本文選題：膿毒癥 + 盲腸結(jié)扎穿孔(CLP)��； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:膿毒癥是重癥患者的主要死因之一,且發(fā)生率還在不斷增長(zhǎng)。在過(guò)去的十年中,住院患者的重癥膿毒癥發(fā)病率將近翻了一倍,大大超出了先前的預(yù)計(jì)。膿毒癥可以導(dǎo)致許多器官功能障礙,其中就包括急性腎損傷。在ICU中AKI的發(fā)病率為6%,其中由膿毒癥導(dǎo)致的約為50%,還有一些研究指出這個(gè)比例約為45%~70%。并且,由膿毒癥導(dǎo)致AKI的患者預(yù)后很差,死亡率高達(dá)70%。就目前的研究而言,炎癥反應(yīng)在缺血再灌注導(dǎo)致AKI發(fā)生的病理生理學(xué)機(jī)制中,扮演了及其重要的角色。但在腹腔感染膿毒癥所致AKI中,炎癥因子到底是否也起到了重要的作用,其器官損傷是否和宿主的炎癥反應(yīng)密切相關(guān)我們還不甚了解。 本研究探旨在探討建立更加貼近臨床的小鼠膿毒癥繼發(fā)急性腎損傷(Acute Kidney Injury,AKI)模型以及觀察在急性腎損傷時(shí)炎癥介質(zhì)的變化及作用。 方法:本研究利用改進(jìn)的更加貼近臨床的盲腸結(jié)扎穿孔(Cecal Ligation and Puncture, CLP)法復(fù)制小鼠膿毒癥致AKI模型。選取健康雄性C57BL/6小鼠60只,隨機(jī)分為3組,①空白對(duì)照組(Normal group)12只:不給予任何處理,通過(guò)取眼球法采血,隨后直接處死;②假手術(shù)組(Sham group)24只:只開(kāi)腹,翻動(dòng)腸管,牽拉盲腸,關(guān)腹,不結(jié)扎和穿孔盲腸;③盲腸結(jié)扎穿孔組(CLP group)24只:進(jìn)行盲腸結(jié)扎穿孔術(shù)。假手術(shù)組(Sham group)與盲腸結(jié)扎穿孔組(CLP group),術(shù)后37℃“生理鹽水”30ml/kg股部皮下注射,并于術(shù)后3小時(shí)“亞胺培南西司他丁鈉”25mg/kg+NS1.0ml,腹部皮下注射,Q12hr×2天。假手術(shù)組(Sham group)與盲腸結(jié)扎穿孔組(CLP group)分別在術(shù)后6 h、12 h、24 h后通過(guò)取眼球法采血(每組每個(gè)時(shí)間點(diǎn)各8只),空白對(duì)照組(Normal group),直接取血,各組取的血標(biāo)本離心后取血清于-80℃凍存,用ELISA法測(cè)定血清中的肌酐、尿素氮、IL-1、IL-6、TNF-α水平,取腹水做細(xì)菌定量培養(yǎng),并取出腎臟,放入10%中性福爾馬林中,固定24至48小時(shí),石蠟包被,供光鏡檢測(cè)備用,隨即處死取血后的小鼠。 數(shù)據(jù)統(tǒng)計(jì)分析采用SPSS13.0版軟件包(SPSS Company, Chicago,Illinois,USA)進(jìn)行;數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,兩組均數(shù)的比較行兩組獨(dú)立樣本t檢驗(yàn),多個(gè)樣本均數(shù)間比較采用完全隨機(jī)設(shè)計(jì)的單因素方差分析,多個(gè)均數(shù)間兩兩比較采用LSD-t檢驗(yàn),P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1實(shí)驗(yàn)動(dòng)物一般情況的觀察 假手術(shù)組小鼠術(shù)后清醒較快,活動(dòng)靈活,自由飲水,剖腹見(jiàn)少量腹水,無(wú)惡臭。盲腸結(jié)扎穿孔組動(dòng)物術(shù)后蘇醒延遲,精神萎靡,活動(dòng)遲緩,精神較差,豎毛,呼吸急促,剖腹可見(jiàn)淡血性腹水多,惡臭,腸管水腫,有的盲腸末端發(fā)黑。 2各組小鼠腎臟大體形態(tài)觀察和組織病理變化(HE染色)及腎臟組織損傷病理評(píng)分結(jié)果 CLP組小鼠腎臟可見(jiàn)充血明顯而假手術(shù)組和正常對(duì)照組小鼠的腎臟形態(tài)無(wú)明顯異常。 腎臟組織病理變化(HE染色):空白對(duì)照組:腎臟組織正常表現(xiàn);假手術(shù)組:腎小管上皮細(xì)胞無(wú)腫脹及變性,形態(tài)正常。盲腸結(jié)扎穿孔組可觀察到腎小球及腎小管充血明顯,腎小管上皮細(xì)胞體積增大,胞漿呈嗜酸性顆粒狀以及有空泡變性出現(xiàn)。 腎臟組織損傷病理評(píng)分結(jié)果:術(shù)后6、12、24小時(shí)的評(píng)分CLP組均高于假手術(shù)組(P0.05);CLP組術(shù)后6、12、24小時(shí)的評(píng)分相比變化并不明顯(P0.05)相同的結(jié)果也出現(xiàn)在假手術(shù)組(P0.05)。 3各組小鼠腹水的性質(zhì)以及細(xì)菌定量測(cè)定結(jié)果 正常對(duì)照組及假手術(shù)組剖腹見(jiàn)少量腹水,無(wú)惡臭,盲腸結(jié)扎穿孔組小鼠剖腹可見(jiàn)淡血性腹水多,惡臭。 CLP組小鼠,術(shù)后6小時(shí)、12小時(shí)、24小時(shí)腹水細(xì)菌定量結(jié)果均為大于105 cfu/ml;假手術(shù)組和正常對(duì)照組的腹水中均未找到致病微生物存在的證據(jù)。 4各組小鼠血清肌酐含量的比較 術(shù)后6小時(shí),CLP組的血清肌酐值明顯高于假手術(shù)組(P0.05)與正常對(duì)照組(P0.05);術(shù)后12、24小時(shí)CLP組的血清肌酐值與假手術(shù)組(P0.05)與正常對(duì)照組(P0.05)之間沒(méi)有明顯差別;術(shù)后6、12、24小時(shí)假手術(shù)組與正常對(duì)照組之間沒(méi)有明顯差別(P0.05);CLP組術(shù)后6小時(shí)、12小時(shí)與24小時(shí)的血清肌酐含量相比較,各個(gè)時(shí)間點(diǎn)相比較無(wú)明顯差別(P0.05)。 5各組小鼠血清尿素氮含量的比較 術(shù)后6、12、24小時(shí)CLP組的血清尿素氮均明顯高于假手術(shù)組(P0.05)與正常對(duì)照組(P0.05),假手術(shù)組與正常對(duì)照組之間沒(méi)有明顯差別(P0.05);CLP組術(shù)后6小時(shí)、12小時(shí)與24小時(shí)的血清尿素氮相比較,各個(gè)時(shí)間點(diǎn)相比較無(wú)明顯差別(P0.05),但各組均高于相對(duì)于正常對(duì)照組(P0.05)。 6各組小鼠血清IL-1含量的比較 術(shù)后6、12、24小時(shí)CLP組的血清IL-1含量均明顯高于假手術(shù)組(P0.05)與正常對(duì)照組(P0.05),假手術(shù)組與正常對(duì)照組之間沒(méi)有明顯差別(P0.05);CLP組CLP術(shù)后6小時(shí)、12小時(shí)與24小時(shí)的血清IL-1含量相比較,各個(gè)時(shí)間點(diǎn)相比較無(wú)明顯差別(P0.05)。 7各組小鼠血清IL-6含量的比較 術(shù)后6、12、24小時(shí)CLP組的血清IL-6含量均明顯高于假手術(shù)組(P0.001)與正常對(duì)照組(P0.001),假手術(shù)組與正常對(duì)照組之間沒(méi)有明顯差別(P0.05);CLP組CLP術(shù)后6小時(shí)的血清IL-6含量高于術(shù)后24小時(shí)的含量(P=0.049);術(shù)后12小時(shí)分別與術(shù)后6小時(shí)、24小時(shí)相比較無(wú)明顯差別(P0.05),與正常對(duì)照組相比均有明顯區(qū)別(P0.005)。 8各組小鼠血清TNF-α含量的比較 術(shù)后6、12、24小時(shí)CLP組的血清TNF-α含量均明顯高于假手術(shù)組(P0.001)與正常對(duì)照組(P0.001),假手術(shù)組與正常對(duì)照組之間沒(méi)有明顯差別(P0.05);CLP組術(shù)后6小時(shí)、12小時(shí)與24小時(shí)的血清TNF-α含量相比較,各個(gè)時(shí)間點(diǎn)相比較無(wú)明顯差別(P0.05)。 結(jié)論: 1本實(shí)驗(yàn)采用改進(jìn)的更加貼近臨床的CLP法可成功復(fù)制由腹腔感染膿毒癥繼發(fā)AKI的小鼠動(dòng)物模型。 2 CLP術(shù)后血清IL-1、IL-6、TNF-α、肌酐、BUN的含量均增高(P0.05)且腎臟組織病理?yè)p傷評(píng)分也較假手術(shù)組有意義(P0.05),所以炎癥介質(zhì)釋放可能是膿毒癥繼發(fā)AKI的機(jī)制之一。
[Abstract]:Objective: sepsis is one of the major causes of death in severe patients and the incidence is still growing. In the past ten years, the incidence of severe sepsis in hospitalized patients has nearly doubled, greatly exceeding the previous prediction. Sepsis can lead to many organ dysfunction, including acute renal injury. The incidence of AKI in ICU is 6%, about 50% of them are caused by sepsis, and some studies have pointed out that the proportion is about 45%~70%. and that the prognosis of AKI patients with sepsis is poor and the death rate is up to 70%.. As far as the current study is concerned, the inflammatory response plays an important role in the pathophysiological mechanism of AKI induced by ischemia-reperfusion, but in the abdomen. Whether the inflammatory factors have also played an important role in AKI caused by sepsis, and whether the organ damage is closely related to the host's inflammatory response is not well understood.
The purpose of this study is to explore the establishment of Acute Kidney Injury (AKI) model and the changes and effects of inflammatory mediators in acute renal injury.
Methods: Cecal Ligation and Puncture (CLP) was used to reproduce the AKI model of sepsis in mice. 60 healthy male C57BL/6 mice were selected and divided into 3 groups randomly. (1) 12 rats in the blank control group (Normal group) were taken without any treatment. (2) 24 sham operation group (Sham group): only opening, turning the intestine, pulling the cecum, closing the abdomen, without ligation and perforation cecum; (3) 24 of the cecum ligation and perforation group (CLP group): the cecum ligation and perforation, the sham operation group (Sham group) and the cecum ligation and perforation group (CLP group), the postoperative 37 C "physiological saline" in the 30ml/kg femoral subcutaneous injection, and 3 after the operation. Hourly "imiminosinastatin sodium" 25mg/kg+NS1.0ml, abdominal subcutaneous injection, Q12hr x 2 days. The sham operation group (Sham group) and the cecum ligation group (CLP group) after 6 h, 12 h, 24 h after the eyeball extraction (each group of 8 each time each time point), the blank control group (Normal group), direct blood extraction, the blood samples from each group were removed from the blood samples from each group. Serum serum creatinine, urea nitrogen, IL-1, IL-6, TNF- alpha in serum were measured by ELISA, and the kidney was taken out of the ascites, and the kidney was removed in 10% neutral formalin, fixed for 24 to 48 hours, the paraffin envelope was tested for reserve, and then the mice were killed.
The data statistical analysis is carried out with the SPSS13.0 software package (SPSS Company, Chicago, Illinois, USA); the data are expressed with the mean mean standard deviation (x + s), and the two groups are compared with two groups of independent samples t test. The single factor variance analysis of the multiple samples is compared with the complete random design, and the 22 comparison between the multiple average numbers is tested by LSD-t. P0.05 was statistically significant for the difference.
Result:
1 Observation of the general situation of experimental animals
In the sham operation group, the mice were awake faster, more flexible, free drinking water, a small amount of ascites and no odour. The animals in the group of caecum ligation and perforation were delayed, sluggish, slow, poor spirit, erect hair, shortness of breath, and caesarean section showed a lot of hemorrhagic ascites, malodour, intestinal edema, and some cecal end blackening.
2 the gross morphology and histopathological changes (HE staining) and pathological score of kidney tissue in each group of mice were observed.
The kidney in group CLP was obviously congested, while the kidneys in sham operated group and normal control group were not abnormal.
Renal tissue pathological changes (HE staining): blank control group: normal renal tissue, sham operation group: renal tubular epithelial cells without swelling and denaturation, normal form. Cecal perforation group can observe the glomerulus and renal tubule congestion, renal tubular epithelial cell volume increases, cytoplasm is eosinophilic granules and vacuolar degeneration.
The pathological score of renal tissue injury: the 6,12,24 hour score in CLP group was higher than that of the sham group (P0.05), and the score of 6,12,24 hours after operation in group CLP was not obvious (P0.05) and the same results were also found in the sham operation group (P0.05).
3 the ascites quality and quantitative results of bacteria in each group of mice.
In the normal control group and sham operation group, there was a small amount of ascites and no stench.
In group CLP mice, the bacterial quantitative results of 6 hours, 12 hours and 24 hours after operation were more than 105 cfu/ml, and there was no evidence for the existence of pathogenic microbes in the ascitic water of the sham operation group and the normal control group.
4 Comparison of serum creatinine content in mice of each group
6 hours after operation, the serum creatinine value of group CLP was significantly higher than that of sham operation group (P0.05) and normal control group (P0.05), and there was no significant difference between the serum creatinine value of group CLP and the normal control group (P0.05) and the normal control group (P0.05) at 12,24 hours after the operation; there was no significant difference between the 6,12,24 hourly hand group and the normal control group (P0.05) after the operation (P0.05); CLP group operation. There was no significant difference in serum creatinine level between 12 hours and 24 hours after 6 hours (P0.05).
5 Comparison of serum urea nitrogen content in mice of each group
The serum urea nitrogen in group CLP 6,12,24 hours after operation was significantly higher than that of the sham operation group (P0.05) and the normal control group (P0.05). There was no significant difference between the sham operation group and the normal control group (P0.05), and there was no significant difference between the 12 hours after the operation and the 24 hour serum urea nitrogen phase after the operation (P0.05), but all the groups were higher than those in the group CLP (P0.05). Compared with the normal control group (P0.05).
6 Comparison of serum IL-1 content in mice of each group
The serum IL-1 content in group CLP 6,12,24 hours after operation was significantly higher than that of sham operation group (P0.05) and normal control group (P0.05), and there was no significant difference between the sham operation group and the normal control group (P0.05). The serum IL-1 content of the CLP group was compared with the serum IL-1 content of 24 hours after CLP operation in CLP group, and there was no significant difference at each time point (P0.05).
7 Comparison of serum IL-6 content in mice of each group
The serum IL-6 content in group CLP 6,12,24 hours after operation was significantly higher than that in sham operation group (P0.001) and normal control group (P0.001). There was no significant difference between the sham operation group and the normal control group (P0.05). The serum IL-6 content in the CLP group after CLP operation was higher than that of the 24 hours after the operation (P=0.049), and the 12 hours after the operation was compared with the 12 hours after the operation, 24 hours after the operation, respectively. There was no significant difference (P0.05), which was significantly different from that in the normal control group (P0.005).
8 Comparison of the content of TNF- alpha in serum of mice in each group
The content of serum TNF- alpha in group CLP 6,12,24 hours after operation was significantly higher than that of sham operation group (P0.001) and normal control group (P0.001). There was no significant difference between the sham operation group and the normal control group (P0.05). The serum TNF- a content of the CLP group was 6 hours after operation and the serum TNF- alpha content was compared with the 24 hours, and there was no significant difference between each time point (P0.05).
Conclusion:
1 in this experiment, the animal model of sepsis secondary to AKI can be successfully replicated by using the improved CLP method which is closer to the clinical practice.
The serum levels of IL-1, IL-6, TNF- a, creatinine and BUN were all increased after 2 CLP (P0.05) and the renal histopathological damage score was also more significant than that of the sham operation group (P0.05), so the release of inflammatory mediators may be one of the mechanisms of secondary AKI in sepsis.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R459.7;R-332

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 馬云;阿托伐他汀對(duì)中度膿毒癥大鼠模型中IL-17、IL-10及臟器功能的影響[D];蚌埠醫(yī)學(xué)院;2012年

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本文編號(hào)：2064810