CN102485272A

CN102485272A - Application of recombinant human Prx-6 protein in treating burns and scalds

Info

Publication number: CN102485272A
Application number: CN2010105735947A
Authority: CN
Inventors: 李建远
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-12-06
Filing date: 2010-12-06
Publication date: 2012-06-06
Also published as: CN106377764A

Abstract

The invention provides application of recombinant human Prx-6 protein in treating burns and scalds. Specifically speaking, the invention provides application of recombinant human Prx-6 protein in preparation of medicines used for treating burns and scalds. According to results of experiments in the invention, Prx-6 protein plays an important role in promoting wound healing in the process of treatment of burns and scalds.

Description

The purposes of recombined human Prx-6 albumen aspect the treatment burn and scald

Technical field

The present invention relates to biotechnology and medical domain, specifically, relate to the purposes of recombined human Prx-6 albumen aspect the treatment burn and scald.

Background technology

Burn and scald is a commonly encountered diseases, and frequently-occurring disease also is the higher trauma of disability rate.The lighter also brings very big misery to the wounded, and weight person usually damages organ dysfunction, causes very big inconvenience for patient's work and life; Because of the more exposure portion that occurs in the people of burn and scald, the cicatrix after the healing damages the wounded's appearance simultaneously, cause harmful effect also can for patient's psychology; Therefore; Burning/scald to mild or moderate then will shorten healing stage as far as possible, reduces less patient suffering, and reduces cicatrization as far as possible.Should reduce complication such as various visceral organ injuries that perfusion again causes and secondary infection to serious burn and scald the wounded as far as possible.

The normal method that adopts of clinical treatment burn generally is after carrying out the wound surface early time treatment at present, adopts occlusive dressing or adopts exposure method to treat.No matter adopt which kind of method, during debridement, all will carry out the wound surface infection and handle in early days, the anti-infectives that debridement is selected for use mainly contains three types: the one, and Chinese herbal and crude drugs preparations is like Ilex purpurea Hassk.[I.chinensis Sims, Radix Ilicis Pubescentis, Rhizoma Coptidis, Cortex Phellodendri etc.; The 2nd, the chemicals preparation is like hydrogen peroxide, bromo geramine, mafenide; The 3rd, antibiotic formulations such as penicillin, gentamycin.Because most drug all can absorb from wound surface, large tracts of land is used these medicines, and especially concentration is excessive or usually can cause when using powder and poison or visceral lesion.Moreover these medicines generally all have certain zest, usually can increase misery to the patient in use.

Peroxiredoxins (Prxs) is that one type of non-selenium finding recently relies on the peroxidase family protein, extensively is present in the various organisms.Difference (1 or 2) according to the conservative cys number of Prxs albumen can be divided into 1-Cys or 2-Cys subclass.So far, 6 kinds of Prxs albumen in mammalian tissues, have been found.Wherein Prx1-5 is the 2-Cys enzyme, with thioredoxin as electron donor; Prx-6 albumen is 1-Cys enzyme unique in the mammal, is electron donor (Reducing agent) with glutathione (GSH).

The at first separated acquisition in the buphthalmos corpus ciliare of Prx-6 albumen; Have another name called that LTW4, antioxidation albumen 2, Clara cell protein 26, p67 phagocyte oxidase (phox) are conjugated protein, keratinocyte growth factor (KGF) regulator gene 1 etc.; Be made up of 224 aminoacid, molecular weight is 25.1KDa.Analyze this proteic crystal structure, found that a thioredoxin of being made up of 80 aminoacid folds, and wherein comprises four βZhe Dies and two α spirals.

Big quantity research shows that the thioredoxin foldable structure is ubiquity in the albumen (like thioredoxin, glutaredoxin, GPx1 albumen, glutathione S-transferase etc.) of some participation redox reaction.In addition, although the proteic primary structure of Prx-6 and GPx1 has only 17% homology, their tertiary structure has high homology.This structural similarity is indicating these two kinds of albumen being correlated with on function.

Test in recent years from incipient cell and to test in the animal body, increasing research has proved that Prx-6 albumen has the anti-oxidative damage function.The research of cellular level shows, crosses expression Prx-6 albumen and can obviously reduce lipid peroxidation, reduces the cytoplasma membrane damage; Suppress the proteic expression of Prx-6, can be observed tangible lipid peroxidation, adipose membrane damage and apoptosis.The live body Study on Level shows that the Prx-6 transgenic mice has significant protective effect to high oxygen injury; The anti-oxidative damage ability of Prx-6 knock-out mice obviously reduces.But these researchs mainly are about the anti-oxidative damage effect of Prx-6 albumen in heart, lung or skin histology, do not relate to the purposes of Prx-6 albumen aspect the treatment burn and scald.

In sum, still lack the medicine of gratifying treatment burn and scald at present, so this area presses for the new medicine that can be used in the treatment burn and scald of exploitation.

Summary of the invention

The purpose of this invention is to provide the application of recombined human Prx-6 albumen aspect treatment burn and scald.

In first aspect of the present invention, the purposes of a kind of Prx-6 albumen in the medicine of preparation treatment burn and scald is provided.

In another preference, described Prx-6 albumen is people Prx-6 albumen.

In another preference, described Prx-6 albumen is recombiant protein.

In another preference, described Prx-6 albumen comprise aminoacid sequence shown in SEQ ID NO:2 albumen or itself and express the fusion rotein that label (like 6-His) forms.

In second aspect of the present invention, a kind of pharmaceutical composition that is used to treat burn and scald is provided, described pharmaceutical composition contains Prx-6 albumen and pharmaceutically acceptable carrier, and described pharmaceutical composition is an external preparation.

In another preference, described Prx-6 albumen is people Prx-6 albumen.

In another preference, described Prx-6 albumen is recombiant protein.

In another preference, described external preparation is unguentum, spray or drop.

In another preference, described pharmaceutical composition also contains the medicine of other treatment burn and scald.

In the third aspect of the invention, a kind of method of treating burn and scald is provided, comprise step:, on the wound surface of burn and scald, use the pharmaceutical composition described in Prx-6 protein or the second aspect present invention to the object of needs treatment.

In another preference, described treatment comprises: promote wound healing and the inflammatory reaction that reduces wound surface.

In another preference, described to liking mammal (comprising the people).

In another preference, described using comprises and applies or spray.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.

Description of drawings

Fig. 1 has shown Prx-6 gene PCR amplification.Swimming lane 1: blank; Swimming lane 2:Prx-6; M: molecular weight standard article.

Fig. 2 has shown Prx-6 albumen affinity purification chromatogram.Peak 1: foreign protein; Peak 2:Prx-6 destination protein.

Fig. 3 has shown Prx-6 protein purification electrophoretogram (12%SDS-PAGE).Swimming lane 1: molecular weight of albumen standard substance (Fermentas); Swimming lane 2: the Prx-6 albumen after affinitive layer purification and the desalination; Swimming lane 3: peak 1; Swimming lane 4: go up appearance and pass albumen; Swimming lane 5: broken bacterium supernatant (going up the sample of appearance).

Fig. 4 has shown the anti-oxidation protection effect (P＜0.05) of reorganization Prx-6 albumen to the HEF cell.

Fig. 5 has shown the anti-oxidation protection effect (P＜0.05) of reorganization Prx-6 albumen to the H9C2 cell.

Fig. 6 has shown the result that reorganization Prx-6 protein for treatment mice is scalded.Among the figure, each picture (2-9d) is followed successively by from left to right: matched group (2); Prx-6 organizes (2); Prx-6+ MEIBAO group (2); MEIBAO group (2).

The specific embodiment

The inventor is through extensive and deep research, except finding that Prx-6 albumen has antioxidation and can protect the cell such as HEF, finds first that also Prx-6 albumen has the effect that promotes burn and scalding healing unexpectedly, so can be used for treating burn and scald.Accomplished the present invention on this basis.

Particularly, recombined human Prx-6 albumen of the present invention can directly be coated on wound surface, can restrain immediately for burning/scald wound, suppresses to ooze out.At once film forming after being coated with is stopped inside and outside infection, can accelerate new granulation tissue's growth, promotes the burn and scald organization healing, has little, the effective advantages such as control infection, short treating period of zest simultaneously.

Prx-6 albumen

As used herein, term " albumen of the present invention ", " Peroxiredoxins albumen " " prdx6 albumen ", " Prx-6 albumen " interchangeable uses such as " peroxidating redox proteins " all refer to have the albumen or the polypeptide of people Prx-6 aminoacid sequence.They can contain or not contain initial methionine.Should be understood that these terms had both comprised the albumen in people (source), be also included within homologue with function of the same race or homologous protein in other mammals (like Canis familiaris L., cattle, sheep, monkey, rodent (like mice)).In addition, this term comprises wild type and saltant Prx-6 albumen.

The cDNA sequence GenBank accession number of people prx-6 gene: NM_004905.2 is shown in SEQ ID NO:1.The accession number of the aminoacid sequence of people Prx-6 is NP_004896, shown in SEQ ID NO:2.

Should be understood that because the proteic nucleotide sequence of mammiferous prdx6 such as people all is known with aminoacid sequence, can use the DNA recombinant technique of this area routine and obtain its albumen.

One type of preferred especially albumen is the prx-6 analog, i.e. the homologous protein of prx-6 in other mammals (like cattle, sheep, rabbit, Canis familiaris L., monkey, Mus etc.).The coded sequence of the homologous protein of these other species can obtain through the method for hybridizing or increase, and then obtain these homologous proteins through conventional recombination method according to the sequence of prdx6.

Pharmaceutical composition and application process

The present invention also provides pharmaceutical composition, and it contains the pharmaceutically acceptable carrier of prx-6 albumen of the present invention or the excipient of safe and effective amount.This type carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.

For example; Can albumen of the present invention be formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium; Wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can change with being prepared Substance Properties and disease to be treated to some extent.

The pharmaceutical composition for preparing can carry out administration through conventional route, comprising (but being not limited to): topical, oral, intramuscular, intravenous.A kind of preferred mode is to be applied directly to the wound surface of burn and scald and on every side.

Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as unguentum, drop, spray, tablet and capsule can prepare through conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.

In addition, albumen of the present invention also can use with the other treatment agent of treatment burn and scald.

In preference, can albumen of the present invention be pressed 0.1-0.5mg/cm ²The consumption of wound surface directly applies or is sprayed on the wound surface of burn and scald.

Major advantage of the present invention comprises:

(1) develops the new medical application of people Prx-6 albumen, opened up a new application.Empyrosis wound surface can be restrained after applying Prx-6 albumen immediately, suppresses to ooze out, and stops inside and outside infection, can accelerate new granulation tissue's growth, promotes to scald organization healing, has little, the effective control infection of zest simultaneously.

(2) Prx-6 albumen is the albumen that is derived from the people, and safe without toxic side effect safe in utilization has good prospect in medicine.

(3) people Prx-6 albumen is made injection type, spray, drop, unguentum etc., and is easy to use.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise umber and percentages.

Embodiment 1.pET32b (+)/Prx-6 expression vector establishment

Gene order according to coding Prx-6 maturation protein has designed and synthesized a pair of special primer (forward primer: 5 '-tatccatatgcccggaggtctgcttc-3 ' (SEQ ID NO:3); Downstream primer: 5 '-ttactcgagaggctggggtgtgtagcg-3 ' (SEQ ID NO:4)); Restriction enzyme site is respectively NdeI and XhoI

MRNA is a template with the extractive people's epididymis of conventional method, carries out the RT-PCR amplification.The result sees Fig. 1, detects the amplified fragments of the 688bp that conforms to theoretical value.

Separate amplified fragments, carry out double digestion with NdeI and XhoI.Then, the fragment that will pass through double digestion is connected to and is cut in linear pET32b (+) expression vector (available from Novagen) with NdeI and XhoI, transforms conventional escherichia coli TOP10F ' (available from Invitrogen).Through the method screening positive clone of bacterium colony PCR, and the further correctness that confirms that frame is read in sequence and expression of order-checking, recombinant expression plasmid pET32b (+)/Prx-6 obtained.The expressed Prx-6 albumen of this expression vector adds 8 aminoacid LEHHHHHH at the C end, so that separation and purification.

The proteic expression of embodiment 2.Prx-6

With plasmid pET32b (+)/Prx-6 transformed into escherichia coli Origami B (DE3) (available from Novagen), positive colony is inoculated in the LB culture medium that contains the 100ug/ml ampicillin 37 degree shaking table overnight incubation.Transferred in the LB culture medium that contain 100ug/ml ampicillin in by 1: 100 next day, and 37 degree are after shaking tables are cultured to nectar degree OD600=0.6～0.8, add IPTG to final concentration 0.4mM to induce the expression of destination protein Prx-6, centrifugal collection thalline behind the 3-4h.

The proteic purification of embodiment 3.Prx-6

The thalline of centrifugal collection is resuspended in the buffer A (the 20mM phosphate buffer, 150mM sodium chloride, pH7.2), and ultrasonication in the ice bath, it is sample to be purified that the centrifugal 15min of 20000g low temperature (4 degree) gets supernatant.After using buffer A balance nickel affinity column,, remove foreign protein (peak 1) with the buffer A flushing pillar that contains the 50mM imidazoles then, destination protein is eluted (peak 2) (see figure 2) with the buffer A that contains the 300mM imidazoles with the sample upper prop.

With the G-25 desalting column albumen of eluting is replaced in buffer A to remove imidazoles, SDS-PAGE electrophoresis detection at last.Through analyzing, obtained purity at 95% above Prx-6 albumen, the about 26.1kD of molecular weight.(see figure 3).

Embodiment 4. proteic antioxidant activity (HEF cellular oxidation injury experiment)

The HEF cell is human embryonic fibroblast (Zavada et al., Nature New Biology, 240:124-125 (1972) commonly used; Or US 7,811,817).With culture medium (high sugared DMEM+5%FBS) recovery cell, cultivate 48h, treat cell grow to 90% converge after, earlier the Prx-6 albumen (Prdx-6-His fusion rotein) that obtains among the embodiment 3 is added in the cultivating system according to following experimental technique, add H again ₂O ₂, behind the effect 30min, the MTT method detects cell survival rate.

Experimental design: orthogonal test

Factor 1:H ₂O ₂Level (1mM, 0.5mM, 0.25mM, 0.125mM, 0mM)

Factor 2:Prdx-6-His level (0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0mg/ml)

The H of each experimental group ₂O ₂As shown in table 1 with Prdx-6-His concentration.

Table 1

Result such as table 1 are with shown in Figure 4.

H ₂O ₂Pair cell has very strong toxic action, when its concentration is 0.125mM, and cell survival rate only 2.47%.Along with H ₂O ₂Concentration rises, and the damage that causes is also big more.

Cell survival rate significantly increases when having Prx-6-His albumen to exist in the system simultaneously.If when having only buffer to exist, the cell motility rate is compared remarkable reduction with the Prx-6-His protein groups.With H ₂O ₂Concentration is that each group of 0.125mM is an example, has added 0.25mg/ml, 0.5mg/ml Prx-6 albumen, can make survival rate significantly rise to 65% from about 2.5%.

In addition, only can not play a protective role with expressing label 6His.

The above results shows: Prx-6-His albumen is to H ₂O ₂Peroxide injury resistant function is arranged.0.25mg/ml albumen can make the HEF cell resist the H of 0.125mM concentration to a certain extent ₂O ₂Damage.

The proteic antioxidant activity of embodiment 5.Prx-6 (H9C2 cellular oxidation injury experiment)

Test method:

The H9C2 cell is rat myocardial cell (US 7,482,320) commonly used.With culture medium (high sugared DMEM+10%FBS) recovery cell, cultivate 48h, treat cell grow to 90% converge after, earlier the Prx-6 albumen (Prdx-6-His fusion rotein) that obtains among the embodiment 3 is added in the cultivating system according to following experimental technique, add H again ₂O ₂, behind the effect 30min, the MTT method detects cell survival rate.

Experimental design: orthogonal experiment

Factor 1:H ₂O ₂Level (0.5mM, 0.25mM, 0.125mM, 0.0625mM, 0mM)

Factor 2:Prx-6-His level (0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0mg/ml)

Table 2

Result such as table 2 are with shown in Figure 5.

H ₂O ₂Pair cell has very strong toxic action, and cell survival rate significantly increases when having Prx-6-His albumen to exist in the system simultaneously, and along with the proteic concentration of Prx-6 increases the also corresponding increase of survival rate.For example at H ₂O ₂Concentration is under the condition of 0.0625mM, and (from 0～0.5mg/ml), the survival rate of cell increases (from 1.4%～97.38%) along with the increase of Prx-6 concentration.

This shows that Prx-6-His albumen is to H ₂O ₂Peroxide injury resistant function is arranged.0.5mg/ml Prx-6 albumen can make the H9C2 cell resist the H of 0.0625mM concentration to a certain extent ₂O ₂Damage.

Embodiment 6Prx-6 albumen is to the therapeutical effect of burn and scald

6.1 animal origin

Mice 24～30g, Yantai Yuhuangding Hospital's Experimental Animal Center.

6.2 medicine

The Prx-6 albumen of preparation among experimental group: the embodiment 3;

Matched group: MEIBAO SHIRUN SHAOSHANG GAO, lot number Z20000004 (treatment burn medicine commonly used is as the positive control medicine).

6.3 burn model preparation

2.5% pentobarbital sodium (35mg/kg body weight) intraperitoneal injection is wiped out the back and is become mildewed after the anesthesia, be coated with the barium sulfide depilatory, strikes off gently after 3-5 minute, and warm water cleans up the normal raising in back.With quadrat method anesthesia, after the anesthesia mice being lain on the back to be fixed in has square hole and scalds on the plate, places 95 degree hot water 15s, takes out immediately with dry gauze and absorbs remaining hot water, lightens restrictions on behind the 24h.The body fluid of going is simultaneously recovered, and intraperitoneal injection sodium lactate Ringer ' solution 10ml, mouse back cause the three degree scalding models that account for body surface area 10%.

6.4 Therapeutic Method

Scald back 1h medication, change dressings every day 3 times.

Experiment comprises: matched group (being coated with not protein-contg buffer), Prx-6 protein for treatment group (being coated with 1mg/mlPrx-6 albumen), MEIBAO+Prx-6 protein positive matched group (are coated with 1mg/ml Prx-6 albumen earlier; The repaste MEIBAO SHIRUN SHAOSHANG GAO), MEIBAO positive controls (being coated with MEIBAO SHIRUN SHAOSHANG GAO); Scald back 1h medication, change dressings every day 3 times.1mg/ml Prx-6 protein solution is coated with mice burn wound, and consumption is 400ul, is folded to 0.1mg/cm ², MEIBAO SHIRUN SHAOSHANG GAO evenly is applied to wound surface, the about 1mm of thickness.

6.5 experimental result

When the 2nd day (2d) after scald, the 3rd day (3d) and the 5th day (5d), comparatively serious inflammatory reaction has taken place in control group mice, and Prx-6 protein for treatment group mice and the inflammatory reaction of MEIBAO positive controls mice significantly alleviate.During 5d, the control group mice wound is incrustation not, still is in the ulcer state after scald, and Prx-6 protein for treatment group mice and MEIBAO positive controls mice wound form a scab (Fig. 6).(annotate: be followed successively by from left to right among the figure: matched group (2); Prx-6 organizes (2); Prx-6+ MEIBAO group (2); MEIBAO group (2)).

Embodiment 7

Be used to treat the pharmaceutical composition of scald

With recombined human Prx-6 protein Preparation paste making agent, each component content is following: Prx-6 albumen lyophilized powder 0.1%, phosphate buffer 20mM (PH7.0) 85%, 20% glycerin, carbomer 1%, essence 0.3%.

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

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Claims

1. the Prx-6 albumen purposes in the medicine of preparation treatment burn and scald.

2. purposes as claimed in claim 1 is characterized in that, described Prx-6 albumen is people Prx-6 albumen.

3. purposes as claimed in claim 1 is characterized in that, described Prx-6 albumen is recombiant protein.

4. purposes as claimed in claim 1 is characterized in that, described Prx-6 albumen comprise aminoacid sequence shown in SEQ ID NO:2 albumen or itself and express the fusion rotein that label forms.

5. a pharmaceutical composition that is used to treat burn and scald is characterized in that, described pharmaceutical composition contains Prx-6 albumen and pharmaceutically acceptable carrier, and described pharmaceutical composition is an external preparation.

6. pharmaceutical composition as claimed in claim 5 is characterized in that, described Prx-6 albumen is people Prx-6 albumen.

7. pharmaceutical composition as claimed in claim 5 is characterized in that, described Prx-6 albumen is recombiant protein.

8. pharmaceutical composition as claimed in claim 5 is characterized in that, described external preparation is unguentum, spray or drop.

9. a method of treating burn and scald is characterized in that, comprises step: to the object of needs treatment, on the wound surface of burn and scald, use the described pharmaceutical composition of Prx-6 protein or claim 5.

10. method as claimed in claim 9 is characterized in that, described treatment comprises: promote wound healing and the inflammatory reaction that reduces wound surface.