RNAⅢ抑制肽衍生物RIP1183抗菌作用与机理研究

发布时间：2018-05-14 07:01

本文选题：耐甲氧西林的金黄色葡萄球菌 + 群体感应系统抑制剂　；参考：《第四军医大学》2016年博士论文

【摘要】：目的:耐药细菌感染是临床抗感染治疗中极为棘手的问题,也是造成重症感染死亡的首要原因。其中被称为“超级细菌”的耐甲氧西林金黄色葡萄球菌(MRSA)在临床感染中的危害性日趋严重。患者一旦感染MRSA,治疗极为困难,常继发多重感染、脓毒性休克和多器官功能衰竭综合症,感染患者死亡率很高。目前临床上还没有特异性针对MRSA的有效抗菌药物,因此,如何有效控制细菌MRSA感染造成的危害已经成为各国政府高度关注的焦点,寻找和研制有效控制MRSA感染的新药物也成为当今世界各国科学家当务之急的研究目标。群体感应(Quorum sensing,QS)是广泛存在于细菌内的一种调控细菌群体行为的机制,参与细菌生物被膜形成、毒力因子释放、免疫逃逸、以及耐药性发生的重要调控系统。抑制细菌群体感应系统(Quorum sensing system,QSS)可以显著抑制细菌的致病性,但不影响细菌的生长,因而对细菌选择性生长压力小,具有不诱导细菌耐药的独特优点。因此,以QSS为靶标的新型抗菌药物研究,成为不易诱导耐药新型抗菌药物研究的新趋势和新热点,也是新型抗菌药物研究领域最有希望的突破口。附属基因调节子系统(accessory gene regulator,agr)是广泛存在于金黄色葡萄球菌的QSS,该系统激活后,启动其效应分子RNAⅢ的表达,进而调控其下游多种毒力因子的表达。抑制agr系统信号调控通路中的关键分子,可以抑制细菌多种毒力因子的表达,进而减轻细菌致病力。RNAⅢ抑制肽(RNAⅢ-inhibiting peptide,RIP)是一种作用于agr系统的抑制剂。业已证实,RIP能够有效抑制MRSA毒力因子释放,降低感染动物死亡率,减轻细菌生物膜的形成等,因此,RIP作为抗MRSA的有效抗菌药物备受关注。目前已知所有作用于QS的抑制剂在体外均无抑菌或杀菌作用,但在感染动物体内则表现出明显的抗菌作用。然而迄今为止,QS抑制剂体内抗菌的内在规律和机制几乎完全不清楚,这已成为制约新型抗菌药物RIP深入研究重要限速环节。本课题采用氨基酸替换、末端修饰、寡聚化等方法设计合成不同的RIP衍生物,体内外实验评价RIP衍生物抗菌活性,并探讨RIP衍生物体内抗菌作用的机理。方法:1.RIP衍生物的设计与合成以RIP-I的序列YKPITNF为模板,通过氨基酸替换,N-端乙酰化修饰,C-端酰胺化修饰,以及寡聚化修饰的方法,设计合成RIP-V、RIP-L、RIP1181、RIP1182、RIP1183五条RIP衍生物,采用RP-HPLC方法分析其纯度,采用ESI质谱仪或MALDI-TOF质谱仪鉴定合成多肽的分子量,确证合成衍生物多肽序列的正确性。2.RIP衍生物体内抗菌活性的筛选建立HA-MRSA感染小鼠脓毒症模型,分别检测RIP衍生物对感染小鼠生存率、脏器菌落计数、组织病理损伤等指标的影响,筛选出抗菌活性较优的RIP衍生物。3.RIP1183体外抗菌效果评价选择6株葡萄球菌(2株标准株和4株多药耐药菌株)体外培养,测定RIP衍生物RIP1183对6株葡萄球菌的最低抑菌浓度(MIC);细菌生长检测仪测定RIP1183对6株菌的生长抑制情况。4.建立不同感染模型评价RIP1183对小鼠治疗作用选择HA-MRSA(MRSA XJ75302)或CA-MRSA(LAC)菌株,分别腹腔注射、经鼻滴入和皮下接种MRSA方式感染小鼠,制备脓毒症、坏死性肺炎和皮肤感染模型,给小鼠腹腔注射不同剂量的RIP1183,观察RIP1183对小鼠生存时间和生存率的影响;通过小鼠脏器或组织中的细菌计数,观察RIP1183对体内细菌生长的影响;通过小鼠肺脏、肝脏或皮肤组织HE染色,观察RIP1183对组织损伤的保护作用。5.RIP1183体内抗菌作用机理探讨(1)按照环磷酰胺法缺失小鼠的中性粒细胞,并用LAC感染该小鼠建立脓毒症模型,腹腔注射RIP1183,观察中性粒细胞缺乏后对RIP1183抗菌作用的影响。(2)分离人外周血中性粒细胞与RIP1183共孵育,髓过氧化物酶(MPO)检测试剂盒测定中性粒细胞中的MPO,观察RIP1183对中性粒细胞内MPO活性的影响。(3)分离小鼠体内MRSA,采用实时定量PCR方法检测细菌RNAⅢ和毒力因子PVL、HLA、PSMα、PSMβ的表达,观察RIP1183对RNAⅢ和毒力因子表达的影响。(4)LAC细菌或细菌上清或HKSA(热杀死金黄色葡萄球菌),与人中性粒细胞共孵育,Western blot检测坏死性凋亡特征蛋白p MLKL的表达水平,观察LAC对中性粒细胞坏死性凋亡的影响。(5)PSMα和PSMβ缺陷菌上清与与人中性粒细胞共孵育,Western blot检测p MLKL的表达水平,观察PSMα和PSMβ对中性粒细胞坏死性凋亡的影响。(6)合成七种PSM多肽分子与人中性粒细胞共孵育,或提前加入MLKL抑制剂NSA,Western blot检测p MLKL的表达水平;透射电镜观察PSMα1对中性粒细胞形态的影响。(7)LAC及其PSMα缺陷菌建立坏死性肺炎模型,腹腔注射给予感染小鼠RIP1183或坏死性凋亡抑制剂Nec,免疫荧光法检测小鼠肺组织中的中性粒细胞坏死性凋亡、Western blot检测肺泡灌洗液中的中性粒细胞p MLKL的表达水平、流式细胞仪检测感染小鼠肺组织中的中性粒细胞死亡的比例。(8)LAC及其PSMα缺陷菌建立坏死性肺炎模型,腹腔注射给予感染小鼠RIP1183或坏死性凋亡抑制剂Nec,观察对小鼠生存率、肺损伤(HE染色)和肺组织中细菌数的变化。6.RIP1183安全性与药物代谢动力学评价(1)采用最大给药量法,大鼠或犬单次静脉注射RIP1183,观察其对大鼠或犬的一般体征、摄食、体重等指标的影响;(2)小鼠RIP1183静脉注射后,观察其对小鼠中枢神经系统的影响;比格犬RIP1183静脉注射后,观察其对比格犬心血管系统和呼吸系统的影响。(3)比格犬分别单次静脉注射2 mg/kg、4 mg/kg、8 mg/kg的RIP1183后,检测给药后不同时间点比格犬血浆中的药物浓度,计算该药物的药代动力学参数。结果:1.RIP衍生物的合成与鉴定采用固相合成的方法分别合成了RIP-I、RIP-V、RIP-L、RIP1181、RIP1182、RIP1183六条多肽,其中RIP-L和RIP-V,是RIP-I的4位氨基酸替换为亮氨酸或缬氨酸得到的,序列分别为YKPLTNF-CONH2和YKPVTNF-CONH2;RIP1183是将RIP-V乙酰化修饰得到的,序列为CH3CO-YKPVTNF-CONH2;寡聚化RIP-V分子得到RIP1182,同时进行乙酰化修饰得到的RIP1181,序列分别为YKPVTNF-ST-YKPVTNF-CONH2和CH3CO-YKPVTNF-ST-YKPVTNF-CONH2。反向高效液相色谱(RP-HPLC)法鉴定RIP衍生物的纯度均在95%以上;质谱鉴定RIP衍生物的分子量与理论值一致,表明所有合成的RIP衍生物结构正确。2.体内筛选RIP衍生物用HA-MRSA感染小鼠建立脓毒症模型,发现RIP-V显著提高小鼠生存率、减轻肺脏和肝脏病理损伤、减少小鼠脏器中细菌数,RIP-V体内抗感染活性优于RIP-I和RIP-L;RIP-V经末端修饰得到的RIP1183,RIP1183显著提高了脓毒症小鼠的生存率和延长生存时间,其抗菌活性优于RIP-V、RIP1181和RIP1182。结果显示设计合成的RIP衍生物中,RIP1183具有良好抗菌活性。3.RIP1183体外无抗菌作用RIP1183对6株葡萄球菌的MIC值均大于256μg/ml;RIP1183在250-1000μg/ml浓度下,均不能抑制6株葡萄球菌的生长,表明RIP1183体外无抗菌活性。4.RIP1183对MRSA感染小鼠保护作用在HA-MRSA(MRSA XJ75302)和CA-MRSA LAC感染所致脓毒症小鼠模型上,RIP1183分别提高小鼠的生存率达40%和60%,显著降低感染小鼠脏器中的细菌数量,减轻了小鼠脏器的病理损伤;在坏死性肺炎小鼠模型上,RIP1183可以显著降低感染小鼠肺脏的细菌数量,减轻肺水肿和肺病理损伤;皮肤感染模型上,RIP1183显著减少小鼠脓肿的形成,降低感染部位的细菌数量,并促进感染部位创面愈合。5.RIP1183体内抗菌机理探讨(1)RIP1183对中性粒细胞缺失的脓毒症小鼠保护作用消失。(2)RIP1183与中性粒细胞共孵育,不影响中性粒细胞内MPO活性(3)RIP1183可显著降低感染小鼠组织内细菌的RNAⅢ和PSMα、PSMβ等多种毒力因子的表达。(4)细菌LAC分泌上清中的PSM可以诱导中性粒细胞坏死性凋亡特征蛋白p MLKL表达增高,并能够被MLKL抑制剂NSA阻断。(5)与野生菌LAC比较,PSMα和PSMβ缺陷菌上清,降低中性粒细胞的p MLKL表达。(6)PSM多肽分子与中性粒细胞共孵育,PSM增加中性粒细胞中的p MLKL表达水平,并可以被NSA阻断;透射电镜从形态上直接观察到PSMα1可诱导中性粒细胞出现典型的坏死性凋亡特征,且NSA可显著减少坏死性凋亡的中性粒细胞的数目。(7)小鼠给予RIP1183或坏死性凋亡抑制剂Nec或缺失细菌PSM后,免疫荧光结果表明,小鼠肺组织中坏死性凋亡的中性粒细胞显著减少;Western blot结果表明,肺泡灌洗液中中性粒细胞的p MLKL表达水平降低了;流式细胞结果表明,坏死性凋亡的中性粒细胞比例降低。(8)小鼠给予RIP1183或坏死性凋亡抑制剂Nec或缺失细菌PSM后,可以显著提高肺炎小鼠生存率、促进肺脏内细菌的清除和减轻肺病理损伤。6.RIP1183安全性与药物代谢动力学研究(1)大鼠和比格犬分别静脉注射100 mg/kg和50 mg/kg的RIP1183后,未观察到任何毒性反应,该剂量分别为RIP1183有效剂量的28和48倍。(2)在治疗范围内,RIP1183对小鼠中枢系统、比格犬的呼吸系统和心血管系统无明显影响。(3)体内药代动力学实验结果表明,RIP1183在犬体内的血浆消除半衰期在20min左右。结论:1.筛选获得agr系统抑制剂RIP1183,并证实RIP1183是一种安全范围较大,毒性低,抗MRSA活性较优的抗菌剂,犬体内的血浆消除半衰期在20 min左右。2.发现RIP1183能够显著抑制MRSA的RNAⅢ和PSMα、PSMβ等多种毒力因子的表达。3.发现RIP1183体内抗菌作用依赖于白细胞功能的正常,中性粒细胞缺失后,RIP1183对脓毒症的保护作用消失,但是RIP1183无直接激活中性粒细胞的作用。4.提出RIP1183体内抗菌作用机理的假说,即RIP1183通过抑制金黄色葡萄球菌agr群体感应系统,抑制致病毒力因子的表达,减轻病毒力因子所致机体病理损伤;又通过抑制PSMα和PSMβ毒力因子表达,减轻这些毒力因子诱导的中性粒细胞坏死性凋亡,保护中性粒细胞清除细菌的功能。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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