發(fā)布時間：2018-05-14 07:01

  本文選題：耐甲氧西林的金黃色葡萄球菌 + 群體感應(yīng)系統(tǒng)抑制劑��； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：目的:耐藥細(xì)菌感染是臨床抗感染治療中極為棘手的問題,也是造成重癥感染死亡的首要原因。其中被稱為“超級細(xì)菌”的耐甲氧西林金黃色葡萄球菌(MRSA)在臨床感染中的危害性日趨嚴(yán)重。患者一旦感染MRSA,治療極為困難,常繼發(fā)多重感染、膿毒性休克和多器官功能衰竭綜合癥,感染患者死亡率很高。目前臨床上還沒有特異性針對MRSA的有效抗菌藥物,因此,如何有效控制細(xì)菌MRSA感染造成的危害已經(jīng)成為各國政府高度關(guān)注的焦點(diǎn),尋找和研制有效控制MRSA感染的新藥物也成為當(dāng)今世界各國科學(xué)家當(dāng)務(wù)之急的研究目標(biāo)。群體感應(yīng)(Quorum sensing,QS)是廣泛存在于細(xì)菌內(nèi)的一種調(diào)控細(xì)菌群體行為的機(jī)制,參與細(xì)菌生物被膜形成、毒力因子釋放、免疫逃逸、以及耐藥性發(fā)生的重要調(diào)控系統(tǒng)。抑制細(xì)菌群體感應(yīng)系統(tǒng)(Quorum sensing system,QSS)可以顯著抑制細(xì)菌的致病性,但不影響細(xì)菌的生長,因而對細(xì)菌選擇性生長壓力小,具有不誘導(dǎo)細(xì)菌耐藥的獨(dú)特優(yōu)點(diǎn)。因此,以QSS為靶標(biāo)的新型抗菌藥物研究,成為不易誘導(dǎo)耐藥新型抗菌藥物研究的新趨勢和新熱點(diǎn),也是新型抗菌藥物研究領(lǐng)域最有希望的突破口。附屬基因調(diào)節(jié)子系統(tǒng)(accessory gene regulator,agr)是廣泛存在于金黃色葡萄球菌的QSS,該系統(tǒng)激活后,啟動其效應(yīng)分子RNAⅢ的表達(dá),進(jìn)而調(diào)控其下游多種毒力因子的表達(dá)。抑制agr系統(tǒng)信號調(diào)控通路中的關(guān)鍵分子,可以抑制細(xì)菌多種毒力因子的表達(dá),進(jìn)而減輕細(xì)菌致病力。RNAⅢ抑制肽(RNAⅢ-inhibiting peptide,RIP)是一種作用于agr系統(tǒng)的抑制劑。業(yè)已證實,RIP能夠有效抑制MRSA毒力因子釋放,降低感染動物死亡率,減輕細(xì)菌生物膜的形成等,因此,RIP作為抗MRSA的有效抗菌藥物備受關(guān)注。目前已知所有作用于QS的抑制劑在體外均無抑菌或殺菌作用,但在感染動物體內(nèi)則表現(xiàn)出明顯的抗菌作用。然而迄今為止,QS抑制劑體內(nèi)抗菌的內(nèi)在規(guī)律和機(jī)制幾乎完全不清楚,這已成為制約新型抗菌藥物RIP深入研究重要限速環(huán)節(jié)。本課題采用氨基酸替換、末端修飾、寡聚化等方法設(shè)計合成不同的RIP衍生物,體內(nèi)外實驗評價RIP衍生物抗菌活性,并探討RIP衍生物體內(nèi)抗菌作用的機(jī)理。方法:1.RIP衍生物的設(shè)計與合成以RIP-I的序列YKPITNF為模板,通過氨基酸替換,N-端乙�；揎�,C-端酰胺化修飾,以及寡聚化修飾的方法,設(shè)計合成RIP-V、RIP-L、RIP1181、RIP1182、RIP1183五條RIP衍生物,采用RP-HPLC方法分析其純度,采用ESI質(zhì)譜儀或MALDI-TOF質(zhì)譜儀鑒定合成多肽的分子量,確證合成衍生物多肽序列的正確性。2.RIP衍生物體內(nèi)抗菌活性的篩選建立HA-MRSA感染小鼠膿毒癥模型,分別檢測RIP衍生物對感染小鼠生存率、臟器菌落計數(shù)、組織病理損傷等指標(biāo)的影響,篩選出抗菌活性較優(yōu)的RIP衍生物。3.RIP1183體外抗菌效果評價選擇6株葡萄球菌(2株標(biāo)準(zhǔn)株和4株多藥耐藥菌株)體外培養(yǎng),測定RIP衍生物RIP1183對6株葡萄球菌的最低抑菌濃度(MIC);細(xì)菌生長檢測儀測定RIP1183對6株菌的生長抑制情況。4.建立不同感染模型評價RIP1183對小鼠治療作用選擇HA-MRSA(MRSA XJ75302)或CA-MRSA(LAC)菌株,分別腹腔注射、經(jīng)鼻滴入和皮下接種MRSA方式感染小鼠,制備膿毒癥、壞死性肺炎和皮膚感染模型,給小鼠腹腔注射不同劑量的RIP1183,觀察RIP1183對小鼠生存時間和生存率的影響;通過小鼠臟器或組織中的細(xì)菌計數(shù),觀察RIP1183對體內(nèi)細(xì)菌生長的影響;通過小鼠肺臟、肝臟或皮膚組織HE染色,觀察RIP1183對組織損傷的保護(hù)作用。5.RIP1183體內(nèi)抗菌作用機(jī)理探討(1)按照環(huán)磷酰胺法缺失小鼠的中性粒細(xì)胞,并用LAC感染該小鼠建立膿毒癥模型,腹腔注射RIP1183,觀察中性粒細(xì)胞缺乏后對RIP1183抗菌作用的影響。(2)分離人外周血中性粒細(xì)胞與RIP1183共孵育,髓過氧化物酶(MPO)檢測試劑盒測定中性粒細(xì)胞中的MPO,觀察RIP1183對中性粒細(xì)胞內(nèi)MPO活性的影響。(3)分離小鼠體內(nèi)MRSA,采用實時定量PCR方法檢測細(xì)菌RNAⅢ和毒力因子PVL、HLA、PSMα、PSMβ的表達(dá),觀察RIP1183對RNAⅢ和毒力因子表達(dá)的影響。(4)LAC細(xì)菌或細(xì)菌上清或HKSA(熱殺死金黃色葡萄球菌),與人中性粒細(xì)胞共孵育,Western blot檢測壞死性凋亡特征蛋白p MLKL的表達(dá)水平,觀察LAC對中性粒細(xì)胞壞死性凋亡的影響。(5)PSMα和PSMβ缺陷菌上清與與人中性粒細(xì)胞共孵育,Western blot檢測p MLKL的表達(dá)水平,觀察PSMα和PSMβ對中性粒細(xì)胞壞死性凋亡的影響。(6)合成七種PSM多肽分子與人中性粒細(xì)胞共孵育,或提前加入MLKL抑制劑NSA,Western blot檢測p MLKL的表達(dá)水平;透射電鏡觀察PSMα1對中性粒細(xì)胞形態(tài)的影響。(7)LAC及其PSMα缺陷菌建立壞死性肺炎模型,腹腔注射給予感染小鼠RIP1183或壞死性凋亡抑制劑Nec,免疫熒光法檢測小鼠肺組織中的中性粒細(xì)胞壞死性凋亡、Western blot檢測肺泡灌洗液中的中性粒細(xì)胞p MLKL的表達(dá)水平、流式細(xì)胞儀檢測感染小鼠肺組織中的中性粒細(xì)胞死亡的比例。(8)LAC及其PSMα缺陷菌建立壞死性肺炎模型,腹腔注射給予感染小鼠RIP1183或壞死性凋亡抑制劑Nec,觀察對小鼠生存率、肺損傷(HE染色)和肺組織中細(xì)菌數(shù)的變化。6.RIP1183安全性與藥物代謝動力學(xué)評價(1)采用最大給藥量法,大鼠或犬單次靜脈注射RIP1183,觀察其對大鼠或犬的一般體征、攝食、體重等指標(biāo)的影響;(2)小鼠RIP1183靜脈注射后,觀察其對小鼠中樞神經(jīng)系統(tǒng)的影響;比格犬RIP1183靜脈注射后,觀察其對比格犬心血管系統(tǒng)和呼吸系統(tǒng)的影響。(3)比格犬分別單次靜脈注射2 mg/kg、4 mg/kg、8 mg/kg的RIP1183后,檢測給藥后不同時間點(diǎn)比格犬血漿中的藥物濃度,計算該藥物的藥代動力學(xué)參數(shù)。結(jié)果:1.RIP衍生物的合成與鑒定采用固相合成的方法分別合成了RIP-I、RIP-V、RIP-L、RIP1181、RIP1182、RIP1183六條多肽,其中RIP-L和RIP-V,是RIP-I的4位氨基酸替換為亮氨酸或纈氨酸得到的,序列分別為YKPLTNF-CONH2和YKPVTNF-CONH2;RIP1183是將RIP-V乙�；揎椀玫降�,序列為CH3CO-YKPVTNF-CONH2;寡聚化RIP-V分子得到RIP1182,同時進(jìn)行乙酰化修飾得到的RIP1181,序列分別為YKPVTNF-ST-YKPVTNF-CONH2和CH3CO-YKPVTNF-ST-YKPVTNF-CONH2。反向高效液相色譜(RP-HPLC)法鑒定RIP衍生物的純度均在95%以上;質(zhì)譜鑒定RIP衍生物的分子量與理論值一致,表明所有合成的RIP衍生物結(jié)構(gòu)正確。2.體內(nèi)篩選RIP衍生物用HA-MRSA感染小鼠建立膿毒癥模型,發(fā)現(xiàn)RIP-V顯著提高小鼠生存率、減輕肺臟和肝臟病理損傷、減少小鼠臟器中細(xì)菌數(shù),RIP-V體內(nèi)抗感染活性優(yōu)于RIP-I和RIP-L;RIP-V經(jīng)末端修飾得到的RIP1183,RIP1183顯著提高了膿毒癥小鼠的生存率和延長生存時間,其抗菌活性優(yōu)于RIP-V、RIP1181和RIP1182。結(jié)果顯示設(shè)計合成的RIP衍生物中,RIP1183具有良好抗菌活性。3.RIP1183體外無抗菌作用RIP1183對6株葡萄球菌的MIC值均大于256μg/ml;RIP1183在250-1000μg/ml濃度下,均不能抑制6株葡萄球菌的生長,表明RIP1183體外無抗菌活性。4.RIP1183對MRSA感染小鼠保護(hù)作用在HA-MRSA(MRSA XJ75302)和CA-MRSA LAC感染所致膿毒癥小鼠模型上,RIP1183分別提高小鼠的生存率達(dá)40%和60%,顯著降低感染小鼠臟器中的細(xì)菌數(shù)量,減輕了小鼠臟器的病理損傷;在壞死性肺炎小鼠模型上,RIP1183可以顯著降低感染小鼠肺臟的細(xì)菌數(shù)量,減輕肺水腫和肺病理損傷;皮膚感染模型上,RIP1183顯著減少小鼠膿腫的形成,降低感染部位的細(xì)菌數(shù)量,并促進(jìn)感染部位創(chuàng)面愈合。5.RIP1183體內(nèi)抗菌機(jī)理探討(1)RIP1183對中性粒細(xì)胞缺失的膿毒癥小鼠保護(hù)作用消失。(2)RIP1183與中性粒細(xì)胞共孵育,不影響中性粒細(xì)胞內(nèi)MPO活性(3)RIP1183可顯著降低感染小鼠組織內(nèi)細(xì)菌的RNAⅢ和PSMα、PSMβ等多種毒力因子的表達(dá)。(4)細(xì)菌LAC分泌上清中的PSM可以誘導(dǎo)中性粒細(xì)胞壞死性凋亡特征蛋白p MLKL表達(dá)增高,并能夠被MLKL抑制劑NSA阻斷。(5)與野生菌LAC比較,PSMα和PSMβ缺陷菌上清,降低中性粒細(xì)胞的p MLKL表達(dá)。(6)PSM多肽分子與中性粒細(xì)胞共孵育,PSM增加中性粒細(xì)胞中的p MLKL表達(dá)水平,并可以被NSA阻斷;透射電鏡從形態(tài)上直接觀察到PSMα1可誘導(dǎo)中性粒細(xì)胞出現(xiàn)典型的壞死性凋亡特征,且NSA可顯著減少壞死性凋亡的中性粒細(xì)胞的數(shù)目。(7)小鼠給予RIP1183或壞死性凋亡抑制劑Nec或缺失細(xì)菌PSM后,免疫熒光結(jié)果表明,小鼠肺組織中壞死性凋亡的中性粒細(xì)胞顯著減少;Western blot結(jié)果表明,肺泡灌洗液中中性粒細(xì)胞的p MLKL表達(dá)水平降低了;流式細(xì)胞結(jié)果表明,壞死性凋亡的中性粒細(xì)胞比例降低。(8)小鼠給予RIP1183或壞死性凋亡抑制劑Nec或缺失細(xì)菌PSM后,可以顯著提高肺炎小鼠生存率、促進(jìn)肺臟內(nèi)細(xì)菌的清除和減輕肺病理損傷。6.RIP1183安全性與藥物代謝動力學(xué)研究(1)大鼠和比格犬分別靜脈注射100 mg/kg和50 mg/kg的RIP1183后,未觀察到任何毒性反應(yīng),該劑量分別為RIP1183有效劑量的28和48倍。(2)在治療范圍內(nèi),RIP1183對小鼠中樞系統(tǒng)、比格犬的呼吸系統(tǒng)和心血管系統(tǒng)無明顯影響。(3)體內(nèi)藥代動力學(xué)實驗結(jié)果表明,RIP1183在犬體內(nèi)的血漿消除半衰期在20min左右。結(jié)論:1.篩選獲得agr系統(tǒng)抑制劑RIP1183,并證實RIP1183是一種安全范圍較大,毒性低,抗MRSA活性較優(yōu)的抗菌劑,犬體內(nèi)的血漿消除半衰期在20 min左右。2.發(fā)現(xiàn)RIP1183能夠顯著抑制MRSA的RNAⅢ和PSMα、PSMβ等多種毒力因子的表達(dá)。3.發(fā)現(xiàn)RIP1183體內(nèi)抗菌作用依賴于白細(xì)胞功能的正常,中性粒細(xì)胞缺失后,RIP1183對膿毒癥的保護(hù)作用消失,但是RIP1183無直接激活中性粒細(xì)胞的作用。4.提出RIP1183體內(nèi)抗菌作用機(jī)理的假說,即RIP1183通過抑制金黃色葡萄球菌agr群體感應(yīng)系統(tǒng),抑制致病毒力因子的表達(dá),減輕病毒力因子所致機(jī)體病理損傷;又通過抑制PSMα和PSMβ毒力因子表達(dá),減輕這些毒力因子誘導(dǎo)的中性粒細(xì)胞壞死性凋亡,保護(hù)中性粒細(xì)胞清除細(xì)菌的功能。
[Abstract]:Objective: drug resistant bacteria infection is a very difficult problem in clinical anti infection treatment, and it is also the primary cause of severe infection death. Among them, methicillin resistant Staphylococcus aureus (MRSA), known as "superbacteria", is becoming more and more dangerous in clinical infection. When patients are infected with MRSA, the treatment is very difficult and often secondary to multiple. Infection, septic shock and multiple organ failure syndrome, the mortality of the infected patients is very high. There is no specific antibacterials for MRSA in clinic. Therefore, how to effectively control the harm caused by bacterial MRSA infection has become the focus of the governments of various countries to find and develop new drugs to effectively control MRSA infection. Quorum sensing (QS) is a kind of mechanism that widely exists in bacteria to regulate bacterial population behavior, and is involved in the formation of bacterial biofilm, release of virulence factors, immune escape, and drug resistance, and the inhibition of bacterial quorum induction. Quorum sensing system (QSS) can significantly inhibit the pathogenicity of bacteria, but does not affect the growth of bacteria. Therefore, it has a small pressure on the selective growth of bacteria and has the unique advantage of not inducing bacterial resistance. Therefore, a new research on the new antibacterial drugs with QSS as the target is a new trend and new trend in the research of new drug resistant drugs which are not easy to induce resistance. Hot spots are also the most promising breakthrough in the research field of new antibacterial drugs. The accessory gene regulator (agr) is a QSS that widely exists in Staphylococcus aureus. After the system is activated, it starts the expression of its effector molecule RNA III, and then regulates the expression of a variety of virulence factors in the downstream of the system and inhibits the agr system signal. The key molecules in the regulatory pathway can inhibit the expression of various virulence factors of bacteria, and then reduce the bacterial pathogenicity.RNA III inhibitory peptide (RNA III -inhibiting peptide, RIP) as an inhibitor that acts on the agr system. It has been proved that RIP can effectively inhibit the release of MRSA virulence factors, reduce the mortality of infected animals and reduce the bacterial biofilm. As a result, RIP has been paid much attention to as an effective antibacterials for anti MRSA. All of the inhibitors of QS are known to have no bacteriostasis or bactericidal effect in vitro, but there are obvious antibacterial effects in infected animals. However, the inherent laws and mechanisms of anti bacteria in QS inhibitors are almost completely unknown to date. RIP has become a new antiseptic drug to study the important speed limiting link. This subject uses amino acid replacement, terminal modification, oligomerization and other methods to design and synthesize different RIP derivatives. In vivo and in vivo and in vitro, the antibacterial activity of RIP derivatives is evaluated and the mechanism of antibacterial activity in RIP derivatives is discussed. Method: design and synthesis of 1.RIP derivatives Taking the sequence YKPITNF of RIP-I as a template, the five RIP derivatives of RIP-V, RIP-L, RIP1181, RIP1182, RIP1183 were designed and synthesized by amino acid replacement, acetylation modification of N- terminal, C- terminal amidation modification, and oligomeric modification. The purity of the RIP was analyzed by RP-HPLC method. The molecular weight of synthetic peptides was identified by ESI mass spectrometer or mass spectrometer. To confirm the correctness of the sequence of synthetic derivatives of polypeptide, the screening of antibacterial activity in.2.RIP derivatives to establish a model of sepsis in mice with HA-MRSA infection, and to detect the effects of RIP derivatives on the survival rate of infected mice, the count of organ colony and tissue pathological damage, and to screen out the antibacterial activity of RIP derivative.3.RIP1183 in vitro. 6 strains of Staphylococcus (2 standard strains and 4 strains of multidrug resistant strains) were cultured in vitro, and the minimum inhibitory concentration (MIC) of RIP derivative RIP1183 on 6 strains of Staphylococcus was measured. The bacterial growth detector was used to determine the growth inhibition of 6 strains of bacteria by RIP1183 and to evaluate the therapeutic effect of HA-MRSA (M) on the therapeutic effect of RIP1183 on mice. RSA XJ75302) or CA-MRSA (LAC) strains, respectively, intraperitoneally injected, infecting mice by nasal drip and subcutaneous inoculation MRSA, preparing sepsis, necrotizing pneumonia and skin infection model, giving mice intraperitoneal injection of different doses of RIP1183, observing the effect of RIP1183 on the survival time and survival rate of mice, and through the bacteria meter in mouse organs or tissues. The effect of RIP1183 on the growth of bacteria in vivo was observed. HE staining of lung, liver or skin tissue in mice was used to observe the protective effect of RIP1183 on tissue damage in.5.RIP1183 (1) the deletion of neutrophils in mice by cyclophosphamide method, and LAC infection in the mice to establish a sepsis model and intraperitoneal injection of RIP118 3, observe the effect of neutrophils deficiency on the antiseptic effect of RIP1183. (2) the neutrophils were incubated with RIP1183, and the MPO assay kit was used to determine the MPO in neutrophils, and the effect of RIP1183 on the activity of MPO in neutrophils was observed. (3) the MRSA in mice was isolated in vivo, and the real-time quantitative PCR method was used. The expression of RNA III and virulence factor PVL, HLA, PSM a, PSM beta, and the effect of RIP1183 on the expression of RNA III and virulence factors. (4) LAC bacteria or bacterial supernatant or HKSA (heat killing Staphylococcus aureus), incubated with human neutrophils, Western blot detection of the expression level of necrotizing apoptotic characteristic protein p The effect of cell necrosis apoptosis. (5) PSM alpha and PSM beta defect bacteria supernatant and human neutrophils co incubation, Western blot detection of P MLKL expression level, observe the effect of PSM A and PSM beta on neutrophil necrotic apoptosis. (6) synthesis of seven PSM polypeptide molecules and human neutrophils, or early MLKL inhibitor NSA, Western The expression level of P MLKL was detected by blot; the effect of PSM alpha 1 on the morphology of neutrophils was observed by transmission electron microscopy. (7) LAC and its PSM alpha deficiency bacteria were established to establish necrotizing pneumonia model, intraperitoneal injection of RIP1183 or necrotic inhibitor Nec in infected mice, and immunofluorescence to detect the necrotic apoptosis of neutrophils in the lung tissue of mice, Western blot The expression level of neutrophil P MLKL in the alveolar lavage fluid was detected. The ratio of neutrophils in the lung tissues of the infected mice was detected by flow cytometry. (8) LAC and its PSM alpha deficiency bacteria were established to establish necrotizing pneumonia model. The mice were injected with RIP1183 or Nec of necrotic apoptosis inhibitor in the infected mice. The survival rate of mice and lung injury were observed. (HE staining) and changes in the number of bacteria in the lung tissue.6.RIP1183 safety and pharmacokinetics evaluation (1) use the maximum dose method, rat or dog single intravenous injection of RIP1183, to observe the effects on the general signs, feeding, weight and other indexes of rats or dogs; (2) after the injection of RIP1183 in mice, the central nervous system of mice was observed. Influence on the cardiovascular system and respiratory system of beagle dogs after intravenous injection of RIP1183. (3) after a single intravenous injection of 2 mg/kg, 4 mg/kg, and 8 mg/kg RIP1183 in a beagle dog, the drug concentration in the plasma of beagle dogs at different time points was detected and the pharmacokinetic parameters of the drug were calculated. Results: 1.RIP derivation. The synthesis and identification of RIP-I, RIP-V, RIP-L, RIP1181, RIP1182, RIP1183 six peptides, respectively, RIP-L and RIP-V, are obtained by the substitution of 4 amino acids from RIP-I to leucine or valine, and the sequences are YKPLTNF-CONH2 and YKPVTNF-CONH2. H3CO-YKPVTNF-CONH2; oligomeric RIP-V molecules obtained RIP1182 and RIP1181 obtained by acetylation, and the purity of RIP derivatives was identified by YKPVTNF-ST-YKPVTNF-CONH2 and CH3CO-YKPVTNF-ST-YKPVTNF-CONH2. reverse high performance liquid chromatography (RP-HPLC), respectively. The molecular weight and theoretical value of the RIP derivatives were identified by mass spectrometry. The results showed that all the synthetic RIP derivatives were correctly screened by.2. in vivo to establish a sepsis model in mice with HA-MRSA infection. It was found that RIP-V significantly improved the survival rate of mice, reduced the pathological damage of the lung and liver, reduced the number of bacteria in the organs of the mice, and the anti infection activity of RIP-V in the body was better than that of RIP-I and RIP-L; RIP-V was modified by the end modification. RIP1183, RIP1183 significantly improved the survival rate and prolongation of the survival time of sepsis mice, its antibacterial activity was better than RIP-V, RIP1181 and RIP1182. results showed that in the synthesized RIP derivatives, RIP1183 had good antibacterial activity and no antibacterial activity.3.RIP1183 in vitro, the MIC value of RIP1183 to 6 strains of Staphylococcus were greater than 256 mu g/ml; RIP1183 was 250 The growth of 6 Staphylococcus strains could not be inhibited under the concentration of -1000 micron g/ml, indicating that the protective effect of RIP1183 in vitro on MRSA infected mice was in the mice model of sepsis caused by HA-MRSA (MRSA XJ75302) and CA-MRSA LAC infection. RIP1183 increased the survival rate of mice by 40% and 60% respectively, and significantly reduced the organs of infected mice. The number of bacteria reduces the pathological damage of the mouse organs, and in the mice model of necrotizing pneumonia, RIP1183 can significantly reduce the number of bacteria in the lungs of the mice, reduce the pulmonary edema and lung pathological damage. On the skin infection model, RIP1183 significantly reduces the formation of mice abscess, reduces the number of bacteria in the infected parts, and promotes the infection site. Study on the antibacterial mechanism of.5.RIP1183 in vivo (1) the protective effect of RIP1183 on septic mice with neutrophils disappeared. (2) RIP1183 and neutrophils were incubated without affecting the MPO activity in neutrophils (3) RIP1183 can significantly reduce the expression of RNA III and PSM a, PSM beta and other virulence factors in infected mice. (4) PSM in the bacterial LAC secretory supernatant could induce increased expression of neutrophil necrotizing apoptosis characteristic protein P MLKL and could be blocked by MLKL inhibitor NSA. (5) PSM A and PSM beta defect bacteria were supernatant, and P MLKL expression of neutrophils was reduced. (6) PSM polypeptide molecules were incubated with neutrophils and increased neutrophils. The expression level of P MLKL can be blocked by NSA, and transmission electron microscopy directly observed that PSM alpha 1 can induce typical necrotizing apoptosis in neutrophils from morphologically, and NSA can significantly reduce the number of neutrophils with necrotic apoptosis. (7) mice are immune to RIP1183 or bad dead apoptosis inhibitor Nec or lack of bacterial PSM, immunization The fluorescence results showed that the necrotic neutrophils in the lung tissue of the mice decreased significantly, and the Western blot results showed that the expression level of P MLKL in the neutrophils in the alveolar lavage decreased, and the flow cytometry showed that the proportion of neutrophils in necrotic apoptosis was reduced. (8) the mice were given RIP1183 or necrotic apoptosis inhibitor Nec or After the absence of bacterial PSM, the survival rate of pneumonia mice could be improved significantly, the bacterial clearance in the lungs was promoted and the.6.RIP1183 safety and pharmacokinetics of lung pathological injury were reduced. (1) no toxic reaction was observed after intravenous injection of 100 mg/kg and 50 mg/kg respectively in rats and beagles, and the dose was RIP1183 effective, respectively. 28 and 48 times the dose. (2) in the range of treatment, RIP1183 had no significant effect on the respiratory system and cardiovascular system of the central system in mice. (3) the pharmacokinetics test in vivo showed that the plasma half-life of RIP1183 in the dog was around 20min. Conclusion: 1., the agr system inhibitor RIP1183 was obtained, and the RIP1183 was one. High safety range, low toxicity and better anti MRSA activity, the plasma elimination half life of the dog was about 20 min.2. found that RIP1183 could significantly inhibit RNA III and PSM a, PSM a, PSM beta and other virulence factors in MRSA, the expression of.3. found that the antibacterial activity of RIP1183 in vivo depended on the normal function of leukocyte, after the deletion of neutrophils, RIP 1183 the protective effect of sepsis disappeared, but RIP1183 did not activate neutrophils directly..4. proposed the hypothesis of the mechanism of RIP1183 in vivo, that is, RIP1183 inhibits the expression of virulence factor by inhibiting the agr quorum induction system of Staphylococcus aureus, and reduces the pathological damage caused by viral force factors. The expression of PSM alpha and PSM beta virulence factors can alleviate the necrotic apoptosis of neutrophils induced by these virulence factors and protect neutrophils from bacteria.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 閔峰;宋閩寧;黃文琪;范榮華;張麗;吳衛(wèi)兵;;壞死性淋巴組織炎誤診一例[J];肝臟;2008年02期

2 康明德;;藥物引起顆粒性白細(xì)胞缺乏癥、鵝口瘡及壞死性舌炎一例[J];中原醫(yī)刊;1984年01期

3 黃如明;急性出血性壞死性腎盂炎一例報告[J];臨床放射學(xué)雜志;1991年04期

4 賈全凡,徐開倫,羅通勇;上頜竇海綿狀血管瘤誤診為壞死性息肉致術(shù)中大出血1例[J];中國耳鼻咽喉顱底外科雜志;2002年04期

5 朱志超;冰硼散加味治療壞死性脈管炎[J];四川中醫(yī);1988年08期

6 高芳;1例急性造血功能停滯并出血壞死性牙齦炎的護(hù)理[J];護(hù)理學(xué)雜志;1998年01期

7 王立珍,祖愛春;亞急性壞死性淋巴結(jié)炎1例[J];實用兒科臨床雜志;2003年11期

8 殷明德;咽復(fù)發(fā)性壞死性粘膜腺周圍炎一例[J];中華耳鼻咽喉科雜志;1994年06期

9 方梅;復(fù)發(fā)性壞死性粘膜腺周圍炎1例[J];蚌埠醫(yī)學(xué)院學(xué)報;1996年04期

10 王付增;程存拴;原云峰;王曉民;;胃壁壞死性穿孔和大面積壞死性瘺18例[J];中原醫(yī)刊;2006年19期

相關(guān)會議論文 前1條

1 李偉人;孔豫蘇;黃家華;;梭狀芽孢桿菌致軟組織壞死性感染7例[A];第七屆中國醫(yī)師協(xié)會美容與整形醫(yī)師大會論文集[C];2010年

相關(guān)重要報紙文章 前2條

1 潘峰;治壞死性脈管炎[N];民族醫(yī)藥報;2004年

2 山東畜牧獸醫(yī)職業(yè)學(xué)院畜牧系 宋釗;雞壞死性腸類的診治[N];河北科技報;2007年

相關(guān)博士學(xué)位論文 前1條

1 周穎;RNAⅢ抑制肽衍生物RIP1183抗菌作用與機(jī)理研究[D];第四軍醫(yī)大學(xué);2016年

相關(guān)碩士學(xué)位論文 前2條

1 張利;高糖環(huán)境對人臍靜脈血管內(nèi)皮細(xì)胞壞死性凋亡及IL-6表達(dá)的影響[D];遵義醫(yī)學(xué)院;2016年

2 宦海霞;雛番鴨壞死性肝炎的病原鑒定及特性研究[D];揚(yáng)州大學(xué);2002年

，

本文編號：1886819