糖尿病小鼠调节肺炎克雷伯菌肝脓肿发病机制的分子机制

发布时间：2018-05-05 09:47

本文选题：肝脓肿 + 糖尿病　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究目的1.分析肝脓肿患者的临床特点,观察经皮穿刺引流术对肝脓肿的治疗效果,分析影响疗效的因素。2.肺炎克雷伯菌感染后,比较糖尿病小鼠和正常小鼠肝脏炎症反应病理学、细胞因子及炎症信号通路蛋白表达量差异,探讨糖尿病小鼠肺炎克雷伯菌肝脓肿发病率高的分子机制。研究方法1.收集上海某三甲医院介入血管外科从2011年1月至2016年1月收治的121例肝脓肿患者临床资料。对确诊肝脓肿患者,充分抗感染的同时,CT评估病灶液化情况,在CT引导下行经皮肝穿刺引流术,分析患者的一般资料、实验室检查、脓肿大小及位置、合并症、致病菌、治疗后的病死率、并发症率、住院时间及其影响因素。2.(1)DM模型构建:有48只健康雄性的ICR小鼠,8周龄。从中随机选取24只小鼠,用STZ腹腔注射,以120mg/kg的剂量,构建糖尿病模型,作为糖尿病组,另外24只小鼠腹腔注射等量柠檬酸盐缓冲液,作为正常组。1周后,用血糖仪监测血糖,如果小鼠血糖值20mmol/L,视为糖尿病模型小鼠。(2)肝脓肿模型构建:DM模型构建成功后1个月,制备3×10*7CFU/50ul肺炎克雷伯菌细菌混悬液,随机选取糖尿病组和正常组小鼠各12只,按每只小鼠口腔灌胃3×10*8 CFU肺炎克雷伯菌的剂量感染小鼠。2组各另外8只小鼠口腔灌胃等剂量的PBS溶液。(3)实验分组:肺炎克雷伯菌-糖尿病组、肺炎克雷伯菌-正常组、PBS-糖尿病组、PBS-正常组。(4)肝组织病理学检查:在灌胃72h以后,在无菌条件下取出小鼠肝脏。病理学观察比较pbs-糖尿病组、pbs-正常组、肺炎克雷伯菌-糖尿病组、肺炎克雷伯菌-正常组肝细胞水肿程度、炎症细胞聚集情况、形成脓肿情况。(5)elisa法比较4组小鼠肝脏炎症细胞因子il-1β、il-2、il-6、il-10、mip-1α、tnfα的表达量,westernblot观察4组小鼠肝脏iκΚα和iκκβ、iκbα、nf-Κb及磷酸化值的表达量。研究结果1.共有121例肝脓肿患者纳入研究,均做了脓液细菌培养或血培养。其中检出肺炎克雷伯菌60例(49.59%),大肠杆菌3例(2.5%)。肝脓肿合并糖尿病60例(50.42%)。2例患者穿刺引流后死亡,病死率为1.65%,影响患者死亡率的因素为高龄、基础疾病,脓肿直径及实性成分;2例患者穿刺引流后出现肝周脓肿、腹壁脓肿,并发症率为1.65%,对并发症积极处理后达到临床治愈,影响并发症率的因素主要为手术操作欠妥;119例患者均达到临床治愈,治愈率为98.34%,平均住院时间为(15.13±6.07)天。影响住院时间的危险因素为:脓肿个数x6(r=0.232,p=0.021),脓肿大小x7(r=0.26,p=0.005),白细胞计数x8(r=0.238,p=0.009)。进一步分析显示影响因素与住院时间相关性的多元线性回归方程有统计学意义(p0.05),多元回归方程为:y=-3.438+3.055x6+0.527x7+0.297x8,f=5.819,r2=0.416。性别、年龄、糖尿病、致病菌、脓肿位置对住院时间的影响无统计学差异(p0.05)。2.stz注射一周后,用血糖仪检测糖尿病组24只小鼠血糖值,均20mmol/l。观察糖尿病组小鼠,毛黄、稀疏,多饮、多食、多尿,消瘦,呈现明显的“三多一少”症状。糖尿病组小鼠血糖较正常组明显升高(p0.05),糖尿病组与正常组小鼠体重无明显差异(p0.05)。肺炎克雷伯菌灌胃感染小鼠后,无菌条件下取小鼠肝脏,he染色实验结果表明:肺炎克雷伯菌-糖尿病组脓肿形成个数较肺炎克雷伯菌-正常组增多(p0.05)。elisa实验结果表明:肺炎克雷伯菌-糖尿病组肝脏IL-1β、IL-2、IL-6、MIP-1α、TNF-α的表达量明显高于肺炎克雷伯菌-正常组(P0.05),肺炎克雷伯菌-糖尿病组肝脏IL-1β、IL-2、IL-6、MIP-1α、TNF-α表达量高于PBS-糖尿病组(P0.05),肺炎克雷伯菌-正常组肝脏IL-1β、IL-2、IL-6、MIP-1α、TNF-α表达量高于PBS-正常组(P0.05)。Western blot实验结果表明:肺炎克雷伯菌-糖尿病组P-IΚΚβ、P-IΚΒα、P-NFΚΒ表达量明显高于肺炎克雷伯菌-正常组(P0.05),肺炎克雷伯菌-糖尿病组P-IΚΚα、P-IΚΚβ、P-IΚΒα、P-NFΚΒ表达量高于PBS-糖尿病组(P0.05),肺炎克雷伯菌-正常组P-IΚΚα、P-IΚΚβ、P-IΚΒα、P-NFΚΒ表达量高于PBS-正常组(P0.05)。研究结论1.CT引导下经皮肝穿刺引流联合抗生素能有效治疗肝脓肿,具有病死率低,治愈率高,并发症率低、住院时间短等优点,值得临床推广应用。糖尿病确实是诱发肝脓肿的高危因素,但当血糖水平控制在正常范围内时,糖尿病可以不影响住院时间。2.本实验成功构建了ICR小鼠1型糖尿病模型及小鼠的肺炎克雷伯菌肝脓肿模型,并且证实了糖尿病小鼠肺炎克雷伯菌肝脓肿发病率增高,探索了其分子机制是细胞NF-ΚΒ信号通路的过度激活扩大了炎症反应进程,加重了肝细胞坏死程度,导致脓肿发病率增高。
[Abstract]:Objective 1. to analyze the clinical characteristics of patients with liver abscess, observe the therapeutic effect of percutaneous drainage for liver abscess, analyze the factors that affect the curative effect of Klebsiella pneumoniae infection, compare the pathological changes of liver inflammation in diabetic mice and normal mice, the difference of the expression of cytokines and inflammatory signaling pathway protein in diabetic mice and normal mice, and explore diabetes. Molecular mechanism of high incidence of Klebsiella pneumoniae liver abscess in mice. Method 1. the clinical data of 121 patients with liver abscess treated from January 2011 to January 2016 in a three a hospital of Shanghai were collected from 121 cases of liver abscess. CT was used to evaluate the liquefaction of the lesions and the percutaneous liver puncture under the guidance of CT. Puncture drainage, analysis of the patient's general data, laboratory examination, abscess size and location, complication, pathogenic bacteria, mortality, complication rate, hospitalization time and influencing factors.2. (1) DM Model Construction: 48 healthy male ICR mice, 8 weeks of age. 24 mice were randomly selected from them, STZ intraperitoneal injection, 120mg/kg dose, configuration of 120mg/kg The diabetes model was built as a diabetic group. The other 24 mice were intraperitoneally injected with equal amount of citrate buffer. After.1 weeks in the normal group, blood glucose was monitored with blood glucose meter. If the blood glucose value of mice was 20mmol/L, the model mice were treated as diabetic model mice. (2) construction of liver abscess model: 1 months after the construction of DM model, 3 x 10*7CFU/50ul Klebsiella pneumoniae was prepared. The bacterial suspension was randomly selected and 12 mice were randomly selected from the diabetes group and the normal group. According to the dose of 3 x 10*8 CFU Klebsiella pneumoniae in each mouse oral gavage, 8 other mice were infected with PBS solution in the other 8 mice. (3) the experimental group: Klebsiella pneumoniae group, Klebsiella pneumoniae normal group, PBS- diabetic group, PBS - normal group. (4) pathological examination of liver tissue: after 72h, the mice liver was removed under aseptic condition. Pathological observation compared pbs- diabetes group, pbs- normal group, Klebsiella pneumoniae diabetes group, Klebsiella pneumoniae - normal group hepatocyte edema degree, inflammatory cell aggregation and abscess condition. (5) comparison of 4 groups by ELISA method The expression of inflammatory cytokines IL-1 beta, IL-2, IL-6, IL-10, MIP-1 alpha, TNF alpha in rat liver. Westernblot was used to observe the expression of I kappa and I kappa beta, I kappa B, I kappa B alpha, nf- I and phosphorylation in the 4 groups of mice. 1. of the study results were included in the study of 121 patients with liver abscess, and 60 cases of Klebsiella pneumoniae were detected (4 9.59%), 3 cases (2.5%) of Escherichia coli (2.5%). 60 cases of liver abscess with diabetes (50.42%).2 patients died after puncture and drainage, the fatality rate was 1.65%. The factors affecting the mortality of the patients were age, basic disease, abscess diameter and solid component; 2 patients had liver Zhou Nongzhong, abdominal abscess, complication rate 1.65% after puncture and drainage, and the complications were positive. The main factors affecting the complication rate were poor operation, 119 patients all reached clinical cure, the cure rate was 98.34%, the average hospitalization time was (15.13 + 6.07) days. The risk factors affecting the hospitalization time were X6 (r=0.232, p= 0.021), abscess size X7 (r=0.26, p=0.005), and leukocyte count X8 (r=0.238, p=). 0.009). Further analysis showed that the multivariate linear regression equation of the correlation between the influencing factors and the time of hospitalization had statistical significance (P0.05). The multiple regression equation was: y=-3.438+3.055x6+0.527x7+0.297x8, f=5.819, r2=0.416. sex, age, diabetes, pathogenic bacteria and abscess position had no statistical difference (P0.05).2.stz injection. After week, the blood glucose values of 24 mice in diabetic group were measured by blood glucose meter, and all the diabetic mice were observed by 20mmol/l.. The diabetic mice, hair yellow, sparsely, polydipsia, polyuria and emaciation showed obvious symptoms of "little more than three". The blood sugar of diabetic mice was significantly higher than that of the normal group (P0.05), and there was no significant difference between the diabetic group and the normal group (P0.05). The results of HE staining showed that the number of abscess formation in Klebsiella pneumoniae group was higher than that of Klebsiella pneumoniae normal group (P0.05).Elisa experimental results showed that the expression of IL-1 beta, IL-2, IL-6, MIP-1 A and TNF- a in Klebsiella pneumoniae diabetes group was significantly higher than that of Klebsiella pneumoniae diabetes group. The expression of IL-1 beta, IL-2, IL-6, MIP-1 alpha, TNF- alpha in Klebsiella pneumoniae normal group (P0.05) and Klebsiella pneumoniae diabetes group was higher than that of PBS- diabetic group (P0.05), and the expression of IL-1 beta, IL-2, IL-6, MIP-1 alpha in the normal group of Klebsiella pneumoniae was higher than that in the normal group. The expression of P-I in diabetic group is significantly higher than that of Klebsiella pneumoniae (P0.05), and the expression of P-I in Klebsiella pneumoniae (Klebsiella pneumoniae) is higher than that in the PBS- diabetic group (P0.05), and that of Klebsiella pneumoniae (P0.05) is higher than that in the PBS- diabetic group (P0.05). The expression of P-I is higher than that of the normal group. (P0.05). Conclusion 1.CT guided percutaneous transhepatic drainage combined with antibiotics can effectively treat liver abscess with low mortality, high cure rate, low complication rate, short hospitalization time and so on. It is worthy of clinical application. Diabetes is indeed a high risk factor for inducing liver abscess, but when blood sugar levels are controlled within the normal range, diabetes can be used. .2. model of type 1 diabetes in ICR mice and Klebsiella pneumoniae liver abscess model in mice was successfully constructed without affecting the time of hospitalization, and it was proved that the incidence of Klebsiella pneumoniae liver abscess increased in diabetic mice, and its molecular mechanism was that the excessive activation of cell NF- signaling pathway increased the process of inflammation and aggravated the process of inflammation. The degree of necrosis of liver cells leads to an increased incidence of abscesses.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.4;R587.1

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