本文選題：糖尿病　切入點(diǎn)：糖尿病足　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.通過比較糖尿病足(diabetic foot,DF)患者創(chuàng)面感染銅綠假單胞菌(Pseudomonas aeruginosa,PA)及感染控制期創(chuàng)面組織中自噬相關(guān)蛋白表達(dá)差異,探究PA對(duì)自噬的影響。2.研究感染多重耐藥銅綠假單胞菌(Multi-drug resistant pseudomonas aeruginosa,MDRPA)以及合并缺血對(duì)自噬的影響。3.PA的Ⅲ型分泌系統(tǒng)(typeⅢsecretion system,T3SS)分泌不同毒力蛋白對(duì)自噬的影響。方法:1.收集2016年3月-2017年2月于天津醫(yī)科大學(xué)代謝病醫(yī)院糖尿病足科住院治療的24例DF感染PA患者的臨床資料;根據(jù)是否感染MDRPA分為兩組,根據(jù)是否缺血分為兩組,標(biāo)準(zhǔn)化治療感染控制前后分為感染期和感染控制期。2.所有納入患者創(chuàng)面清創(chuàng)后深部取材,并將所取組織分為3部分。一部分用于細(xì)菌培養(yǎng)及藥敏試驗(yàn)、創(chuàng)面菌落計(jì)數(shù)并凍存菌株,通過PCR鑒定PA的T3SS所攜帶的毒力基因。一部分用于制作石蠟切片:HE染色觀察患者創(chuàng)面組織病理學(xué)改變;免疫組織化學(xué)染色,半定量檢測(cè)患者創(chuàng)面組織內(nèi)細(xì)胞自噬相關(guān)蛋白LC3(Microtubule-associated protein1 light chain 3)、Beclin-1和P62的平均光密度值;免疫熒光雙染色,定性檢測(cè)患者創(chuàng)面組織中巨噬細(xì)胞的自噬水平。最后一部分蛋白印跡定量測(cè)定LC3-II/I、Beclin-1和P62的蛋白表達(dá)量。3.感染控制后同上述方法測(cè)量上述指標(biāo)。結(jié)果:1.DF患者感染期與感染控制期比較,感染控制期炎癥指標(biāo)(白細(xì)胞計(jì)數(shù)、中性粒細(xì)胞百分比、血沉和超敏C反應(yīng)蛋白)較感染期下降、創(chuàng)面菌落計(jì)數(shù)下降、果糖胺下降、創(chuàng)面面積縮小(P0.05),余一般資料未見差異(P0.05)。MDRPA組炎癥指標(biāo)(中性粒細(xì)胞百分比、血沉和超敏C反應(yīng)蛋白)以及創(chuàng)面內(nèi)細(xì)菌菌落數(shù)較N-MDRPA組高(P0.05)。缺血組較非缺血組ABI低、創(chuàng)面面積大(P0.05)。2.與感染期相比,感染控制期創(chuàng)面組織中新生的毛細(xì)血管、成纖維細(xì)胞較多,炎癥細(xì)胞較少;免疫組織化學(xué)法顯示創(chuàng)面組織中LC3和Beclin-1表達(dá)較高、P62表達(dá)較少(P0.05);免疫熒光顯示芽組織LC3與巨噬細(xì)胞標(biāo)記物CD14 Merge后,巨噬細(xì)胞自噬水平較高;蛋白印跡顯示LC3-II/I和Beclin-1表達(dá)較高、P62表達(dá)較低(P0.05)。與MDRPA組相比,N-MDRPA組炎癥細(xì)胞浸潤(rùn)較少,創(chuàng)面組織中新生的毛細(xì)血管、成纖維細(xì)胞相比無明顯差異;免疫組織化學(xué)法顯示LC3的表達(dá)較高(P0.05),Beclin-1和P62表達(dá)無統(tǒng)計(jì)學(xué)意義(P0.05);免疫熒光顯示芽組織LC3與巨噬細(xì)胞特異性標(biāo)記物CD14 Merge后,巨噬細(xì)胞自噬水平較高;蛋白印跡法顯示創(chuàng)面組織中LC3-II/I表達(dá)較高(P0.05),Beclin-1、P62的表達(dá)無統(tǒng)計(jì)學(xué)意義(P0.05)。與缺血組相比,非缺血組創(chuàng)面組織中新生的毛細(xì)血管、成纖維細(xì)胞以及炎癥細(xì)胞較多;免疫組織化學(xué)法顯示創(chuàng)面組織中LC3和Beclin-1表達(dá)較高、P62表達(dá)較低(P0.05);免疫熒光顯示芽組織LC3與巨噬細(xì)胞特異性標(biāo)記物CD14 Merge后,巨噬細(xì)胞自噬水平較高;蛋白印跡法顯示創(chuàng)面組織中LC3-II/I和Beclin-1表達(dá)較高、P62表達(dá)較低(P0.05)。3.感染期real-time PCR檢測(cè)送檢細(xì)菌均攜帶毒力基因exo S。結(jié)論:PA感染控制以及代謝紊亂改善后,自噬水平升高,這提示嚴(yán)重的代謝紊亂可能下調(diào)自噬,PA可能通過某種機(jī)制下調(diào)甚至逃避細(xì)胞自噬,MDRPA感染時(shí),細(xì)胞自噬水平進(jìn)一步降低,說明MDRPA逃避自噬的能力可能更強(qiáng)。合并缺血的DFI自噬水平進(jìn)一步降低,適度誘導(dǎo)自噬或許可以作為治療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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2

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