CN104069128A - Application of poly vinyl pyrrolidone (PVP) modified nano-silver and imidazole medicines in preparation of drug tolerance resistant fungus active combined medicines - Google Patents

Abstract

The invention provides an application of poly vinyl pyrrolidone (PVP) modified nano-silver and imidazole medicines in preparation of drug tolerance resistant fungus active combined medicines. The invention shows that fluconazole or voriconazole and PVP modified nano-silver are used jointly to have synergetic drug tolerance resisting fungus effect. If drug combination is carried out on the imidazole drug resistant strain CA10 (MIC>256mg/L), the minimum inhibitory concentration can be lowered to 1mg/L, and the drug resistance is reversed.

Description

Nanometer silver and the application of azole drug in preparing overriding resistance fungi activity combination medicine that PVP modifies

Technical field

The invention belongs to medical technical field, be specifically related to nanometer silver (Ag-NPs) and azole drug fluconazol or the application of voriconazole in preparing overriding resistance fungi activity combination medicine that PVP modifies.

Background technology

Over nearly 20 years, increase along with cancer radiation, chemotherapy, organ transplantation, HIV sufferers, the long-term extensive use of broad ectrum antibiotic and immunosuppressant, immuuoeorapromised host is on the increase, the incidence rate of deep fungal infection sharply raises, and fungal infection has become the major disease patient's of above-mentioned immunologic hypofunction one of major causes of death.Antifungal drug be applied in large quantities for a long time clinical in, fastbacteria has become main cause [the Ruhnke M of clinical antifungal therapy failure, Eigler A, Tennagen I, Geiseler B, Engelmann F, Trautmann is of Fluconazole-Resistant Strains of Candida albicans in Patients with Recurrent Oropharyngeal Candidosis and Human Immunodeficiency Virus Infection.J Clin Microbiol.32:2092 – 2098. M.1994.Emergence].Multiple beads disease, Cryptococcus histolyticus, Aspergillus fumigatus are still modal deep pathomycetes.In hospital's blood preparation, the recall rate of candidiasis is 7.6%, ranks the 4th of all pathogen, first of pathomycete.In Candida, modal pathogenic species is Candida albicans.

At present can be for the antifungal drug Limited Number of selection of clinical, conventional polyene antibiotics (amphotericin B), triazole antifungal agent thing (fluconazol), propylamine antifungal drug (terbinafine), flucytosine and the echinocandin-class antifungal compound etc. of mainly containing.Wherein, fluconazol, because of good bioavailability, less untoward reaction and moderate price, becomes clinical practice antifungal drug the most widely.But because fluconazol only has the effect of Antifungi, so in treatment long-term and that repeat, make fungus produce fast-developing drug resistance.And fluconazol drug resistance fungal has cross resistance to other antifungal drugs, make ketoconazole, itraconazole, even amphotericin B is also felt simply helpless to increasing fastbacteria, this is that fungus Infection is treated failed main cause.

The strategy of reply Antifungal resistance mainly contains: (1) developing new drug.But new drug development process somewhat expensive, the time is tediously long and drug candidate mortality is high; (2) drug combination.Combining with antifungal drug is one of current clinical strategy that overcomes the most easily antifungal agent resistance.The theory advantage of therapeutic alliance is: the different phase of different pharmaceutical Antifungi metabolism and produce synergism, and a kind of drug effect is in cell wall or cell membrane and increase another kind of medicine and permeate in cell; Act on different molecule target position; Complementation on pharmacokinetics/pharmacodynamics; Single pharmaceutical quantities reduces and reduction toxic and side effects; Reduce the generation of drug resistance etc.Drug combination mainly concentrates on and treats cryptococcosis, Invasive candidiasis and invasive aspergillosis clinically.

Summary of the invention

In order to overcome the deficiencies in the prior art, the nanometer silver and the application of azole drug in preparing overriding resistance fungi activity combination medicine that the object of the present invention is to provide PVP to modify.

Wherein, described azole drug is selected from one or both in fluconazol and voriconazole.

Wherein, described drug resistance fungal is drug resistance Candida albicans.

The present invention has used standard micro-dilution method to measure drug susceptibility, use checkerboard type micro-dilution method, agar diffusion method and time-kill curve method evaluation Ag-NPs and fluconazol or the external relation of voriconazole use in conjunction overriding resistance Candida albicans, result shows to show: the anti-azole Resistant strain of Ag-NPs and fluconazol or voriconazole use in conjunction has synergism relation.

Synergism cytological mechanism is: Ag-NPs increase fungal cell membrane is damaged, and fluconazol and voriconazole can increase the quantity that Ag-NPs adheres to fungal cell membrane, and Ag-NPs and azole coupling can suppress normal Budding process etc. significantly.

Synergism molecular mechanism is: the nanometer silver that PVP modifies and azole drug produce synergism by acting on ergosterol biosynthesis pathway related gene and efflux pump expressing gene.

The present invention also provides a kind of pharmaceutical composition, comprises nanometer silver and azole drug that PVP modifies, and described azole drug is selected from one or both in fluconazol and voriconazole.

Beneficial effect: in order to overcome Candida albicans drug resistance phenomenon, the present invention utilizes existing drug regimen that the application in preparing overriding resistance fungi activity combination medicine of nanometer silver that azole drug and PVP modify is provided.

Particularly, the present invention, with respect to prior art, has following outstanding advantage and effect:

(1) the present invention shows, during nanometer silver use in conjunction that fluconazol or voriconazole and PVP modify, and the effect that can produce collaborative overriding resistance fungus.To azole Resistant strain CA10 (MIC>256mg/L), drug combination can make its minimum inhibitory concentration be reduced to 1mg/L, thus reversing drug resistance.

(2) the present invention shows, the medicine that acts on ergosterol biosynthesis pathway and suppress the gene expression of cell membrane efflux pump may be the molecular mechanism of associating antifungic action, for new drug development and old medicine newly use possible research direction is provided.

(3) nano silver gel is because it is non-antibiotics broad-spectrum high efficacy antibacterial, for prevention and the treatment of gynaecopathia clinically.Nanometer silver stability after polyvinylpyrrolidone (PVP) is modified is higher.Effective equally to drug resistance fungal during with azole use in conjunction, and can reduce dosage separately, thus reduce adverse effect, expand the scope of application of medicine, the generation of slowing down drug resistance.

Accompanying drawing explanation

Fig. 1 is the sign collection of illustrative plates of AgNPs.Wherein, A is the Electronic Speculum data of AgNPs separately or after interpolation azole drug; B is AgNPs ultraviolet absorption spectrum; C is the crystal diffraction data of AgNPs; D be AgNPs separately or add after azole drug hydraulic diameter and zeta potential value.

Fig. 2 is AgNPs and the analysis of azole use in conjunction overriding resistance Candida albicans CA10 synergism.Wherein, A is the equivalence curve after fluconazol and AgNPs coupling; B has sterilization synergy after dull and stereotyped drop method hint fluconazol and AgNPs coupling; C is agar diffusion method experimental result; D is killing curve experimental result.Bacterial strain uses therefor is Resistant strain CA10, and FLC represents fluconazol; VOR represents voriconazole.

Fig. 3 is to the modal change of Candida albicans after fluconazol and AgNPs coupling.Wherein, A is the adhesive attraction of AgNPs to fungal cell, fluconazol concentration 4mg/L, AgNPs concentration 2mg/L and their use in conjunction; B is the adhesive attraction of fluconazol concentration affects AgNPs to fungal cell; C is the comparison of relative bud ratio, fluconazol concentration 4mg/L, AgNPs concentration 2mg/L and their use in conjunction.Sprout is defined as the cell that is less than parent 1/2 size. ^**p<0.01。

Fig. 4 is that fluconazol and AgNPs coupling absorb the impact of propidium iodide on Candida albicans.Wherein, A is that drug combination absorbs the impact of propidium iodide (PI) on Candida albicans; B is the rate of change that after drug treating, Candida albicans absorbs propidium iodide.Fluconazol concentration is 4mg/L, and AgNPs concentration is 4mg/L. ^**p<0.01。

Fig. 5 is that quantitative PCR is measured fluconazol and the impact of AgNPs coupling on gene expression.Fluconazol concentration is 4mg/L, and AgNPs concentration is 4mg/L.**p<0.01。

Fig. 6 is fluconazol and the impact of AgNPs coupling on Quantitative Determination of Ergosterol.Wherein, A is ergosterol ultraviolet absorption spectrum; B is fluconazol and the impact of AgNPs coupling on Quantitative Determination of Ergosterol.Fluconazol concentration is 4mg/L, and AgNPs concentration is 4mg/L. ^**p<0.01.

The specific embodiment

Below in conjunction with accompanying drawing, the present invention is made and being further illustrated.

The AgNPs building-up process reference literature that the present invention uses carries out [Guo D, Zhu L, Huang Z, et al.Anti-leukemia activity of PVP-coated silver nanoparticles via generation of reactive oxygen species and release of silver ions.Biomaterials2013; 34:7884-94].The AgNPs purity 99.99% making.

The structural characterization of AgNPs, see Fig. 1:

(1) maximum absorption band that ultraviolet full wavelength scanner draws is at 416nm;

(2) crystal diffraction data have four typical peak values of nanometer silver, 111,200,220,311;

(3) electron microscopic observation statistics size, AgNPs particle size distribution with add the size after azole drug, the degree of polymerization does not have difference statistically;

(4) Darwin's particle size analyzer determination AgNPs (1mg/mL) particle diameter 34.92 ± 2.1nm, AgNPs particle diameter 34.67 ± 1.7nm and 35.12 ± 2.4nm after interpolation fluconazol and voriconazole;

(5) zeta current potential-5.35 ± 1.4mV of AgNPs, zeta current potential-4.23 ± 1.5mV and-4.23 ± 1.2mV after the interpolation fluconazol of 8mg/L and the voriconazole of 8mg/L, adding of azole drug significantly do not affect the stable of AgNPs.

The external collaborative experiment of embodiment 1

Extracorporeal sensitivity experiment and the checkerboard type micro-dilution method reference literature National Committee for Clinical Laboratory Standards.Methods for antifungal disk diffusion susceptibility testing of yeasts of antifungal drug; Approved guideline M44-A.NCCLS, Wayne, PA, USA, 2004.Concrete operations are as follows:

(1) bacterium solution preparation and sensitivity analysis

After the albicans strain of-70 ℃ of preservations thaws, be inoculated on YPD solid medium, cultivate 24h for 37 ℃.Get well-developed single bacterium colony and be again inoculated into YPD above, cultivate 24h for 35 ℃, to obtain pure culture.Select diameter to be greater than 5 of the bacterium colonies of 1mm, used 0.85% sterile saline to make bacterial suspension.Bacteria suspension was vibrated for 15 seconds on agitator, and with blood cell counting plate counting bacterium number, adjusting bacteria suspension concentration is 1～5 * 10 ⁶cfu/ml.Used 1000 times of RPMI-1640 culture fluid dilution bacterium liquid, as the bacteria suspension of inoculation, take and guarantee that the final concentration of the rear every pore fungi liquid of inoculation is 0.5～2.5 * 10 ³cfu/ml.96 orifice plates were put in 37 ℃ of constant incubators after 48 hours, and the MIC value of and Quality Control bacterial strain good when growth control is within CLSI M27-A2 scheme given range time, by the optical density (OD) at microplate reader mensuration 600nm place.OD value/growth control the OD in rate of growth=each hole, is made as so that conk is more than 80% suppressed the minimal inhibitory concentration that standard judges each strain bacterium.

(2) checkerboard type micro-dilution method (Checkerboard microdilution assay)

According to the checkerboard type micro-dilution method of CLSI M27-A2 scheme, determined the minimum inhibitory concentration of each medicine.According to its minimum inhibitory concentration, determine drug level scope used in this experiment, and become 4 times of working concentrations with RPMI-1640 fluid medium dilution medicine.By concentration order from high in the end, absorb azole drug 50 μ l, add respectively the 12-2 row of 96 orifice plates, in the 1st row, add the fluid medium of 50 μ l; By concentration order from high to low, draw AgNPs medicinal liquid 50 μ l, add respectively the A-G of 96 orifice plates capable, the corresponding 50 μ l fluid mediums that add during H is capable.Then the bacteria suspension 0.5～2.5 * 10 of concentration will be mixed up ³cfu/ml100 μ l adds in each hole.H1 does not contain the growth control of medicine.77 kinds of combinations of two kinds of medication combined medication variable concentrations have so just been produced.96 orifice plates are placed in to 37 ℃ of constant incubators to be cultivated after 48 hours and measures and record result with microplate reader 600nm place.All experiments in triplicate.For the Evaluation of Germicidal Efficacy of drug combination, each sample in 96-orifice plate is got to 5 microlitre points by the method for dull and stereotyped drop and drips on YPD flat board, then in 37 ℃ of constant incubators, cultivate 48 hours after their bactericidal effect of photographic analysis.

(3) evaluation and decision method

The non parametric method of LA model adopts classification Mlc index (fractional inhibitory concentration index, FICI), and formula is ∑ FIC=FIC _a+ FIC _b=(MIC A, coupling/MIC A, alone)+(MIC B, coupling/MIC B is alone).

Determine FIC index: all FIC value in calculating data table, when in tables of data, all FIC values are all less than 4, now that value of FIC minimum is exactly FIC value; Otherwise FIC value maximum is FIC.FIC≤0.5, shows as synergism; FIC>4 is antagonism; In between the two, be uncorrelated.The less expression two medicine synergism of FIC index stronger [Odds FC.2003.Synergy, antagonism, and what the chequerboard puts between them.J Antimicrob Chemother.52:1].

Isobologram: pass through checkerboard method, the minimum inhibitory concentration of each medicine is drawn according to isobologram, if the MIC value after two medicine associatings drops to 1/4 of single medicine MIC value and can think that two medical instruments have synergy, the summation of the FICs of this meaning coupling medicine is equal to or less than 0.5.According to the shape of curve, can judge collaborative (convex), antagonism (spill) and irrelevant (straight line) [Te Dorsthorst DTA, Verweij PE, Meletiadis J, Bergervoet M, Punt NC, Meis FGM, Mouton JW.2002.In vitro interaction of flucytosine combined with amphotericin B or fluconazole against thirty-five yeast isolates determined by both the fractional inhibitory concentration index and the response surface approach.Antimicrob Agents Chemother.46:2982 – 2989].

(4) result (in Table 1) of sensitivity analysis and checkerboard type micro-dilution method

Sensitivity analysis result, in Table 1, shows: AgNPs between 16-32mg/L, does not have notable difference to azole sensitive organism and azole fastbacteria to the MIC value scope of the Candida albicans of the clinical separation of 10 strain.

Checkerboard type micro-dilution method result, in Table 1, shows: to Resistant strain CA10, CA135 and CA137, drug combination can significantly reduce the MIC of medicine, and as for CA10 bacterial strain, after coupling, the MIC value of fluconazol is reduced to 1mg/L from 256mg/L; The MIC value of voriconazole is reduced to 0.25mg/L from 64mg/L, and MIC value can reduce more than more than 200 times.Their interaction in vitro relation is to be collaborative interactively to Resistant strain; To sensitive strain, be collaborative or incoherent interactively.

Table 1 checkerboard type micro-dilution method is measured AgNPs and is evaluated with the anti-clinical separated Candida albicans of 10 strain of azole drug and FICI

Wherein,

^amIC is minimum inhibitory concentration;

^bfICI is classification Mlc index;

^csYN representative is collaborative; ANT represents antagonism; IND represents uncorrelated.Collaborative: FICI value≤0.5, antagonism: FICI value >4.0, uncorrelated FICI value 0.5～4.

^dfLC, fluconazol;

^evOR, voriconazole.

The rate of growth data of fluconazol and AgNPs use in conjunction overriding resistance Candida albicans CA10, in Table 2.AgNPs and fluconazol concentration range are all 0-8mg/L, and there is the drug regimen of 77 variable concentrations centre.

By calculating rate of growth, be less than 20% corresponding drug level, and calculating FICI, to be worth minima be 0.02, is less than 0.5, prompting AgNPs and fluconazole application are collaborative interactivelies.

Table 2 FICI index analysis AgNPs and the anti-azole Resistant strain of fluconazole medication CA10

FLC, fluconazol; FICI, classification Mlc index.

In Fig. 2, A figure has shown the Equivalence analysis of fluconazol and AgNPs drug combination overriding resistance Candida albicans CA10, and its isoeffect curve is spill, illustrates that both couplings are collaborative relations.

In Fig. 2, B figure has shown the bactericidal effect after dull and stereotyped drop, and result shows after both couplings to have the collaborative effect of sterilization.

(5) agar diffusion method (Agar diffusion assay): picking clinical drug-resistant bacterial strain CA10 monoclonal is inoculated in 10mlYPD fluid medium, and 37 ℃, 200rpm shakes incubated overnight, then gets this bacterium liquid and again activates.Get 1ml bacterium liquid, centrifugal, resuspended with normal saline, and with blood counting chamber counting, with normal saline dilution and adjust bacterial concentration to 5 * 107CFU/ml.Get 100 μ l bacterium liquid and join 10ml preheating containing mixing and pour into rapidly in preprepared YPD solid culture ware in the YPD culture medium of 0.75% agar, on its surface, put the aseptic filter paper sheet (diameter 6mm) that contains various dose medicine, blank is containing corresponding equivalent solvent.After being cultivated to 48 hours in 37 ℃ of incubators, agar culture dish observes inhibition zone state.

Agar diffusion method experimental result:

In Fig. 2, C figure has shown the experimental result that the method for utilization agar diffusion is verified.For fastbacteria CA10, azole drug fluconazol 10 μ g, voriconazole 4 μ g do not have obvious bacteriostasis on YPD, the antibacterial action of 2 μ g AgNPs also a little less than, but drug combination can increase inhibition zone size significantly, and inhibition zone in asepsis growth.

(6) time-kill curve (Time-killing test)

Clinical drug-resistant Candida albicans CA10 is activated to twice, 37 ℃ in YPD culture fluid, and 200rpm shaken cultivation, makes fungus in later stage exponential phase of growth.Get this bacterium liquid to 1ml, centrifugal and resuspended with normal saline, cytometry to be adjusted to nectar degree be 108cfu/ml.This bacterium liquid is diluted to 104cfu/ml with RPMI-1640, and is divided into 4 parts, and every part of 20ml adds respectively corresponding medicine to final concentration: PVP-AgNPs concentration is 4mg/L, and fluconazol and voriconazole are all 1mg/L; Blank adds the DMSO of same volume, and in all bacterium liquid, DMSO content is all lower than 1%.Then in 37 ℃ of cultivations, after 0,12,24,48 and 72 hour, measure.

Assay method: viable bacteria counting method: from each pipe bacterium liquid, get 100 μ l liquid, then with 10 times of serial dilutions of 0.9% normal saline it, from the bacterium liquid of different extension rates, respectively get 50 μ l and be evenly coated with and be laid on YPD solid culture primary surface.Plate is counted bacterium colony number cultivate 48 hours in 37 ℃ of fungal culture casees after, and the clump count of growing on YPD plate of take reaches 20 as minimum.Calculate the viable bacteria number (CFU) that falls, with Log10CFU/ml, the time is made to curve.Evaluate the dependency of the two.

The effect evaluation method of drug combination: powerhouse compares with alone antifungal drug activity, confirms that two medical instruments have synergism when two medicines share rear bacteria concentration drop-out value >=2Log10CFU/ml; Powerhouse compares with alone antifungal drug activity, when share rear bacteria concentration drop-out value≤2Log10CFU/ml, two medicines confirm that two medical instruments have irrelevant effect [Haynes MP, Buckley HR, Higgins ML, Pieringer RA.1994.Synergism between the Antifungal Agents Amphotericin B and Alkyl Glycerol Ethers.Antimicrob Agents Chemother.38:1523 – 1529].

Killing curve experimental result:

In Fig. 2, D figure has shown the experimental result that the method for operate time-killing curve is verified.After 72 hours, matched group Candida albicans CA10 cell number 8.4LogCFU/mL, alone group of CA10 cell number of PVP-AgNPs is 8.23LogCFU/mL, the alone processing of fluconazol CA10 cell number 6.8LogCFU/mL, and after PVP-AgNPs-fluconazole medication processing, CA10 cell number is 4.56LogCFU/mL.Compare with alone medicine, after fluconazol and PVP-AgNPs use in conjunction, LogCFU/mL bacterium is counted decline >2LogCFU/mL, and checking drug combination has collaborative effect.After 72 hours, the independent medication group of voriconazole Candida albicans CA10 cell number 7.1LogCFU/mL, after voriconazole and PVP-AgNPs use in conjunction, LogCFU/mL bacterium is counted decline 2.2LogCFU/mL, and checking voriconazole and PVP-AgNPs use in conjunction have collaborative effect.

Embodiment 2 cytology's synergistic mechanism aspect researchs

(1) research method that AgNPs adheres to fungal cell: 1 * 10 ⁷cFU/mL Candida albicans CA10 treated with medicaments is after 24 hours, at 100 times of oily Microscopic observations and take pictures.Data statistics ImageJ software analysis, each sample is at least analyzed 100 fungal cells.

Result shows: adding of fluconazol can increase the adhesive attraction of AgNPs to fungal cell, and with the increase of concentration, adhering to ratio increases (Fig. 3 A and 3B), and drug combination can reduce fungal cell's bud ratio (Fig. 3 C) significantly.

(2) propidium iodide (PI) absorption analysis research method: the indication probe that adopts propidium iodide (PI) to increase as fungal cell's membrane permeability, PI can not pass through living cells film, but but can to core, dye through damaged cell membrane, Candida albicans after drug treating and the PI37 of 5mg/L ℃ are cultivated 15 minutes in the dark, and PBS rinsing 3 is taken pictures inferior to fluorescence microscopy Microscopic observation.

Result shows: drug combination can significantly increase PI red fluorescence amount, and after prompting drug combination, fungal cell's membrane permeability increases (Fig. 4 A and 4B).

Embodiment 3 Study on Molecular Mechanism

(1) method of quantitative fluorescent PCR is measured expression (ERG3, ERG5, the ERG1 of related gene in ergosterol biosynthesis pathway, ERG25, ERG6, ERG11) and fungal cell membrane efflux pump related gene (MDR1, CDR1, CDR2) 1.1 sample treatment

Picking Resistant strain CA10 monoclonal is inoculated in 30ml YPD culture fluid, 37 ℃ of overnight incubation, and the centrifugal 5min of 3000g, with the resuspended adjustment nectar of fresh YPD degree 1.0 * 10 ⁶cfu/ml is medicine group and matched group respectively.37 ℃ of concussions are cultivated 12 hours, and 4 ℃ centrifugal.

1.2TES preparation

10mM Tris.Cl pH7.5; 10mM EDTA; 0.5% (W/V) SDS water preparation of polluting without RNA enzyme, room temperature storage.

1.3 hot phenol methods are extracted RNA

Bacterial sediment is resuspended with the ice-cold PBS of 1ml, transfers in the Eppendorf pipe that 1.5ml E.C. 3.4.21.64 processed 3000g, centrifugal 10min.Discard feelings, bacterial sediment is even with 400 μ lTES solution suspendibles, then adds 400 μ l acidic phenol, with the fierce vibration of vortex agitator 10s.Be transferred in 65 ℃ of water-baths, hatch 30-60min, every 5min, with the fierce vibration of vortex agitator 10s.Take out, ice bath 5min, 4 ℃ in the centrifugal 5min of 10000g.In the Eppendorf pipe that upper water phase transfer was processed to 1.5ml E.C. 3.4.21.64, add chloroform protein precipitation.Fierce vibration, 4 ℃ in the centrifugal 5min of 10000g.Upper strata is shifted in the Eppendorf pipe that 1.5ml E.C. 3.4.21.64 processed and added in the isopropyl alcohol of 2/3 volume, 4 ℃ of standing 2h.4 ℃ of centrifugal 8min of 10000g.Abandon supernatant, 70% ice-cold ethanol suspendible for precipitation, quick oscillation washing RNA precipitation.4 ℃ of centrifugal 8min of 10000g, siphon away supernatant as far as possible up hill and dale, drying at room temperature, the water dissolution that precipitation is polluted without RNA enzyme with 15 μ l.Sample is measured absorption value at 260nm and 280nm respectively, determines the purity of RNA, and calculates the quality of RNA, and RNA is in-80 ℃ of preservations.At whole RNA leaching process, strictly prevent the pollution of RNA enzyme.

1.4 reverse transcription reactions synthesize cDNA

By reverse transcription test kit description, carry out reverse transcription, total RNA reverse transcription is become to cDNA.Step:

1) get the total RNA2 μ of the fungus g of extracting, add Oligo (dT) 1 μ l, add without RNA enzyme deionized water and be settled to 12 μ l.Mix rear centrifugal 3～5sec.

2) 70 ℃ of degeneration 5min, cooled on ice 30sec, centrifugal 3～5sec.

3) by reactant mixture ice bath, then add following component: 5 * buffer4 μ l, RNA enzyme inhibitor (20U/ μ l) 1 μ l, dNTPs (10mmol/L) 2 μ l, mix rear centrifugal 3～5sec.

4) 37 ℃ of water-baths, after 5 minutes, add reverse transcriptase (20U/ μ l) 1 μ l, and cumulative volume is 20 μ l.

5) 37 ℃ of reaction 60min of reaction mixture, at 70 ℃ of deactivation 10min, quenching on ice ,-20 ℃ save backup.

Quantitative fluorescent PCR

Take cDNA as template, and the 18S rRNA gene of take carries out pcr amplification as internal reference.The primer sequence is as following table 3.

Table 3 qPCR Auele Specific Primer

Reaction system:

Amplification condition: 95 ℃ of denaturation 5min;

95 ℃ of degeneration 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 45s, totally 35 circulations.

72 ℃ are extended 5min again.

Negative control: do not add cDNA template and carry out pcr amplification.

Result shows: PVP-AgNPs and fluconazole medication can be raised ERG5 and ERG6 gene significantly, lowers CDR1 gene.

(2) assay method of ergosterol and result:

Assay method reference literature [the Prasad T of ergosterol, Chandra A, Mukhopadhyay CK, Prasad is link between iron and drug resistance of Candida spp.:iron depletion enhances membrane fluidity and drug diffusion R.2006.Unexpected, leading to drug-sensitive cells.Antimicrob Agents Chemother.50:3597 – 3606.] basis on slightly improve, concrete grammar is as follows:

Picking Resistant strain CA10 monoclonal inoculation in 30ml YPD culture fluid, 37 ℃ of overnight incubation, the centrifugal 5min of 3000g, with the resuspended adjustment nectar of fresh YPD degree 1.0 * 10 ⁶cfu/ml is medicine group and matched group respectively.37 ℃ of concussions are cultivated 12 hours, and collecting cell claims to extract sterol after wet bacterium weight.The PBS and freshly prepared saponifier (containing 90% alcoholic solution of the 15% sodium hydroxide) 6ml that add 2.5ml, mix 80 ℃ of water-bath saponification 60min.Add petroleum ether (boiling range 30-60 ℃) 6ml and extract 2 times, merge extractive liquid,, adding distil water 6ml washing 1 time, ether layer volatilizes under nitrogen, saponification fat not, add cyclohexane extraction and dissolve that to make liquor capacity be the wet bacterium of 1ml/g ,-20 ℃ of preservations.

Ultraviolet determination: have four characteristic peaks at wavelength 240nm-300nm place.Ergosterol and 24 (28)-dehydroergosterols (dehydroergosterol, DHE) have absworption peak at 281.5nm place, but only have 24 (28)-DHE to have absworption peak at 230nm place.The degree of ergosterol can deduct by 24 total (28)-DHE and ergosterol (by calculating the absorption at 281.5nm place) 24 (28)-DHE absorptions at 230nm place.Pass through equation:

Ergosterol %+24 (28)-DHE%=[(A _281.5/ 290 * F)]/thalline weight in wet base;

24 (28)-DHE%=[(A ₂₃₀/ 518) * F]/thalline weight in wet base;

Ergosterol %=[(A _281.5/ 290 * F)]/thalline weight in wet base-[(A ₂₃₀/ 518) * F]/thalline weight in wet base;

Wherein F is extension rate, and 290 and 518 is E value (percentage ratio of every centimetre), by the crystal structure decision of ergosterol and 24 (28)-DHE.Experiment in triplicate.

Result shows: the content that can reduce ergosterol 25% for AgNPs individual processing Candida albicans Resistant strain CA10, fluconazol can cause the minimizing of ergosterol 86.2%, and AgNPs compares with arbitrary alone medicine (Fig. 6 B) with fluconazole application (Fig. 6 A), the content of ergosterol significantly reduces, and can make ergosterol reduce 92%.

Claims (4)

1. Nanometer silver and the application of azole drug in preparing overriding resistance fungi activity combination medicine that 1.PVP modifies.
2. 2. application as claimed in claim 1, is characterized in that: described azole drug is selected from one or both in fluconazol and voriconazole.
3. 3. application as claimed in claim 1, is characterized in that: described drug resistance fungal is drug resistance Candida albicans.
4. 4. a pharmaceutical composition, is characterized in that: comprise nanometer silver and azole drug that PVP modifies, described azole drug is selected from one or both in fluconazol and voriconazole.