本文選題：慢性腎臟病 + 膿毒癥��； 參考：《復(fù)旦大學(xué)》2014年博士論文

【摘要】：第一部分“二次打擊”膿毒癥繼發(fā)肺炎大鼠模型的構(gòu)建及炎癥特點(diǎn)分析研究背景膿毒癥是繼發(fā)于感染的系統(tǒng)炎癥反應(yīng)綜合征(systemic inflammatory response syndrome, SIRS),由于其高發(fā)病率和高死亡率,一直是研究的熱點(diǎn)。臨床和動(dòng)物研究結(jié)果均顯示,免疫反應(yīng)的變化對(duì)膿毒癥的預(yù)后起著重要的作用。既往觀點(diǎn)認(rèn)為,機(jī)體在細(xì)菌內(nèi)毒素攻擊后,可產(chǎn)生大量的炎癥細(xì)胞因子,引起全身炎癥反應(yīng),并誘發(fā)多器官功能衰竭。動(dòng)物研究結(jié)果發(fā)現(xiàn),膿毒癥模型的動(dòng)物多由于炎癥早期“促炎因子風(fēng)暴”導(dǎo)致死亡；相較之下,臨床患者常可幸免于早期感染,但卻因膿毒癥的持續(xù)存在或既發(fā)感染的出現(xiàn),導(dǎo)致病情急劇加重、住院時(shí)間延長,甚至死亡率明顯升高；這可能與膿毒癥病程中出現(xiàn)的代償性抗炎反應(yīng)(Compensatory anti-inflammatory response syndrome, CARS)導(dǎo)致機(jī)體處于“免疫麻痹”(或稱免疫抑制)狀態(tài)相關(guān)。研究目的本研究的目的是通過構(gòu)建膿毒癥大鼠模型,觀察膿毒癥各發(fā)病階段炎癥反應(yīng)的改變,認(rèn)識(shí)“免疫麻痹”出現(xiàn)時(shí)機(jī)及免疫應(yīng)答能力。同時(shí),模擬臨床膿毒癥繼發(fā)感染的發(fā)病情況,構(gòu)建“二次打擊”模型,即在膿毒癥“免疫麻痹”時(shí)期繼發(fā)肺部感染,探究二次打擊對(duì)機(jī)體的損害與免疫狀態(tài)的相關(guān)性。研究方法36只約250g SD雄性大鼠采用盲腸結(jié)扎穿孔術(shù)(cecal ligation puncture, CLP)建立膿毒癥模型(其中CLP組30只,正常對(duì)照組6只)。隨機(jī)選取膿毒癥大鼠12只分別在CLP術(shù)后第4天或第7天分別經(jīng)鼻腔注射肺炎鏈球菌200μl(細(xì)菌濃度為1×1010 CFU/ml)導(dǎo)致繼發(fā)肺部感染,從而構(gòu)建“二次打擊”模型。觀察單純CLP組及“二次打擊”組大鼠術(shù)后生存狀態(tài)及死亡時(shí)間。分別留取單純CLP術(shù)后第1、4、7天及“二次打擊”組大鼠給菌后1天的血清及脾臟組織標(biāo)本。分別檢測(cè)各種大鼠的血常規(guī)、外周血菌落計(jì)數(shù)及生化指標(biāo)。流式細(xì)胞術(shù)(FCM)計(jì)數(shù)脾臟樹突狀細(xì)胞、CD4+、CD8+及調(diào)節(jié)性T細(xì)胞比例；ELISA法檢測(cè)血清炎癥因子水平；HE染色及TUNEL檢測(cè)評(píng)估脾臟組織損害和細(xì)胞凋亡程度。研究結(jié)果觀察各模型大鼠術(shù)后狀態(tài),CLP術(shù)后大鼠出現(xiàn)呼吸急促、嗜睡、腹部膨隆等,且在術(shù)后12h內(nèi)即開始出現(xiàn)死亡。“二次打擊”組大鼠在鼻腔給菌后6h內(nèi)即出現(xiàn)呼吸音粗、紫紺等肺部感染表現(xiàn)。單純膿毒癥組大鼠生存率為73.33%(11/15)術(shù)后第4天給菌組大鼠生存率為26.67%(4/15),較單純CLP組明顯升高(P0.05)；而術(shù)后第7天給菌組較單純膿毒癥組死亡率無明顯差異(P=0.467)。外周血白細(xì)胞計(jì)數(shù)的結(jié)果示,膿毒癥大鼠在CLP術(shù)后dl白細(xì)胞數(shù)量達(dá)高峰(15.57±2.77×109/L),且外周血中菌落計(jì)數(shù)量達(dá)高峰；而術(shù)后d4給菌組細(xì)菌清除力較同時(shí)期單純CLP組顯著下降(1.78 vs.0.25 log CFU/ml, P0.05)。同時(shí),CLP術(shù)后d1,大鼠的肝腎功能指標(biāo)升高,但“二次打擊”組分別與同時(shí)期膿毒癥組均無明顯差異。各組大鼠脾臟流式細(xì)胞術(shù)檢測(cè)DC比例在術(shù)后第1天達(dá)高峰(0.87±0.31%),CLP術(shù)后d4組,DC、CD4+、CD8+T細(xì)胞比例顯著下降。調(diào)節(jié)性T細(xì)胞比例的高峰出現(xiàn)在術(shù)后第4天(3.14±0.74%),術(shù)后d7逐漸恢復(fù)正常。“二次打擊”組與同時(shí)期單純膿毒癥組比較無明顯差異血清TNF-α、IL-6、IL-1β水平的高峰均出現(xiàn)在膿毒癥發(fā)生后24h,而HMGB-1的高峰出現(xiàn)在術(shù)后第4天(1.76±0.71 ng/m1)�？寡滓蜃覫L-10、TGF-βl水平的高峰出現(xiàn)在膿毒癥中,后期下降；sTNFR-I在CLP術(shù)后d7仍維持于高濃度(1.56±0.39 pg/ml)。術(shù)后d4給菌組大鼠炎癥因子較同時(shí)期單純膿毒癥組無明顯差異,術(shù)后d7給菌組IL-1β及TGF-β1較同時(shí)期膿毒癥大鼠升高(P0.05)。HE染色和TUNEL檢測(cè)結(jié)果示膿毒癥可導(dǎo)致大鼠脾臟組織結(jié)構(gòu)破壞顯著,凋亡顯著。CLP術(shù)后d4給菌組與同時(shí)期單純膿毒癥組相比,凋亡細(xì)胞數(shù)明顯增加(74.48個(gè)/HP vs.52.99個(gè)/HP,P0.05)；而術(shù)后d7給菌組較同時(shí)期膿毒癥組差異無明顯差異。研究結(jié)論采用盲腸結(jié)扎穿孔術(shù)可成功構(gòu)建大鼠膿毒癥模型,且可出現(xiàn)膿毒癥各發(fā)病階段相應(yīng)的癥狀和體征,導(dǎo)致菌血癥的出現(xiàn)及肝腎功能的損害。膿毒癥發(fā)生后,免疫細(xì)胞的數(shù)量減少、抗炎因子水平升高及脾臟凋亡加重,提示大鼠出現(xiàn)“免疫麻痹”狀態(tài)。模擬臨床膿毒癥繼發(fā)肺炎的“二次打擊”模型,較單純膿毒癥模型大鼠死亡率明顯升高,細(xì)菌清除力下降,可能與該時(shí)期機(jī)體存在的免疫麻痹相關(guān)。第二部分合并慢性腎臟病大鼠膿毒癥模型的構(gòu)建及炎癥分析研究背景針對(duì)膿毒癥的治療方法,從動(dòng)物模型轉(zhuǎn)化到臨床應(yīng)用經(jīng)常無法成功,這可能是由于大部分臨床膿毒癥患者常合并一種或多種基礎(chǔ)疾病,導(dǎo)致簡單的動(dòng)物模型無法完全模擬人類膿毒癥的發(fā)病情況。既往研究發(fā)現(xiàn),潛在的腎臟損傷可加重膿毒癥,導(dǎo)致死亡率升高及腎臟損害的進(jìn)一步加重,可能的機(jī)制包括尿毒癥誘導(dǎo)的白細(xì)胞功能障礙、腎臟清除率下降所致的炎癥細(xì)胞因子聚集或其他合并癥等。因此,免疫功能的變化可能是合并慢性腎臟病(chronic kidney disease, CKD)患者膿毒癥死亡率較普通人群顯著升高的重要原因之一。研究目的本研究的目的是通過構(gòu)建合并慢性腎臟病(CKD)大鼠膿毒癥模型,觀察CKD大鼠膿毒癥各時(shí)期炎癥反應(yīng)的變化,并與單純膿毒癥模型相比較,了解并分析CKD大鼠在膿毒癥各病程階段免疫狀態(tài)的變化及免疫抑制的程度。研究方法24只約250g SD雄性大鼠采用5/6腎臟切除術(shù)(先行2/3左腎切,一周后行右腎全切)構(gòu)建慢性腎臟病(CKD)模型。對(duì)成功建模的CKD大鼠再行盲腸結(jié)扎穿孔術(shù)(CLP)構(gòu)建合并CKD大鼠膿毒癥模型。觀察實(shí)驗(yàn)術(shù)后生存狀態(tài)及死亡時(shí)間,并留取CKD大鼠CLP術(shù)后第0、1、4及7天的血清及脾臟組織標(biāo)本(各亞組大鼠數(shù)量n=6)。分別檢測(cè)各組大鼠的血常規(guī)、外周血菌落計(jì)數(shù)及生化指標(biāo)。流式細(xì)胞術(shù)(FCM)計(jì)數(shù)脾臟樹突狀細(xì)胞(DC)、CD4+、CD8+T細(xì)胞及調(diào)節(jié)性T細(xì)胞比例；ELISA法檢測(cè)血清炎癥因子水平;HE染色及TUNEL檢測(cè)評(píng)估脾臟組織損害和細(xì)胞凋亡程度。研究結(jié)果CKD大鼠發(fā)生膿毒癥后的生存率較單純膿毒癥組下降(40% vs.73.33%, P=0.094)。CKD大鼠基礎(chǔ)白細(xì)胞較正常大鼠高(10.21 X 109/L vs.4.04 ×109/L,P0.05),且在CLP術(shù)后第1天達(dá)峰值(14.55±2.88×109/L)。合并CKD大鼠術(shù)后菌血癥較單純膿毒癥組加重(1.17±0.68 log CFU/ml,較單純CLP d4組0.25±0.28log CFU/ml, P0.05)。CKD大鼠發(fā)生膿毒癥后的肝腎功能指標(biāo)顯著升高,在膿毒癥后期,Scr、BUN水平持續(xù)升高,提示腎功能恢復(fù)的延遲。CKD大鼠樹突狀細(xì)胞在CLP術(shù)后d4較基礎(chǔ)值顯著下降(0.75% vs.1.23%,P0.05),且在術(shù)后d7持續(xù)下降(0.44±0.08%,較CKD+CLP d4組P0.05);重復(fù)測(cè)量分析法比較CKD+CLP與單純CLP組,結(jié)果示DC數(shù)量存在顯著差異(F=19.39, P0.05)。CD4+、CD8+T細(xì)胞在CKD+CLP d4組也顯著降低(較基礎(chǔ)值P0.05),而Treg細(xì)胞膿毒癥發(fā)生后呈持續(xù)升高至CLP術(shù)后d7(與d4組比較,5.06% vs.4.12%,P0.05)。與單純CLP模型比較,兩種模型不同時(shí)間點(diǎn)CD8+T和Treg細(xì)胞比例存在差別(P0.05),而CD4+T細(xì)胞無統(tǒng)計(jì)學(xué)差異。CKD大鼠血清促炎及抗炎因子基礎(chǔ)水平均較正常大鼠明顯升高。膿毒癥發(fā)生后,促炎因子升高幅度低于單純膿毒癥組。抗炎因子在膿毒癥發(fā)生后也明顯升高,且sTNFR-I的高峰持續(xù)至術(shù)后第7天(2.44 vs.1.50 pg/ml, P0.05);與單純CLP組大鼠相比,抗炎因子水平在各發(fā)病階段均高于單純膿毒癥組(P0.05)。主成分分析結(jié)果示在膿毒癥中期,抗炎因子逐漸占據(jù)優(yōu)勢(shì),提示機(jī)體處于“免疫麻痹”狀態(tài),且合并CKD可加重免疫功能的損害,導(dǎo)致免疫抑制的加重及恢復(fù)的延遲。TUNEL檢測(cè)結(jié)果示,CKD+CLP大鼠細(xì)胞凋亡較單純CLP組明顯加重(術(shù)后d4兩組模型比較,67.40 vs.52.99個(gè)/HP,P0.05),也提示了CKD大鼠在膿毒癥發(fā)生中后期存在免疫抑制的加重。研究結(jié)論該研究成功構(gòu)建了合并慢性腎臟病大鼠膿毒癥模型,復(fù)制了CKD患者膿毒癥高死亡率的臨床特征,較單純膿毒癥動(dòng)物模型更符合臨床實(shí)際。CKD大鼠存在基礎(chǔ)炎癥(“微炎癥”)狀態(tài),膿毒癥中期加重免疫功能損傷,導(dǎo)致“免疫麻痹”加重及恢復(fù)的遷延,最終導(dǎo)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R459.7;R-332

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1 顧承東;;2012年《國際嚴(yán)重膿毒癥和膿毒癥休克治療指南》解讀[A];中華醫(yī)學(xué)會(huì)急診醫(yī)學(xué)分會(huì)第十六次全國急診醫(yī)學(xué)學(xué)術(shù)年會(huì)論文集[C];2013年

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