發(fā)布時間：2018-05-10 04:50

  本文選題：納米材料 + 細菌耐藥性　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：細菌耐藥性的擴散是一個嚴重威脅全球公共衛(wèi)生安全的重要問題,尤其是2010年,在南亞和我國發(fā)現(xiàn)新的“超級細菌”后,人們更加關(guān)注細菌耐藥性問題。細菌獲得抗生素抗性基因,一方面是由于抗生素過度使用和濫用產(chǎn)生的抗生素選擇壓力造成細菌產(chǎn)生抗生素抗性突變；另一方面是細菌通過水平轉(zhuǎn)移的方式獲得抗生素抗性基因。后者是細菌獲得耐藥性的主要方式。細菌抗生素抗性基因往往編碼在可移動質(zhì)粒上,通過質(zhì)粒接合轉(zhuǎn)移在細菌間進行傳播。質(zhì)粒介導(dǎo)的細菌耐藥基因接合轉(zhuǎn)移對細菌耐藥性快速擴散起到了至關(guān)重要作用。許多環(huán)境因素都能夠影響質(zhì)粒的接合轉(zhuǎn)移過程。 納米材料由于具有新穎的物理、化學(xué)和生物學(xué)特性,已經(jīng)被應(yīng)用于多個領(lǐng)域,尤其是作為高效吸附劑和氧化劑,正用于水處理行業(yè)。然而,納米材料在環(huán)境中的殘留對環(huán)境、生物以及人體的負面影響正在引起人們重視。納米材料也正在成為一種新的環(huán)境污染物。大量研究表明,納米材料可產(chǎn)生大量活性氧,破壞細菌細胞膜,影響蛋白質(zhì)和基因,載帶DNA或RNA分子進入動物和植物細胞�，F(xiàn)在,水環(huán)境中存在著大量耐藥菌和納米材料,納米材料是否促進耐藥基因在細菌間傳播還不清楚。 本研究基于以上事實提出,水中納米材料可作用于細菌細胞膜和/或調(diào)控基因表達,促進細菌耐藥基因接合轉(zhuǎn)移的假說,并建立質(zhì)粒介導(dǎo)的接合轉(zhuǎn)移模型,研究水中常見納米材料對細菌耐藥基因接合轉(zhuǎn)移的影響及規(guī)律,分析納米材料作用過程中各種因素的影響規(guī)律,利用正交實驗的方法分析其中的主要因素,從生物化學(xué)、細胞生物學(xué)和分子生物學(xué)多個角度探討了納米材料促進細菌耐藥基因接合轉(zhuǎn)移機制。主要研究結(jié)果如下： 利用RP4質(zhì)粒建立了四個接合轉(zhuǎn)移模型,包括耐藥基因在大腸桿菌種屬內(nèi)、大腸桿菌到沙門氏菌、大腸桿菌到糞腸球菌、糞腸球菌到糞腸球菌的轉(zhuǎn)移；利用RK2質(zhì)粒建立了耐藥基因在大腸桿菌內(nèi)的轉(zhuǎn)移模型；利用pCF10建立了耐藥基因在糞腸球菌內(nèi)的轉(zhuǎn)移模型。建立的6個耐藥基因接合轉(zhuǎn)移模型,既包括了耐藥基因在種屬內(nèi)的接合轉(zhuǎn)移,又包括了耐藥基因的跨種屬轉(zhuǎn)移。研究結(jié)果表明,建立的6個耐藥基因接合轉(zhuǎn)移模型比較穩(wěn)定,可以用來評價納米材料對耐藥基因接合轉(zhuǎn)移的影響。 納米TiO2、納米SiO2、納米Fe2O3和納米Al2O3都能促進RP4質(zhì)粒從大腸桿菌到沙門菌的轉(zhuǎn)移。納米材料濃度為5mmol/L時,與空白對照和大顆粒材料相比,可使RP4接合轉(zhuǎn)移分別提高89倍、10倍、20倍和142倍,納米Al2O3的促進作用最強。不同接合轉(zhuǎn)移體系中,納米材料都有促進作用。5mmol/L納米Al2O3可使RP4從大腸桿菌到大腸桿菌、革蘭陰性菌到革蘭陽性菌、糞腸球菌到糞腸球菌的接合頻率轉(zhuǎn)移分別增加200倍、50倍和100倍。納米Al_2O_3對RK2和pCF10接合轉(zhuǎn)移的促進作用可分別達到170倍和120倍。這些數(shù)據(jù)說明,納米材料對接合質(zhì)粒介導(dǎo)的耐藥基因轉(zhuǎn)移的促進作用可能是一個普遍現(xiàn)象。而且相同成分的大顆粒材料對耐藥基因的接合轉(zhuǎn)移無明顯的促進作用,也說明對接合轉(zhuǎn)移的促進作用來自納米材料的納米結(jié)構(gòu)。 以納米Al_2O_3作為代表材料研究了接合轉(zhuǎn)移中的影響因素。結(jié)果表明,隨著濃度升高,納米Al_2O_3對RP4接合轉(zhuǎn)移促進作用增強。納米Al_2O_3濃度為5mmol/L時達到最高,可以提高RP4接合轉(zhuǎn)移100倍以上。納米Al_2O_3濃度高于5mmol/L時,促進作用減弱,但是與空白對照組和大顆粒組比較,仍能顯著促進RP4接合轉(zhuǎn)移。納米Al_2O_3的作用效果隨濃度變化有峰值出現(xiàn),原因可能是納米材料,尤其是高濃度納米材料能使細胞產(chǎn)生嚴重的氧化應(yīng)激反應(yīng),造成細菌大量死亡。利用數(shù)學(xué)模型排除細菌死亡影響,5mmol/L納米Al_2O_3能提高接合轉(zhuǎn)移常數(shù)5個數(shù)量級。納米Al_2O_3作用下,隨著接合菌密度升高和接合時間延長,接合子數(shù)量逐漸增加,與空白對照和大顆粒組比較,增加顯著。納米Al_2O_3作用后,接合溫度低于20℃時,隨溫度升高生成的接合子數(shù)量增加；溫度高于20℃時,接合子生成數(shù)量增加不明顯。接合體系的pH值對接合子生成無明顯影響。在富營養(yǎng)條件下(LB培養(yǎng)基),接合子生成的數(shù)量更多。利用正交實驗分析各因素的作用情況發(fā)現(xiàn),各因素對接合子生成影響的重要次序為：初始菌密度納米Al_2O_3濃度接合時間接合溫度初始菌密度與納米濃度的交互作用。納米Al_2O_3濃度已成為僅次于初始菌密度的影響因素。在最適宜的條件下能夠提高RP4在PBS中跨種屬轉(zhuǎn)移效率200倍,提高RP4在LB中同種屬內(nèi)的轉(zhuǎn)移效率250倍。 納米Al_2O_3促進RP4接合轉(zhuǎn)移機制研究發(fā)現(xiàn)：納米Al_2O_3能促使細菌細胞產(chǎn)生大量OH·,激發(fā)細菌的氧化抗氧化反應(yīng)。透射電鏡與原子力顯微鏡的結(jié)果也表明,納米Al_2O_3對細菌細胞膜產(chǎn)生影響,細胞膜表面變得不光滑,甚至部分細胞膜發(fā)生溶解。濃度越高,納米Al_2O_3對細菌細胞膜影響越嚴重。大顆粒Al_2O_3卻無類似的結(jié)果,說明納米Al_2O_3的納米結(jié)構(gòu)是產(chǎn)生細胞膜損傷的主要因素。納米Al_2O_3對細菌細胞膜的影響為供體菌和受體菌的細胞膜融合和DNA跨膜轉(zhuǎn)運提供了便利條件。納米Al_2O_3通過抑制korA和korB的mRNA表達,激活了trbBp基因的mRNA表達,進而促使供體菌與受體菌間形成“接合橋”,促進了接合轉(zhuǎn)移的第一個過程。電鏡結(jié)果也證實納米Al_2O_3使形成的“接合橋”數(shù)量增加。同時納米Al_2O_3通過抑制korB和trbA的mRNA的表達,激活了trfAp基因的mRNA表達,促使RP4質(zhì)粒的轉(zhuǎn)移與復(fù)制,促進了接合轉(zhuǎn)移的第二個過程。 總的來說,納米材料能通過影響細菌細胞膜狀態(tài),影響與接合轉(zhuǎn)移過程相關(guān)的調(diào)控基因mRNA表達而促進了接合質(zhì)粒的接合轉(zhuǎn)移。 本課題利用生命科學(xué)的新技術(shù),研究材料領(lǐng)域的納米粒子對環(huán)境安全領(lǐng)域的細菌耐藥性接合轉(zhuǎn)移的影響規(guī)律與機制,是典型的學(xué)科交叉研究,也是相關(guān)學(xué)科的前沿領(lǐng)域,將開辟納米材料環(huán)境安全研究的新領(lǐng)域。本課題的研究結(jié)果將進一步增加人們對細菌耐藥性轉(zhuǎn)移與獲得的認識程度,豐富相關(guān)的科學(xué)知識,同時也將提高人們對環(huán)境中殘留納米材料所引起的環(huán)境與生態(tài)風險的認識。本研究提示我們應(yīng)更加認真的對納米材料進行相關(guān)的安全性評價和謹慎地使用納米材料,以便控制或減少由納米材料導(dǎo)致的環(huán)境中耐藥菌的形成與增加。
[Abstract]:The spread of bacterial drug resistance is an important issue that poses a serious threat to global public health safety , especially in 2010 , after the discovery of new " super - bacteria " in South Asia and China , people pay more attention to bacterial drug resistance .
Bacterial antibiotic resistance genes are often encoded on a movable plasmid and propagated through a plasmid joint transfer between bacteria . The plasmid - mediated drug resistance gene - binding transfer plays a crucial role in the rapid diffusion of bacterial drug resistance . Many environmental factors can influence the junction transfer process of the plasmid .

Nano - materials have been used in many fields due to their novel physical , chemical and biological characteristics , especially as high - efficiency adsorbents and oxidants , and are being used in water treatment industries . However , nano - materials are becoming a new environmental pollutant . Nano - materials are also becoming a new environmental pollutant . Many researches have shown that nano - materials can produce large amounts of active oxygen , destroy bacterial cell membranes , affect proteins and genes , carry DNA or RNA molecules into animals and plant cells . Nowadays , there are a large number of drug - resistant bacteria and nano - materials in the water environment .

Based on the above facts , it is suggested that nano - materials in water can act on the expression of bacterial cell membrane and / or regulate gene expression , promote the transfer of drug - resistant genes , and establish a plasmid - mediated junction transfer model .

Four conjugative transfer models were constructed by using RP4 plasmid , including the transfer of drug resistance genes in E . coli , E . coli to Salmonella , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli and E . coli .
The transfer model of resistance gene in E . coli was established by using RK2 plasmid .
The transfer model of drug resistant gene was established by using pCF10 . Six drug resistant gene transfer models were established , which included both drug resistance gene and drug resistance gene transfer . The results showed that the 6 drug resistant gene transfer models were stable and could be used to evaluate the effect of nano - material on drug resistance gene transfer .

Nano - TiO2 , nano - SiO2 , Fe2O3 and Al2O3 can promote the transfer of RP4 plasmid from E . coli to Salmonella .

The effects of nano Al _ 2O _ 3 on the bonding transfer of RP4 were studied . The results showed that the effect of nano - Al _ 2O _ 3 was higher than that of the blank control group and the large - particle group . The effect of nano - Al _ 2O _ 3 was higher than that of the control group and the large - particle group .
Under the condition of rich nutrition ( LB medium ) , the number of zygotes was increased . In the condition of rich nutrition ( LB medium ) , the number of zygotes was much more . It was found that the influence factors of the initial bacterial density were the interaction of initial bacterial density with nano - concentration . Under optimum conditions , the efficiency of RP4 was 200 - fold higher than that in PBS and 250 - fold increase in RP4 ' s transfer efficiency in LB medium .

The effect of nano Al _ 2O _ 3 on the cell membrane of bacteria was investigated . The results of TEM and AFM showed that the nano - Al _ 2O _ 3 had an effect on cell membrane damage .

In general , the nano - material can promote the junction transfer of the bonding plasmid by influencing the state of the cell membrane of the bacteria , affecting the expression of the regulating gene mRNA related to the junction transfer process .

The research results of this project will further increase people ' s understanding of bacterial drug resistance transfer and acquisition , enrich relevant scientific knowledge , and raise people ' s awareness of environmental and ecological risks caused by residual nano materials in the environment .

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：TB383.1;R318.08

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