摘要
新型氮杂环卡宾银配合物(N-heterocyclic carbene silver, Ag-NHC)兼具优异的稳定性、水溶性和杀菌活性,是具有巨大应用前景的抗菌候选物。
目的
探讨合成的1,3-二苄基Ag-NHC [1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene silver (I) acetate, SBC3]对大肠杆菌的抗菌活性及作用机制。
方法
采用可见分光光度法检测SBC3的抗菌活性;使用透射电子显微镜观察SBC3处理后大肠杆菌DHB4菌株的形态学变化;通过5,5′-二硫代双(2-硝基苯甲酸) [5,5′-dithiobis-(2-nitrobenzoic acid), DTNB]实验检测SBC3处理后DHB4胞内硫氧还蛋白(thioredoxin, Trx)及硫氧还蛋白还原酶(thioredoxin reductase, TrxR)的活性和谷胱甘肽(glutathione, GSH)的含量;利用流式细胞术检测SBC3对DHB4胞内活性氧(reactive oxygen species, ROS)含量的影响,并通过还原剂二硫苏糖醇(dithiothreitol, DTT)进行挽救实验。通过在实验室连续传代,获得SBC3耐受性大肠杆菌菌株SBC3-resistant strains (SRSs)。其中SRS3、SRS4和SRS7菌株对SBC3的最小抑菌浓度(minimal inhibitory concentration, MIC)依次为24、32和56 μg/mL,分别为野生菌株MIC的3倍、4倍和7倍。将对应的菌株分别命名为SRS3、SRS4和SRS7,并使用这3种菌株复测上述指标;采用Western blotting检测SBC3对Trx1及S-谷胱甘肽化蛋白(S-glutathionylated proteins, S-PSSG)表达量的影响。
结果
SBC3对实验菌株的MIC为8.0-30.0 μg/mL;SBC3处理后的DHB4菌株发生肿胀,并伴有内容物流出;SBC3可显著抑制DHB4胞内Trx和TrxR的活性,明显降低GSH的含量,同时升高ROS水平。此外,SBC3处理后的SRS3、SRS4和SRS7菌株胞内Trx和TrxR的蛋白活性降低,GSH含量和S-PSSG的蛋白表达水平降低,但与DHB4菌株相比有不同程度的升高。
结论
SBC3通过靶向大肠杆菌的硫醇依赖的氧化还原系统(thiol-dependent redox system, TDRS)系统发挥其抗菌作用,为SBC3作为新型抗菌剂的开发设计提供了新思路。
抗生素应用于抗感染治疗已将近一个世纪,而抗生素的广泛使用甚至滥用加剧了细菌抗生素耐受性的获得性进
银离子(silver ion, A
氮杂环卡宾化合物(N-heterocyclic carbene, NHC)得益于其特殊的电子结构、较高的反应活性、形成配合物后的稳定性以及绿色环保可循环再利用性,被用于A
本研究旨在阐述Matthias Tacke团队合成的Ag-NHC,即1,3-二苄基Ag-NHC (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene silver (I) acetate, SBC
SBC3作为一种可通过快速释放A
1 材料与方法
1.1 材料
1.1.1 菌株
大肠杆菌DHB4、鲍曼不动杆菌ATCC-19606、阴沟肠杆菌ATCC-700323、肺炎克雷伯杆菌ATCC-700603、铜绿假单胞菌ATCC-27853和金黄色葡萄球菌ATCC-25923均购自中国普通微生物菌种保藏管理中心(China general Microbiological Culture Collection Center, CGMCC)。实验室连续传代获得以DHB4为来源的SBC3耐受性菌株SRS3、SRS4和SRS7。
本研究检测SBC3对菌株的最小抑菌浓度及菌落形成实验中所使用的细菌初始菌量均为OD600=0.4后按1:1 000的比例稀释混匀,其余实验初始菌量为OD600=0.4。因此,各实验中使用的SBC3浓度会根据起始菌量的差异而不同。
1.1.2 主要试剂和仪器
SBC3,Matthias Tacke团队合成;EbSe,Selleck Chemicals公司;BCA蛋白定量试剂盒,北京普利莱基因技术有限公司;2′,7′-二氯荧光素二乙酸酯、硫氧还蛋白、谷胱甘肽还原酶、二甲基亚砜,Sigma-Aldrich公司;二硫苏糖醇,生工生物工程(上海)股份有限公司;大肠杆菌属anti-glutathione单抗、anti-PSSG单抗,ViroGen公司;硫氧还蛋白还原酶、大肠杆菌属thioredoxin 1蛋白,IMOC Crop公司;山羊抗小鼠IgG,艾博抗(上海)贸易有限公司;兔抗绵羊IgG,北京百奥莱博科技有限公司。
全自动酶标仪,ThermoFisher Scientific公司;恒温摇床,上海捷呈实验仪器有限公司;台式高速离心机,Eppendorf公司;超声波细胞破碎仪,宁波新芝生物科技股份有限公司;电子恒温水浴锅,上海齐欣科学仪器有限公司;化学发光成像仪,上海勤翔科学仪器有限公司;电泳仪电源,北京市六一仪器厂;分析型流式细胞仪,Beckman Coulter公司;脱色摇床,海门市其林贝尔仪器制造有限公司;透射电子显微镜,Hitachi公司。
1.2 可见分光光度法检测SBC3的抗菌活性
可见分光光度法是根据被测菌液对可见波段范围单色光的吸收或反射光谱特性进行细菌定量分析的一种方法。在600 nm波长下,酶标仪(分光光度计)对浊度的反应较为灵敏。OD600是指在600 nm波长下测得的吸光值,本研究用于测量细菌生长情况或菌液密度。
大肠杆菌DHB4、鲍曼不动杆菌ATCC-19606、阴沟肠杆菌ATCC-700323、肺炎克雷伯杆菌ATCC-700603、铜绿假单胞菌ATCC-27853和金黄色葡萄球菌ATCC-25923于37 ℃、220 r/min培养过夜后,分别用LB液体培养基将培养至对数生长期(OD600=0.6-0.8)的菌液OD600调至0.4。按照1:1 000的比例稀释混匀后,用不同浓度的SBC3处理(环丙沙星作为阳性对照)。混匀后接种于96孔板,每孔200 μL,设置4个复孔。将96孔板置于37 ℃恒温培养箱中培养24 h,并在酶标仪上连续监测0、2、4、8、16、24 h的OD600。
1.3 菌落形成实验检测SBC3对DHB4菌株的杀伤效果
大肠杆菌DHB4于37 ℃、220 r/min培养12 h后,用LB液体培养基将培养至对数生长期(OD600=0.6-0.8)的菌液OD600调至0.4,按照1:1 000的比例稀释混匀。随后用不同浓度的SBC3处理(DMSO作为对照),置于37 ℃温箱静置16 h,孵育完毕后,将处理好的菌液依次倍比稀释至1
1.4 透射电子显微镜观察SBC3对DHB4菌株形态学的影响
将培养至对数生长期(OD600=0.6-0.8)的菌液用LB液体培养基调节其OD600为0.4,用不同浓度(48 μg/mL、54 μg/mL)的SBC3处理10 min,以0.8% DMSO 为对照。于4 ℃、4 000 r/min离心10 min获得细胞,弃去上清液后用PBS重悬清洗,去除胞外残留药物及LB培养基。清洗后再次离心获得细胞,继续清洗,重复3次后去除上清液,用PBS重悬细胞,并用2.5%戊二醛固定。在80 kV工作电压下,于透射电子显微镜下以7 000×、15 000×放大倍数观察E. coli形态在给药前后的变化。
1.5 流式细胞术检测SBC3对DHB4胞内ROS产量的影响
大肠杆菌DHB4于37 ℃、220 r/min培养12 h后,利用LB液体培养基将培养至对数生长期(OD600=0.6-0.8)的菌液OD600调至0.4,用SBC3处理30 min;4 000 r/min离心10 min收集沉淀,用PBS重悬3次,最后重悬于800 μL PBS中,并用终浓度为10 mmol/L的H2DCF-DA在37 °C染色30 min。用流式细胞仪检测并记录FITC通道的平均荧光强度,最佳激发波长为488 nm,最佳发射波长为525 nm。
1.6 5,5′-二硫代双(2-硝基苯甲酸) [5,5′- dithio-bis-(2-nitrobenzoic acid), DTNB]法检测SBC3对DHB4菌株TDRS的影响
TrxR/Trx与GSH活性测定中,TrxR催化还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate hydrogen, NADPH)还原DTNB生成2-硝基-5-巯基苯甲酸(5-mercapto-2-nitrobenzoic acid, TNB)和烟酰胺腺嘌呤二核苷磷酸(nicotinamide adenine dinucleotide phosphate, NAD
1.6.1 细菌预处理
大肠杆菌DHB4于37 ℃、220 r/min培养12 h后,用LB液体培养基将培养至对数生长期(OD600=0.6-0.8)的菌液的OD600调至0.4。使用SBC3进行处理后混匀,37 ℃恒温孵育30 min。4 ℃、5 000 r/min离心3 min,用等体积的PBS重悬3次后弃去上清。
1.6.2 细菌蛋白制备
用150 μL 50 mmol/L Tris-HCl和2 mmol/L EDTA重悬细菌沉淀,加入10 μL蛋白酶抑制剂和40 μL 5 mmol/L溶菌酶混匀,置于37 ℃水浴锅中30 min。利用超声破碎仪裂解细菌(2 mm变幅杆,20%-25%功率,超声8 min),置于4 ℃、12 000 r/min离心15 min,吸取上清,进行蛋白定量。
1.6.3 Trx活性
取25 μg蛋白,用超纯水定容至20 μL,加入200 μmol/L NADPH、2 mmol/L DTNB、50 mmol/L Tris-HCl (pH 7.5)、2 mmol/L EDTA、100 nmol/L TrxR,于OD412处连续检测30 min。
1.6.4 TrxR活性
取25 μg蛋白,用超纯水定容至20 μL,加入200 μmol/L NADPH、2 mmol/L DTNB、50 mmol/L Tris-HCl (pH 7.5)、2 mmol/L EDTA、5 μmol/L Trx,于OD412处连续检测30 min。
1.6.5 GSH含量
取25 μg蛋白,用超纯水定容至20 μL,加入200 μmol/L NADPH、2 mmol/L DTNB、50 mmol/L Tris-HCl (pH 7.5)、2 mmol/L EDTA、50 nmol/L GR,于OD412处连续检测30 min。
1.7 耐药菌的筛选与培养
在实验室通过连续传代筛选以DHB4为基础的SBC3耐受性菌株,获得SRS3、SRS4、SRS7菌株,这3株菌对SBC3的MIC分别为DHB4的3倍、4倍和7倍。取少量菌液进行四区平板划线,于37 ℃培养过夜(12 h)后,挑取单克隆接种于装有2 mL液体培养基的EP管中,置于37 ℃、220 r/min恒温摇床中培养12 h至对数生长期用于后续实验。
1.8 流式细胞术检测SBC3对SRSs胞内ROS产量的影响
大肠杆菌DHB4、SRS3、SRS4、SRS7 37 ℃培养过夜后,按照1.5节方法进行后续操作。
1.9 DTNB实验检测SBC3对SRSs胞内TDRS的影响
大肠杆菌DHB4、SRS3、SRS4、SRS7 37 ℃培养过夜后,按照1.6节方法进行后续操作。
1.10 Western blotting检测SBC3对DHB4及SRSs菌株Trx1表达水平的影响
大肠杆菌DHB4与SRS3、SRS4、SRS7 37 ℃培养过夜后,按照1.6.1和1.6.2节方法进行细菌预处理与蛋白制备。
取25 μg蛋白,用超纯水定容至20 μL,加入5 μL 5×Loading Buffer混匀后,100 ℃煮沸10 min。在12%聚丙烯酰胺凝胶中进行分组加样,60 V恒压电泳30 min后,转为120 V继续电泳。180 mA恒流转膜60 min,取出PVDF膜置于5 g/L脱脂牛奶中封闭2 h,TBST清洗1次。一抗(Trx)在4 ℃、30 r/min孵育过夜,TBST洗膜3次,每次15 min;二抗(兔抗羊)室温、80 r/min孵育1 h,TBST洗膜3次,每次15 min。将PVDF膜置于ECL显影液中1 min后,于显影仪中进行Trx1蛋白显影并拍照。
1.11 Western blotting检测SBC3对DHB4及SRSs菌株S-PSSG表达量的影响
大肠杆菌DHB4与SRS3、SRS4、SRS7 37 ℃培养过夜后,按照1.6.1和1.6.2节方法进行细菌预处理与蛋白制备。
每组取25 μg蛋白,后续操作与1.10方法一致。封闭洗涤完成后,一抗(PSSG)在4 ℃、30 r/min孵育过夜,TBST洗膜3次,每次15 min;二抗(鼠抗羊)室温、80 r/min孵育1 h,TBST洗膜3次,每次15 min,将PVDF膜置于ECL显影液中1 min后,于显影仪中进行S-PSSG蛋白显影并拍照。
1.12 统计学方法
实验结果采用统计软件SPSS 20进行分析。数值以mean±SD表示,组间比较采用t检验(Student’s t-test);以P<0.05表示差异具有统计学意义。
2 结果与分析
2.1 SBC3具备良好的抗菌活性
为了评价SBC3的抗菌效果,首先检测了其抗菌活性,并以临床常用抗生素环丙沙星(ciprofloxacin, CIP)作为阳性对照。连续监测24 h发现(

图1 SBC3抗菌活性。A:SBC3对大肠杆菌DHB4的抗菌效果;B:SBC3对鲍曼不动杆菌ATCC-19606的抗菌效果;C:SBC3对阴沟肠杆菌ATCC-700323的抗菌效果;D:SBC3对肺炎克雷伯菌ATCC-700603的抗菌效果;E:SBC3对铜绿假单胞菌ATCC-27853的抗菌效果;F:SBC3对金黄色葡萄球菌ATCC-25923的抗菌效果。P<0.05表示差异显著,Student’s t-test,n=4。
Figure 1 Antimicrobial activity of SBC3. A: Antibacterial effect of SBC3 against Escherichia coli DHB4; B: Antibacterial effect of SBC3 against Acinetobacter baumannii ATCC-19606; C: Antibacterial effect of SBC3 against Enterobacter cloacae ATCC-700323; D: Antibacterial effect of SBC3 against Klebsiella pneumoniae ATCC-700603; E: Antibacterial effect of SBC3 against Pseudomonas aeruginosa ATCC-27853; F: Antibacterial effect of SBC3 against Staphylococcus aureus ATCC-25923. P<0.05 indicates significant difference, Student’s t-test, n=4.
2.2 SBC3可破坏大肠杆菌细胞正常形态
大肠杆菌DHB4过夜培养后,调整OD600为0.4后进行菌落形成实验(

图2 SBC3对DHB4形态的影响。A:菌落形成实验评价SBC3对DHB4的杀伤效果;B:透射电子显微镜下观察SBC3对DHB4形态的影响。
Figure 2 Effect of SBC3 on the morphology of DHB4. A: Colony formation assay to evaluate the killing effect of SBC3 on DHB4; B:Transmission electron microscopy to observe the effect of SBC3 on the morphology of DHB4.
进一步通过透射电子显微镜观察(
2.3 SBC3可导致大肠杆菌胞内ROS产量升高
利用SBC3处理对数生长期的DHB4,通过H2DCF-DA荧光探针检测细菌胞内ROS水平变化。与对照组相比(

图3 SBC3对DHB4胞内活性氧含量的影响。A:流式细胞术检测SBC3对DHB4胞内ROS含量的影响;B:SBC3处理组与SBC3+DTT组的ROS含量的平均荧光强度(MFI)。P<0.05表示差异显著,Student’s t-test,n=4。
Figure 3 Effect of SBC3 on the intracellular ROS content of DHB4. A: Effect of SBC3 on ROS content in DHB4 in vivo detected by flow cytometry; B: Mean fluorescence intensity (MFI) of ROS content in SBC3-treated vs. SBC3+DTT groups. P<0.05 indicates significant difference, Student’s t-test, n=4.
2.4 SBC3可损伤大肠杆菌TDRS系统
TDRS系统是大肠杆菌胞内最重要的抵御氧化压力的硫醇依赖的氧化还原系统。通过DTNB法检测SBC3对DHB4胞内的Trx、TrxR活性和GSH含量的影响。如

图4 SBC3对大肠杆菌TrxR、Trx活性及GSH含量的影响。A:SBC3对DHB4胞内TrxR的影响;B:SBC3对DHB4胞内Trx的影响;C:SBC3对DHB4胞内GSH含量的影响。P<0.05表示差异显著,Student’s t-test,n=4。
Figure 4 Effect of SBC3 on TrxR and Trx activities and GSH content in E. coli. A: Effect of SBC3 on DHB4 intracellular TrxR; B: Effect of SBC3 on DHB4 intracellular Trx; C: Effect of SBC3 on DHB4 intracellular GSH content. P<0.05 indicates significant difference, Student’s t-test, n=4.
2.5 SRSs对SBC3的耐受性相对于野生菌株显著增高
通过实验室连续定向传代,获得SBC3耐药性菌株SRSs。为了评价耐药株对SBC3的耐药性,通过96孔板实验检测了SBC3对耐药株的抗菌活性(

图5 SBC3作用下SRSs的生长曲线。A:SBC3对大肠杆菌DHB4的抗菌活性;B:SBC3对大肠杆菌耐药株SRS3的抗菌活性;C:SBC3对大肠杆菌耐药株SRS4的抗菌活性;D:SBC3对大肠杆菌耐药株SRS7的抗菌活性。P<0.05表示差异显著,Student’s t-test,n=4。
Figure 5 Growth curves of SRSs in the presence of SBC3. A: Antibacterial activity of SBC3 against E. coli DHB4; B: Antibacterial activity of SBC3 against SRS3; C: Antibacterial activity of SBC3 against SRS4; D: Antibacterial activity of SBC3 against SRS7. P<0.05 indicates a significant difference, Student’s t-test, n=4.
2.6 耐SBC3菌株SRSs胞内ROS含量更低
细菌胞内ROS基线水平是评价其抵御杀菌型抗生素抗菌能力的关键指标之一。使用H2DCFA荧光探针对SRS3、SRS4、SRS7进行染色后(

图6 流式细胞术检测SRSs胞内ROS水平。A:流式细胞术检测DHB4及SRSs经SBC3处理后的胞内细菌数量和ROS含量;B:流式细胞术检测细菌胞内ROS含量的平均荧光强度。P<0.05表示差异显著,Student’s t-test,n=4。
Figure 6 Detection of intracellular ROS levels in SRSs by flow cytometry. A: Flow cytometry detection of intracellular bacterial counts and ROS levels of DHB4 and SRSs after treatment with SBC3; B: Flow cytometry detection of the mean fluorescence intensity of bacterial intracellular ROS levels. P<0.05 indicates a significant difference, Student’s t-test, n=4.
2.7 SBC3可影响耐SBC3菌株SRSs胞内TDRS活性
Trx系统作为大肠杆菌的抗氧化系统,可通过清除胞内多余的ROS避免氧化应激导致的细胞损伤。通过DTNB法检测SBC3对SRS3、SRS4、SRS7胞内的Trx/TrxR活性与GSH含量的影响,以DHB4为阳性对照(

图7 SBC3作用后SRSs中TDRS活性变化。A:SBC3对SRSs胞内Trx活性的影响;B:SBC3对SRSs胞内TrxR活性的影响;C:SBC3对SRSs胞内GSH含量的影响。P<0.05表示差异显著,反之为差异无统计学意义,常用无显著差异(no significance, ns)表示,当Student’s t-test,n=4。
Figure 7 Changes in TDRS activity in SRSs after the treatment of SBC3.A: Effect of SBC3 on SRSs’ intracellular Trx activity; B: Effect of SBC3 on SRSs’ intracellular TrxR activity; C: Effect of SBC3 on SRSs’ intracellular GSH content. P<0.05 indicates significant difference, and the ns indicates that difference is not statistically significant, Student’s t-test, n=4.
2.8 SBC3对SRSs蛋白表达的影响
通过Western blotting检测发现(

图8 SBC3对Trx1蛋白表达量的影响。A:统计Western blotting检测的细菌胞内Trx1表达水平;B:Western blotting检测的细菌胞内Trx1表达水平;C:SDS-PAGE结果图作为上样内参(考马斯蓝染色)。ns为差异无统计学意义,Student’s t-test,n=3。
Figure 8 Effect of SBC3 on Trx1 protein expression level. A: Statistics of bacterial intracellular Trx1 expression level detected by Western blotting; B: Bacterial intracellular Trx1 expression level detected by Western blotting; C: SDS-PAGE result graphs were used as the internal reference for up-sampling (stained with Kaomas blue). ns indicates that difference is not statistically significant, Student’s t-test, n=3.
2.9 SBC3可降低耐SBC3菌株SRSs胞内S-PSSG水平
GSH系统是与大肠杆菌Trx系统相互支撑的抗氧化系统。谷胱甘肽与反应性蛋白半胱氨酸共价结合,这一过程称为S-谷胱甘肽化(PSSG)。通过观察每条泳道蛋白的整体表达水平来评价细菌胞内蛋白发生S-PSSG修饰的水平。结果显示(

图9 SBC3对S-PSSG蛋白表达量的影响。A:统计Western blotting检测的细菌胞内S-PSSG表达水平;B:Western blotting检测的细菌胞内S-PSSG表达水平;C:SDS-PAGE结果图作为上样内参(考马斯蓝染色)。P<0.05表示差异显著,Student’s t-test,n=3。
Figure 9 Effect of SBC3 on the expression of S-PSSG protein. A: Statistics of bacterial intracellular S-PSSG expression level detected by Western blotting; B: Bacterial intracellular S-PSSG expression level detected by Western blotting; C: SDS-PAGE result graphs were used as the internal reference for up-sampling (stained with Kaomas blue). P<0.05 indicates significant difference, Student’s t-test, n=3.
3 讨论与结论
本研究探讨了SBC3的抗菌作用及其机制。ROS是细胞内重要的信号分子,但过量的ROS可导致氧化应激,进而引发细胞损伤。在调节氧化还原平衡的众多系统中,大肠杆菌主要通过TDRS,即Trx和GSH系统,来维持胞内ROS稳态。本研究发现,SBC3可通过显著抑制Trx和TrxR的活性、降低GSH含量和S-PSSG表达水平,导致大肠杆菌DHB4胞内ROS水平上升,破坏细菌的氧化还原平衡,从而发挥抗菌活性。
本研究阐述了SBC3对耐药性的影响。通过实验室连续传代筛选得到的SBC3耐药性大肠杆菌菌株SRS3、SRS4和SRS7,显示出对SBC3的耐药性。耐药株的ROS含量明显低于敏感株DHB4,且SBC3处理后耐药株SRS4和SRS7的Trx/TrxR活性与GSH含量以及Trx1表达量无明显变化,而S-PSSG表达量显著下调。这表明即使在细菌具有较高耐药性时,SBC3仍可作用于GSH系统发挥抗菌作用,在处理多重耐药性革兰氏阴性菌时更具优势,进一步证实了TDRS系统是SBC3抗菌作用的靶标。
本研究进一步评估了SBC3的抗菌潜力。目前,具有抗菌活性的Ag-NHC已被证实具有用于新型药物设计的潜
总之,本研究深入探讨了新型Ag-NHC配合物SBC3对大肠杆菌的抗菌机制。鉴于抗生素耐药性日益严峻的问题,新型抗菌药物的开发显得尤为迫切。SBC3因其优异的稳定性、水溶性和杀菌活性成为潜在的抗菌候选物。Zou
尽管本研究证实SBC3可靶向TDRS发挥杀菌作用,但其具体分子机制仍然未知。因此,本课题组对已获得的SRSs进行了全基因组测序(whole genome sequencing, WGS),期望能发现一些新的氧化还原网络相关分子。初步的WGS结果分析发现,编码三甲胺-N-氧化还原酶特异性伴侣蛋白的torD发生了AGC→AAC的突变,编码环氧辫苷还原酶的queG发生了GTC→GTT和TTC→TTT的突变。然而,由于氧化还原家族成员众多且功能互为替补,要准确判断参与其中的新分子,仍需进一步的实验验证。我们期望在后续研究中取得突破,并及时进行报道。
作者贡献声明
谭超:研究构思和设计、数据收集和处理、论文撰写和修改;伍中宝:协助实验操作、论文撰写和修改、论文讨论;沈舒楚:协助实验操作、数据收集和处理;黎国春:论文讨论;Matthias Tacke:提供技术支持;王君:参与论文讨论、提供技术支持;邹黎黎:研究构思和设计、参与论文讨论、论文撰写和修改。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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