自噬对糖尿病足患者感染铜绿假单胞菌作用的研究

发布时间：2018-03-07 00:11

本文选题：糖尿病　切入点：糖尿病足　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.通过比较糖尿病足(diabetic foot,DF)患者创面感染铜绿假单胞菌(Pseudomonas aeruginosa,PA)及感染控制期创面组织中自噬相关蛋白表达差异,探究PA对自噬的影响。2.研究感染多重耐药铜绿假单胞菌(Multi-drug resistant pseudomonas aeruginosa,MDRPA)以及合并缺血对自噬的影响。3.PA的Ⅲ型分泌系统(typeⅢsecretion system,T3SS)分泌不同毒力蛋白对自噬的影响。方法:1.收集2016年3月-2017年2月于天津医科大学代谢病医院糖尿病足科住院治疗的24例DF感染PA患者的临床资料;根据是否感染MDRPA分为两组,根据是否缺血分为两组,标准化治疗感染控制前后分为感染期和感染控制期。2.所有纳入患者创面清创后深部取材,并将所取组织分为3部分。一部分用于细菌培养及药敏试验、创面菌落计数并冻存菌株,通过PCR鉴定PA的T3SS所携带的毒力基因。一部分用于制作石蜡切片:HE染色观察患者创面组织病理学改变;免疫组织化学染色,半定量检测患者创面组织内细胞自噬相关蛋白LC3(Microtubule-associated protein1 light chain 3)、Beclin-1和P62的平均光密度值;免疫荧光双染色,定性检测患者创面组织中巨噬细胞的自噬水平。最后一部分蛋白印迹定量测定LC3-II/I、Beclin-1和P62的蛋白表达量。3.感染控制后同上述方法测量上述指标。结果:1.DF患者感染期与感染控制期比较,感染控制期炎症指标(白细胞计数、中性粒细胞百分比、血沉和超敏C反应蛋白)较感染期下降、创面菌落计数下降、果糖胺下降、创面面积缩小(P0.05),余一般资料未见差异(P0.05)。MDRPA组炎症指标(中性粒细胞百分比、血沉和超敏C反应蛋白)以及创面内细菌菌落数较N-MDRPA组高(P0.05)。缺血组较非缺血组ABI低、创面面积大(P0.05)。2.与感染期相比,感染控制期创面组织中新生的毛细血管、成纤维细胞较多,炎症细胞较少;免疫组织化学法显示创面组织中LC3和Beclin-1表达较高、P62表达较少(P0.05);免疫荧光显示芽组织LC3与巨噬细胞标记物CD14 Merge后,巨噬细胞自噬水平较高;蛋白印迹显示LC3-II/I和Beclin-1表达较高、P62表达较低(P0.05)。与MDRPA组相比,N-MDRPA组炎症细胞浸润较少,创面组织中新生的毛细血管、成纤维细胞相比无明显差异;免疫组织化学法显示LC3的表达较高(P0.05),Beclin-1和P62表达无统计学意义(P0.05);免疫荧光显示芽组织LC3与巨噬细胞特异性标记物CD14 Merge后,巨噬细胞自噬水平较高;蛋白印迹法显示创面组织中LC3-II/I表达较高(P0.05),Beclin-1、P62的表达无统计学意义(P0.05)。与缺血组相比,非缺血组创面组织中新生的毛细血管、成纤维细胞以及炎症细胞较多;免疫组织化学法显示创面组织中LC3和Beclin-1表达较高、P62表达较低(P0.05);免疫荧光显示芽组织LC3与巨噬细胞特异性标记物CD14 Merge后,巨噬细胞自噬水平较高;蛋白印迹法显示创面组织中LC3-II/I和Beclin-1表达较高、P62表达较低(P0.05)。3.感染期real-time PCR检测送检细菌均携带毒力基因exo S。结论:PA感染控制以及代谢紊乱改善后,自噬水平升高,这提示严重的代谢紊乱可能下调自噬,PA可能通过某种机制下调甚至逃避细胞自噬,MDRPA感染时,细胞自噬水平进一步降低,说明MDRPA逃避自噬的能力可能更强。合并缺血的DFI自噬水平进一步降低,适度诱导自噬或许可以作为治疗DF合并PA感染的新手段。
[Abstract]:Objective: through the comparison of 1. diabetic foot (diabetic foot, DF) of Pseudomonas aeruginosa in patients with wound infection (Pseudomonas aeruginosa, PA) differential expression of autophagy and infection control period of wound tissue associated protein, explore the effect of PA on autophagy in multi drug resistant Pseudomonas aeruginosa infection of.2. (Multi-drug resistant Pseudomonas aeruginosa, MDRPA) and group III the combined effects of ischemia on autophagy type.3.PA secretion system (type secretion system, T3SS) secretion of different virulence protein on autophagy. Methods: 1. March 2016 -2017 year in February in diabetic foot department metabolic disease hospital of Medical University Of Tianjin hospital treatment of 24 cases of DF infection in PA patients with clinical data; according to whether the infection of MDRPA is divided into two according to whether the ischemia group, divided into two groups, the standard treatment for infection control and divided into infection and infection control of.2. of all patients after deep wound debridement Department of materials, and the samples were divided into 3 parts. One part is used for bacterial culture and sensitivity test, wound colony count and frozen strain, virulence genes identified by PCR PA carried by T3SS. A part for making paraffin section: To observe changes of wound tissue with pathological HE staining; immunohistochemistry staining, semi quantitative detection in patients with wound tissue cell autophagy related protein LC3 (Microtubule-associated protein1 light chain 3), the average optical density of Beclin-1 and the value of P62; double immunofluorescent staining, the level of autophagy in wound tissue of patients with qualitative detection of macrophages. The last part of Western blot quantitative determination of LC3-II/I, the expression of Beclin-1 and P62 protein with the method to measure the amount of.3. index after controlling infection. Results: 1.DF infection and infection control of patients, infection control period of inflammation index (white blood cell count, neutrophil The percentage of ESR and high sensitivity C reactive protein) than the infection period decreased wound colony count decreased, fructosamine decreased wound area (P0.05), but there was no difference in general information (P0.05) group.MDRPA inflammatory index (neutrophil percentage, ESR and high sensitivity C reactive protein) and wound bacterial colonies higher than group N-MDRPA (P0.05). The ischemic group compared with non ischemic group ABI low, the wound area (P0.05) of.2. compared with the period of infection, neonatal infection control period in the wound tissue capillaries into more fiber cells, less inflammatory cell; immunohistochemical method showed higher LC3 and Beclin-1 expression in the wound tissue, P62 low expression (P0.05); immunofluorescence showed bud tissue LC3 and macrophage marker CD14 Merge, macrophage autophagy level is higher; Western blot showed that LC3-II/I and Beclin-1 high expression, P62 expression was lower (P0.05). Compared with MDRPA group, N-MDRPA group Less inflammatory cells infiltration, wound tissue in newborn capillaries, fibroblasts showed no significant difference; immunohistochemistry showed high expression of LC3 (P0.05), Beclin-1 and P62 expression had no statistical significance (P0.05); immunofluorescence showed bud tissue LC3 and macrophage cell specific markers CD14, Merge, macrophage autophagy high level; Western blot showed high expression of LC3-II/I in the wound tissue (P0.05), Beclin-1, no statistical significance of the expression of P62 (P0.05). Compared with the ischemia group, ischemia group and non newborn wound tissue in capillaries, fibroblasts and many inflammatory cells; immunohistochemistry showed high expression of Beclin-1 and LC3 in wound tissue in the expression of P62 is lower (P0.05); immunofluorescence showed bud tissue LC3 and macrophage specific marker CD14 Merge, macrophage autophagy level is higher; the Western blot showed a High LC3-II/I and Beclin-1 expression in tissue, the expression of P62 was low (P0.05).3. infection detection inspection bacterial real-time PCR virulence gene exo S. conclusion: PA infection control and improve the metabolic disorders, autophagy level increased, suggesting that severe metabolic disorders may be regulating autophagy, through a mechanism of down-regulation of PA may even escape autophagy, MDRPA infection, autophagy decreased further illustrate the ability of MDRPA to escape autophagy may be stronger. With ischemia DFI autophagy decreased further, moderately induced autophagy may be the treatment of DF with PA infection methods.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

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