大鼠脓毒症“免疫麻痹”模型的构建及炎症特点的初步分析

发布时间：2018-07-03 15:38

本文选题：慢性肾脏病 + 脓毒症　；参考：《复旦大学》2014年博士论文

【摘要】：第一部分“二次打击”脓毒症继发肺炎大鼠模型的构建及炎症特点分析研究背景脓毒症是继发于感染的系统炎症反应综合征(systemic inflammatory response syndrome, SIRS),由于其高发病率和高死亡率,一直是研究的热点。临床和动物研究结果均显示,免疫反应的变化对脓毒症的预后起着重要的作用。既往观点认为,机体在细菌内毒素攻击后,可产生大量的炎症细胞因子,引起全身炎症反应,并诱发多器官功能衰竭。动物研究结果发现,脓毒症模型的动物多由于炎症早期“促炎因子风暴”导致死亡；相较之下,临床患者常可幸免于早期感染,但却因脓毒症的持续存在或既发感染的出现,导致病情急剧加重、住院时间延长,甚至死亡率明显升高；这可能与脓毒症病程中出现的代偿性抗炎反应(Compensatory anti-inflammatory response syndrome, CARS)导致机体处于“免疫麻痹”(或称免疫抑制)状态相关。研究目的本研究的目的是通过构建脓毒症大鼠模型,观察脓毒症各发病阶段炎症反应的改变,认识“免疫麻痹”出现时机及免疫应答能力。同时,模拟临床脓毒症继发感染的发病情况,构建“二次打击”模型,即在脓毒症“免疫麻痹”时期继发肺部感染,探究二次打击对机体的损害与免疫状态的相关性。研究方法36只约250g SD雄性大鼠采用盲肠结扎穿孔术(cecal ligation puncture, CLP)建立脓毒症模型(其中CLP组30只,正常对照组6只)。随机选取脓毒症大鼠12只分别在CLP术后第4天或第7天分别经鼻腔注射肺炎链球菌200μl(细菌浓度为1×1010 CFU/ml)导致继发肺部感染,从而构建“二次打击”模型。观察单纯CLP组及“二次打击”组大鼠术后生存状态及死亡时间。分别留取单纯CLP术后第1、4、7天及“二次打击”组大鼠给菌后1天的血清及脾脏组织标本。分别检测各种大鼠的血常规、外周血菌落计数及生化指标。流式细胞术(FCM)计数脾脏树突状细胞、CD4+、CD8+及调节性T细胞比例；ELISA法检测血清炎症因子水平；HE染色及TUNEL检测评估脾脏组织损害和细胞凋亡程度。研究结果观察各模型大鼠术后状态,CLP术后大鼠出现呼吸急促、嗜睡、腹部膨隆等,且在术后12h内即开始出现死亡。“二次打击”组大鼠在鼻腔给菌后6h内即出现呼吸音粗、紫绀等肺部感染表现。单纯脓毒症组大鼠生存率为73.33%(11/15)术后第4天给菌组大鼠生存率为26.67%(4/15),较单纯CLP组明显升高(P0.05)；而术后第7天给菌组较单纯脓毒症组死亡率无明显差异(P=0.467)。外周血白细胞计数的结果示,脓毒症大鼠在CLP术后dl白细胞数量达高峰(15.57±2.77×109/L),且外周血中菌落计数量达高峰；而术后d4给菌组细菌清除力较同时期单纯CLP组显著下降(1.78 vs.0.25 log CFU/ml, P0.05)。同时,CLP术后d1,大鼠的肝肾功能指标升高,但“二次打击”组分别与同时期脓毒症组均无明显差异。各组大鼠脾脏流式细胞术检测DC比例在术后第1天达高峰(0.87±0.31%),CLP术后d4组,DC、CD4+、CD8+T细胞比例显著下降。调节性T细胞比例的高峰出现在术后第4天(3.14±0.74%),术后d7逐渐恢复正常。“二次打击”组与同时期单纯脓毒症组比较无明显差异血清TNF-α、IL-6、IL-1β水平的高峰均出现在脓毒症发生后24h,而HMGB-1的高峰出现在术后第4天(1.76±0.71 ng/m1)。抗炎因子IL-10、TGF-βl水平的高峰出现在脓毒症中,后期下降；sTNFR-I在CLP术后d7仍维持于高浓度(1.56±0.39 pg/ml)。术后d4给菌组大鼠炎症因子较同时期单纯脓毒症组无明显差异,术后d7给菌组IL-1β及TGF-β1较同时期脓毒症大鼠升高(P0.05)。HE染色和TUNEL检测结果示脓毒症可导致大鼠脾脏组织结构破坏显著,凋亡显著。CLP术后d4给菌组与同时期单纯脓毒症组相比,凋亡细胞数明显增加(74.48个/HP vs.52.99个/HP,P0.05)；而术后d7给菌组较同时期脓毒症组差异无明显差异。研究结论采用盲肠结扎穿孔术可成功构建大鼠脓毒症模型,且可出现脓毒症各发病阶段相应的症状和体征,导致菌血症的出现及肝肾功能的损害。脓毒症发生后,免疫细胞的数量减少、抗炎因子水平升高及脾脏凋亡加重,提示大鼠出现“免疫麻痹”状态。模拟临床脓毒症继发肺炎的“二次打击”模型,较单纯脓毒症模型大鼠死亡率明显升高,细菌清除力下降,可能与该时期机体存在的免疫麻痹相关。第二部分合并慢性肾脏病大鼠脓毒症模型的构建及炎症分析研究背景针对脓毒症的治疗方法,从动物模型转化到临床应用经常无法成功,这可能是由于大部分临床脓毒症患者常合并一种或多种基础疾病,导致简单的动物模型无法完全模拟人类脓毒症的发病情况。既往研究发现,潜在的肾脏损伤可加重脓毒症,导致死亡率升高及肾脏损害的进一步加重,可能的机制包括尿毒症诱导的白细胞功能障碍、肾脏清除率下降所致的炎症细胞因子聚集或其他合并症等。因此,免疫功能的变化可能是合并慢性肾脏病(chronic kidney disease, CKD)患者脓毒症死亡率较普通人群显著升高的重要原因之一。研究目的本研究的目的是通过构建合并慢性肾脏病(CKD)大鼠脓毒症模型,观察CKD大鼠脓毒症各时期炎症反应的变化,并与单纯脓毒症模型相比较,了解并分析CKD大鼠在脓毒症各病程阶段免疫状态的变化及免疫抑制的程度。研究方法24只约250g SD雄性大鼠采用5/6肾脏切除术(先行2/3左肾切,一周后行右肾全切)构建慢性肾脏病(CKD)模型。对成功建模的CKD大鼠再行盲肠结扎穿孔术(CLP)构建合并CKD大鼠脓毒症模型。观察实验术后生存状态及死亡时间,并留取CKD大鼠CLP术后第0、1、4及7天的血清及脾脏组织标本(各亚组大鼠数量n=6)。分别检测各组大鼠的血常规、外周血菌落计数及生化指标。流式细胞术(FCM)计数脾脏树突状细胞(DC)、CD4+、CD8+T细胞及调节性T细胞比例；ELISA法检测血清炎症因子水平;HE染色及TUNEL检测评估脾脏组织损害和细胞凋亡程度。研究结果CKD大鼠发生脓毒症后的生存率较单纯脓毒症组下降(40% vs.73.33%, P=0.094)。CKD大鼠基础白细胞较正常大鼠高(10.21 X 109/L vs.4.04 ×109/L,P0.05),且在CLP术后第1天达峰值(14.55±2.88×109/L)。合并CKD大鼠术后菌血症较单纯脓毒症组加重(1.17±0.68 log CFU/ml,较单纯CLP d4组0.25±0.28log CFU/ml, P0.05)。CKD大鼠发生脓毒症后的肝肾功能指标显著升高,在脓毒症后期,Scr、BUN水平持续升高,提示肾功能恢复的延迟。CKD大鼠树突状细胞在CLP术后d4较基础值显著下降(0.75% vs.1.23%,P0.05),且在术后d7持续下降(0.44±0.08%,较CKD+CLP d4组P0.05);重复测量分析法比较CKD+CLP与单纯CLP组,结果示DC数量存在显著差异(F=19.39, P0.05)。CD4+、CD8+T细胞在CKD+CLP d4组也显著降低(较基础值P0.05),而Treg细胞脓毒症发生后呈持续升高至CLP术后d7(与d4组比较,5.06% vs.4.12%,P0.05)。与单纯CLP模型比较,两种模型不同时间点CD8+T和Treg细胞比例存在差别(P0.05),而CD4+T细胞无统计学差异。CKD大鼠血清促炎及抗炎因子基础水平均较正常大鼠明显升高。脓毒症发生后,促炎因子升高幅度低于单纯脓毒症组。抗炎因子在脓毒症发生后也明显升高,且sTNFR-I的高峰持续至术后第7天(2.44 vs.1.50 pg/ml, P0.05);与单纯CLP组大鼠相比,抗炎因子水平在各发病阶段均高于单纯脓毒症组(P0.05)。主成分分析结果示在脓毒症中期,抗炎因子逐渐占据优势,提示机体处于“免疫麻痹”状态,且合并CKD可加重免疫功能的损害,导致免疫抑制的加重及恢复的延迟。TUNEL检测结果示,CKD+CLP大鼠细胞凋亡较单纯CLP组明显加重(术后d4两组模型比较,67.40 vs.52.99个/HP,P0.05),也提示了CKD大鼠在脓毒症发生中后期存在免疫抑制的加重。研究结论该研究成功构建了合并慢性肾脏病大鼠脓毒症模型,复制了CKD患者脓毒症高死亡率的临床特征,较单纯脓毒症动物模型更符合临床实际。CKD大鼠存在基础炎症(“微炎症”)状态,脓毒症中期加重免疫功能损伤,导致“免疫麻痹”加重及恢复的迁延,最终导致死亡率升高。
[Abstract]:The first part of the "two hits" model of sepsis secondary pneumonia rat model and the analysis of the inflammatory characteristics of the background sepsis is secondary to the infection of systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS). Because of its high incidence and high mortality, it has always been a hot spot of research. Clinical and animal research results. The changes in the immune response have been shown to play an important role in the prognosis of sepsis. In the past, it is believed that after the bacterial endotoxin attack, the body can produce a large number of inflammatory cytokines, cause systemic inflammatory response and induce multiple organ failure. Animal studies have found that the animals in the sepsis model are mostly "proinflammatory" in the early stage of inflammation. "Factor storm" leads to death; in contrast, clinical patients are often exempted from early infection, but due to the persistent presence of sepsis or the emergence of a existing infection, the disease is exacerbated, prolonged hospitalization and even higher mortality; this may be associated with a compensatory anti inflammatory response (Compensatory anti-infla) in the course of sepsis. Mmatory response syndrome, CARS) causes the body to be in the state of "immune paralysis" (or immunosuppressive). The purpose of this study is to observe the changes in the inflammatory response at each stage of sepsis by constructing the rat model of sepsis, to recognize the opportunity and immune response of "immune paralysis". In the case of sepsis secondary infection, a "two hit" model was constructed, that is, the secondary pulmonary infection in the period of sepsis "immune paralysis", and to explore the correlation between the damage to the body and the immune state of the two attack. 36 250g SD male rats were treated with cecum ligation and perforation (cecal ligation puncture, CLP) to establish sepsis. The disease model (30 in group CLP and 6 in normal control group). 12 rats of sepsis were randomly selected to cause secondary pulmonary infection by injection of Streptococcus pneumoniae (1 * 1010 CFU/ml) by nasal cavity fourth days or seventh days after CLP operation, respectively, and then constructed the "two hit" model, and observed the simple CLP group and the two strike group rats. The state of survival and the time of death after operation. The serum and spleen tissue specimens were collected for 1 days after the simple CLP operation and 1 days after the "two strike" group, respectively. The blood routine, the colony count and the biochemical indexes of the peripheral blood were measured respectively. The flow cytometry (FCM) counted the splenic dendritic cells, CD4+, CD8+ and the regulatory T cell ratio. The level of serum inflammatory factors was detected by ELISA method; HE staining and TUNEL detection were used to evaluate the damage of spleen tissue and the degree of apoptosis. The results of the study were to observe the postoperative state of the rats, and the rats after CLP had a sudden breathing, drowsiness, abdominal swelling and so on, and the death occurred within 12h after the operation. The rats in the "two strike" group were given the nasal cavity. The survival rate of the rats in the simple sepsis group was 73.33% (11/15) after the operation, and the survival rate of the rats was 26.67% (4/15) at the fourth day after the operation (4/15), which was significantly higher than that in the simple CLP group (P0.05), but there was no significant difference in the mortality rate between the bacteria group and the pure sepsis group (P=0.467) on the seventh day after the operation (P=0.467). The count results showed that the number of DL leukocytes in the sepsis rats reached the peak after CLP (15.57 + 2.77 x 109/L), and the number of colonies in the peripheral blood reached the peak, and the clearance of the bacteria in the D4 group after the operation was significantly lower than that of the same CLP group (1.78 vs.0.25 log CFU/ml, P0.05). Meanwhile, D1 after CLP, the liver and kidney function index of the rats increased, but " There was no significant difference between the two percussion group and the concurrent sepsis group. The proportion of DC in the spleen flow cytometry in each group was at the peak of first Tianda (0.87 + 0.31%) after operation. The proportion of D4, DC, CD4+ and CD8+T cells decreased significantly after CLP. The peak of the proportion of regulatory T cells appeared at fourth days (3.14 + 0.74%) after the operation, and the D7 was gradually restored to normal after operation. There was no significant difference in serum TNF- a, IL-6 and IL-1 beta levels between the "two percussion" group and the simple sepsis group at the same time. The peak of the level of IL-6 and IL-1 beta appeared after the occurrence of sepsis 24h, while the peak of HMGB-1 appeared on the day after the operation (1.76 + 0.71 ng/m1). The high peak of the anti inflammatory factor IL-10, TGF- beta L level appeared in sepsis and later decreased; sTNFR-I in CLP operation. After the operation, the D7 remained at a high concentration (1.56 + 0.39 pg/ml). The inflammatory factors in the D4 group were not significantly different from those in the same period. After the operation, IL-1 beta and TGF- beta 1 in the D7 group were higher than the same time sepsis rats (P0.05).HE staining and TUNEL detection results showed that sepsis could lead to significant destruction of the spleen tissue structure in rats, and the apoptosis was significant. After CLP, the number of apoptotic cells increased significantly (74.48 /HP vs.52.99 /HP, P0.05) in the group of D4 to the same period (/HP vs.52.99 /HP, P0.05), but there was no significant difference between the postoperative D7 and the sepsis group in the same period. The corresponding symptoms and signs resulted in the emergence of bacteremia and the damage of liver and kidney function. After the sepsis, the number of immune cells decreased, the level of anti-inflammatory factors and the aggravation of spleen apoptosis increased, suggesting that the rats appeared "immune paralysis". The model of "two strikes" in the simulated clinical sepsis secondary pneumonia was larger than that of the simple sepsis model. The mortality of rats increased significantly and the bacterial clearance decreased, which may be related to the immune paralysis that existed in this period. The construction of the sepsis model in the second part of the chronic renal disease rats and the analysis of the inflammatory analysis are often unsuccessful for the treatment of sepsis, which may be due to the large part of the animal model. Clinical sepsis often combined with one or more basic diseases that lead to a simple animal model that can not fully simulate the incidence of human sepsis. Previous studies have found that potential kidney damage can aggravate sepsis, lead to higher mortality and further increase in renal damage. Possible mechanisms include leukocytes induced by uremia. Dysfunction, accumulation of inflammatory cytokines or other complications caused by a decrease in renal clearance. Therefore, the changes in immune function may be one of the important reasons for the significant increase in the mortality of sepsis in patients with chronic kidney disease (CKD). A rat sepsis model of chronic kidney disease (CKD) was used to observe the changes of inflammatory response in each period of sepsis in CKD rats. Compared with the simple sepsis model, the changes of immune state and the degree of immunosuppression of CKD rats in each stage of sepsis were analyzed and the degree of immunosuppression was analyzed. 24 250g SD male rats were treated with 5/6 nephrectomy. A model of chronic kidney disease (CKD) was constructed after a week of 2/3 left kidney resection. A model of successfully modeled CKD rats with cecum ligation and perforation (CLP) was constructed with a CKD rat sepsis model. The survival and death time of the CKD rats were observed and the serum and spleen tissue specimens of CKD rats after CLP operation (each of the subtypes of the serum and spleen) were observed. The number of rats in group n=6). Blood routine, colony count and biochemical indexes of peripheral blood were measured respectively. Flow cytometry (FCM) was used to count the proportion of spleen dendritic cells (DC), CD4+, CD8+T cells and regulatory T cells; ELISA method was used to detect the level of serum inflammatory factors; HE staining and TUNEL detection were used to evaluate the damage of spleen tissue and the degree of apoptosis. Results the survival rate of CKD rats after sepsis was lower than that of pure sepsis group (40% vs.73.33%, P=0.094), the basal leucocytes of.CKD rats were higher than normal rats (10.21 X 109/L vs.4.04 x 109/L, P0.05), and first Tianda peak (14.55 + 2.88 x 109/L) after CLP operation. After operation, the bacteremia in CO and CKD rats was worse than that of the pure sepsis group (1.17 +. 0.68 log CFU/ml, compared with the simple CLP D4 group, 0.25 + 0.28log CFU/ml, P0.05), the liver and kidney function indexes of the rats were significantly higher after sepsis. In the late stage of sepsis, the level of Scr and BUN increased continuously. The continuous decrease (0.44 + 0.08%, P0.05 in group CKD+CLP D4), CKD+CLP and CLP group were compared, the results showed that the number of DC was significantly different (F=19.39, P0.05).CD4+ (F=19.39, P0.05), and CD8+T cells in CKD+CLP D4 group was also significantly decreased (compared with the base value). Vs.4.12%, P0.05). Compared with the simple CLP model, the proportion of CD8+T and Treg cells at different time points of the two models was different (P0.05), but there was no statistical difference in CD4+T cells. The level of serum proinflammatory and anti-inflammatory factors in.CKD rats was significantly higher than that of normal rats. After the occurrence of sepsis, the elevation of proinflammatory factors was lower than that of the simple sepsis group. The factor was also significantly higher after sepsis, and the peak of sTNFR-I continued to seventh days after the operation (2.44 vs.1.50 pg/ml, P0.05). Compared with the simple CLP group, the level of anti inflammatory factors was higher than that of the simple sepsis group (P0.05). The principal component analysis showed that the anti inflammatory factors gradually took advantage in the middle of sepsis, suggesting the body. In the state of "immune paralysis", and the combination of CKD can aggravate the damage of immune function, and lead to the delayed.TUNEL detection results of the aggravation of the immunosuppression and the recovery, the apoptosis of CKD+CLP rats is significantly higher than that in the simple CLP group (compared with the D4 two groups after the operation, 67.40 vs.52.99 /HP, P0.05), which also suggests that the CKD rats are in the middle and late stages of the sepsis. The study concluded that the study successfully constructed a model of sepsis in rats with chronic renal disease and replicated the clinical characteristics of the high mortality rate of sepsis in CKD patients. Compared with the simple sepsis animal model, it was more consistent with the clinical practice of.CKD rats with basic inflammation ("micro inflammation"), and the immune function was aggravated in the middle of sepsis. Injury leads to the aggravation of immune paralysis and the delay of recovery, leading to higher mortality.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7;R-332

【引证文献】