CN109731148A - A kind of antibacterial Heat Conduction Material - Google Patents

Abstract

The present invention provides a kind of antibacterial heat conducting coatings, which is characterized in that antibacterial heat conducting coating includes graphene oxide and antibacterial peptide, and the mass ratio of graphene oxide and antibacterial peptide is 1:25-1:75.Antibacterial heat conducting coating of the invention can mitigate the postoperative infection phenomenon of organ transplant, and stable interior environment can be provided for organ, and there are good market prospects and public and social interest to be worth.

Description

A kind of antibacterial Heat Conduction Material

Technical field

The invention belongs to anti-biotic material manufacturing technology fields, and in particular to a kind of antibacterial heat conducting coating.

Background technique

Organ during the transplantation process, usually saves it using machine perfusion instrument, and machine perfusion saves instrument Working principle is metabolite to be eliminated using controllable continuous preservation liquid, and provide nutrition and oxygen to organ, in other words, The simulation that environment in instrument is perfused is the key that isolated organ successfully saves.Therefore, temperature stability and filling inside instrument is perfused Biofacies content, the antibiotic property of note instrument internal material have vital effect for the performance of organ survival and function.Tradition Perfusion instrument generallys use resin as cabinet design material.Although resin has preferable thermal insulation effect, external environment temperature is reduced The influence of degree, still, perfusion liquid also bring along the variation of temperature in the process that inside is irrigated, due to the heat transfer efficiency of resin It is poor, when the temperature control system that instrument is perfused being caused to carry out temperature control to inside, it is easy to appear big inertia, large dead time and nonlinear temperature Variation, to influence the state of organ.Meanwhile for preventing bacterium infection, tradition is all that antibiosis is added in perfusion liquid Element such as Carbapenems, tigecycline, polymyxin, phosphonomycin, aminoglycoside, fluoroquinolones, has anti-pseudomonad The beta-lactam of category, preparation containing Sulbactam etc., still, common antibiotic either uses in perfusion liquid, still The problem of being applied to patient after surgery, all suffering from resistance.

Currently, by organ transplant postoperative infection occur pathogenic distribution the study found that postoperative infection infects Pathogen is with Grain-negative (G^-) bacterium is in the majority, common separation bacterium be Klebsiella Pneumoniae (Klebsiella Pneumoniae, KP), pseudomonas aeruginosa, escherichia coli, stenotrophomonas maltophilia, Grain-positive (G⁺) most common in bacterium, it is secondly dung Enterococcus.It is used generally for the treatment that post-transplantation infects using the combination of antibiotic, for example, the super wide spectrum β-of light moderate production is interior Amidase enterobacteriaceae bacterium, which infects, selects Piperacillin/Tazobactam Sodium, Sulbactam/Cefoperazone etc. in combination with drug sensitivity tests, treats Carbapenems can be changed to when imitating bad；The preferably preferred Carbapenems of severe infection, after treating clinical stability can depression of order ladder be Beta-lactam antimicrobial/beta-lactamase inhibitor mixture.However, continuing on with antibiotic, the bacterium of infection goes out Serious drug resistance is showed, so as to cause the postoperative means less effective using antibiotic treatment.

Graphene is the two dimensional crystal for the monoatomic layer thickness that carbon atom is formed in hexa-atomic circle permutation.2004, single layer Graphene is successfully separated from graphite by Novoselov and Geim, and since then, graphene becomes scientific circles' millions of people and looks steadily Purpose focus.The optics of graphene, electricity and heat performance be all it is outstanding, all cause people pole in each scientific domain Big interest (such as: nano electron device, transparent conductive film) has obtained apparent progress [4-5] in terms of many now.To list Layer graphene is studied while also resulting in concern of the people to other carbon-based materials, wherein most noticeable is graphite oxide Alkene (Graphene oxide, GO).GO is the Graphene derivative with oxygen-containing functional group, is graphite in acid condition by oxygen Obtained from change, basal plane and edge have hydroxyl, epoxy group and carboxyl.GO has good water dispersible, some organic molten In agent can also stable dispersion, the oxygen-containing functional group that basal plane and edge have enriches the surface-active of graphene, makes it easier to It is modified and functionalization, its huge specific surface area and good biocompatibility, GO possess more than other carbon-based materials in addition Mostly potential advantage, these attractive special performances make GO be highly suitable to be applied for field of biomedicine, become instantly Most one of the material of prospect.Prior art report, graphene-supported antibacterial material, such as silver ion, can be prepared into coating, The effect of antibacterial is played, still, the antibacterial effect of inorganic matter is not good enough.

Nineteen nineties, biologist have found that the immune system of day silkworm chrysalis produces when with microorganism induction day silkworm chrysalis A kind of polypeptides matter is given birth to, which has bactericidal activity.It is cecropin (Ceropins) that scientist, which names this polypeptide, Cecropin is formally recognized by people as first antibacterial peptide in the world.Since then, the innate immunity of organism causes vast The research and concern of scholar.In short decades, people have had found from microorganism, animals and plants, human body etc. not of the same race The natural antibacterial peptide of category.At present, it is found that natural antibacterial peptide quantity considerably beyond 1000 kinds.Natural antibacterial peptide Amino acid residue numbers are generally 15-50, and have many advantages, such as to heat rear stability height, and dissolubility is preferable in water.It is anti- Bacterium peptide distributes widely in nature, and has broad spectrum antibacterial, many antibacterial peptides are not only to Gram-negative bacteria, Gram-positive Bacterium has bactericidal effect, and also makees with certain inhibition to certain fungies, protist or even tumour cell and virus With, and part natural antibacterial peptide is smaller to eukaryocyte side effect simultaneously.Due to its unique antibacterial mechanisms, it is not easy to lure The drug resistance for sending out bacterium is expected to become the novel antibacterial drug with great potential.Currently, in the prior art for graphite The research that alkene loads antibacterial peptide is less, and being prepared into coating to reach the research of antibacterial purpose is even more to be rarely reported, and such painting Whether layer can be used for the perfusion system of organ transplant, belong to research blank in currently available technology.

Therefore, obtaining one kind keeps organ perfusion's internal system temperature stable and can prevent bacterium in organ perfusion process It infects, so that the antibacterial heat conducting coating for inhibiting post-transplantation to infect, is current urgent problem.

Summary of the invention

Postoperative infection, and bacterial infection drug resistance are easy to appear in order to solve the organ transfer operation in the presence of the prior art Property is strong, the problem of using antibiotic treatment less effective, and the internal temperature stability in machine perfusion system work process Not high problem, the present inventor are used to prepare machine perfusion system using coating prepared by graphene oxide and antibacterial peptide System, can be good at solving the above problems, in consideration of it, the present invention provides a kind of antibacterial heat conducting coatings, which is characterized in that antibacterial Heat conducting coating includes graphene oxide and antibacterial peptide, and the mass ratio of graphene oxide and antibacterial peptide is 1:25-1:75.

In one embodiment, the graphene oxide be reduced form graphene oxide, by graphene oxide with One hydration hydrazine reaction obtains.

In one embodiment, the antibacterial peptide is fused polypeptide, and the fusion of preferably cecropin A and thanatin is more Peptide.Cecropin A (cecropinA) is cationic antibacterial peptide from silkworm, and protein structure is parents' αhelix, tool Have a sterilizing ability of wide spectrum, thermal stability it is good and to normal cell without killing effect.Studies have found that the N-terminal sequence of cecropin A Play a significant role for its antibacterial activity.The sequence of cecropin A is RWKIFKKIEK MGRNIRDGIV KAGPAIEVLG SAKAI (totally 35 amino acid).Thanatin (Thanatin) is in insect spot ventral spine benefit stinkbug (Podisusmaculiventris) The small molecule antibacterial peptide of middle discovery, structure is simple, has inhibiting effect to gram positive bacteria, gram-negative bacteria and certain fungies. The sequence of the thanatin is GSKKPVPIIY CNRRTGKCQR M (totally 21 amino acid).By the 1-7 ammonia of cecropin A Base acid residue and the 4-19 amino acid residue of thanatin are merged, and are prepared into fused polypeptide, sequence is RWKIFKKKPV PIIYCNRRTG KCQ (totally 23 amino acid).The fused polypeptide is for Acinetobacter bauamnnii, the sour Cray of production The bacteriums such as primary bacterium, pseudomonas aeruginosa, Burkholderia cepacia, Klebsiella Pneumoniae, staphylococcus aureus have good (" expression and its activity research of the recombinant antimicrobial peptide in Pichia pastoris ", Yang Guimao etc., international laboratory medicine are miscellaneous for inhibitory effect Will, in June, 2018, the 12nd phase of volume 39,1439-1442).

In one embodiment, the fused polypeptide is the fused polypeptide (RWKIFKKKPV of cecropin A and thanatin PIIYCNRRTG KCQ) derived sequence, preferably with the fused polypeptide have 60%, 70%, 80%, 90%, 95%, The sequence of 99% identity, or there is one or more, for example, 2,3,4,5, the sequence of amino acid mutation.

In general, having higher antibacterial activity compared to the antibacterial peptide containing lysine containing arginic antibacterial peptide, containing coloured The antibacterial peptide of propylhomoserin more can be effectively in conjunction with bacterial cell membrane than the antibacterial peptide containing phenylalanine.This may be because of smart ammonia For acid compared to lysine, the positive charge delocalization degree on amino is bigger, it is possible to can enhance quiet with negatively charged bacterial membrane Electric attraction effect.Therefore, in one embodiment, by cecropin A and thanatin fused polypeptide (RWKIFKKKPV PIIYCNRRTG KCQ) the 3rd, the 7th of sequence, the 8th lysine (K) replaces with arginine (R), by the 5th benzene Alanine (F) replaces with tryptophan (W), to obtain antibacterial peptide: RWRIWKRRPV PIIYCNRRTG RCQ.

In one embodiment, the antibacterial peptide is prepared by way of Peptide systhesis, for example, liquid phase synthesis Method, solid-phase synthesis, Fmoc method, tBoc method etc., or Peptide synthesizer is used, for example, the ABI336 type polypeptide of American AB I company Synthesizer.This field conventional gene Engineering operation can also be used, is recombinantly expressed in host cell, for example, in yeast It is prepared in expression system, such as document " expression and its activity research of the recombinant antimicrobial peptide in Pichia pastoris ", Yang Guimao etc., International laboratory medicine magazine, in June, 2018, the 12nd phase of volume 39,1439-1442, disclosed method.

In one embodiment, a kind of antibacterial Heat Conduction Material is provided, the antibacterial Heat Conduction Material is by the thermally conductive painting of antibacterial Layer is coated on Heat Conduction Material, it is preferred that the Heat Conduction Material is metal material, it is furthermore preferred that the Heat Conduction Material is aluminium conjunction Gold.

In one embodiment, the antibacterial Heat Conduction Material the preparation method comprises the following steps:

1), Heat Conduction Material is immersed in the dopamine hydrochloride solution of 2mg/ml, pH value to 8-8.5, is reacted under room temperature Dry after sufficiently being cleaned with distilled water after 16 hours, the step for repeating, three times, obtains poly-dopamine coating；

2), the material for preparing step 1), is immersed into the Poly-L-Lysine Solution of 5mg/ml and reacts 3-6 hours, with steaming Distilled water cleaning, it is dry, it obtains poly and relies ammonia coating；

3), by graphene oxide and a hydration hydrazine reaction, reduced form graphene oxide is obtained, by reduced form graphene oxide It is prepared into the solution of 0.1mg/ml, the concentration of addition is in the antibacterial peptide solution of 2.5mg/ml, reduced form graphene oxide and anti- The volume ratio of bacterium peptide is 1:1-1:4；After being sufficiently mixed 2-4h, centrifugation disperses again, obtains the reduced form oxidation of load antibacterial peptide Graphene solution；

4), material prepared by step 2) is immersed in the solution of step 3) preparation, adsorbs 15-45min, distilled water is clear It washes, obtains the reduced form graphite oxide ene coatings of single layer antimicrobial peptide；It repeats step 4) 4-6 times, preferably 5 times, obtains thermally conductive The reduced form graphite oxide ene coatings of multilayer load antibacterial peptide are covered on material.

Preferably, the volume ratio of the reduced form graphene oxide and antibacterial peptide is 1:2.5.

In one embodiment, the antibacterial heat conducting coating or antibacterial Heat Conduction Material are used to prepare organ machine perfusion system System or organ machine perfusion instrument, it is preferred that the organ is selected from heart, organ, kidney, pancreas, marrow.

Compared with prior art, present invention has the advantage that coating of the invention is by graphene oxide-loaded antibacterial peptide It is prepared, graphene has good thermal conductivity, temperature control system can be made rapidly to control internal temperature, reduces temperature The fluctuation of degree provides good temperature environment for organ.Graphene oxide biocompatibility is high, harmless to cytotoxic, bears The antibacterial peptide of load to pathogen have good inhibiting effect and be not present drug resistance problems, prevent organ in filling process by To infecting for pathogen.Antibacterial heat conducting coating or antibacterial Heat Conduction Material of the invention can prevent postoperative infection phenomenon, and can Stable interior environment is provided for organ, there are good market prospects and public and social interest to be worth.

Detailed description of the invention

Fig. 1: graphene oxide-loaded antimicrobial coating surface topography and the pattern SEM of fractograph figure, wherein Fig. 1 a is table Face pattern, Fig. 1 b are the pattern of fractograph；

Fig. 2: flat-plate bacterial colony figure of the bacterium after GO-CT-1-C coating surface and the culture of control group surface, wherein Fig. 2 a is Flat-plate bacterial colony figure after the culture of pseudomonas aeruginosa control group surface；Fig. 2 b is pseudomonas aeruginosa in GO-CT-1-C coating table Flat-plate bacterial colony figure after the culture of face；Fig. 2 c is the flat-plate bacterial colony figure after the culture of Klebsiella Pneumoniae control group surface；Fig. 2 d is lung Flat-plate bacterial colony figure of the scorching klebsiella after GO-CT-1-C coating surface culture；Fig. 2 e is staphylococcus aureus control group table Flat-plate bacterial colony figure after the culture of face；Fig. 2 f is flat-plate bacterial colony of the staphylococcus aureus after GO-CT-1-C coating surface culture Figure；

Fig. 3: fluorescent staining figure of the endothelial cell after GO-CT-1-C coating surface culture；

Fig. 4: the schematic diagram for the machine perfusion system that the present invention is saved for organ, wherein 1: Central Processor Module, 2: Peristaltic pump, 3: organ perfusion's liquid memory, 4: input pipe C, 5: membrane oxygenator, 6: input pipe A, 7: input pipe B, 8: device Official's storage device, 9: output duct A, 10: perfusion liquid filter device, 11: output duct B；12: fluid collection device, 13: liquid Meter, 14: organ room temperature machine perfusion system, 15: monitoring data collection device, 16: temperature sensor, 17: pressure sensing Device, 18: flow sensor, 19: data processing equipment, 20: control assembly, 21: information carrying means；

Fig. 5: liver perfusate memory or liver storage device result schematic diagram, wherein 22: cold and hot regulation pipe, 23: ABS resin, 24: aluminium alloy, 25: coating GO-CT-1-C.

Specific embodiment

Below by way of preferred forms of the invention to the structure of the machine perfusion system saved for organ of the invention It is described in detail at effect, still, the following contents should not be construed as limiting the scope of the invention.

The preparation of 1 recombinant antimicrobial peptide of embodiment

It is synthesized using recombinant antimicrobial peptide of the polypeptide synthesis to mutation, the polypeptide of synthesis is named as CT-1.Polypeptide closes At use the full-automatic solid-phase synthesis of F-moc, the specific method is as follows:

The synthesis of 1.1 polypeptides

Using the model of 433A automated synthesizer (ABI, Foster City, CA) in the shielded polypeptide of assembled on resin. At room temperature, peptide resin is incubated for 2.5 hours in suspension, Deprotection.Suspension system is by 10 milliliters of TFA, 0.75 gram of benzene What phenol, 0.25 milliliter of 1,2- dithioglycol, 0.5 milliliter of thioanisole and 0.5 milliliter of water formed.By filtering, from more Peptide deprotects separation resin in based mixtures.Thick polypeptide precipitates in the diethyl ether solution that 150ml is pre-chilled, and with 10% glacial acetic acid work Chromatographic purifying is carried out in -25 column of sephadex G for eluant, eluent.Then, the component containing polypeptide is caught and is lyophilized, and makes It is 80% or so with the purity that high performance liquid chromatography measures thick polypeptide.

1.2 peptide purifications and characterization

By the C18 liquid-phase chromatographic column of the direct loading of filtrate to Zorba, wherein preparative high-performance liquid chromatographic is used to pump (Waters 2000 series,Milford,MA).C18 column first uses buffer solution A (aqueous solution of 0.1%TFA) prerinse column, 10-40% buffer solution B (the second cyanogen solution of 0.1%TFA) then is used, 40 minutes linear gradients are carried out with the speed of 8mL/min Elution.Obtained fraction is the concentrate of the CT-1 containing 90%, then uses and loads onto 9.4 × 250mm Zorbax C18 liquid phase color The semipreparative reversed-phase high performance liquid chromatography of spectrum column is further purified.Finally, in -25 chromatographic column of sephadex G with 20% acetic acid solution is eluent, and final product is shifted in acetate solution from tfa salt solution transfer.The purity of polypeptide is by dividing What the reversed-phase high performance liquid chromatography of analysis type was assessed, obtained final product-polypeptide purity is 98%.

It is determined with sequence composition of the Ultraflex III TOF/TOF mass spectrograph to polypeptide.The polypeptide prepared Sequence is as shown in table 1,

Table 1: the polypeptide sequence prepared

Title	Sequence
		CT-1	RWRIWKRRPV PIIYCNRRTG RCQ

Embodiment 2 covers the preparation of the Heat Conduction Material of graphene oxide-loaded antibacterial peptide coating

The graphene oxide-loaded antibacterial peptide coating of reduced form is first prepared in this implementation, in the way of self assembly, by coating It coats to Heat Conduction Material surface, wherein Heat Conduction Material selects aluminium alloy, obtains and covers graphene oxide-loaded antibacterial peptide coating Aluminum alloy materials, it is specific the preparation method is as follows:

The surface modification of 2.1 Heat Conduction Materials

By the aluminium alloy after cleaning-drying, it is first immersed in the dopamine hydrochloride solution of 2mg/ml, is adjusted with NaOH PH value is used under room temperature after reaction overnight to 8.5, and after distilled water sufficiently cleans, drying at room temperature is dry, above-mentioned steps in triplicate, Enabling aluminum alloy to surface modification has poly-dopamine, then has the aluminum alloy materials of poly-dopamine to be immersed into 5mg/ml's surface modification It reacts 5 hours, is sufficiently cleaned after taking-up with distilled water, drying at room temperature in Poly-L-Lysine Solution, obtaining surface modification has poly The aluminum alloy materials of lysine.

The preparation of the reduced form graphene oxide solution of 2.2 load antibacterial peptides

The graphene oxide solution for configuring 0.1mg/ml first takes 100ml graphene oxide solution that 1mg mono- is added and is hydrated Hydrazine, 80 DEG C are sufficiently stirred reaction 2 hours, and centrifugal drying obtains redox graphene, and ultrasonic disperse is at 0.2mg/ml's again Solution；

The recombinant antimicrobial peptide prepared in embodiment 1 is configured in the composite antibacterial peptide solution of 2.5mg/ml, by oxygen reduction Graphite alkene solution is added to composite antibacterial peptide solution, and wherein the volume ratio of reduced form graphene oxide and antibacterial peptide is 1:1-1: 4；After being sufficiently mixed 3.5h, centrifugation disperses again, obtains the reduced form graphene oxide solution of load recombinant antimicrobial peptide.

The preparation of the Heat Conduction Material of the 2.3 graphene oxide-loaded antibacterial peptide coatings of covering

The aluminium alloy material that face prepared by step 2.1 is modified with poly-D-lysine is immersed in solution prepared by step 2.2, Room temperature adsorbs 30min, and distilled water cleaning is dry, obtains the reduced form graphite oxide ene coatings of single layer antimicrobial peptide；Repeat the step Rapid 5 times, obtain the aluminum alloy materials of the reduced form graphite oxide ene coatings of surface covering multilayer load antibacterial peptide.

2.4 scanning electron microscope (SEM) analysis

Using the graphene oxide coating surface shape prepared in JSM-6700F field emission scanning electron microscope observation 2.3 The appearance structure (acceleration voltage 10kV) of looks and fractograph.Before test, the processing of sample surfaces elder generation metal spraying.

As shown in Figure 1, the reduced form graphene oxide for the surface covering multilayer load antibacterial peptide that aluminum alloy materials are formed applies Layer, can be observed the feature pleated structure of graphene oxide, have sharp edge；It can be seen that aluminium from fractograph form Alloy material surface forms certain thickness coating.

The antibacterial effect of the coating of recombinant antimicrobial peptide and the graphene oxide preparation of 3 different proportion of embodiment compares

In the present embodiment, in order to probe into different proportion recombinant antimicrobial peptide and graphene oxide preparation coating antibacterial Effect, finds out the best concentration ratio of recombinant antimicrobial peptide and graphene oxide, aoxidizes stone respectively with recombinant antimicrobial peptide and reduced form Black alkene volume ratio is 1:1,1:2,1:2.5, and the concentration of 1:3, the method for embodiment 2 prepares the aluminium alloy material of corresponding applying coating Expect that GO-CT-1-A, GO-CT-1-B, GO-CT-1-C, GO-CT-1-D, experimental setup control group are the aluminium alloy of uncoated coating Material, specific table 2 as shown in table 2: the coating preparation of different antibacterial peptides and graphene proportion

The measurement of 3.1 antibiotic properties

By colony counting method to the antibacterial of GO-CT-1-A, GO-CT-1-B, GO-CT-1-C, GO-CT-1-D, control group Property is measured, specific steps are as follows:

Using pseudomonas aeruginosa, Klebsiella Pneumoniae, staphylococcus aureus (S.aureus) as gram-negative The type species of property bacterium and gram-positive bacteria carry out the antibiotic property of test sample.Before each antibacterial experiment, all vierics Ware, phosphate buffer solution (PBS) will carry out high pressure steam sterilization at 120 DEG C.

It is 10 by 200 μ L concentration⁶The bacterial solution of cfu/mL is dripped respectively in GO-CT-1-A, GO-CT-1-B, GO-CT-1- C, GO-CT-1-D makes bacterial solution spread over sample surfaces, then the ultrasonic vibration culture 3h at 37 DEG C.

The bacterium of sample surfaces after ultrasonic vibration culture is rinsed with 10mL PBS, takes 100 μ L to rinse thin Bacterium solution is equably coated in solid culture primary surface, and places it in 37 DEG C of constant incubators, culture 18~for 24 hours after, observation training The growing state for supporting primary surface bacterium colony, the coating anti-microbial property matched to same antibacterial peptide and graphene are evaluated.

Specific experiment result is as shown in Table 3 and Fig. 2

Table 3: the coating bacteriostasis rate of different ratio compares

From the results shown in Table 3, compared to control group, the heat conduction material coated with antibacterial peptide and graphite oxide ene coatings Material all has preferable fungistatic effect, especially antibacterial peptide and graphene oxide for Gram-negative bacteria and gram-positive bacteria Mass ratio be 1:62.5 prepared by antimicrobial coating, have 90% or more antibiotic rate for Gram-negative bacteria, for leather Lan Shi positive bacteria can also reach 70% or so bacteriostasis rate, achieve preferable effect.

The biocompatibility of embodiment 4GO-CT-1-C detects

In the present embodiment, in order to detect the biocompatibility of antibacterial peptide and graphite oxide ene coatings, to be coated with antibacterial peptide Mass ratio with graphene oxide is the Heat Conduction Material of antimicrobial coating prepared by 1:62.5 as substrate, and Human Umbilical Vein Endothelial Cells carry out Culture, further using the growth situation of dead cell stain reagent observation endothelial cell living, specific experiment operation is as follows.

The detection of 4.1 cytotoxicities

By cell with 2 X 10⁴Density be inoculated into the cated Heat Conduction Material surface of covering, cultivate 3 days, after culture With PBS rinsing three times, be protected from light in 37 DEG C of constant incubators with coloring agent dyeing 1 hour, 1 hour after with PBS rinse 2 times, so Cell is observed with laser scanning co-focusing microscope afterwards, is taken pictures.

4.2 experimental result

As shown in Figure 3, it can be seen that endothelial cell is good in coating surface growing state, and dead cell is not observed, Show that the normal growth breeding of coating Human Umbilical Vein Endothelial Cells has not significant impact, tentatively judges that coating Human Umbilical Vein Endothelial Cells are not shown Apparent cytotoxic effect.

Embodiment 5 prepares organ perfusion's system using the Heat Conduction Material of coating GO-CT-1-C

In the present embodiment, organ perfusion's system is prepared using the Heat Conduction Material of coating GO-CT-1-C, which can To be suitable for the in vitro room temperature machine perfusion of liver, specific configuration is as follows:

As shown in Figure 4,5, in perfusion system 14, Central Processor Module 1 is connect with peristaltic pump 2, passes through central processing Control assembly 20 in device assembly 1 controls the output power of peristaltic pump 2, and the peristaltic pump 2 is controlled by the variation of output power The injection pressure and flow velocity of perfusion liquid, further, peristaltic pump 2 is connect with liver perfusate memory 3, and the liver perfusate is deposited Reservoir 3 is inside and outside two layers, and interlayer is provided with cold and hot regulation pipe 22.The outer layer is the preparation of ABS resin 23.The internal layer is closed by aluminium Prepared by gold 24, internal layer is coated with the coating GO-CT-1-C 25, specific configuration such as Fig. 5 prepared in embodiment 3 towards cavity side It is shown.The cold and hot regulation pipe 22 is controlled by control assembly 20, for adjusting the temperature of perfusion liquid；Liver perfusate memory 3 In be provided with temperature sensor 16, for monitoring the temperature of liver perfusate；Liver perfusate memory 3 passes through input pipe C4 It is connect with membrane oxygenator 5, flow sensor 18 is provided between liver perfusate memory 3 and membrane oxygenator 5, for monitoring liver The oxygenation efficiency of the overall flow rate of dirty perfusion liquid, the membrane oxygenator 5 is carried out by the control assembly 20 in Central Processor Module 1 Control；Membrane oxygenator 5 is connect by input pipe A6 and input pipe B7 with liver storage device 8, and the input pipe A6 is Hepatic arterial infusion conduit is provided with flow sensor 18 and pressure sensor 17, for monitoring the flow velocity of hepatic arterial infusion With perfusion hydraulic coupling；The input pipe B7 is portal vein input pipe, is again provided with flow sensor 18 and pressure thereon Sensor 17, for monitoring the flow velocity and perfusion hydraulic coupling of portal vein perfusion；It is provided with opening in liver storage device 8, inputs Conduit A6 and input pipe B7 can enter 8 inside of liver storage device and connect with the liver of Plantlet in vitro.The liver storage Device 8 is inside and outside two layers, and interlayer is provided with cold and hot regulation pipe 22.The outer layer is the preparation of ABS resin 23.The internal layer is closed by aluminium Prepared by gold 24, internal layer is coated with the coating GO-CT-1-C 25, specific configuration such as Fig. 5 prepared in embodiment 3 towards cavity side It is shown.The cold and hot regulation pipe 22 is controlled by control assembly 20, for adjusting the interior environment temperature of liver storage device 8；Liver Temperature sensor 16 is provided in storage device 8, for monitoring interior environment temperature.

The liver storage device 8 is connect by output duct A9 with perfusion liquid filter device 10, in liver storage device 8 It is provided with opening, output duct A9 can enter inside liver storage device to be connect with the liver of Plantlet in vitro；The perfusion liquid Filter device 10 is connect with liver perfusate memory 3, and the perfusion liquid that filtering is completed is recycled to liver perfusate memory 3； The liver storage device 8 is connect by output duct B11 with fluid collection device 12, for collecting liver in filling process The bile of secretion；It is provided with liquid meter 13 in the fluid collection device 12, is divided in filling process for measuring liver The Amount of Bile secreted.

Further, perfusion system 14 is provided with monitoring data collection device 15, and the monitoring data collection device 15 is collected Monitoring data caused by temperature sensor 16, pressure sensor 17 and flow sensor 18 and liquid meter 12, and will receive The data of collection are transmitted to Central Processor Module 1.The Central Processor Module 1 is provided with data processing equipment 19, is used for Monitoring data collected by monitoring data collection device 15 is handled, Central Processor Module 1 further includes information carrying means 21, can The data processing equipment 19 is handled data parameters as a result, passing to external corresponding reception device.

The temperature stability of 6 organ perfusion's system of embodiment detects

In the present embodiment, in order to examine in embodiment 5, using organ perfusion's internal system of coated aluminium alloy material preparation The steadiness of temperature supervises the internal temperature of liver storage device and liver perfusate memory in filling process Control, compares the fluctuation situation of temperature in filling process, specific as follows:

6.1 experimental method

Using naringenin derivative room temperature liver perfusate as test perfusion liquid, the set temperature of system is perfused are as follows: 4 DEG C, 20 DEG C, 36.5 DEG C, environment temperature is indoor environment temperature, and about 23-26 DEG C, perfusion overall flow rate is 450ml/min.

After perfusion system starts 10min, the internal temperature of liver storage device and liver perfusate memory is supervised Control, each 10min detection temperature is primary, infusion time 1h.

Control group is set, and the perfusion system of control group uses the aluminum alloy materials without coating to prepare, other structures setting It is same as Example 5.

6.2 experimental result

5 institute of the maximum temperature and minimum temperature of the measurement of liver storage device and liver perfusate memory such as table 4 and table Show.

The maximum temperature and minimum temperature of 4 liver storage device of table

The maximum temperature and minimum temperature of 5 liver perfusate memory of table

From table 4, the result that table 5 is shown can be seen that the perfusion system relative to control group, coated with GO-CT-1-C coating The internal temperature of system, liver storage device and liver perfusate memory is more stable, and temperature difference is about ± 0.1 DEG C, it is seen then that Graphene itself has good thermal conductivity, and GO-CT-1-C coating is conducive to the conduction of heat fast and stable, for internal system The stabilization of temperature plays the role of good.

Embodiment 7 carries out storage in vitro to liver using the perfusion system containing GO-CT-1-C coating

1 experimental animal

Male China miniature pig 20,30 ± 3Kg of weight.It is divided into two groups, experimental group and control group, every group each 10.Institute Some experimental animals are treated by humanitarianism, meet U.S. National Institutes promulgation " management of laboratory animal and use refer to South ".

2. method

(1) experimental animal pre-operative anxiety is prohibited water 8 hours.Intramuscular injection yellow Jackets carry out induced anesthesia, weigh weight And it records.Dorsal position four limbs are taken to be fixed on operating table, high flow capacity oxygen uptake, preserved skin connects precordial leads cardiac monitoring, pigtail end Connect blood oxygen saturation probe.Scalp acupuncture is established peripheral vein access through auricular vein and is sufficiently fixed, before trachea cannula, chlorination Scoline injection (1mg/kg) anesthesia induction judges that connection Anesthesia machine is ventilated after being inserted into correct position, keeps inspiratory/expiratory 1: 2.It remains of flaccid muscles through peripheral vein access interruption Vecuronium Bromide, give Propofol maintenance anesthesia.

(2) liver obtains: " ten " word notch successively into abdominal cavity, is dissected ligamentum hepatoduodenale, is given after free choledochus out With ligation, proximal end plugs in catheter, separates out arteria hepatica, and portal vein is simultaneously suspended in midair, and whole section of arteria hepatica is completely free.And start to dissociate Splenic vein starts free liver inferior caval vein out after the completion of splenic vein is free, then dissociate suprahepatic vena cava.After dissociating successfully from Splenic vein is intubated to portal vein and completes, and starts free abdominal aorta, after the completion of abdominal aorta is free and is intubated, is injected intravenously heparin 12500 unit whole body test tube of hepari, after the completion of test tube of hepari, row liver perfusion uses UW liquid trans-portal vein groundwater increment about 500ml, abdomen Aortic perfusion amount about 1000ml, biliary tract rinse about 150ml.Liver surface covers sterile ice bits constantly to help liver in filling process It hepatectomizes after the completion of cooling perfusion, is placed in fill and be modified in 4 DEG C of sterile basins of protection liquid.

(3) preheating of machine perfusion system: open system maintains perfusion liquid temperature and the temperature of liver locker room 36.5 DEG C, keep Perfusion preservation system operating etc. to be accessed for liver.

(4) in vitro liver accesses perfusion system, wherein experimental group accesses machine perfusion system prepared by embodiment 5, right The aluminum alloy materials without coating are used to prepare according to the perfusion system of group, other structures setting is same as Example 5.

(5) Perfusion preservation: after the completion of connecting, adjustment arteria hepatica pressure 80~120mm Hg, portal venous pressure 10~ 20mm Hg, perfusion total flow are maintained at 400-500ml/min, and oxygen flow/partial pressure of oxygen is maintained at 2-3L/min, and keeps being perfused Liquid temperature is 36.5 DEG C.Portal vein, arteria hepatica flow and pressure are monitored by pressure sensor and flow sensor respectively in real time, The output power for adjusting peristaltic pump maintains the injection pressure and flow velocity of setting, maintains portal vein and artery blood flow in scope of design It is interior.The method that every 3h takes equivalent to replace releases 400ml perfusion liquid and adds the fresh perfusion liquid of 400ml, the perfusion liquid used for Naringenin derivative room temperature liver perfusate, infusion time 4h.

The transplanting and postoperative infection situation of 8 Plantlet in vitro organ of embodiment

In order to further detect the postoperative infection situation of in vitro liver transplant in embodiment 7, by be perfused 4h after liver into Row transplanting, specific experiment are as follows:

8.1 experimental animal

Male China miniature pig 20,30 ± 3Kg of weight.Random two groups of grouping, every group 10, as liver transplant by Body, wherein A group: experimental group liver transplant is received；B group: control group liver transplant is received.

8.2 transplantation method

Into abdominal cavity, it is dynamic successively to dissect choledochus, liver for sessile receptor pig, anesthesia, preserved skin, disinfection, drape, " people " notched cut Arteries and veins and portal vein are suspended in midair after the completion of dissection, and start to dissect liver superior and inferior vena cava, and dissociate arteria hepatica, and successively blocks It cuts off arteria hepatica (into no liver phase), portal vein, infrahepatic vena cava and liver superior and inferior vena cava, after extracing organ, starts rapidly Implantation is for liver；The liver superior and inferior vena cava that coincide is immediately begun to, after the completion of coincideing, starts to rinse portal vein and be coincide, open door Vein and semi-open liver superior and inferior vena cava, the relatively steady rear Full-open liver superior and inferior vena cava of monitoring vital sign (terminate without liver Phase), infrahepatic vena cava is opened after next successively coincideing again, arteria hepatica coincide after completing the above blood vessel, observation organ secretion Bile (new liver function recovery) out, and start the choledochus that coincide, indwelling T-type Tube Drain goes out bile, successively closes abdomen, and it closes abdomen and completes, T Type Tube Drain goes out bile, and vital sign is relatively steady, and operation is completed, and monitors vital sign, during which gives appropriate antibiotic, hormone And the corresponding energy therapy of supplement, gradually simultaneously defecation removes ECG monitor after not occurring obvious rejection for feed.

The detection method of organ state after 8.3 transplanting

After transplanting, conventional antibiotic anti-infective therapy (Ceftriaxone) is carried out to experimental group and control group and general again may be used Happy, learn and prednisone triple immunosuppressive therapy after transplanting 7 days, extracts transplant organ experimental group and control group respectively Whole blood, phlegm, bile, ascites, tracheal secretion carry out Bacteria culturing separation, antibiotics susceptibility test.Bacteria Culture instrument is purchased from the U.S. BiotonMerieux company, Bacteria Identification apply semi-automated analysis instrument and France's life purchased from U.S. BectonDinson company The API system of object Mei Liai company.>=2 positives of all continuous appearance are simultaneously closed for that can be diagnosed as after liver transplantation when identical bacterial strain And bacterium infection.

8.4 experimental result

15 plants of bacteriums are isolated from the inspection sample of experimental group liver transfer operation pig, it is specific as shown in table 6.

6 experimental group pork liver of table transplants positive sample Bacteria Detection situation

29 plants of bacteriums are isolated from the inspection sample of control group liver transfer operation pig, it is specific as shown in table 7.

7 control group pork liver of table transplants positive sample Bacteria Detection situation

It can be seen that the perfusion system containing GO-CT-1-C coating either in the type of infectious bacteria from the result of table 6-7 On, or in the quantity of infectious bacteria, be less than control group, especially in Gram-negative bacteria pseudomonas aeruginosa and Klebsiella Pneumoniae has apparent inhibiting effect, this explanation, GO-CT-1-C coating is beneficial to prevent the postoperative sense of organ transplant Dye.

Above the present invention is described in detail with a general description of the specific embodiments, but in the present invention On the basis of, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, not These modifications or improvements on the basis of deviation spirit of that invention, fall within the scope of the claimed invention.

SEQUENCE LISTING

<110>Jiaxing Lai Pusheng medical science and technology Co., Ltd

<120>a kind of antibacterial Heat Conduction Material

<130> CP11902069C

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 35

<212> PRT

<213>cecropin A

<400> 1

Arg Trp Lys Ile Phe Lys Lys Ile Glu Lys Met Gly Arg Asn Ile Arg

1 5 10 15

Asp Gly Ile Val Lys Ala Gly Pro Ala Ile Glu Val Leu Gly Ser Ala

20 25 30

Lys Ala Ile

35

<210> 2

<211> 21

<212> PRT

<213>thanatin

<400> 2

Gly Ser Lys Lys Pro Val Pro Ile Ile Tyr Cys Asn Arg Arg Thr Gly

1 5 10 15

Lys Cys Gln Arg Met

20

<210> 3

<211> 23

<212> PRT

<213>fused polypeptide

<400> 3

Arg Trp Lys Ile Phe Lys Lys Lys Pro Val Pro Ile Ile Tyr Cys Asn

1 5 10 15

Arg Arg Thr Gly Lys Cys Gln

20

<210> 4

<211> 23

<212> PRT

<213>antibacterial peptide

<400> 4

Arg Trp Arg Ile Trp Lys Arg Arg Pro Val Pro Ile Ile Tyr Cys Asn

1 5 10 15

Arg Arg Thr Gly Arg Cys Gln

20

Claims (10)

1. a kind of antibacterial heat conducting coating, which is characterized in that antibacterial heat conducting coating includes graphene oxide and antibacterial peptide, graphite oxide The mass ratio of alkene and antibacterial peptide is 1:25-1:75.

2. antibacterial heat conducting coating described in claim 1, which is characterized in that the antibacterial peptide is melting for cecropin A and thanatin Close polypeptide.

3. antibacterial heat conducting coating of any of claims 1 or 2, which is characterized in that the sequence of the fused polypeptide is RWRIWKRRPV PIIYCNRRTG RCQ。

4. antibacterial heat conducting coating described in claim 2-3 any one, which is characterized in that the matter of graphene oxide and antibacterial peptide Amount is than being 1:62.5.

5. antibacterial heat conducting coating described in claim 1-4 any one, which is characterized in that the graphene oxide is reduced form Graphene oxide.

6. purposes of the antibacterial heat conducting coating described in claim 1-5 any one in preparation antibacterial Heat Conduction Material.

7. a kind of antibacterial Heat Conduction Material, which is characterized in that the antibacterial Heat Conduction Material will be described in claim 1-5 any one Antibacterial heat conducting coating, which is coated on, to be caused on material, it is preferred that and it is described that material is caused to be metal material, it is furthermore preferred that described cause Material is aluminium alloy.

8. the preparation method of antibacterial Heat Conduction Material as claimed in claim 7, which is characterized in that the specific steps of the preparation method Are as follows:

1), Heat Conduction Material is immersed in the dopamine hydrochloride solution of 2mg/ml, pH value to 8-8.5, it is small that 16 is reacted under room temperature The step for Shi Hou, drying after sufficiently being cleaned with distilled water, repetition, three times, obtains poly-dopamine coating；

2), the material for preparing step 1), is immersed into the Poly-L-Lysine Solution of 5mg/ml and reacts 3-6 hours, use distilled water Cleaning, it is dry, it obtains poly and relies ammonia coating；

3), by graphene oxide and a hydration hydrazine reaction, reduced form graphene oxide is obtained, is prepared by reduced form graphene oxide At the solution of 0.2mg/ml, it is added in the antibacterial peptide solution that concentration is 2.5mg/ml, reduced form graphene oxide and antibacterial peptide Volume ratio be 1:1-1:4；After being sufficiently mixed 2-4h, centrifugation disperses again, obtains the reduced form graphite oxide of load antibacterial peptide Alkene solution, it is preferred that the volume ratio of the reduced form graphene oxide and antibacterial peptide solution is 1:2.5；

4), material prepared by step 2) is immersed in the solution of step 3) preparation, adsorbs 15-45min, distilled water cleaning obtains Obtain the reduced form graphite oxide ene coatings of single layer antimicrobial peptide；It repeats step 4) 4-6 times, it is negative to obtain covering multilayer on Heat Conduction Material The reduced form graphite oxide ene coatings of antimicrobial peptide.

9. the purposes of antibacterial Heat Conduction Material described in antibacterial heat conducting coating described in claim 1-5 any one or claim 7, It is characterized in that, the antibacterial heat conducting coating or antibacterial Heat Conduction Material are used to prepare organ machine perfusion system or organ machinery fills Infuse instrument.

10. the purposes of machine perfusion system as claimed in claim 9, which is characterized in that the organ is selected from heart, liver, kidney It is dirty.