~5d比较差异均无显著性;5~7d凋亡细胞再次增加,白髓和红髓中见大量TUNEL标记阳性的凋亡细胞;CD4+T细胞含量再次减少,而CD8+T细胞含量明显增加,CD4+/CD8+比值倒置,与正常对照组比较差异具有显著性(P均<0.01)。(8)重症肺炎模型模型小鼠3-5d组,肺组织匀浆液和血清TNF-α含量较对照组显著上升约3-5倍,具有极显著统计学差异(P<0.01)。(9)重症肺炎小鼠模型3-5d组、5-7d组,DCs孵育上清液中IL-10水平较对照组明显上升,统计学差异显著(P<0.05)。(10)重症肺炎小鼠模型,各组肺组织CDllc+DCs数量明显增多,12-24h组CDllc+细胞突起明显增多,集中分布在血管旁及肺泡间隔中,5-7d组CDllc+细胞达到最高值,但细胞结构模糊,部分细胞凋亡。(11)重症肺炎小鼠模型,CD205阳性细胞数在4h组与正常对照组差异无统计学意义(P>0.05),而12-24h组显著升高,与正常对照组差异具有统计学意义(P<0.01),CD205与CD11c比值明显升高至1:1左右,5-7d组,CD205阳性细胞数比较12-24h组显著下降(P<0.05)。(12)重症肺炎小鼠模型,12-24h组,CD86阳性细胞显著升高,与正常对照组和5-7d组比较差异具有统计学意义(P<0.01)。CD86阳性细胞在5-7d较12-24h组明显下降,差异具有显著意义(P<0.05)。结论:(1)重症社区获得性肺炎患者外周血树突细胞亚群明显减低。DCs表面共刺激分子CD80、CD86表达较健康对照组明显下降,提示SCAP患者外周血树突细胞处于未成熟状态,导致此类患者抗原呈递功能下降,免疫应答能力不足,机体抗感染能力降低。DCs表面共抑制分子PD-L1表达增加,表明SCAP患者存在免疫抑制,是导致其感染控制不佳,甚至发生继发感染的重要原因。(2)重症肺炎小鼠脾脏DCs经历了由早期免疫增强到晚期免疫抑制的演变过程,感染后早期脾脏树突状细胞免疫活化和晚期的免疫抑制与SCAP病情密切相关。(3)重症肺炎小鼠早期,肺间质DCs大量募集、活化,进而导致免疫反应亢进,炎症反应失调。重症肺炎小鼠晚期,肺间质DCs活性减低(表达CD205和CD86比例下降),通过分泌大量负反馈调节细胞因子IL-10,诱导并促进了免疫抑制阶段的发生、发展。更多还原

【Abstract】 Background: Severe community-acquired pneumonia(severe community- acquired pneumonia, SCAP) high mortality, poor prognosis, a major cause of death of patients is infection, and systemic inflammatory response after infection, and then cause sepsis(sepsis),severe sepsis(severe sepsis), septic shock(sepsis shock), triggered multiple organ dysfunction. To participate in the immune system involved, so the study of severe pneumonia immunological mechanisms has attracted great attention from researchers.Dendritic cells(dendritic cells, DC) is the body for Free power system is an important component. As antigen presenting cells, which initiate and control the adaptive immune response against respiratory pathogens. In addition, they function in a variety of the innate immune system, reducing the gap between innate and adaptive immunity. However, changes in severe community acquired pneumonia and its role in peripheral blood dendritic cell subsets and their phenotype is still not very clear.Objective: observational study of patients with severe community-acquired pneumonia in peripheral blood dendritic cell phenotype and its associated functional molecules, analyze the correlation with clinical indicators of severe pneumonia among patients. In addition, by observing the different time points of severe pneumonia model mice spleen, lung changes in dendritic cells, and to explore dendritic cells(DCs) and phenotypic subgroup of severe community-acquired pneumonia in early immune hyperthyroidism and late stages of immunosuppression and its effects.Methods: Flow cytometry SCAP patients and healthy controls frequency and peripheral blood cell surface m DC and p DC expression levels of costimulatory molecules CD80, CD86 and co-inhibitory molecule PD-L1, and to analyze the frequency and DC oxygenation in patients with SCAP index(Pa O2 / Fi O2), blood lactate(Lac) correlation. The72 male SPF Kunming mice were randomly divided into severe pneumonia group(n = 36)and healthy controls(n = 36), each group was randomly divided into six groups(4h group,12 h group, 1d group, 3d group, 5d group, 7d group), severe pneumonia group and the healthy control group were 6 mice per group. By intratracheal injection of Klebsiella pneumoniae method to produce a mouse model of severe pneumonia. In saline alternative Klebsiella pneumoniae was at the hour injection. Sacrificed at different time points in each group of mice, the removal of the lungs and spleen of mice, according to the different groups separately formalin-fixed, cryopreservation, enzymatic digestion or homogenized. Spleen pathological changes detected severe pneumonia group HE staining of mice at different time points, immunohistochemistry was observed in the spleen of mice CD205, CD86 distribution of dendritic cells, flow cytometry assay expression CDllc / CD86 positive cells mark and changes in peripheral blood T cell subsets, with terminal deoxyribonucleotide transferase-mediated biotinylated d UTP nick labeling technique(TUNEL) of severe pneumonia at different time points mice spleen dendritic cells undergo apoptosis original bit detection. Determination of TNF-α concentration in serum of mice were lung homogenates and by the ELISA method. The mice were observed by light microscopy gross pathological changes in lung tissue, and its CD86, CD205 immunohistochemistry, CDllc immunofluorescence labeled mouse lung tissue DCs. Density gradient centrifugation mouse lung tissue mononuclear cells using MACS MACS mouse lung tissue mononuclear cells DCs, DCs were identified identification and DCs cell surface phenotype by flow cytometry techniques. Lung low density mononuclear cells isolated by density gradient centrifugation,macs sorting DCS flow cytometry DCS identification and immunophenotypic analysis.Detection of IL-10 content in DCs cells by ELISA assay.Results:(1) SCAP peripheral blood dendritic cell subsets m DC and p DC relatively healthy control group significantly decreased the expression of frequency(patient group were 0.37 ±0.19 and 0.19 ± 0 12, the control group was 0.51 ± 0.18 and 0.29 ± 0.13, P <0.05).(2) severe pneumonia in patients with m DC frequency and oxygenation index(Pa O2 /Fi O2), blood lactate(Lac) was negatively correlated(r =-0.5878, P <0.0001; r =-0.4628, P= 0.003).(3) SCAP peripheral blood of patients with co-stimulatory dendritic cell surface molecules CD80, CD86 expression relative to healthy controls frequency decreased. Related cosuppression molecule PDL1 relatively healthy control group, the expression of the frequency increase.(4) normal mouse spleen DC content is less, mainly in the spleen marginal zone.Experimental 4h severe pneumonia murine splenic DC cells were significantly hyperplasia and functionally active, CD86 + / CDIlc + DC accounted spleen mononuclear cells was significantly increased compared there was a significant difference(P <0.01) and the normal group; a significant increase in CD205 cells, especially in fringe significant, patchy distribution, the normal control group, the difference was significant(P <0.01); CD86 cells was significantly increased, with the most abundant edges, diffuse, visible DC s brown staining of the membrane was branch shape to extend around, and the normal control group,the difference was significant(P <0.01); white pulp and red pulp with scattered number of TUNEL-positive cells were distributed small pieces increased significantly, showing a large,spotty or nested distribution; CD4 + T cells were significantly decreased, while the CD8 + T cells were significantly increased, CD4 + / CD8 + ratio decreased, and the normal control group, the difference was significant(P <0.05).(5) The model of severe pneumonia in mice 12 ~ 24 h group CD86 + / CDIlc + DC ratio decreased slightly, compared with normal group there are significant differences(P <0.05);CD205 cells slightly reduced compared with 4 h group, distribution is sparse, but still significant more than the normal control group(P <0.05); CD4 + T cell levels continue to the next bottoming out, and CD8 + T cell levels also decreased, CD4 + / CD8 + ratio decreased,and the normal control group, the difference was significant(P <0.01). 3 ~ 5d groups CD86+ / CDllc + DC expression ratio close to normal; CD205 breakdown close to normal control group.(6) The model of severe pneumonia in mice 3 ~ 5d apoptosis of spleen cells than 12 ~24h decreased slightly more than the normal control group; 3-5d CD4 + T cell levels increased, CD4 + / CD8 + ratio rebounded, but still lower than the normal control group.(7) The model of severe pneumonia in mice 5 ~ 7d group splenic CD86 + / CDIlc + DCIX expression was significantly decreased compared there was a significant difference(P <0.01)and normal group; cells in the CD205 increased significantly again, flaky diffuse, and normal control group and experimental group 3 ~ 5d and the difference was significant(P<0.05); CD86 cytoplasmic membrane and some dark brown, no dendritic membrane protrusion, positive cells content than the 3 ~ 5d group slightly increased, 4h increase far less obvious, CD + 86 cells with normal control group and experimental group 3 ~ 5d difference was not significant; 5 ~ 7d apoptotic cells increased again, see the white pulp and red pulp large number of TUNEL-positive withered apoptotic cells; CD4 + T cell levels decrease again, and CD8 + T cells were significantly increased, CD4 + / CD8 + ratio upside down,with the normal control group, the difference was significant(P <0.01).(8) The model of severe pneumonia mouse model 3-5d group, lung tissue fluid and serum TNF-α content than the control group significantly increased by about 3-5 times, with a very significant difference(P<0.01).(9) The mouse model of severe pneumonia group 3-5d, 5-7d group, DCs incubated supernatant significantly increased IL-10 levels than the control group, the difference was statistically significant(P<0.05).(10) A mouse model of severe pneumonia, lung tissue significantly increased the number of CDllc + DCs, 12-24 h group CDllc + cell processes increased significantly concentrated in the blood vessels in the peri-alveolar septa, 5-7d group CDllc + cells reached the highest value, but fuzzy cell structure partly apoptosis.(11) A mouse model of severe pneumonia, CD205 positive cells in 4h group and the normal control group, the difference was not statistically significant(P>0.05), and significantly increased 12-24 h group, a statistically significant difference with the control group(P <0.01), CD205 and CD11 c were significantly increased to about 1: 1, 5-7d group,CD205-positive cells was significantly decreased comparison 12-24h(P<0.05).(12) The mouse model of severe pneumonia, 12-24 h group, CD86-positive cells was significantly increased, with statistical significance(P<0.01) and normal control group and5-7d group differences. CD86-positive cells was significantly decreased compared 12-24 h in5-7d, the difference was significant(P<0.05).Conclusions:(1) Patients with severe community-acquired pneumonia in peripheral blood dendritic cell subsets was significantly reduced. Costimulatory molecules CD80, CD86 expression decreased, suggesting the presence of dendritic cells in patients with SCAP mature dysfunction, antigen-presenting capacity decreased, resulting in reduced immune response.Cosuppression molecule PDL1 expression increased, indicating that SCAP patients immunosuppression is a major cause of poor infection control, and even the occurrence of secondary infection.(2) severe pneumonia in immunocompetent mice spleen DCs undergo a process of evolution from the inhibition to hyperactivity, early after infection spleen dendritic cells in immune activation and immune suppression advanced disease is closely related to SCAP.(3) Early severe pneumonia in mice, raising a large number of interstitial lung DCs,activation, leading to hyperactive immune response, inflammation disorders. Late in mice with severe pneumonia, interstitial lung DCs reduce the activity(expression of CD86 and CD205 declining proportion), a lot of negative feedback regulation of the cytokine IL-10 secretion by inducing immune suppression stage and promote the occurrence and development.更多还原