CN110974961B - Nano composite material for removing bacterial biofilm by enhancing photo-thermal based on enzymatic degradation and preparation method and application thereof - Google Patents
Nano composite material for removing bacterial biofilm by enhancing photo-thermal based on enzymatic degradation and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano composite material for enhancing photothermal bacteria biofilm removal based on enzymatic degradation and a preparation method and application thereof2Nanosheet (i.e., MnO)2-amylase-PEG-ICG NSs, MAPI for short). The material can degrade Extracellular Polymeric Substance (EPS) of the biomembrane through the amylase, destroy the structure of the biomembrane and further enhance the photothermal sterilization effect of the ICG. The MAPI provided by the invention has good stability and excellent killing effect on bacteria in biomembranes, has low cytotoxicity and meets the basic conditions of biomedical application.
Description
Technical Field
The invention belongs to the technical field of nano anti-biofilm, and particularly relates to a nano composite material for removing a bacterial biofilm by enhancing photo-thermal degradation based on enzyme degradation, and a preparation method and application thereof.
Background
Bacterial infections constitute a serious threat to human health, causing over 200 million diseases and over 23,000 deaths annually in the united states. Among them, about 80% of human bacterial infectious diseases are associated with biofilms formed on living tissues. In biofilms, bacteria are bound together by self-synthesized EPS, which is generally composed of polysaccharides, proteins, and extracellular dna (edna). Under the protection of this viscous and firm framework, bacteria within the biofilm are significantly more resistant to antibiotics, host immune defenses and environmental stresses than free bacteria. Bacterial biofilms, if not eradicated completely, often lead to persistent infections and even death. Therefore, how to effectively remove the bacterial biofilm is an urgent problem to be solved in the medical field.
In addition, in recent years, abuse of antibiotics causes drug resistance of bacteria, so that the curative effect of the antibiotics is greatly reduced. Therefore, new antibacterial agents having excellent effects are receiving increasing attention. With the continuous development of nano biomedicine, new therapies for bacterial infection diseases are generated: combines biological method and nanometer technology, designs novel nanometer antibacterial material, and can better treat bacterial biofilm infection. The current major strategies for nanomaterials to treat bacterial biofilms include (IEEE trans. nanobiosci, 2016, 15, 294.): (1) the nano material is used as a carrier to deliver the drug to the interior of the biological membrane; (2) the nano material is used as an antibacterial agent, and proteins or DNA of bacteria are damaged by releasing metal ions; (3) killing bacteria within the biofilm by hyperthermia based on the magnetocaloric or photothermal properties of the nanomaterial; (4) modifying the surface of the nano material with negatively charged molecules to enhance the permeability of the nano material to a biological membrane and the like. However, the current nanotechnology has several problems: lack of targeting ability to biological membranes, large side effects and obstruction of EPS result in poor efficacy.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nano composite material for removing bacterial biofilm by enhancing photo-thermal degradation based on enzyme degradation, a preparation method and application thereofMnO2The nano-sheet is used as a carrier, and loaded with amylase with the function of degrading EPS, PEG for improving the biocompatibility of the material and ICG with excellent photo-thermal property, so as to prepare the MAPI nano-sheet. MnO in MAPI nanosheets2Degrading under the acidic environment of the biological membrane to release the amylase and the ICG, degrading the polysaccharide in the EPS to destroy the polysaccharide structure in the bacterial biological membrane EPS, and simultaneously enhancing the near infrared photothermal sterilization effect, thereby realizing the effective removal of the bacterial biological membrane.
In order to achieve the purpose, the invention adopts the technical scheme that:
a nano composite material for removing bacterial biomembrane by enhancing photo-thermal based on enzyme degradation is MnO modified by amylase, polyethylene glycol PEG and indocyanine green ICG2Nanoplate MAPI.
A preparation method of a nano composite material for removing bacterial biofilm by enhancing photo-thermal based on enzyme degradation comprises the following steps:
step 1: preparation of MnO2An aqueous solution of a nanomaterial;
step 2: MnO of2Adding the aqueous solution of the nano material into the aqueous solution of amylase amyloylase, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifugation in water, and continuously centrifuging and purifying to obtain MnO2-an amylase nanoplate;
and step 3: MnO of2Adding a polyethylene glycol (PEG) aqueous solution into an aqueous solution of the-amylase nanosheet, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifuging in water, and continuously centrifuging and purifying to obtain MnO2-an amylase-PEG nanoplatelet;
and 4, step 4: MnO of2Adding an aqueous solution of an-amylase-PEG nano-sheet into an ICG aqueous solution of indocyanine green, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifugation into water, and continuously centrifuging and purifying to obtain MnO2-amylase-PEG-ICG nanoplatelets.
Preferably, in said step 1, MnO2The nano material is MnO2Nanosheets, MnO2The particle size of the nano-sheet is 20-200 nm.
Preferably, the amylase amThe ylase is one or more of alpha-amylase, beta-amylase, gamma-amylase and isoamylase; the polyethylene glycol PEG is mPEG-SH, mPEG-COOH or mPEG-NH2One or more of HCl and mPEG-OH.
Preferably, in said step 2, MnO2The concentration of the aqueous solution of the nano material is 1-100 mug/mL, and the concentration of the aqueous solution of the amylase is 10-1000 mug/mL; the MnO2The volume ratio of the aqueous solution of the nano material to the aqueous solution of the amylase is 1: 0.5 to 5.
Preferably, in said step 3, MnO2The concentration of an aqueous solution of the-amylase nano-sheet is 1-100 mu g/mL, the concentration of an aqueous solution of the polyethylene glycol PEG is 10-1000 mu g/mL, and MnO is added2-the volume ratio of aqueous solution of amylase nanosheet to aqueous solution of polyethylene glycol PEG is 1: 0.5 to 5.
Preferably, in said step 4, MnO2The concentration of an aqueous solution of the-amylase-PEG nano-sheet is 1-100 mu g/mL, the concentration of an aqueous solution of the indocyanine green ICG is 2-200 mu g/mL, and MnO is added2The volume ratio of the aqueous solution of the amylase-PEG nanosheets to the aqueous solution of indocyanine green (ICG) is 1: 0.5 to 5.
Preferably, in the steps 2 to 4, stirring is carried out at room temperature by using a magnetic stirrer, and the stirring time is 0.5 to 12 hours; centrifuging at 6000-18000 rpm for 45 min; the conditions of centrifugal purification are 6000-18000 rpm, the time of centrifugal purification is 45 min, and the times of centrifugal purification are 3 times.
The application of a nano composite material for enhancing photothermal bacteria biofilm removal based on enzymatic degradation is characterized in that the nano composite material has the antibacterial performance of killing bacteria in a biofilm and removing the bacteria biofilm.
Further, MnO is added when the bacterium is methicillin-resistant Staphylococcus aureus MRSA2The action concentration of the-amylase-PEG-ICG nano-sheet is 5-100 mug/mL.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention does not contain antibiotics, eliminates bacteria through the photothermal effect of ICG and is not easy to cause the problem of bacterial drug resistance.
(2) Bacterial biofilm microenvironment response performance. MnO2The nano-sheet can be degraded under the weak acid condition of the bacterial biomembrane, realizes the controllable release of the amyloase and the ICG, has the specific recognition capability on the bacterial biomembrane, and is beneficial to reducing the toxic and side effect on normal cells in the bacterial removal process.
(3) And bacterial biofilm EPS degradation performance. The amylase can degrade polysaccharide components in EPS, can destroy the integral structure of a biological membrane, reduces the barrier effect of the biological membrane on a photothermal therapy reagent, and is beneficial to exposing bacteria in the surrounding environment, thereby enhancing the photothermal sterilization effect of ICG.
(4) Low toxic by-effect. The invention adopts MnO2The nanosheet is used as a carrier, Mn ions after degradation are easily eliminated from a human body, natural amylase can be degraded by a human body, ICG is a reagent approved by the United states Food and Drug Administration (FDA), and the potential toxicity is low.
Drawings
Fig. 1 is a schematic diagram of the results of example 1 of the present invention to verify biocompatibility of MAPI nanoplates;
fig. 2 is a schematic diagram of the results of example 2 of the invention demonstrating the anti-biofilm performance of MAPI nanoplates, wherein: (a) and (c) are the crystal violet staining photograph and the corresponding biofilm biomass of example 2 for verifying the biofilm resistance of the MAPI nanosheets, respectively, (b) and (d) are the plating photograph and plating data statistics, respectively, of example 2 for verifying the biofilm resistance of the MAPI nanosheets;
fig. 3 is a Scanning Electron Microscope (SEM) image of inventive example 3 demonstrating anti-biofilm performance of MAPI nanoplates.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
MnO2Preparation of-amylase-PEG-ICG (MAPI) nanoplates
1. MnO2Preparation of nanosheets
0.6 g of MnCl was taken2·4H2O (3 mM) crystals in 50 mLInto the flask, 10 mL of H was added2And O. 12 mL of TMA. OH (tetramethylammonium hydroxide, 12 mM) solution was taken, and 2 mL of H was added2O2(30% wt) and diluted to 20 mL with water. The two solutions are mixed, the solution changes from colorless transparency to brown, and the mixture is stirred on a magnetic stirrer to react for 12 hours. After the reaction was complete, the solution was removed, centrifuged at 3000 rpm for 10 min, and the lower layer was precipitated. And washed three times with ethanol and water under the centrifugation condition, and finally added with aqueous solution to reach the constant volume of 30 mL. 10 mL of cleaned MnO220 mL of H was added2O, performing ultrasonic treatment for 10 h (power: 40%, time interval: 5 s) by using a probe ultrasonic instrument, centrifuging the obtained solution at 12000 rpm for 45 min, discarding the lower-layer precipitate, centrifuging the supernatant at 18000 rpm for 1.5 h, taking the lower-layer precipitate, and repeatedly centrifuging for three times to finally obtain MnO2Nanosheets.
2. MnO2Preparation of (E) -amylase (MA) nanoplates
Adding 5 mL of MnO 25 mL of an aqueous amyl ase solution (1 mg/mL) is added into the aqueous nanosheet solution (100. mu.g/mL), and the mixture is placed on a magnetic stirrer to be stirred for 12 hours at room temperature. And centrifuging the obtained solution at 18000 rpm for 45 min, and purifying for 3 times to obtain the MA nanosheet.
3. MnO2Preparation of (E) -amylase-PEG (MAP) nanosheets
5 mL of MA nanosheet aqueous solution (100. mu.g/mL) was added to 5 mL of PEG aqueous solution (1 mg/mL), and the mixture was stirred on a magnetic stirrer at room temperature for 12 hours. And centrifuging the obtained solution at 18000 rpm for 45 min, and purifying for 3 times to obtain the MAP nano-sheet.
4. MnO2Preparation of-amylase-PEG-ICG (MAPI) nanoplates
5 mL of MAP nanoplate aqueous solution (100. mu.g/mL) was added to 5 mL of ICG aqueous solution (200. mu.g/mL), and the mixture was stirred on a magnetic stirrer at room temperature for 4 hours. And centrifuging the obtained solution at 18000 rpm for 45 min, and purifying for 3 times to obtain MAPI nanosheets.
In the examples, the amyloase is selected as alpha-amyloase and the PEG is selected as mPEG-NH2HCl。
Example 2
Biocompatibility of MAPI nanosheets
HeLa cells were cultured in DMEM medium containing FBS (10%), penicillin (80U/mL) and streptomycin (0.08 mg/mL). The collected log phase cells were added to a 96-well plate (100. mu.L per well, cell density adjusted to 103-104Pore) placed in a 5% CO atmosphere2And incubated in an incubator at 37 ℃. After the cells adhered to the wall, the culture medium was removed, and 100. mu.L of MAP (MnO) containing different concentrations was added to each well20, 5, 10, 20, 40, 80, 160. mu.g/mL, amylase: 0, 18, 36, 72, 144, 288, 576. mu.g/mL) and MAPI (MnO)20, 5, 10, 20, 40, 80, 160. mu.g/mL, 0, 18, 36, 72, 144, 288, 576. mu.g/mL for amylase, 0, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2. mu.g/mL for ICG, 5 replicate wells per concentration, and then placed in DMEM medium filled with 5% CO2Incubated at 37 ℃ in an incubator protected from light for 24 h. Thereafter, 20. mu.L of MTT solution (5 mg/mL, i.e., 0.5% MTT) was added to each well, and incubation in the incubator for 4 h with exclusion of light was continued. The incubation was terminated, the supernatant was aspirated, 150. mu.L of dimethyl sulfoxide was added to each well to dissolve the crystals completely, and the absorbance at 490 nm was measured for each well using a microplate reader. The HeLa cell activity calculation formula of the method is as follows: s = C/C0X 100%, wherein C represents the OD of the test sample490,C0OD of blank control490。
As can be seen from the results shown in fig. 1, the relative cell viability of HeLa cells after 24 hours of incubation with MAP and MAPI was measured by MTT, and it was found that the cell viability was maintained above 88%. The experimental result shows that MAP NSs and MAPI NSs have excellent biocompatibility.
Example 3
Effect of MAPI nanosheet on removing bacterial biofilm
A single colony of methicillin-resistant staphylococcus aureus (MRSA, ATCC 43300) is streaked in 5 mL of liquid LB culture medium, and is incubated and grown for 10-12 h (rotating speed: 220 rpm, temperature: 37 ℃) under a constant temperature shaking table to obtain MRSA suspension in logarithmic phase. The bacterial suspension was washed three times with physiological saline (centrifugation conditions: 10000 rpm, 3 min) and quantified with a microplate reader (OD)600If = 0.1, the bacterial liquid concentration is 1 × 107CFU/mL). The washed bacterial solution was diluted to 1X 10 with LB medium containing 1% glucose8CFU/mL, respectively adding 96-well plate (200. mu.L per well) and 6-well plate (3 mL per well) containing silicon wafer, and incubating in 37 ℃ incubator for 2d to obtain MRSA biofilm.
(A) Crystal violet dyeing method
Referring to FIGS. 2a and 2c, the supernatant was removed from the biofilm grown in a 96-well plate, and 100. mu.L of physiological saline and MnO were added to each well, respectively2The cells were incubated in 37 ℃ incubator for 3 h with amylose, ICG, MAP, MAPI, MBP, and MBPI. For the illumination set, a 785 nm laser was used at 0.8W/cm2The light is used for 10 min. The supernatant was removed and 100. mu.L of formalin was added to each well. After fixation for 10 min, the supernatant was removed and 100. mu.L of 0.2% crystal violet stain was added to each well. After staining for 30 min, each well was washed three times with physiological saline and then imaged under an inverted microscope, the imaging results are shown in fig. 2 a. Thereafter, 200. mu.L of ethanol was added to each well to dissolve the crystal violet, and after 3 h the absorbance at 590 nm of each well was measured by a microplate reader, and the bacterial biomass was as shown in FIG. 2 c.
As can be seen from the results shown in FIGS. 2a and 2c, MnO can be observed2、amylase、ICG、MAP、MAPI、MBP(MnO2BSA-PEG, BSA without degradation of the biofilm), MBPI (MnO2BSA-PEG-ICG) in the absence of or under light (785 nm laser, 0.8W/cm)2Light illumination for 10 min) effect of degrading MRSA biofilm (biofilm can be stained purple by crystal violet, while degraded biofilm is stained colorless). Wherein, under the condition of no illumination, the amyloase, MAP and MAPI can all cause certain degradation effect on the biological membrane, which indicates that the amyloase is modified to MnO2The enzyme activity of the nanometer material is not damaged after the nanometer material is coated. Meanwhile, the degradation effect of MAPI on MRSA biomembranes is more obvious compared with that of MAPI which is not subjected to light treatment, which shows that the photothermal effect of MAPI can enhance the degradation effect of MAPI on biomembranes. For MAPI and MBPI with the same photothermal effect, the degradation effect of the MAPI and the MBPI on a biological membrane is obviously better after the MAPI and the MBPI are subjected to light treatment, and the result shows that the amylase canThe degradation of the biomembrane by the photo-heat is enhanced.
(B) Dilution plate method
Referring to FIGS. 2b and 2d, the supernatant was removed from the biofilm grown in 96-well plates, and 100. mu.L of physiological saline and MnO were added to each well2、amylase、ICG、MAP、MAPI、MBP(MnO2-BSA-ICG)、MBPI(MnO2BSA-ICG-ICG) (materials were all suspended in physiological saline) and incubated in an incubator at 37 ℃ for 3 h. For the illumination set, a 785 nm laser was used at 0.8W/cm2The light is used for 10 min. Removing supernatant, adding 200 μ L physiological saline into each well, mixing, adding into 1.5 mL centrifuge tube, and performing gradient dilution 10 and 10 respectively2、103、104、105、106Doubling, then diluting 100. mu.L stock solution by 104、105、106The multiplied bacterial solution was added to a glass petri dish containing a solid LB medium for plating quantification.
As can be seen from the results shown in FIG. 2d, MnO was present under non-illuminated conditions2The amylose, ICG, MAP, MAPI, MBP and MBPI have almost no bactericidal effect on MRSA biological membranes; under the condition of illumination, the sterilizing effect of MAPI on MRSA biomembrane can reach 2 orders of magnitude, the sterilizing effect of MBPI on MRSA biomembrane can reach about 1 order of magnitude, and MAPI with the same photo-thermal effect is higher than that of MBPI by one quantity and about, which shows that the degradation biomembrane of amylase can enhance the photo-thermal sterilizing effect of MAPI.
(C) Scanning Electron Microscope (SEM) observation method
Referring to FIG. 3, the supernatant of the grown biofilm was removed, and 1 mL of physiological saline and MnO were added to each well, respectively2MAP, MAPI, MBP, MBPI (materials suspended in physiological saline, MnO)278 μ g/mL, amylose 280 μ g/mL, ICG 25 μ g/mL) and using a 785 nm laser at 0.8W/cm2The mixture is irradiated for 10 min and placed in a constant temperature box at 37 ℃ for incubation for 3 h. Removing supernatant, adding 1 mL formalin into each well, fixing for 10 min, gradient dehydrating with ethanol of different concentrations (30%, 50%, 70%, 80%, 90%, 95%), treating for 15 min, and treating with 100% ethanolThe treatment is carried out twice, and each time is 20 min. The treated samples were imaged by SEM.
From the results shown in fig. 3, it can be seen that MAPI and MBPI cause significant damage to the cell structure of MRSA under the illumination condition, and at the same time, MAPI with the same photothermal effect has a better bactericidal effect than MBPI, indicating that the amyloase-degrading biofilm can enhance the photothermal bactericidal effect of MAPI.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (8)
1. A nano composite material for enhancing photothermal removal of bacterial biofilm based on enzymatic degradation is characterized in that the nano composite material is MnO modified by amylase, polyethylene glycol PEG and indocyanine green ICG2Nanoplate MAPI.
2. A preparation method of a nano composite material for removing bacterial biofilm by enhancing photo-thermal degradation based on enzyme degradation is characterized by comprising the following steps:
step 1: preparation of MnO2An aqueous solution of a nanomaterial;
step 2: MnO of2Adding the aqueous solution of the nano material into the aqueous solution of amylase amyloylase, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifugation in water, and continuously centrifuging and purifying to obtain MnO2-an amylase nanoplate;
and step 3: MnO of2Adding a polyethylene glycol (PEG) aqueous solution into an aqueous solution of the-amylase nanosheet, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifuging in water, and continuously centrifuging and purifying to obtain MnO2-an amylase-PEG nanoplatelet;
and 4, step 4: MnO of2Adding an aqueous solution of an-amylase-PEG nano-sheet into an ICG aqueous solution of indocyanine green, stirring, centrifuging the obtained solution, dispersing the precipitate obtained by centrifugation into water, and continuing to carry out centrifugal purification to obtain MnO2-amylase-PEG-ICG nanoplatelets.
3. The method for preparing the bacterial biofilm-removal nanocomposite material based on enzymatic degradation-enhanced photothermal removal according to claim 2, wherein in step 1, MnO is added2The nano material is MnO2Nanosheets, MnO2The particle size of the nano-sheet is 20-200 nm.
4. The method for preparing the nanocomposite for enhancing photothermal removal of a bacterial biofilm based on enzymatic degradation according to claim 2, wherein the amylase amyloylase is one or more of alpha-amylase, beta-amylase, gamma-amylase and isoamylase; the polyethylene glycol PEG is mPEG-SH, mPEG-COOH or mPEG-NH2One or more of HCl and mPEG-OH.
5. The method for preparing a bacterial biofilm nanocomposite capable of enhancing photothermal removal based on enzymatic degradation as claimed in claim 2, wherein in step 2, MnO is added2The concentration of the aqueous solution of the nano material is 1-100 mug/mL, and the concentration of the aqueous solution of the amylase is 10-1000 mug/mL; the MnO2The volume ratio of the aqueous solution of the nano material to the aqueous solution of the amylase is 1: 0.5 to 5.
6. The method for preparing the bacterial biofilm removal nanocomposite material based on enzymatic degradation enhanced photothermal removal according to claim 2, wherein in step 3, MnO is added2The concentration of an aqueous solution of the-amylase nano-sheet is 1-100 mu g/mL, the concentration of an aqueous solution of the polyethylene glycol PEG is 10-1000 mu g/mL, and MnO is added2-the volume ratio of aqueous solution of amylase nanosheet to aqueous solution of polyethylene glycol PEG is 1: 0.5 to 5.
7. The method for preparing the bacterial biofilm removal nanocomposite material based on enzymatic degradation enhanced photothermal removal according to claim 2, wherein in step 4, MnO is added2The concentration of the aqueous solution of the-amylase-PEG nano-sheet is 1 to 1100 mu g/mL, the concentration of the aqueous solution of indocyanine green ICG is 2-200 mu g/mL, MnO is added2The volume ratio of the aqueous solution of the amylase-PEG nanosheets to the aqueous solution of indocyanine green (ICG) is 1: 0.5 to 5.
8. The method for preparing the nanocomposite for removing the bacterial biofilm by the enzyme degradation-enhanced photothermal removal according to claim 2, wherein in the steps 2 to 4, the stirring is performed at room temperature by using a magnetic stirrer, and the stirring time is 0.5 to 12 hours; centrifuging at 6000-18000 rpm for 45 min; the conditions of centrifugal purification are 6000-18000 rpm, the time of centrifugal purification is 45 min, and the times of centrifugal purification are 3 times.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018053646A1 (en) * | 2016-09-23 | 2018-03-29 | Klox Technologies Inc. | Biophotonic compositions, methods, and kits for inhibiting and disrupting biofilms |
CN108186676A (en) * | 2018-03-05 | 2018-06-22 | 南京邮电大学 | A kind of nano-antibacterial gel of treat wound infection and promoting healing and preparation method thereof |
-
2019
- 2019-12-19 CN CN201911318058.XA patent/CN110974961B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018053646A1 (en) * | 2016-09-23 | 2018-03-29 | Klox Technologies Inc. | Biophotonic compositions, methods, and kits for inhibiting and disrupting biofilms |
CN108186676A (en) * | 2018-03-05 | 2018-06-22 | 南京邮电大学 | A kind of nano-antibacterial gel of treat wound infection and promoting healing and preparation method thereof |
Non-Patent Citations (2)
Title |
---|
"Near-Infrared Light-Enhanced Protease-Conjugated Gold Nanorods As A Photothermal Antimicrobial Agent For Elimination Of Exotoxin And Biofilms";Wei Li等;《International Journal of Nanomedicine》;20191004;第14卷;第8047-8058页 * |
"负载吲哚菁绿二氧化锰纳米片光热特性及影响因素研究";杭银辉等;《影像研究与医学应用》;20190831;第3卷(第16期);第255-256页 * |
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