CN101541829A - Chymotrypsin from lucilia sericata larvae and its use for the treatment of wounds - Google Patents

Abstract

The use of larval enzymes, particularly a chymotrypsin, is described herein. The enzymes are usable in the treatment of wounds for debridement and for cell regeneration.

Description

Quimotrase and the purposes in the treatment wound thereof from lucilia sericata

The present invention relates to a kind of larval enzyme.More particularly, the present invention relates to one or more can be from the larval enzyme of lucilia sericata (Lucilia sericata) acquisition, and described enzyme can be used for tissue regeneration and wound healing.

The beneficial effect that larva is invaded and harassed some wound just is being observed many years ago very much.Yet,, the mechanism that this process relates to is not further studied up to becoming medical science for the strains that infects popular especially before the problem in the wound healing field.

Research has before seen that larval enzyme (removes downright bad, infect or external material from wound) as the antibiotic additive of tradition or co-adjuvant (adjuvants) in the wound debridement, in control infection (prevent or treat infections) thus and nearest make the growth of new tissue of wound and the effect in the closure of wound (healing) in the promotion tissue regeneration.Employed larval enzyme can be removed any drainage or the secretory product of larva body surface by cleaning larva, obtains from the hemolymph of whole larva or homogenate, carries out or do not carry out the cleaning of larva before this.The effect of cultivating larva under aseptic or non-aseptic state has also been investigated in research before, uses the effect of homogenate, hemolymph or drainage/secretory product, and the effect at larva age (newly hatching or an age or two ages).Find that all of these factors taken together can both influence the character or the activity of the product that is obtained from larva.

Inventor of the present invention pays close attention to the larva product recently, and in particular for the wound healing characteristic of the enzyme of larva product integral part, described enzyme is by promoting that tissue growth promotes wound closure.Surprisingly, inventor of the present invention has been found that one or more Quimotrases of finding in drainage/secretory product (surface cleaning thing) at lucilia sericata larvae play a crucial role in tissue forms: such enzyme often and tissue disruption (breakdown) interrelate.

Correspondingly, the invention provides a kind of isolating Quimotrase or its analogue or its synthesized form that comes from insect larvae.

Advantageously, Quimotrase of the present invention has shown as the character of bringing into play dual and contradiction, and it not only (removes downright bad, infect or external material) in the wound debridement have effect, and this is a proteolytic enzyme character in accordance with expectation; Also in inoblast adhesion (adhesion) (being tissue growth) effect is arranged, this is the character that proteolytic enzyme is all beyond one's expectations.Quimotrase of the present invention has selective active; Its removal or some tissue of degrading, wound eschar for example, but do not remove or degrade institute in a organized way, for example healthy or new tissue.Have reason to expect this or no matter tissue will be degraded or remove to every kind of proteolytic enzyme and its type, for example betide the situation of the calliphorid dermamyiasis that is caused by lucilia cuprina, wherein the Huai Si tissue with health is degraded.The debridement of the same enzyme and the dual nature that promotes cell to grow are uncommon and are surprising to those skilled in the art, because these characteristics ought to be conflicting.

Term used herein " eschar " is intended to define the tissue of any death, its surface from skin comes off, and is included in burn, gangrene, ulcer (especially pressure ulcer), infects (especially fungi infestation), be exposed to after the tissue of anthrax or any necrosis late period.

Insect is lucilia sericata (Lucilia sericata) preferably.

Quimotrase of the present invention preferably is taken at the drainage/secretory product that maybe can be taken at insect larvae.Yet identical composition may be present in hemolymph or the homogenate, and can obtain from these sources.Quimotrase can for example obtain by cleaning larva and collecting cleaning medium.Inventor of the present invention is contained constitutive expression and derivable composition at drainage/secretory product (ES) of finding insect larvae before, comprises enzyme, hormone etc.For this reason, the growth conditions of preferred larva remains constant.In the most preferred embodiment of the present invention, collect and what use is the drainage/secretory product that grows in the new hatching larva under the sterile state.

Preferably, Quimotrase has the sequence as shown in Fig. 1, Fig. 2 or Figure 13, and perhaps it keeps the biologically active polypeptide fragment of natural or complete chymotrypsin activity, and its avtive spot quilt in Fig. 1,2 and 13 is clear and definite.Preferably, the definite avtive spot of institute or have high homology among this fragment and Fig. 1,2 and 13 with coding chymotryptic peptide segmental DNA, for example the avtive spot homology should be at least 90%, preferably more than 95%, more preferably has 99% homology.Ideally, fragment should with peptide avtive spot or consistent with coding chymotryptic peptide segmental DNA.Associated dna sequence is shown among Fig. 1,2 and 12.

The Quimotrase of these sequences has shown has tissue regeneration character and debridement activity, but do not degrade or tissue digestive health or that live, consider its be derived from the cause of disease body lucilia cuprina of calliphorid dermamyiasis (a kind of tissue deterioration process) Quimotrase (even its to health or living tissue also have well-known tissue degradation character) have high homology, this is particularly wondrous.This homology is showed in Fig. 1 and 2.

Inventor of the present invention has been found that the tissue remodeling of ES and regeneration properties are just disappearing or be suppressed after the serpin phenylmethylsulfonyl fluoride (PMSF) that suppresses trypsin-like and chymotrypsin-like serine protease is cultivated, but is not then disappearing after the 4-that only suppresses trypsin-like serine protease (amidino groups phenyl) sulfonyl methane fluorine (APMSF) is cultivated or be suppressed.

Therefore, can sum up, be responsible among the ES that extracellular matrix is reinvented or active important even composition core of tissue remodeling and tissue regeneration is Quimotrase or chymotrypsin-like enzyme.This has obtained confirmation by the order-checking that will illustrate below, has wherein measured the sequence of the Quimotrase with double properties.As implement as shown in the scheme, the tissue regeneration character of ES has disappeared when this Quimotrase is removed, and this Quimotrase is removed the wound eschar, and therefore identical Quimotrase has the double properties of tissue regeneration and removing.

Quimotrase of the present invention is not only by debridement or improvement debridement, and also by promoting the inoblast migration, matrix is reinvented and changed over the fibrocyte form and promotes aspect its healing purposes is arranged at the treatment wound.The fact that these reverse effects (proteolysis and albumen generate) are present in the same enzyme is surprising to those skilled in the art, and from prior art, can't predict, especially consider that its Quimotrase with the lucilia sericata that comes to cause calliphorid dermamyiasis (this sick can destroy downright bad and living tissue simultaneously) has high homology.

Correspondingly, the invention provides the purposes of Quimotrase at the composition that is used for healing a wound.

The present invention also provides the purposes of the medicine that Quimotrase is used for healing a wound in preparation, and the method for using chymotrypsin protein enzyme treatment wound.Preferably, the Quimotrase that uses in composition or medicine has the sequence among Fig. 1, Fig. 2 or Figure 13, or by the dna sequence encoding among Fig. 1,2 or 12.

Alternatively, the active fragments of Quimotrase can be used for such composition or medicine.Quimotrase, or its fragment can be natural or synthetic.It preferably is obtained from the larva of lucilia sericata.Wound of the present invention defines below, can infected or not infection.Preferably, wound is chronic wound, and can have resistance to traditional treatment.

Wound can be reinvented or treat by changing over the fibrocyte form by promoting matrix by promoting fibroblastic migration.Preferably, wound carries out debridement by Quimotrase of the present invention.

On the other hand, the present invention also provides a kind of wound dressings (dressing), and this dressing comprises the said Quimotrase of previous section of the present invention.Correspondingly, the present invention also comprises a kind of method for the treatment of wound, and this method is included in the step of using above-mentioned dressing on the wound that needs treatment.

Inventor of the present invention finds ES is hatched with Trypsin inhibitor SBTI (STI), removed ability that ES is enhanced to the fibrocyte migration with and the ability of the extracellular matrix protein of degrading.Similarly, when the activity of Quimotrase was removed, though as described below, some tryptic activity had kept, its proteolysis (debridement) activity change.The albumen of being removed by STI is checked order.Find that these sequences are Quimotrase or chymotrypsin-like sequence,, and confirmed that this enzyme is relevant with activity as herein described with its autoploid as shown in Fig. 1,2 and 13.

Correspondingly, the present invention also provides the Quimotrase with sequence as shown in Fig. 1, Fig. 2 or Figure 13.The present invention also provides the dna sequence dna of the Quimotrase of coding shown in Fig. 1,2 and 13, and the dna sequence dna (Figure 12) that is presented at lucilia sericata chymotrypsinogen sequence in the pBac-3 carrier multiple clone site dna sequence dna.The dna fragmentation in coding chymotrypsin activity site (seeing Figure 13) also constitutes a part of the present invention.

Term as used herein " wound " is intended to define any infringement to skin, epidermis or reticular tissue, no matter the damage or disease causes, include but are not limited to, hurt, stab, operative incision, ulcer, pressure sore, burn (comprising) because hot, freezing, chemical, electricity and radiation-induced burning, corium abrades or wound, osteomyelitis and plastic surgery wound.Wound can be infected.In addition, wound can be chronic or acute.Chronic wounds is derived from various situations and comprises diabetes foot ulcer, venous leg ulcers, the surgical incision of infection, the wound of Cosmetics Surgery, osteomyelitis and pressure sore.

Quimotrase of the present invention can be natural maybe can be the synthesized form of natural enzyme for example shown in Figure 13.The synthesized form of these enzymes can for example use the expression of recombinant plasmid system or use any known peptide synthetic method or the device as shown in following example from the sequence traditional method that provides.The active fragments of enzyme, promptly enzyme keeps the fragment of enzyme function, and particularly those comprise the avtive spot determined among Figure 13, also are considered to a part of the present invention as their dna fragmentation of coding.

Quimotrase of the present invention can use with extract, its can be unprocessed or purify or its can with comprise solvent for example, thinner, buffer reagent, vehicle, stablizer, wetting Agent for Printing Inks, vehicle, wedding agent, co-adjuvant, sanitas, anti-caking agent, souring agent, jelling agent, emulsifying agent, tinting material, perfume compound with and analogue, particularly those are used for locality prescription, the pharmaceutical composition combination of conventional additive.In a preferred embodiment, Quimotrase will use locally, but and not mean that using of oral or other drug administration by injection pattern just is excluded outside scope of the present invention.Therefore, in preferred mode of administration, topical application, Quimotrase of the present invention can be with the delivery carrier of washing fluid, suspension, sanitising agent, emulsifiable paste, emulsion, gel, ointment, ointment, ointment, pulvis or solid or liquid.Optionally, Quimotrase can be fit to Quimotrase can be combined or make capsule with the material that the mode of slowly-releasing or sustained release is transported to wound.One of this suitable examples of material is poly-(lactide-co-glycolide) (PLGA) particle, and it can be customized to the delivery mode release peptide with a kind of control.Most preferably, Quimotrase can combine with dressing and be applied to wound.The example of such dressing comprise combine slowly-releasing hydrocolloid particle that contains Quimotrase or the stage of the sponge that contains Quimotrase or layering dressing; optionally cover with traditional dressing, Smith et al 2006 is seen in the description of these examples.The type of the hydrocolloid dressing that uses is that trade mark is sold with " Granuflex " for example at present, thereby can improve Quimotrase is discharged into the wound.

Quimotrase of the present invention can be unprocessedly maybe can be to use traditional albumen method of purification to purify.Quimotrase can pass through for example amidation of COOH, uses noncoding irregular amino acid to replace and/or carry out the CO-NH amido linkage with isostere (isotere) and replaces and protect it to suffer aminopeptidase or other enzymic activitys.In addition, Quimotrase, especially synthetic or other nascent Quimotrase can be by hydroxylation, glycosylation, sulphating, phosphorylations, or other secondarys or three-stage process, especially work as such secondary or three-stage process and given enzyme stability or improved solubility or other desired characteristics.Especially preferredly be that the synthesized form of enzyme reaches the conformation near natural enzyme through secondary or three-stage process, can reduce enzymic activity unless do like this.

In addition, inventor of the present invention finds the drainage/secretory product of insect larvae, and lucilia sericata especially can be moulded as tissue culture or periplast and keep cell.That is to say that the drainage/secretory product of lucilia sericata can replace for example serum use of bovine serum, keep the viability of cell.When such cell with transplanted or grafting to wound or when middle because it has eliminated this particularly important of the potential source of infection, for example with the relevant PrPC of Creutzfeldt-Jakob disease (CJD), or use the needs of serum to eliminate the disease transmission by removing.

Therefore the present invention also provides a kind of media that is used to keep viable cell, and this media comprises the drainage/secretory product of insect larvae.Preferably, this larva is a lucilia sericata.

Typically, lucilia sericata larvae or lucilia sericata maggot are applied to the chronic wounds of traditional treatment failure.Clinical observation proves that maggot has removed necrotic tissue (debridement), promotes sterilization and quicken granulation tissue to form (Sherman et al, 2000; Wollina et al, 2002).In order to be illustrated in these effects mechanism afterwards, inventor of the present invention has investigated the enzymic activity that is present in maggot secretory product and/or the movement (ES) (address is because the material that maggot continues to ooze out may be to be derived from secretory product and/or excremental like this) and has showed as an independent institute, be discharged into wound (Schmidtchen et al, 2003).Serine protease, the evidence of metalloprotease and l-asparagine protease activity are found (Chambers et al, 2003).In addition, the serine protease that exists among the ES shows degraded multiple common extracellular matrix (ECM) composition (Chambers et al, 2003).

Inventor of the present invention has yet tested maggot ES for interactional effect (Horobin et al between human body dermal fibroblast and extracellular matrix (ECM) composition, 2003,2005), wherein they have played keying action (Eckes et al, 2000) aspect the formation organizing.By combine (Giancotti and Ruoslahti, 1999) with cell-membrane receptor, ECM provides scaffolding for contact guidance, is controlled to the fibrocyte adhesion and instructs cell migration (Clark, 1996; Greilingand Clark, 1997).Be derived from the proteolytic enzyme that comprises fibroblastic many sources, regulate such interaction.This can influence fibroblast proliferation (Abe et al, 2000 by the direct activation cell surface receptor; Dery and Bunnett, 1999) and revascularization (Blair et al, 1997) or pass through round-about way, the proteolytic degradation product of ECM composition wherein, be apparent that most fibronectin, induce inoblast migration and chemotaxis (Greiling and Clark, 1997; Livantet al, 2000), epithelium regeneration (Gianelli et al, 1997) and tissue remodeling (Gould et al, 1997; Werb et al, 1980).Our result has been shown as under the existence that sticks in ES on surface of fibrocyte and fibronectin and collagen protein parcel and has reduced (Horobin et al, 2003).Recently, by using two-dimentional analyzed in vitro, we have showed that the inoblast migration passes the serine protease that fibronectin is present among the ES and quicken (Horobin et al, 2005), inventor for example of the present invention has connected with the inoblast migration of passing plane surface that has strengthened this kind of enzyme is active.Yet evidence shows, describe as them, cell in two dimension with the three-dimensional live body environment of being familiar with at them in different (Abbott, 2003 that show; Cukierman et al, 2001; Friedl and Br δ cker, 2000).For example, on plane surface, cell shows a kind of flat lamelliform outward appearance.On the contrary, home position observation to cell be star and extend dendritic processes.They also show the different associativity of matrix on every side, are called 3D matrix adhesion (Cukierman et al, 2001).In two dimension, in default of the resistance to progressive cell paste on plane surface, therefore moving and passing certain surface mainly is adhesion and the function of going the adhesion incident.Yet in three-dimensional, the matrix obstacle forces its form of cell change, makes its change shape and/or enzyme effect degraded ECM composition to make things convenient for its motion.Therefore inventor of the present invention has been developed the said three-dimensional body outer analysis, observes the fibroblastic migration and the form of replying ES therein.

Like this, inventor of the present invention concentrates on research to the said three-dimensional body outer analysis with their research, observes the fibroblastic migration and the form of replying ES therein.Setting up such model of representing the microenvironment that cell exists more accurately in live body, is in order to understand ECM in wound healing process better, interactional importance between resident cell and the ES.It also provides the basis that is used for development system, and movable therein corium and epidermic cell are contained in support, and be aqueous, and biodegradable and bioactive class periplast is transported to open wound and helps healing.Developed being included under the imputed migration optimum concn, be embedded in detection (Greiling and Clark, 1997 of the isolated species of the initial human foreskin fibroblast (HFF) in the colloid that forms by collagen protein and fibronectin; Friedl and Br δ cker, 2000).The collagen protein colloid is extensive use of in the class active somatic cell is cultivated, and has been considered to represent the just breeding (Friedl and Br δ cker, 2000) of biophysics structure of corium.In addition, being embedded in collagen protein gelationus cell has shown and has adopted the extension (Cukierman et al, 2001) that has the dendroid network of some similarity with class original position morphology.In fibronectin is comprised in, because this molecule plays remarkable effect (Greiling and Clark, 1997) at the guiding cell in the wound space migration.The result shows that ES is accelerated into the fibrocyte migration by a kind of dose-dependent mode and passes colloid.This may reinvent and guide more by enhancing matrix, and the cellular form of good distribution obtains promoting.As with showing in the example below, to compare with relevant contrast, the concentration of ES has significantly increased migrating cell quantity and its distance that moves from the cell droplet when being 1 and 5 μ g/ml.These concentration of ES have also been induced the cellular form of good distribution, and under low population density, the ES of 5 μ g/ml has promoted intercellular matrix protofibril to arrange.On the contrary, the ES of 10 μ g/ml, the maximum concentration of being tested suppresses the migration of cell but has changed the form of cell really.When the concentration of ES is 0.1 μ g/ml, show effect when testing in the training period hardly.

By these observation, can sum up the performance that ES promotes matrix reorganization and cell tractive force.In migration detected, the performance of traction was by separating the back and characterizing in the contraction that colloid is exposed to cell drop volumes in the gelationus liquefaction process of ES of 10 μ g/ml with the colloid of the ES that contains 5 μ g/ml.When cell was observed under than low inoculum density, the existence of tractive force characterized by appearance straight, that arrange the good matrix primitive fiber that is in tight state between cell, may arrange like this owing to the performance of opposite tractive force.It also characterizes by the cellular form of good distribution.As Harris and colleague (1981) inoblast that is embedded in the collagen protein colloid is observed, inoblast traction " being obviously different with simple contraction as muscle " is because " when the cell compression and stretched when being enclosed in their on every side collagen proteins cell elongation rather than shortened ".

Strengthened matrix by inoblast and reinvented, and realized that so the promoted ES of migration may be illustrated by colloid compression (compaction) model that is proposed by Bellows et al (1981).By more dissimilar cellular contraction and tissue collagen albumen gelationus ability, these investigators have determined a series of in-order colloid compression stage as shown in table 1.By our observation, obviously ES has started first three stage of colloid compression, promotes cell attachment and dispersion and the arrangement of facilitating matrix (as collagen protein) fiber.Like this, reach cell migration to the quadravalence section.Matrix reinvent and cell migration between the research of contact by Sawhney and Howard (2002) obtained reinforcement, the collagen protein " band (strap) " that they find to be formed between the cell cluster (clusters) that is embedded in the collagen protein colloid is facilitated advancing of cell and is instructed cell to closing on cell cluster migration (contact guidance).

Table 1

By the in order step of people such as Bellows in the colloid compression of suggestion in 1981

Step	Explanation
			1	Cell combines with collagen protein
2	The dispersion of cell in collagen fabric matrix
		3	Tissue and arrangement by the cell processes collagen fabric
4	Cell migration
		5	The foundation of iuntercellular contact
6	This arrangement develops into three-dimensional, histioid Cellular Networks.

Relatively can be clear by these, cell shows similarly morphological feature as by the viewed cell that is embedded in the collagen protein colloid of other investigators and matrix reorganization when ES exists.Why such other researchs do not comprise the product that comes from maggot, therefore must ask behavior and not be observed in we have the cell of contrast of ES.Answer may be relevant with serum, contain blood plasma as the comparative study in detecting, and blood plasma is left out here.Tomasek et al (1992) provides blood plasma to promote the interactional evidence of cell-matrix, and they find to remove serum before fibroblastic collagen protein colloid and suppressed the degree that the colloid on resident cells limit is subsequently shunk discharging being full of of constraint.Serum is added in the colloid, in case they are released the contraction that will cause immediately.Other investigator has also observed dependence (Steinberg et al, 1980 of matrix reorganization to serum; Guidry and Grinnell, 1985).In serum, found platelet derived growth factor (PDGF), beta transforming growth factor (TGF β) and bioactive lipid medium phosphatidic acid (LPA), and show that they have participated in the reorganization of stimulation matrix (Montesano and Orci, 1988; Grinnell et al, 1999; Roy et al, 1999; Toews et al, 2002; Kondo et al, 2004).

Therefore, the ES of maggot might contain found activeconstituents in serum.Other possibility is the serine protease that is present among the ES, and it has showed that before it is accelerated into fibrocellular migration (Horobin et al, 2005), may make contributions by cutting off membrane-bound protease activated acceptor (PARs).The gang of g protein coupled receptor is activated after the enzymatic lysis by serine protease (for example zymoplasm and trypsin-like enzyme).Such behavior has exposed the nitrogen end of the ligand that acceptor connecting, after it with cracked receptors bind and activation.Activatory PARs with influence cell shape, secretory product, integral protein activation, metabolic reaction is transcribed and is responded and the signal transduction chain coupling (Cottrell et al, 2002) of cell mobility.Work has before shown that zymoplasm promotes generation (Kolodney and Wysolmerski, 1992 of shrink tension by protease activated PAR in the inoblast of chicken embryos in the collagen protein colloid; Pilcher et al, 1994; Chang et al, 2001).What is interesting is, notice that the serine protease that appears among the ES has activity (Chambers et al to zymoplasm and plasmin substrate Tosyl-Gly-Pro-Arg-AMC, 2003), this means that ES may give play to inoblast is similar to the effect the same to zymoplasm.Any class plasmin activity also may be appropriate, because plasmin has shown the proenzyme precursor of activation various kinds of cell excretory matrix metalloproteinase (MMPs), thus the reorganization make contributions (Mignatti et al, 1996) to localized matrix.

Maggot ES has shown has activity to unsettled peptide under the urokinase-type activity.Urokinase is a kind of serine protease of the urokinase fibrinogen activator (uPA) that is otherwise known as, and it transforms fibrinogen the fibrinogen of its activated form.It also combines with fibrinogen activator acceptor (uPARs), and research has shown that it is by inoblast expression (Mignattiet al, 1996; Ellis et al, 1993; Behrendt et al, 1993).In case combination, uPARs is positioned the adhesion site of focus, and adjusts integral protein regulatory function (Wang et al, 1995; Chapman and Wei, 2001; Porter and Hogg, 1998), thus the mechanism of the more swarm cell phenotypes of a kind of guiding is provided.Certain, uPAR has been in the news and has claimed to have signal effect (Odekon et al, 1992 in cell migration, adhesion and chemotaxis; Waltz et al, 1993; Gyetko et al, 1994).Therefore have reason to infer that the variation that class urokinase activity among the ES also can the pair cell behavior contributes.

Other of ES may promote that the interactional behavior of inoblast-matrix may be relevant with the ability of its degraded gelatinous matrix composition.Show that according to observations the concentration that ES exists is high more, colloid is fast more to change in its appearance, becomes more translucently, and when the concentration of ES reaches the highest, becomes the similar liquids shape.We have also confirmed serine protease degrade collagen albumen and the fibronectin (Chambers et al, 2003) among the ES in work before.The gelationus degraded can cause loosening of mechanical tension, because the contact between matrix fiber and the tissue culture ware surface may be etched and microfiber webs is broken.Some have developed tension force cultivates the evidence that a lot of researchs of the investigator of power watch-dog provide the inoblast that is embedded in the collagen protein colloid can respond to and mechanical tension be made change.They find that inoblast keeps active tension dynamic balance, and reaction is to adjust interior matrix tension force (Brown et al, 1998 of giving birth to the direction that the outside is exerted pressure opposite; Eastwood et al, 1994).Therefore, the increase of exerting pressure causes the minimizing that the cell adjustment is shunk, and vice versa.Perhaps such discovery is given the credit in this reaction, and promptly mechanical tension influences the generation level of MMP, and matrix allows cell to react (Lambert et al, 2001) thereby reinvent on every side by release proteolytic enzyme.Like this, the mechanical tension that causes of gelationus proteolytic degradation lax may irritation cell shrinks and reinvents the matrix around it and take the form that is easier to move.To be biologically active peptides discharge from the collagen protein and the fibronectin of having degraded another the possibility of result, and it has shown can influence inoblast adhesion and migration (Greiling and Clark, 1997; Livant et al, 2000).

Inoblast can be responded to and the change of mechanical tension be reacted, though clearly illustrate that from our observation ES stimulates inoblast to reinvent matrix, may cause the enhancing of intercellular contact.This is because each cell can be responded to the difference of partial matrix mechanical stress because opposing causes the arrangement of matrix primitive fiber from the pulling force of other cells.So to what close on, even remote relatively, the coordination that the increase of cell perception can cause reacting between the cell is improved, and makes contributions to strengthening migration.Certain, Sheetz, Felsenfeld and Gabraith (1998) have proposed the cell migration matrix of similar coordination, and wherein cell is indicated its motion according to the direction and the hardness of ECM thiozell.

Although ES benefits aspect reinventing strengthening migration and matrix, the protease activity that importantly is present in ES is not an over-drastic, because their reaction causes breaking of matrix comprehensively.For the ease of contact guidance and migration, need to keep enough micro-fiber structures, this is that the denseest ES of 10 μ g/ml detects explanation by the inventor's to containing concentration.Here, ES not only suppressed the migration also apace colloid is degraded to stickiness, liquid.Be clear that having the optimum concn of ES may be the cytoactive that wound healing is contributed to promote.

Generally speaking, our result shows, the first, and ES promotes the inoblast migration.The second, when serum-free, ES is essential to the form that helps cell and keep and extend good distribution.The 3rd, still when serum-free, ES promotes the reorganization of ECM, especially at intercellular.The 4th, ES implements these effects by directly the ECM proteolysis being modified and/or directly changed over fibrocellular phenotype by interacting with cell receptor, has so just replaced the demand to serum.Our ability of possessing skills is come separating active substances from ES (Chambers et al, 2003; Horobin et al, 2003,2005), and will therefore continue to identify these active substances and mechanism wherein.

As shown in the data in the example II, the Quimotrase that extracts from larva ES has debridement activity (seeing Figure 21) because of the albumen of its dissolving wound eschar.Research and order-checking by inhibitor find that it is a kind of Quimotrase.

Embodiment of the present invention will be described now, and carry out reference and explanation with appended accompanying drawing.Wherein

Fig. 1 and Fig. 2 have showed that (2) of complete (1) and part show the sequence table of lucilia sericata gene order, and protein translation and with the homology of lucilia cuprina chymotrypsinogen, wherein the DNA codon is a lowercase, protein translation is a capitalization; There is the capitalization of underscore to represent in the lucilia sericata sequence when exclusive amino-acid residue relatively the time with nearest lucilia cuprina sequence of the same clan; Bold text is represented the avtive spot residue; Italic text representation signal peptide; The cracking site that the arrow representative is possible, the signal peptide of symbol cleavable; Terminator codon represented in asterisk; There is the lowercase of underscore to represent untranslated zone (UTR); The lowercase of runic band underscore is represented the polypurine tail, and the polypurine signal represented in the literal of italic band underscore.

Fig. 3 has illustrated how three-dimensional vitro detection makes up.1. the improved Eagle ' s of Dulbecco ' s (D-MEM) substratum that contains 1.5mg/ml collagen protein and 30 μ g/ml fibronectins is poured 58mm group training ware into, and thin smooth glue-line congeals under 37 ℃.2. will contain 1 * 10 ⁷The droplet of D-MEM/ collagen protein/fibronectin of HFF cell/ml places on first glue-line, and the glue that congeals under 37 ℃.3. second layer D-MEM/ collagen protein/fibronectin is poured on the cell droplet, and the glue that congeals under 37 ℃.4. the cell culture medium that does not contain FCS is poured on the glue.5. the detection of complete combination is illustrated on the cross section, shows how all cells in the droplet is surrounded by the matrix colloid fully, therefore must move to three-dimensional.

Fig. 4 has illustrated the inoblast migration from 2 μ l colloid droplets is how to carry out external quantitative analysis from differing micro image in three-dimensional vitro detection.1. just detected the droplet of the inoblast inoculation after the combination (0 hour cultivate).2.24 the same droplet after hour cultivation.3. the fibroblastic droplet of inoculation in 0 hour cultivates, color is contrast furnishing black, is superimposed upon from the image of cultivating in 24 hours.4. have only those cells that migration has been come out from droplet in 24 hours to be shown, they can be counted.The distance that each cell moves is calculated by measuring vector length, signs in synergetic 0 hour edge of image from the forward position of each cell.Inoculating the Edge Distance of fibroblastic droplet on cultivating in 0 hour is also measured by drawing a picture around the image at synergetic 0 hour.The micron bar is represented 300 μ m.

Fig. 5 showed 2 μ l in the three-dimensional vitro detection inoculated fibroblastic colloid droplet followed by analyze combination (0 hour) or at a, there is not ES (0ES) or has 0.1 μ g/mlES (0.1ES) or 5 μ g/ml ES (5ES), there is not ES (0ES) in perhaps b or exists under the situation of 1 μ g/mlES (1ES) or 10 μ g/ml ES (10ES) and cultivates representative phase difference image after 24 hours or 48 hours.Under all situations, the micron bar is represented 300 μ m.

Fig. 6 has showed that 2 μ l in the three-dimensional vitro detection have inoculated the presentation graphics at fibroblastic colloid droplet edge, have highlighted cell in morphologic difference.Image differs, unless other explanations are arranged.A. wherein there is not ES (0ES) in cellular control unit and is exposed to 5 μ g/mlES (5ES) and cultivates contrast between the cell after 24 hours; B. cellular control unit (0ES) and be exposed to the maximum intensity projection that 1 μ g/ml ES (1ES) cultivates the z series optical cross section of contrast between the cell after 48 hours-confocal, cell dyes with FITC-Phalloidine (Actin muscle) and iodate pyridine (nuclear); Cellular control unit (0ES) and be exposed to contrast between the cell after 10 μ g/ml ES (10ES) cultivate 24 hours (c) or 48 hours (d).A., c, the micron bar among the d. represent 50 μ m (on) or 20 μ m (descending), in b., represent 50 μ m.

Fig. 7 has showed in the three-dimensional vitro detection that the colloid droplet inoblast that inoculated cell from 2 μ l was 24 hours migration.The result expresses (n=5) with the average quantity ± SEM of every millimeter edge migration of droplet.The migration of (contrast) or have 0.1 μ g/ml ES (0.1ES) or 5 μ g/ml ES (5ES) when a. not having ES.The migration of (contrast) or have the migration of 1 μ g/ml ES (1ES) or 10 μ g/ml ES (10ES) when b. not having ES.

Fig. 8 has showed that in three-dimensional vitro detection inoblast has inoculated the average moving distance of the droplet migration of cell from each 2 μ l.The value of having showed five repetition droplets.The distance that solid phase (experiment 1) (contrasts #1) or moves under the situation that has 0.1 μ g/ml ES or 5 μ g/ml ES when there is not ES in representative.The distance that opening shape (experiment 2) (contrasts #2) or moves under the situation that has 1 μ g/ml ES or 10 μ g/ml ES when there is not ES in representative.

Fig. 9 has showed representational phase difference image, and it has showed that at inoculum density be 3 * 10 ⁵Inoblast in the 20 μ l colloid droplets of cell/ml is immediately following detecting combination (cultivating in 0 hour) afterwards or after cultivating 24 hours or 48 hours.Cell does not exist ES (0ES) (contrast) or the outward appearance under existence 1 μ g/ml ES (1ES) or 5 μ g/ml ES (5ES).Black arrow is represented between the cell when having arranged as the connectivity fiber of rope sample becomes visual.In all cases, the micron bar is represented 20 μ m.

Figure 10 be two be illustrated in 24 hours and cultivate after, about the proteolytic activity of every kind of enzyme concn, the mixing of ES, trypsinase, Quimotrase and trypsinase/Quimotrase is to the figure of inoblast with the influence of the surperficial adhesion that has been coated with one deck FN.Such activity is assessed from the 7-amino-methyl tonka bean camphor (AMC) of the synthetic peptide substrates release of a. trypsinase or b. Quimotrase by monitoring.The adhesion of cell is Triphosaden (ATP) analysis on basis in order to Lampyridea luciferase element.The result represents with the average percentage ± 1SD (n=3) of the cytoadherence of control group.

Figure 11 is that fibronectin is at the gel electrophoresis photo of 37 ℃ of cultivations after 24 hours.Value is represented molecular weight standard (kDa).1. has only FN.2.FN+try/chy。3.FN+chy。4.FN+try。5.FN+ES。Every kind of enzyme concn shows the identical activity level to relevant trypsinase or chymotrypsin protein enzyme substrates.Try=0.32 μ g/ml trypsinase.Chy=0.02 μ g/ml Quimotrase.ES＝1μg/ml。

Figure 12 is the dna sequence dna table that is presented at the lucilia sericata chymotrypsinogen sequence of pBac-3 carrier multiple clone site dna sequence dna, and wherein pBac-3 carrier sequence has added underscore.

Figure 13 is the protein sequence of the dna sequence dna prediction among Figure 12, and wherein the avtive spot residue is represented with black matrix.

Figure 14 is the figure that is presented at tryptic/chymotrypsin activity after the S300 gel-filtration.

Figure 15 is a series of photo display are determined each active peak value by gelatin SDS-PAGE substrate gel analysis a typical proteolysis curve.

Figure 16 is presented at the figure that uses C1 to merge the albumen curve of thing (pool) back Trypsin inhibitor SBTI agarose affinity chromatography.

Figure 17 is a gel photograph, be presented at use C1 merge behind the thing proteolytic activity and with the proteic inhibitor properties of Trypsin inhibitor SBTI agarose bonded.

Figure 18 is presented at the figure that uses T2 to merge the albumen curve of Trypsin inhibitor SBTI agarose affinity chromatography behind the thing.

Figure 19 is a gel photograph, be presented at use T2 merge behind the thing proteolytic activity and with the proteic inhibitor properties of Trypsin inhibitor SBTI agarose bonded.

Figure 20 is the photo that shows 2 clotting glue, and it has been showed in the potential variation of handling the albumen curve of back wound eschar with lucilia sericata ES product.The potential zone that changes is circled.Figure 20 a shows the eschar that is untreated, and the eschar that Figure 20 b display process is spent the night.

Figure 21 is the photo that shows 4 clotting glue, and it has been showed in the potential variation for the treatment of the albumen curve of back wound eschar with lucilia sericata ES product.The potential zone that changes is circled.Figure 21 a shows the eschar that is untreated, and 20b handles with Quimotrase peak value (C1), and 20c handles with the first tryptic peak value (T1), and 20d is with the second tryptic peak value (T2).

Embodiment

Embodiment 1-tissue regeneration

Promote into fiber adhesion

Lucilia sericata larvae drainage/secretory product is collected and is characterized

Drainage/secretory product (ES) is from aseptic, the lucilia sericata larvae (LarvE of fresh hatching ^TM, Zoobiotic Ltd UK) collects as previously mentioned.Briefly, the larva of 400 work was cleaned 30 minutes with the aseptic phosphate buffered saline buffer (PBS) of 1ml down in room temperature (RT), to obtain the ES product.The protein concentration of ES and proteolytic activity are used Bio-Rad respectively, and (Hercules, CA) (Horobin et al, 2003) are measured in protein detection kit and fluorescein isothiocyanate (FITC)-casein digestion.These combine, and to reflect protein concentration be 161.74 μ g/ml, and activity specific is every mg ES albumen 6.04 * 10 ⁶The relative fluorescence primitive unit cell.

Initial human foreskin inoblast (HFF) cell cultures

HFF cell (TCS

UK) at Eagle ' s substratum (the D-MEM) (Gibco that contains Dulbecco ' s improvement ^TM, Invitrogen Ltd, UK), 10% calf serum (FCS) (

UK), antimicrobial/antimycotic solution (

) (100 unit/ml penicillin, 100 μ g/ml Streptomycin sulphates and 0.25 μ g/ml amphotericin B) 2mM L-glutaminate ( ) the T75 bottle (Nunc, Life Technologies Ltd, UK) in monolayer culture.Cell is maintained at 37 ℃ and contains 5%CO ₂Damp atmosphere in.

Three-dimensional vitro detection-inoblast migration

Preparation contains the D-MEM that doubles common cell cultures concentration (seeing above), the stoste of antimicrobial/antimycotic and L-glutaminate.After freezing, stoste with contain 3mg/ml ox type i collagen albumen (ICN Biomedicals, Ohio, USA), 60 μ g/ml ox inoblasts ( ) and the frozen soln of larva ES or blank PBS mixing on ice with 1: 1 ratio.The ultimate density that obtains is 1 * D-MEM, 1.5mg/ml collagen protein and 30 μ g/ml inoblasts.The final protein concentration of larva ES shown in sign, is 0.1 μ g/ml, 1 μ g/ml, 5 μ g/ml or 10 μ g/ml.These analyze as shown in Figure 3, and group is incorporated in this explanation: D-MEM-collagen protein/inoblast mixture of 1000 μ l, aforesaid, be introduced into 58mm tissue culture ware (Nunc, Life Technologies Ltd) in, then congeal into smooth at 37 ℃, approach the successive glue-line.HFF cell (6 passage) is by trypsinized and in being suspended among the D-MEM that contains 10%FCS with trypsinase.They granulate by centrifugal, and resuspending is in the D-MEM that does not contain FCS afterwards.After using Hematocyte Counter estimation cell quantity, cell is formed granular and with 1 * 10 once more ⁷The concentration resuspending of cell/ml is in D-MEM/ collagen protein/fibronectin colloidal mixt.Every five droplet that contain 2 these cell suspending liquids of μ l are placed in the culture dish on the gel coat, and 37 ℃ of cultivations until its glue that congeals into.D-MEM/ collagen protein/fibronectin mixture of other 1000 μ l is poured on the cell droplet it is covered fully, and the glue that congeals under 37 ℃.Finally, the gelatinous antimicrobial/antifungal medicament that contains, the D-MEM that does not contain FCS of the same concentrations of L-glutaminate and blank PBS or larva ES is added into the top layer gel coat.The detection of combination is containing 5%CO under 37 ℃ ₂Damp atmosphere in cultivate certain hour.Whole process keeps sterile state.

In whole 48 hours, each experimental session all passes through reverse Leica (UK) DM IRB microscope to be observed with differing pair cell.The low power image that shows each cell droplet in the whole zone that is contained in each gel is used to quantize the cell migration after the cultivation in 24 hours.Here, having used Microsoft Paint Shop Pro 6 that the image of each cell droplet of 0 hour is superimposed upon 24 hours cultivates on each identical cell droplet image of back.Then use Leica QUIPS software that these composographs are analyzed, as shown in Figure 4.Surround the distance at the edge of each cell droplet when at first, calculating 0 hour.After this quantity of having moved out and left the cell at initiating cell droplet edge after cultivating in 24 hours is counted.For the droplet Edge Distance of correct for variations, the quantity of migrating cell is represented with every millimeter initiating cell droplet of cell boundary edge.Each cell migration goes out the slant range at edge and also measures.

After 48 hours cultivate, complete gel with 4% Paraformaldehyde 96 (TAAB, Aldermaston UK) fix 20 minutes, afterwards with contain 1% bovine serum albumin (BSA) (

) PBS clean three times.Add with ice-cooled 20mM N-(2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) that contain; 4-(2-hydroxyethyl) piperazine-1-ethyl sulfonic acid (HEPES), 300mM sucrose, 50mM sodium-chlor, 3mM magnesium chloride and 0.5%Triton X-100 (all from

) perviousness solution (pH7.4) and kept 10 minutes.Gel cleans with 1%BSA again.The 0.08%FITC-Phalloidine (

) solution (in the ethanol) is with 1%BSA1:100 dilution, adds gel afterwards.After 30 minutes, as cleaning gel before.Add contain 10 μ g/ml iodate pyridines (PI) (

) BSA (1%), and before cleaning gel once more, kept 1 minute.

After FITC-Phalloidine/PI was painted, gel was blotted carefully to remove excess liquid and to use Bio-Rad fluorescence mounting medium and cover plate sealing.Use confocal Leica TCS4D system to add that the upright fluorescent microscope of Leica DMRBE makes the gel video picture.The maximum strength image that obtains cell droplet edge is to observe the form of the cell that moves.Also obtain the Z series optical section of gel and contrast with the optical section of after its combination (cultivating in 0 hour), fix immediately and painted independent detection is obtained.Do like this is to occur in all directions in order to confirm from the cell migration of droplet.

Statistical study

Represent the value of each every millimeter edge of cell droplet migrating cell quantity in each is handled, all to carry out repetition, and change into its square root.These then use GraphPad Prism ^TMSoftware carries out one-way analysis of variance (ANOVA) and the check of Dunnett ' s multiple comparisons.Each droplet repeated establishment during the migration slant range that cell reaches was handled by each.Then geometric mean makes up by appropriate processing classification to guarantee normal distribution by logarithm (log) conversion for they.Use GraphPad Prism ^TMMiddle available list factor ANOVA and the check of Dunnett ' s multiple comparisons are analyzed the mean value of combination.For all situations, confirm identical variance and statistical significance is defined as P≤O.05.

Three-dimensional vitro detection-inoblast form and I of matrix organization

In an independent experiment, three-dimensional vitro detection makes up by aforementioned, but small change is arranged.Here, contain 3 * 10 ⁵20 μ l droplets of the low cell density of cell/ml become the part of each detection.Detect with 1 μ g/ml ES, 5 μ g/ml ES or blank PBS (contrast) handle.Cultivate the back at 0,24,48 hour and use phase microscope observation of cell form.Also note the outward appearance of gel matrix.

Three-dimensional vitro detection-inoblast migration

Making up three-dimensional vitro detection is in order to check fibroblastic migration.And then detect after the combination (0 hour), or the confocal microscopy image of the gel after cultivating in 48 hours has confirmed, first, when 0 hour cultivates, outside the cell droplet, do not observe cell and, second, cell moves to come out from the cell droplet and not only occurs in horizontal direction but also occur in vertical direction after 48 hours, and this has confirmed the three-dimensional essence (Horobin AJ PhD thesis, 2004) of migration again.

Quantizing owing to during the cell migration of larva ES, carried out two independent experiments.An effect that has compared 0.1 μ g/ml ES and 5 μ g/ml ES to the contrast of no ES.Another has checked the effect of 1 μ g/ml ES and 10 μ g/ml ES when comparing with another contrast.In each experiment, use be from identical bottle and the cell that goes down to posterity.

Obtain image with the cell migration of check from each cell droplet.Range estimation is presented at 24 hours cultivate after, the cell migration when having 5 μ g/ml ES shows as that the most extensive (Fig. 5 is a).From morphology, what these cells were also cashed out better dispersion of cell in must those contrasts and outstanding longer greater amt extends on every side that (Fig. 6 a) in the gel.Cultivate by 48 hours, the gel that is exposed to 5 μ g/ml ES is transparent and becomes and separate with panel surface.The cell droplet shrinks, and matrix is inwardly drawn, thereby increased intramatrical tension force between the droplet.By this, the gel that is exposed to 1 μ g/ml ES shows than contrast relocation site separately farther (Fig. 5 b) and has formed the form of good distribution, and the gel (Fig. 6 b) of length around extending into.By contrast, diminished on the cell drop volumes that exists under the 10 μ g/ml ES, some regions contract of cell becomes tight black material (Fig. 5 b).This is with before gelatinous solution and be that limpid viscous liquid state connects at present.When comparing, the form that is exposed to the cell of 10 μ g/ml ES obviously need not.Here, after the cultivation in 24 hours, cell shows a lot of tiny extensions, and wherein some grows (Fig. 6 c) very much.After cultivating in 48 hours, cell has shunk, and becomes circle, but they are derived by tiny intercellulars of growing with other and keep direct physical to contact (Fig. 6 d).

Use can watch the whole low power image of each droplet to come total cell migration from each droplet is carried out quantitatively.After cultivating in 24 hours, analyze and confirmed to compare with relevant contrast, 5 μ g/ml ES (P＜0.01) on the quantity of cell migration (Fig. 7) (Fig. 8) have significantly increased the migration of cell with mobile distance last (P＜0.001).The ES of 1 μ g/ml has also significantly increased cell migration (P on P on the cell quantity＜0.01 and the move distance＜0.05) (Fig. 7,8) in its contrast.Comparatively speaking, compare 10 μ g/ml ES with its contrast and significantly suppressed cell migration (P on P on the cell quantity＜0.01 and the move distance＜0.01) (Fig. 7,8).Be exposed to 0.1 μ g/ml ES show suppressed slightly cell migration to quantity (to the contrast P＜0.05) (Fig. 7).Yet 0.1 μ g/mlES has no significant effect (P＞0.05) (Fig. 8) to migration distance compared with the control, and its pair cell form does not have (data not shown goes out) yet.Can notice the experiment 1 in the contrast cell migration quantity (Fig. 7 a) be higher than the experiment 2 in the contrast (Fig. 7 b).Difference between this experiment may be to bring a little difference of cell concn in the cell droplet owing to the mistake of the inevitable level that causes with Hematocyte Counter estimation cell quantity.Yet the variation in this potential source exists only between the experiment, and not in experiment (is the cell of same count piece because make up each detection employed in each experiment).Therefore, in each experiment, remain effective between comparative result rather than the experiment.

Three-dimensional vitro detection-inoblast form and II of matrix organization

Containing that the fibroblastic three-dimensional vitro detection of low concentration is combined is in order to check the tissue of fibroblastic form and matrix.Soon there is ES or do not exist the cell under the ES situation to look like similar (Fig. 9) after detecting combination.Yet by 24 hours cultivation, cell did not exist the cell of ES to compare with those when having 1 μ g/ml ES and particularly having 5 μ g/ml ES to have taked the more form of good distribution, had longer tenuigenin extension.When having 5 μ g/ml ES, arrange the connectivity matrix primitive fiber of good similar string between observation of cell.After cultivating in 48 hours, the difference between the detection becomes more obvious.By this, cell has become round (Fig. 9) when not having ES.Simultaneously, be exposed to the form of the cell maintenance good distribution of 1 μ g/ml ES or 5 μ g/ml ES.The connectivity matrix primitive fiber that is exposed to the similar string that the iuntercellular of the observation of 5 μ g/ml ES arranges has become more detailed and numerous.In addition, it is limpider that matrix seems, the random network of primitive fiber is so unobvious.

Comparing embodiment I

Inventor of the present invention has carried out further experiment to compare the effect of ES Quimotrase and commercially available BCTR.Therefore ES compares with the chymotrypsin protein enzyme formulations with commercially available trypsinase with the effect of fibronectin adhesion inoblast.This be by on the surface of fibronectin parcel at the ES of various concentration, the inoculation inoblast is finished under the situation that commercially available trypsinase or commercially available Quimotrase exist.After cultivation reached 48 hours, sample was sucked away All Media, and leniently cleaned, thereby removed the cell that is not attached to the surface.Use Triphosaden (ATP) to detect in order to quantize to be retained in surperficial cell relative populations according to survey ATP concentration.Contrasting ES and containing in the tryptic effect that the trypsinase specific substrate is had suitable activity level, ES adheres to the surface more effectively to being reduced to fibrocyte.For example, after cultivating in 24 hours, 5 μ g/ml ES with cytoadherence be reduced to contrast 16.6% (Figure 10 a).Commercially available has the active trypsin 1.6 μ g/ml of 103.2%ES to trypsinase characteristic fluorogenic substrate Tosyl-Gly-Pro-Arg-AMC) cytoadherence is reduced to 34.9% of contrast.After cultivating in 48 hours, the cytoadherence in the presence of 5 μ g/ml ES has been reduced to 9.3% of contrast, and the commercially available trypsinase of 1.6 μ g/ml has reduced to cytoadherence 24.7% of contrast.

Commercially available Quimotrase (0.1 μ g/ml), it shows 94.1% 5 μ g/ml ES activity to Quimotrase specificity fluorescent substrate (Suc-Ala-Ala-Pro-Phe-AMC), does not show the ability (Figure 10 b) that changes over the fibrocyte adhesion.Even the commercially available Quimotrase of 0.2 μ g/ml, it shows 204.9% 5 μ g/ml ES activity to the Quimotrase specific substrate, and the inoblast adhesion is not had effect.The inoblast that is exposed to commercially available trypsinase and commercially available Quimotrase does not simultaneously show any change greater than the adhesion that only is exposed to the commercially available tryptic cell of same concentrations.

These results show the enzyme that is present in for example Quimotrase among the ES than independent commercially available trypsinase or commercially available Quimotrase or its be combined in change in the fibrocyte adhesion more effective.Research has before shown that ability that ES changes over the fibrocyte adhesion is and it changes the ability relevant (Horobin et al 2003 is the same) on the surface of fibronectin parcel by proteolytic degradation.These results show that ES may be than commercially available trypsinase or commercially available Quimotrase more effective aspect the degraded extracellular matrix protein so.This confirms with gel electrophoresis.Here, dilution larva ES, commercially available trypsinase, commercially available Quimotrase or commercially available trypsinase have trypsinase specificity or the identical active solution of Quimotrase specific substrate with acquisition with commercially available Quimotrase bonded sample.Ox fibronectin (100 μ g/ml I) was exposed to these solution 24 hours under 37 ℃ before being dissolved in 12% polyacrylamide gel.As directed, when comparing with two kinds of commercially available enzymes, ES is degraded to fibronectin main littler fragment (Figure 11) more widely.In addition, being present in proteolytic ferment among the ES has shown and has increased fibroblastic migration (Horobin et al 2005 is the same).This can connect with the variation of inoblast adhesion.In the wound, the acceleration of inoblast migration can promote granulation tissue to grow into the wound gap.Like this, the enzyme that is derived from ES may not only prove those the more effective debridement agents than in the market, also may increase the wound healing response simultaneously.

Embodiment 2-debridement

The purifying of maggot Quimotrase

The Sephacryl S300 chromatography of lucilia sericata ES product

With Sephacryl S-300HR filling glass chromatography column (1.5cm * 50cm) and with PBS balance (flow velocity 0.33ml/min).This post is calibrated and uses widely the gel-filtration standard substance, and (200-12.5kDa Sigma) determines void column.Approximately 2ml lucilia sericata ES product (0.5mg) joins in the post, then collect the eluant of 50 parts of void columns (2ml/ part), and use fluorogenic substrate Suc-Ala-Ala-Pro-Phe-AMC and Tosyl-Gly-Pro-Arg-AMC to detect Quimotrase and tryptic activity respectively.See Figure 14 and 15, but Quimotrase/tryptic activity after its demonstration S300 gel-filtration.Observe typical 3 proteolytic activity peaks, for two of trypsinase enrichments (being called T1 and T2) and for 1 of chymotrypsin activity enrichment (being called C1).Figure 15 has also shown typical each proteolysis curve by the active peak of gel SDS-PAGE substrate gel analysis mensuration.

Use of the detection of the synthetic peptide substrates of fluorescence to Quimotrase/tryptic activity

Quimotrase/tryptic activity is estimated by 7-amino-4-methylcoumarin (AMC) that monitoring discharges from Suc-Ala-Ala-Pro-Phe-AMC (Quimotrase) and Tosyl-Gly-Pro-Arg-AMC (trypsinase).The substrate that every part 50 μ l dilute in PBS with 150 μ l (5 μ M) is cultivated.Sample was cultivated 30 minutes down at 37 ℃, used Dynex MFX microplate photofluorometer to measure its fluorescence (365nm excites, and 465nm launches detection) afterwards.Emission surpassed 30 minutes unit and fluorescence unit quantitaes after proteolytic activity was used in and deducts the time zero reading.

Use gel substrate SDS-PAGE to detect Quimotrase/tryptic activity

Use Kumar ﹠amp; The method that Pritchard (1992) describes is carried out substrate SDS-PAGE.Preparation comprises 0.1% (w/v) gelatin and is dissolved in interior 12% (w/v) SDS-PAGE glue of glue.This glue is heated to 55 ℃ and dissolves gelatin.Every part the 10 μ l and the irreducibility sample buffer of equal volume (0.5M Tris, pH6.8,5%SDS (w/v), 20% glycerol (w/v), 0.01% tetrabromophenol sulfonphthalein) were cultivated 30 minutes under mixing and being incorporated in 37 ℃.These parts then are placed in the independent hole that is formed on the level gel, and sample is electrophoresis under constant current 20mA.Behind the electrophoresis, glue cleans under the room temperature in 2.5%Triton X-100 and made the enzyme renaturation in 20 minutes, as Lacks ﹠amp; Springhorn (1980) is described.This is glueing joint in water and to clean 20 minutes, at last under 37 ℃ in PBS overnight incubation.Proteolytic activity is by measuring with coomassie brilliant blue R250 dyeing glue, and observed is white space on blue background.

By Trypsin inhibitor SBTI agarose affinity chromatography purifying Quimotrase

The salt concn that the C1 of chymotrypsin activity merges thing is adjusted to 0.5M sodium-chlor and is used for PBS, the 3ml Trypsin inhibitor SBTI agarose column of 0.5M sodium-chlor balance (flow velocity 0.2ml/min).This post PBS, 0.5M sodium-chlor clean up to the absorption of damping fluid at the 280nm place by this post and reach zero.Conjugated protein usefulness 0.7% thanomin elution, 1ml partly is collected, and use at once 2M Tris.Cl (1: 1v/v) neutralization, concentrate and to PBS dialysed overnight (Figure 16).Use fluorogenic substrate and gelatin SDS-PAGE to detect the proteic proteolytic activity (Figure 17) of elution.

Figure 17 has showed proteic hydrolytic activity and the inhibitor properties that is attached to the Trypsin inhibitor SBTI agarose after using C1 to merge thing.From the protein cleavage substrate Suc-Ala-Ala-Pro-Phe-Arg-AMC of Trypsin inhibitor SBTI agarose column elution and only show and suppressed by PMSF.Do not see trypsinase substrate Tosyl-Gly-Pro-Arg-AMC is had activity.

When analyzing, observe at least 3 proteolytic activities significantly zone (the mark arrow) with gelatin substrate SDS-PAGE.

By Trypsin inhibitor SBTI agarose affinity chromatography purification of trypsin

Similar, the T2 of trypsinase part merges logistics and crosses the Trypsin inhibitor SBTI post.The part of elution is neutralized immediately from the post, concentrates and to PBS dialysed overnight (Figure 19).

Figure 19 has showed proteic hydrolytic activity and the inhibitor properties that is attached to the Trypsin inhibitor SBTI agarose after using T2 to merge thing.From the protein cleavage trypsinase substrate Tosyl-Gly-Pro-Arg-AMC of Trypsin inhibitor SBTI agarose column elution and show by PMSF and APMSF and suppress.Do not see chymotrypsin protein enzyme substrates Suc-Ala-Ala-Pro-Phe-Arg-AMC is had activity.When analyzing, observe 1 proteolytic activity significantly with (the mark arrow) by gelatin substrate SDS-PAGE.

The ES product is to the effect of wound eschar

About 1mg wound eschar and 10 μ g lucilia sericata ES product overnight incubation.Reprocessed and the untreated eschar of cultivation be dissolved in Biorad etc. electricity (iso-electric) focus on rehydrated (focusing re-hydration) damping fluid (8M urea, 2%CHAPS 50mMDTT, 0.2% amphotericeledrolyte).Use Biorad2D albumen " cleaning " test kit to be further purified albumen, the particle of acquisition is dissolved in the rehydrated damping fluid of isoelectrofocusing again.

The ES product is tested with the 2D gel analysis to the effect of wound eschar.Explanation use ' ReadyStrip ' 7cm IPG strips pH3-10 (Biorad) according to service manual focuses on the albumen of about 100 μ g in first dimension.The second dimension SDS-PAGE carries out (Schagger and Vonjagow, 1987) with 10%tricene gel.Gel Coomassie blue R250 dyeing Figure 20.

Quimotrase/trypsinase merges the effect of thing to the wound eschar

Approximately 1mg wound eschar and 100 μ l lucilia sericata C1, T1 or T2 merge the thing overnight incubation.After the cultivation, that handled handles as mentioned above with untreated eschar.

C1, T1 or T2 merge thing the effect of wound eschar are then tested with the 2D gel analysis.About as mentioned above 100 μ g albumen focus on 2 dimensions.Gel Coomassie blue R250 dyeing Figure 21.

The clone of lucilia sericata Quimotrase gene

The total length of the open reading frame of lucilia sericata chymotrypsinogen (ORF) increases by PCR, uses the forward primer add the N/col restriction site (5 '-CTGCCATGGTCATGAAATTCTTAATAGTT-S ') and adds the reverse primer (5 '-GACGCTAGCATAAGAAATTCCGGTGTG-3 ') in Nhe site.The fragment that obtains is cloned the downstream of pBac-3 polyhedrin promotor into and order-checking (Figure 12) and has been predicted aminoacid sequence (Figure 13).

Therefore can reach a conclusion, the ES of lucilia sericata comprises the Quimotrase with amino acid shown in this article and dna sequence dna, it passes through to promote the inoblast migration, reinvents and debridement wound and promotion wound healing by changing over fibrocellular form by promoting matrix.

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Claims (25)

1. isolating Quimotrase or its synthesized form that is derived from insect larvae.

2. the Quimotrase of claim 1, wherein this Quimotrase have among Fig. 1 or Figure 13 sequence or by the dna sequence encoding among Fig. 1,2 or 12.

3. the fragment of a Quimotrase, it has the activity identical with the Quimotrase of claim 1 or claim 2.

4. each Quimotrase in the claim 1 to 3, wherein this Quimotrase is obtained from the larva of lucilia sericata.

5. each Quimotrase in the aforementioned claim, wherein this Quimotrase is from the drainage/secretory product of lucilia sericata larvae.

6. each Quimotrase in the aforementioned claim, wherein larva is new hatching.

7. each Quimotrase in the claim 1 to 5, wherein larva is an age.

8. each Quimotrase in the aforementioned claim, wherein larval growth is in gnotobasis.

9. composition that is used for the treatment of wound comprises in the aforementioned claim each Quimotrase.

10. each Quimotrase is used for the treatment of purposes in the medicine of wound in preparation in the claim 1 to 8.

11. the purposes of claim 9 or 10, wherein Quimotrase have among Fig. 1, Fig. 2 or Figure 13 sequence or by the dna sequence encoding among Fig. 1,2 or 12.

12. each purposes in the claim 9 to 11 is wherein used the active fragments of Quimotrase.

13. each purposes in the claim 9 to 12, wherein Quimotrase is a synthetic.

14. each purposes in the claim 9 to 13, wherein Quimotrase is derived from lucilia sericata.

15. each purposes in the claim 9 to 14, wherein wound be selected from incised wound, stab, operative incision, ulcer, pressure sore, burn, the corium scratch or wound, osteomyelitis and plastic surgery wound, described burning comprises because hot, freezing, chemical, electricity and radiation-induced burning.

16. each purposes in the claim 9 to 15, wherein wound infects.

17. each purposes in the claim 9 to 16, wherein wound is chronic.

18. each purposes in the claim 9 to 17, wherein wound is treated by promoting the inoblast migration.

19. each purposes in the claim 9 to 17, wherein wound is treated by promoting matrix to reinvent.

20. each purposes in the claim 9 to 17, wherein wound is treated by changing over fibrocellular form.

21. each purposes in the claim 9 to 20, wherein wound Quimotrase debridement.

22. a dressing that is used for wound, this dressing contain in the claim 1 to 8 each Quimotrase.

23. a pharmaceutical composition, it comprises in the claim 1 to 8 each Quimotrase.

24. a method for the treatment of wound, this method comprise the Quimotrase of the wound of needs treatments being used in the claim 1 to 8 each.

25. a method for the treatment of wound, this method comprise the step of the wound of needs treatments being used the pharmaceutical composition of the composition of dressing, claim 9 of claim 22 or claim 23.

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