小鼠脓毒症继发AKI模型的制备以及炎症介质的变化和作用

发布时间：2018-06-25 05:33

本文选题：脓毒症 + 盲肠结扎穿孔(CLP)　；参考：《河北医科大学》2010年硕士论文

【摘要】： 目的:脓毒症是重症患者的主要死因之一,且发生率还在不断增长。在过去的十年中,住院患者的重症脓毒症发病率将近翻了一倍,大大超出了先前的预计。脓毒症可以导致许多器官功能障碍,其中就包括急性肾损伤。在ICU中AKI的发病率为6%,其中由脓毒症导致的约为50%,还有一些研究指出这个比例约为45%~70%。并且,由脓毒症导致AKI的患者预后很差,死亡率高达70%。就目前的研究而言,炎症反应在缺血再灌注导致AKI发生的病理生理学机制中,扮演了及其重要的角色。但在腹腔感染脓毒症所致AKI中,炎症因子到底是否也起到了重要的作用,其器官损伤是否和宿主的炎症反应密切相关我们还不甚了解。本研究探旨在探讨建立更加贴近临床的小鼠脓毒症继发急性肾损伤(Acute Kidney Injury,AKI)模型以及观察在急性肾损伤时炎症介质的变化及作用。方法:本研究利用改进的更加贴近临床的盲肠结扎穿孔(Cecal Ligation and Puncture, CLP)法复制小鼠脓毒症致AKI模型。选取健康雄性C57BL/6小鼠60只,随机分为3组,①空白对照组(Normal group)12只:不给予任何处理,通过取眼球法采血,随后直接处死;②假手术组(Sham group)24只:只开腹,翻动肠管,牵拉盲肠,关腹,不结扎和穿孔盲肠;③盲肠结扎穿孔组(CLP group)24只:进行盲肠结扎穿孔术。假手术组(Sham group)与盲肠结扎穿孔组(CLP group),术后37℃“生理盐水”30ml/kg股部皮下注射,并于术后3小时“亚胺培南西司他丁钠”25mg/kg+NS1.0ml,腹部皮下注射,Q12hr×2天。假手术组(Sham group)与盲肠结扎穿孔组(CLP group)分别在术后6 h、12 h、24 h后通过取眼球法采血(每组每个时间点各8只),空白对照组(Normal group),直接取血,各组取的血标本离心后取血清于-80℃冻存,用ELISA法测定血清中的肌酐、尿素氮、IL-1、IL-6、TNF-α水平,取腹水做细菌定量培养,并取出肾脏,放入10%中性福尔马林中,固定24至48小时,石蜡包被,供光镜检测备用,随即处死取血后的小鼠。数据统计分析采用SPSS13.0版软件包(SPSS Company, Chicago,Illinois,USA)进行;数据均以均数±标准差(x±s)表示,两组均数的比较行两组独立样本t检验,多个样本均数间比较采用完全随机设计的单因素方差分析,多个均数间两两比较采用LSD-t检验,P0.05为差异有统计学意义。结果: 1实验动物一般情况的观察假手术组小鼠术后清醒较快,活动灵活,自由饮水,剖腹见少量腹水,无恶臭。盲肠结扎穿孔组动物术后苏醒延迟,精神萎靡,活动迟缓,精神较差,竖毛,呼吸急促,剖腹可见淡血性腹水多,恶臭,肠管水肿,有的盲肠末端发黑。 2各组小鼠肾脏大体形态观察和组织病理变化(HE染色)及肾脏组织损伤病理评分结果 CLP组小鼠肾脏可见充血明显而假手术组和正常对照组小鼠的肾脏形态无明显异常。肾脏组织病理变化(HE染色):空白对照组:肾脏组织正常表现;假手术组:肾小管上皮细胞无肿胀及变性,形态正常。盲肠结扎穿孔组可观察到肾小球及肾小管充血明显,肾小管上皮细胞体积增大,胞浆呈嗜酸性颗粒状以及有空泡变性出现。肾脏组织损伤病理评分结果:术后6、12、24小时的评分CLP组均高于假手术组(P0.05);CLP组术后6、12、24小时的评分相比变化并不明显(P0.05)相同的结果也出现在假手术组(P0.05)。 3各组小鼠腹水的性质以及细菌定量测定结果正常对照组及假手术组剖腹见少量腹水,无恶臭,盲肠结扎穿孔组小鼠剖腹可见淡血性腹水多,恶臭。 CLP组小鼠,术后6小时、12小时、24小时腹水细菌定量结果均为大于105 cfu/ml;假手术组和正常对照组的腹水中均未找到致病微生物存在的证据。 4各组小鼠血清肌酐含量的比较术后6小时,CLP组的血清肌酐值明显高于假手术组(P0.05)与正常对照组(P0.05);术后12、24小时CLP组的血清肌酐值与假手术组(P0.05)与正常对照组(P0.05)之间没有明显差别;术后6、12、24小时假手术组与正常对照组之间没有明显差别(P0.05);CLP组术后6小时、12小时与24小时的血清肌酐含量相比较,各个时间点相比较无明显差别(P0.05)。 5各组小鼠血清尿素氮含量的比较术后6、12、24小时CLP组的血清尿素氮均明显高于假手术组(P0.05)与正常对照组(P0.05),假手术组与正常对照组之间没有明显差别(P0.05);CLP组术后6小时、12小时与24小时的血清尿素氮相比较,各个时间点相比较无明显差别(P0.05),但各组均高于相对于正常对照组(P0.05)。 6各组小鼠血清IL-1含量的比较术后6、12、24小时CLP组的血清IL-1含量均明显高于假手术组(P0.05)与正常对照组(P0.05),假手术组与正常对照组之间没有明显差别(P0.05);CLP组CLP术后6小时、12小时与24小时的血清IL-1含量相比较,各个时间点相比较无明显差别(P0.05)。 7各组小鼠血清IL-6含量的比较术后6、12、24小时CLP组的血清IL-6含量均明显高于假手术组(P0.001)与正常对照组(P0.001),假手术组与正常对照组之间没有明显差别(P0.05);CLP组CLP术后6小时的血清IL-6含量高于术后24小时的含量(P=0.049);术后12小时分别与术后6小时、24小时相比较无明显差别(P0.05),与正常对照组相比均有明显区别(P0.005)。 8各组小鼠血清TNF-α含量的比较术后6、12、24小时CLP组的血清TNF-α含量均明显高于假手术组(P0.001)与正常对照组(P0.001),假手术组与正常对照组之间没有明显差别(P0.05);CLP组术后6小时、12小时与24小时的血清TNF-α含量相比较,各个时间点相比较无明显差别(P0.05)。结论: 1本实验采用改进的更加贴近临床的CLP法可成功复制由腹腔感染脓毒症继发AKI的小鼠动物模型。 2 CLP术后血清IL-1、IL-6、TNF-α、肌酐、BUN的含量均增高(P0.05)且肾脏组织病理损伤评分也较假手术组有意义(P0.05),所以炎症介质释放可能是脓毒症继发AKI的机制之一。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R459.7;R-332

【引证文献】