CN1178552A - New difined enzyme mixture for obtaining cells and treating wounds - Google Patents
New difined enzyme mixture for obtaining cells and treating wounds Download PDFInfo
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- CN1178552A CN1178552A CN 96192616 CN96192616A CN1178552A CN 1178552 A CN1178552 A CN 1178552A CN 96192616 CN96192616 CN 96192616 CN 96192616 A CN96192616 A CN 96192616A CN 1178552 A CN1178552 A CN 1178552A
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Abstract
The invention relates to the use of mixtures of defined composition of purified enzymes from Clostridium histolyticum for obtaining in a reproducible, standardized manner, from cells or tissue fragments of human or animal tissues, and to these enzymes and mixtures thereof; in addition it relates to the direct or indirect medical use of these enzymes, alone or as ingredient of mixtures, eg. in wound treatment.
Description
The present invention relates to from clostridium histolyticum (Clostridium histolyticum) having of the enzyme of purifying determine the mixture formed with a kind of repeatably, standardized mode obtains the purposes of cell or tissue fragment from human or animal tissues, and the enzyme of these purifications is used for the application of wound processing.
The method of isolated cell should be repeatably from tissue, and should guarantee injury minimum that the cell that is obtained is caused.Usually, be used to this purpose from the preparation that contains collagenase with uncertain composition of clostridium histolyticum, but above-mentioned target can not be finished reliably with this.Using other zymin or non-enzyme method then is rare relatively poor separating resulting (1), (9) that maybe can only provide.
The preparation that contains collagenase (2) normally used or recommended use is to obtain from the filtrate of the nutrient solution of clostridium histolyticum, except containing multiple collagenase and proteolytic enzyme (3a), (3b), it also contains the split product of these enzymes that form owing to proteolyzing and other composition, and the some of them composition is deleterious and does not know that it is what.
According to the knowledge of present stage, the viable cell that obtains high yield with a kind of single enzyme from clostridium histolyticum is impossible.On the contrary, decompose in order to make to organize effectively, need be from the acting in conjunction of the various enzymes of this bacterium.Yet for the ratio that reaches the required enzyme amount of this purpose also is not disclosed up to now, the user also can not get the definite mixture from the enzyme of clostridium histolyticum.
Based on the problems referred to above, many research organizations efforts be made so that with the enzyme of purifying isolated cell from tissue.Yet these study all fail to cause using pure enzyme or any completely specified mixture.
People such as Suggs (4) have used a kind of mixture separation people's venous endothelial cell, this mixture is made up of the collagenase fraction of having purified and the trypsinase from Pancreas Bovis seu Bubali of having purified.Yet, employed enrichment the collagenase fraction do not have the composition of determining of various enzymes, it also contains a spot of material such as clostripain, and this separating resulting does not have tangible difference with using the resulting result of the preparation that contains collagenase with uncertain composition.The one-component that uses said mixture be impossible obtain gratifying organize dissociated.Yet using trypsinase to be used for cellular segregation neither be no problem, because this proteolytic ferment destroys the protein of cytolemma.Thereby for instance, it has detrimental effect to the Regular Insulin that is connected on liver film and the adipocyte.
The mixture that the collagenase fraction of purifying is used in Hefley (6), (7) separates osteocyte from the skull of mouse.(7) before this, this author has reported the possibility of using the pure collagenase fraction of putting forward and the mixture of neutral protease successfully to separate these cells.Yet, in above-mentioned two researchs, all used elutriant from separator column, wherein the content of the different various collagenase content of Substratspezifitaet and other composition all is unknown.
People such as Wolters (8) use neutral protease and the mixture of " the VII Collagen Type VI enzyme " that provided by Sigma comes detached island cell in the pancreas of rat, although and do not know the kind and the quantity of the various collagenases that contained in this mixture.In addition, it also contains a spot of clostripain and " nonspecific protease ".Neutral protease is used with a kind of highly purified form.The mixture of above-mentioned two kinds of components has produced the live body island cell of a greater number.
The present invention relates to these independent enzymes of highly purified HP collagenase, AZ collagenase and elastoser, also relate to their mixture.
In with the analysis of synthetic six peptide Z-Gly-Pro-Gly-Gly-Pro-Ala as the Gra β mann of substrate and Nordwig (11), the ratio work of HP collagenase is 20U/mg at least, is preferably 50U/mg at least.If be used for medical purpose, it is 100U/mg or higher than work preferably.
In using the analysis of A Zuokao (Azokoll) as the people such as Mandl (12) of substrate, the ratio work of AZ collagenase is 10U/mg at least, is preferably 30U/mg at least.If be used for medical purpose, it is 50U/mg or higher than work preferably.
Be in the analysis that substrate was carried out with the elastin from the ox ligament of neck, the ratio work of elastoser is 2U/mg at least, is preferably 5U/mg at least.If be used for medical purpose, it is 12U/mg or higher than work preferably.
The invention still further relates to the mixture that uses a kind of HP collagenase and elastoser, add or do not add AZ collagenase and/or clostripain, in order to isolated cell from human or animal tissues or fragment of tissue.
The invention still further relates to these enzymes separately or as the direct or indirect application of the component of mixture, be used for medical use, for example be used for wound and handle.
Dissociate in order to be used for tissue, for instance, mixture can be packed in the bottle with freeze-dried, amount wherein is enough to make the liver (weight in wet base of this organ is approximately 9-11g) of a rat to dissociate.
Suitable mixture contains two kinds in the enzyme of following purification at least: HP collagenase (50-300U; Be preferably 70-170U) and elastoser (5-70U; Be preferably 10-25U), add or do not add AZ collagenase (1-20U, preferably 2-8U) and/or clostripain (10-280U, preferably 20-50U).Above-mentioned numeral shows is in the bottle in a unit-for example-the amount that mixture had.
Judging criterion to the purity of employed enzyme is that their ratio is lived under every kind of situation, shows its homogeneity (sds gel electrophoresis, isoelectrofocusing of carrying out on sepharose and electrophoresis) in being normally used for the electrophoresis method of this purpose.The ratio of the enzyme behind purifying value alive is high more than 100 times compared with the ratio of beginning material value alive.
Have following ratio purifying enzyme alive and be used to prepare mixture: than work is the HP collagenase of 20U/mg at least, is the elastoser of 2U/mg at least, is the AZ collagenase of 10U/mg at least, is the clostripain of 10U/mg at least.
Since this class by the mixture of determining to form have can synergistic collagen lytic enzyme, elastolytic enzyme (sic) and proteolytic ferment, they are specially adapted to from the humans and animals tissue with no damage and isolated cell or fragment of tissue effectively.
Because employed preparation is all from the enzyme behind the purifying of known its character in each case, just no longer necessary to the time-consuming and expensive detection of each batch, this is quite favourable.This has saved the work that need carry out for the Function Identification of cell, or workload has been diminished.Because above-mentioned, zooperal quantity can reduce in a lot of fields.
Use the enzyme that does not pass through fine purifying; or use to have and hang down than the enzyme of living, can cause the output of cell in above-mentioned application lower usually, but also can produce common problem: separating resulting lacks repeatability; the consistence that lacks batch may have the existence of the unknown component of disadvantageous effect.
A lot of experimental results of reporting in the document are difficult to make explanations, and reason is the cracking that the proteolytic enzyme of association in preparation, purification or qualification process has caused enzyme.The contribution that each single enzyme has been done experimental result in cellular segregation also is difficult to explain, reason is the enzymic activity that the fragment that produced by collagenase, elastoser and proteolytic enzyme has in some cases kept them.Yet, the degree that substrate specificity sexually revises is not made explanations.In addition, many commodity preparation of containing collagenase only contains the split product of initial collagenase sometimes.Therefore, in the prior art, the producer of preparation or the user of cell isolation method also can not clearly make the standard preparationization that contains collagenase.
Because the mixture of the enzyme of purifying has actively widely when cracking is organized among the present invention, they are applicable to the tissue and the cell of all humans and animals, preferred fragment of tissue and/or cell from:
Bile duct, blood system, body of gland, vascular system, brain, skin, heart, intestines, pancreas islet, liver, lung, stomach, spleen, muscle, umbilical cord, nerve, kidney, pancreas, spinal cord, Tiroidina, terminal ileum, tumor tissues, uterus, digestive tube and tongue.
Use the cell or tissue fragment of described mixture separation to be highly suitable for the transplanting of cell and tissue and to be used for gene therapy (for example pancreas islet, island cell, liver cell, tumour cell, adipocyte), immunotherapy or wound healing.
A crucial purposes of mixture of the present invention is isolating hepatocytes, island cell, endotheliocyte, epithelial cell, adipocyte, ovocyte and tumour cell.Stdn and improvement to aforesaid method in these Application Areass are very favourable.
For example, a kind of mixture that contains two kinds of different collagenases of substrate specificity and a kind of elastoser with definite composition is proved and very is suitable for isolating hepatocytes from rat liver (using embodiment A, D, E) and people's liver (use embodiment F), from rat liver, separate bile duct epithelial cell (using embodiment G), from human umbilical cord, separate endotheliocyte (using embodiment [sic] H), separating tumor cell from people's tumour (use example I) and detached island cell (using embodiment J) from Pancreas Sus domestica.
Compare with the best zymin that contains collagenase with uncertain composition, the separating effect of mixture of the present invention better or at least is equally good.
Mixture of the present invention can be used for substituting all employed preparations that contain collagenase usually, makes the tissue necessary various composition that dissociates because they contain.This has just been avoided the present employed shortcoming that contains the preparation of collagenase, and one of them reason is that mixture of the present invention has consistent composition.
An important applied field of said mixture be with a kind of repeatably, standardized mode, obtain liver cell according to Berry and Friend (9), (10) received method from liver, use therein up to now is the zymin that contains collagenase of not determining composition; And pass through to use the mixture of prior art from the pancreas tissue, to obtain complete island cell better.
Another main Application Areas is in wound healing.Wherein the purity of employed enzyme is high more good more for this.
I. from the preparation and the character of the enzyme of clostridium histolyticum
1.HP collagenase
A. preparation
Use ammonium sulfate precipitation protein
All operations in the enzyme purification process all carries out under 4-8 ℃.
The thick collagenase of 200g is dissolved in the 3l water, then the pulverous ammonium sulfate of 680g is joined in this solution.With centrifugal method protein precipitation A1 is removed; Again 420g ammonium sulfate is added in the supernatant liquor.With centrifugal method current sedimentary protein fraction A2 is separated, this precipitation is dissolved in the 1l water and in water dialyses.(exclusion is limited to 10,000Da) it is concentrated into about 100ml by ultra-filtration membrane then.Then with this concentrated solution freeze-drying.Resulting Powdered protein fraction A2 mainly contains HP collagenase and AZ collagenase.
By metal-chelating affinity chromatogram HP collagenase and AZ collagenase are carried out chromatographic separation
With load Zn is arranged
2+Chelating type agarose 6B HP collagenase and AZ collagenase are carried out chromatographic separation.The concrete practice is that this material is added separator column (in 5 * 100cm), highly is 70cm, with initial damping fluid (500mM sodium-acetate+20mM calcium acetate, pH8.0) balance.With TRIS (three (methylol) aminomethane) pH of initial damping fluid is transferred to 8.4, protein fraction A2 that then will about 1.2g is dissolved in this damping fluid of 15ml and joins on the separator column, washs from separator column with the various compositions of initial damping fluid with protein fraction A2.In the wash-out of using buffer A (500mM sodium-acetate+20mM calcium acetate transfers to 6.3 with acetic acid with pH) to carry out subsequently, what at first collect in separating fraction is the AZ collagenase, is the HP collagenase then.
With ultra-filtration membrane (exclusion is limited to 10,000 Da) HP collagenase and these two kinds of isolating fractions of AZ collagenase are concentrated into 50-100ml respectively, in water, dialyse then, use ultra-filtration membrane (exclusion is limited to 10,000 Da) that they are concentrated into about 10ml again.
The final purification of HP collagenase
The final purification of HP collagenase uses a Pharmacia FPLC equipment, and (HR 10/10, carries out on Pharmacia) at a Mono Q anion exchanger.The concrete practice is that the mixture that 1ml buffer B and 2ml contain from the fraction that contains the HP collagenase of above-mentioned chromatrographic separation step is joined the top of using buffer B (20 mM TRIS/HCl pH7.5) equilibrated separator column.
After with the buffer B washing, (20m M TRIS/HCl+20mM NaCl is pH7.5) with form from the separator column wash-out of HP collagenase with purifying with damping fluid C.
B. character
Why the HP collagenase is named as " HP " is because its cracking six peptide Z-Gly-Pro-Gly-Gly-Pro-Ala very effectively.Therefore it is suitable for the specific detection to vigor.The characteristic of HP collagenase is the collagen that it can only transform inactivation to a small extent, for example gelatin or A Zuokao.Yet it but destroys ox flesh key collagen and makes the synthetic peptide cracking of mentioning among the embodiment 2b with very high transformation efficiency, and the position is between glycine and the glycine or between any amino acid and the glycine.
Should benly be the HP collagenase has the super effect that adds with AZ collagenase (vice versa, promptly any mixture that contains HP and AZ collagenase at least) to the cracking of natural collagen.When being used for tissue and dissociating, external synergistic effect also is crucial (for example Application Example G).
In the analysis of using synthetic substrate Z-Gly-Pro-Gly-Gly-Pro-Ala to carry out, the height ratio work of HP collagenase is 146 U/mg (11).Compare with initial substance, the ratio vigor of enzyme has correspondingly improved about 100 times.
All only form bands of a spectrum in the isoelectrofocusing in sds gel electrophoresis and on sepharose, carried out of the HP collagenase of purifying and the electrophoresis by this way.
Measuring its molecular weight by sds gel electrophoresis is 106,000 Da.Its iso-electric point is pH5.8-6.0.
2.AZ collagenase
A. preparation
All operations all carries out under 4-8 ℃ in the enzyme purification process.First two steps purge process (classification protein precipitation and metal-chelating affinity chromatogram) to the AZ collagenase is described in 1.
The final purification of AZ collagenase
The final purification of AZ collagenase is that (HR 10/10, carries out on Pharmacia) at Mono Q anion exchanger down Pharmacia FPLC equipment auxiliary.The concrete practice be with 1ml damping fluid D (the 20mM calcium acetate, pH7.2) and the 1ml mixture that contains the fraction (from above-mentioned metal-chelating affinity chromatogram) of AZ collagenase join the top of using damping fluid D equilibrated separator column.
After with damping fluid D washing, (the 20mM calcium acetate is pH5.0) with AZ collagenase wash-out from separator column with damping fluid E.
B. character
Why the AZ collagenase is named as " AZ " is because its cracking substrate A Zuokao very effectively.Its characteristic is the collagen that can transform inactivation effectively, and such as gelatin or A Zuokao, but it also can transform ox flesh key collagen.Yet it can not destroy the synthetic peptide of short chain, such as 2-furans acryloyl-Leu-Gly-Pro-Ala, 4-phenylazo-benzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and Z-Gly-Pro-Gly-Gly-Pro-Ala.
In the analysis of use A Zuokao as substrate by people such as Mandl (12) exploitation, the height ratio work of AZ collagenase is 82U/mg.Compare with initial substance, the ratio vigor of enzyme has correspondingly improved about 80 times.
All only form bands of a spectrum in the isoelectrofocusing in sds gel electrophoresis and on sepharose, carried out of the AZ collagenase of purifying and the electrophoresis by this way.
Measuring its molecular weight by sds gel electrophoresis is 111,000 Da.Its iso-electric point is pH 5.9-6.1.
3. elastoser
A. preparation
All operations in the enzyme purification process all carries out under 4-8 ℃.
The first step of elastin enzyme purification is according to the method ammonium sulfate precipitated protein matter that is described among the 1.a.
Protein precipitation A1 is dissolved in the 1l water, it is concentrated into about 170ml and in the calcium acetate solution of 0.1mM, dialyses by ultra-filtration membrane (exclusion is limited to 10,000 Da).By ultra-filtration membrane (exclusion is limited to 10,000 Da) this enzyme solution is concentrated into about 50ml once more subsequently, then with its freeze-drying.
Last purifying carries out on the gel chromatography of SEPHADEX G100 or G200 (Pharmacia).
B. character
The character of elastoser is that it can be with high transformation efficiency cracking elastin.It is 18U/mg than work (referring to 3.C.).Compare with initial substance, the ratio vigor of enzyme has correspondingly improved about 80 times.
All only form bands of a spectrum in the isoelectrofocusing in sds gel electrophoresis and on sepharose, carried out of the elastoser of purifying and the electrophoresis by this way.
The molecular weight of being measured elastoser by sds gel electrophoresis is 35,000 Da.
C. vitality test
The mensuration of enzyme activity uses elastin as substrate.
The elastin of the ox ligament of neck that 20mg is pulverizing be dissolved in 0.4ml damping fluid (50mM TRIS/HCl+10 mM calcium acetate, pH7.2) in, in 37 ℃ water-soluble, be incubated jolting in advance 5 minutes.The elastoser that is dissolved in the 0.1ml damping fluid by adding makes the reaction beginning then, and continues in 37 ℃ of following joltings 10 minutes (amplitude 25mm, 150 rev/mins of speed).The frozen water that adds 3.5ml is then removed unreacted elastin at once in a strainer.The extinction value E of filtrate is measuring on spectrophotometer under the 280nm wavelength.Experimentize with identical method, different is has only and just adds the elastin enzyme solution after elastin removed in mixture, with the extinction value E that obtains like this
BAs blank.Use the same method then and under 280nm, measure the extinction value E of this filtrate
B
The vigor of elastoser is represented with unit (U).1U is defined in the enzyme activity of per minute dissolving 1mg elastin under the given experiment condition.The meltage of elastin is to determine by measuring from the extinction value of filtrate under 280nm of tested mixture.
Calculate than living with following formula:
ΔE＝E-EB〔sic〕
F＝1.376
Factor F determines with the following method: the use elastoser is dissolved the elastin of 10mg specific lot number fully, measures corresponding dullness difference DELTA E.The milligram number that factor F and dullness difference DELTA E are multiplied each other and just obtained institute's dissolved elastin in the tested mixture of every ml.
4. clostripain
A. preparation
Separate clostripain with Ullmann with the method that Jakubke (14) describes.
As follows than the measuring method of living to this enzyme: using 1 of 2mM, 4-dithioerythritol solution activates 3 hours in advance with clostripain solution and uses synthetic substrate α-N-benzoyl-L-arginine ethyl ester (=BAEE) (15) later on.It is approximately 83U/mg than living.
B. character
The characteristic of thiol proteinase clostripain is that it can cut in the arginic back of amino acid L-in polypeptide chain He in the synthetic substrate specifically.
With its molecular weight of SDS cataphoretic determination is 55,000Da.
II. Application Example
What in the following embodiments, A-J showed is the particularly suitable of some mixtures of purifying enzyme when isolated cell and fragment of tissue from animal and human's tissue.What embodiment K and L showed is the suitability of purifying enzyme when being used for wound healing.
The present invention is not limited by these Application Examples.Embodiment A
Use mixture isolating hepatocytes from liver of three kinds of enzymes
Use Berry and the standard method of Friend (9) and the modification method isolating hepatocytes of Seglen (6).Make the Wistar rat anesthesia by i.p. injection vetanarcol (35mg Sodital/kg body weight).After opening the abdominal cavity sleeve pipe is inserted portal vein, open postcava after, under the constant hydrostaticpressure (12cm water column, variable flow), be 7.4 ± 0.05 the solution that is filled with carbogenes with following pH 36-36.8 ℃ of input down: perfusion:
1. 5 minutes about 100ml do not contain Ca
2+The cell damping fluid
2. 5 minutes about 100ml do not contain Ca
2+, contain the cell buffering of EGTA (0.42mM)
Liquid
3. 8 minutes about 200ml do not contain Ca
2+The cell damping fluid
4. the cell damping fluid of 5-30 minute about 100-600ml, wherein be dissolved with purification after
The freeze dried mixture of enzyme.Cell damping fluid: 120.0 mM NaCl, 1.29 mM CaCl
25.50mM D (+)-glucose 1.19 mM KH
2PO
44.81mM KCl 1.20 mM MgSO
415.0mM NaHCO
310.0 mM HEPES carbogenes: contain 95%O
2And 5%CO
2(V/V) gaseous mixture.
Enzyme mixture is the HP collagenase of 130U, the AZ collagenase of 5U and the lyophilized products of 21U elastoser, and it is dissolved in the cell damping fluid of 87.5ml.
After the liver organization deliquescing, just stop perfusion.After having divested the fibrous capsule of liver, liver cell is shaken off down from liver organization and filter with a screen cloth (mesh width 100 μ m).After the filtration, in the environment of carbogenes with cell jolting 20 minutes in 37 ℃ of following water-baths.In the cell damping fluid, carry out three times 2 minutes centrifugal with 50 times universal gravity constant to collect complete liver cell.Discard at each supernatant liquor that will mainly contain dead cell after centrifugal.The liver cell that lives, liver cell to or the content of a plurality of liver cell aggregates in a Burker nucleonics, determine (with the trypan blue staining of 0.08% concentration, 2 minutes) in back that resulting cell mass is being suspended once more by microscope.The result
Use above-mentioned enzyme mixture to carry out a series of experiment (n=4), resulting cell suspending liquid on average contains 88.7% viable cell, and the cell yield of each liver is 360 * 10
6Individual.
The preparation that use has the uncertain composition that contains collagenase of very high specificity hydrolytic collagen vigor compares experiment, and the result has caused serious primary cellular defect and 72.5% viable cell ratio (n=4) only.Embodiment B
Use mixture isolating hepatocytes from liver of two kinds of enzymes
Implementation method is similar to embodiment A, but uses the HP collagenase of following enzyme mixture: 60U and the elastoser of 3U.On average contain 90.5% viable cell in the resulting cell suspending liquid, cell yield is 263 * 10
6Individual (n=2).Embodiment C
Use mixture isolating hepatocytes from liver of four kinds of enzymes
Implementation method is similar to embodiment A, but uses the HP collagenase of following enzyme mixture: 30U, the AZ collagenase of 5U, the elastoser of 3U and the clostripain of 16U.
Resulting cell suspending liquid on average contains 83% viable cell, and cell yield is 232 * 10
6Individual (n=2).Embodiment D
By in four different laboratories, with the result's of mixture isolating hepatocytes from rat liver of the three kinds of enzymes of usefulness that show by the comparison of the uncertain preparation of forming that contains collagenase validity widely
The method of cellular segregation has small difference between different laboratories, but this possibility of result for isolating hepatocytes is important.In order to check the effect of the mixture of mentioning in the embodiment A, in four different laboratories, carried out a large amount of liver cell separating experiments, they all independently use method separately.
Employed enzyme mixture is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in the cell damping fluid of 87.5ml (same embodiment A).
Use following five kinds of parameters that hepatocellular separating resulting is measured: 1. the content of microscopic examination alive liver cell (determining the viable cell % number in all liver cells in the resulting cell suspending liquid) with the trypan blue staining exclusive method, 2. determine isolating all hepatocellular quantity, 3. determine isolating all hepatocellular quantity in every gram animal weight, 4. (cell of all aggregated forms is the % number of benchmark to the content of definite individual cells from resulting cell suspending liquid, promptly compare with two or more accumulative liver cells), 5. set up successfully the dabbling arrangement of time of the required collagenase solution of disintegrated tissue.
In these detect, to compare with traditional preparation, enzyme mixture of the present invention has shown more repeatable result.Embodiment E
Use mixture isolating hepatocytes from rat liver of three kinds of enzymes: the keeping of cell function
In order to prove that mixture of the present invention not only is suitable for obtaining simple result when the isolating hepatocytes but also the function of having considered cell especially, use a kind of preparation that contains collagenase on rat liver, to carry out the comparison of cellular segregation, the composition that said preparation is specially adapted to above-mentioned purpose and does not have to determine.
Use Berry and the standard method of Friend (9) and the modification method of Seglen (16) to come isolating hepatocytes.
Employed enzyme mixture is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in be used for from the cell damping fluid of rat liver isolating hepatocytes (the same embodiment A) of 87.5ml.
Determine the simple separation result of rat hepatocytes by following parameter: with the content of the definite viable cell of trypan blue staining exclusive method, the cell yield of every gram liver, the content of individual cells (is unit with %).
With the following hepatocyte function (21 that relates to; 22) parameter is further determined the quality of resulting cell suspending liquid: ATP content; power consumption (EC); the lidocaine metabolism is an ethyl glycine xylidine (MEGX); and in the substrate of 21 μ M concentration to bile taurine ((3 α; 7 α, 12 α-trihydroxy--5 β-cholane-24-acyl group)-2-amino-ethyl sulfonic acid) absorption.
In any parameter of being investigated, all do not demonstrate these two kinds of significant differences that contain the preparation of collagenase.
No matter according to the evaluation of isolating simple result still according to different cell function-as the special function of differential matter transportation, the special metabolic activity of the perhaps hepatocellular enzyme relevant with Cytochrome P450-evaluation, can demonstrate with the isolating liver cell of enzyme mixture of the present invention and not suffer damage fully.Embodiment F
Use mixture isolating hepatocytes from people's liver of three kinds of enzymes
Use a kind of viviperfuse technology (21) to come isolating hepatocytes according to the method for Berry and Friend (9) and the modification method (16) of Seglen (16).
Employed enzyme mixture is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in the cell damping fluid of 50ml.
Determine the separating resulting of human liver cell with following parameters: with the viable cell content of trypan blue staining exclusive method detection and the cell yield of every gram liver.
The result shows also can be with constant result isolating hepatocytes (content of viable cell is 85-90%) successfully from people's liver with mixture of the present invention.Embodiment G
From rat liver, separate the courage epithelial cell
In a series of experiment (n=4), from rat liver, separate the courage epithelial cell according to the method that is described in the document (18).This requirement is at first removed liver cell with enzyme process from tissue collecting's thing in the tissue of the first step dissociates, separate the courage epithelial cell with trypsinase from remaining tissue residue thing in second step.
Be used to remove the lyophilized products of the elastoser of the AZ collagenase of HP collagenase that hepatocellular enzyme mixture is 130U, 5U and 21U, it is dissolved in the cell damping fluid of 87.5ml.
In all preparations, the epithelial content of living all is higher than 95%, and cell yield produces 4-6 * 10 for each liver
6Individual cell (n=4).Use above-mentioned enzyme mixture to be very easy to trypsinase and separate the courage epithelial cell from remaining tubular system, reason is to be substantially free of liver cell in the resulting residue tissue after the first step.
Used so far obtain the problem that the epithelial method of courage exists and be, residue in the cell in the cell suspending liquid and the cell of Kupffer cell and other type and be difficult to usually from epithelial cell to be separated, remove.
Use the mixture of above-mentioned pure enzyme to save time significantly and to spend, reason is to compare with employed method so far, and dissociating of hepatic tissue obtained tangible improvement, and is completely basically.This shows that the hepatocellular removals as impurity to a large amount of existence usually are gratifying.Embodiment H
Use the mixture of three kinds of enzymes from people's umbilical cord, to separate endotheliocyte
Method with Jaffe (17) is separated endotheliocyte from people's umbilical vein.The concrete practice is that umbilical cord (20～30cm is long) is divided equally into two halves, to wherein pouring into special enzyme solution in couples, is containing 5%CO
2Incubator in 37 ℃ of down insulations 15 minutes.The cell that will come off is removed and is kept in the nascent substratum to be used for detection.Through four days cultivation, after with the non-adherent cell flush away, determine can the splitted viable cell output.
This enzyme mixture is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in the cellular segregation damping fluid of 87.5ml.
Cell inoculation one day after with non-adherent cell (red corpuscle, scavenger cell etc.) flush away.At the 4th day substratum is changed.Usually be not later than reached cell in postvaccinal the 8th day be paved with (＞10
5Individual cell/cm
2).Show by the FACS test, the cell lawn of tiling contains the endotheliocyte more than 95%, the antibody that uses in the test is antagonism antigen (Vonwillebrand thrombin) or the two kind endothelium non-specific surface antigen (EN-4s relevant with blood coagulation factor VIII, PAL-E), also used the fluorescent ligand (dil-Ac-LDL) of removing acceptor in the test.
Surpassed the resulting output of the zymin that contains collagenase with the resulting output of mixture (n=2) of the enzyme of above-mentioned purifying with uncertain composition.Cell density after four days is 1.8 * 10
4Individual/cm
2Cell density value of being starkly lower than of using disclosed up to now method and obtaining.After having used above-mentioned mixture, (only after 5-6 days) are paved with regard to the cell that has reached individual layer in the time more early.
When using different zymin enzymatics to separate endotheliocyte, can damage umbilical vein in no instance.The cell of being removed is separated satisfactorily.Do not observe the cytotoxic phenomenon.After one hour, found the adhesion of endotheliocyte.
Prepared first-generation cell is carried out Function detection (generation of endothelin, the release of LDH).All numerical value that obtain thus are all within normal range.Thereby these cells compare the difference that does not have on the function with the result of control experiment, use the preparation that contains collagenase of uncertain composition to come isolated cell in control experiment.
The mixture of the enzyme behind the employed purifying is significantly in the advantage that improves aspect the cell yield, and this will cause the integrity (cellularstructure and function) of the best of faster formation that the cell of individual layer is paved with and institute's isolated cells.
For example, replace above-mentioned enzyme mixture also to obtain goodish result with the mixture that contains other mixture of HP collagenase and elastoser and contain HP collagenase, AZ collagenase, elastoser and clostripain.Example I
Use mixture separating tumor cell from people's tumour of three kinds of enzymes
With method (23) separating tumor cell from people's tumour.
Employed enzyme mixture is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in the cellular segregation damping fluid of 28.4ml (in the Ringer lactic acid salt/PBS).
To determining of separating resulting by measuring the content of viable cell with the trypan blue staining exclusive method and lymphocyte count being finished.In the isolating direct comparison of pair cell, the definite preparation of forming that contains collagenase of nothing that on four kinds of different tumor tissues, has used enzyme mixture of the present invention and be very suitable for above-mentioned purpose.The result
Enzyme mixture of the present invention can make us very being used to satisfactorily separating tumor cell from people's tumour.
In the variation range of experiment, separating resulting of the present invention is consistent with the selected result who contains the preparation of collagenase and obtain of use, and the latter is very suitable for the tumour cell separation and does not have and determine to form.
According to above-mentioned result, the multiple reticular tissue structure in people's tumor tissues because use enzyme mixture of the present invention can successfully dissociate, it reaches usually or has improved prior art (keeping of the quality of isolating result, isolated cells, method variable), thereby can think that enzyme mixture of the present invention is applicable to dissociating of other animal and human tissue.Embodiment J
Use mixture detached island cell from Pancreas Sus domestica of three kinds of enzymes
For detached island cell from Pancreas Sus domestica, used famous Ricordi preparation method, wherein introduced some important modifications (24), comprising the supervision that improves separating resulting.In above-mentioned situation, after pouring into by ductus pancreaticus, collagenase solution can be to the time of pancreatic tissue effect than length under standardized especially condition.The island cell that has peeled off is entered under lower temperature in the storage receptacle continuously; Dissociate finish after, the island cell that breaks away from of part and its parent comes off fully and is separated by soft mechanical treatment in the pancreas tissue.
The detached island cell has extra high requirement to the preparation that contains collagenase from pancreas, and reason is for the mechanism of successfully separating complete island cell from pancreas and imperfectly understands.
The suitability of definite enzyme mixture of the present invention is confirmed by following embodiment.This shows that purpose is the pancreas (for example people's pancreas) that the tissue of the success of the huge n cell aggregate (pancreas islet) of separation dissociates and is applicable to other species with this tissue morphology too.Some experiences it has been generally acknowledged that the detached island cell is more more difficult than detached island cell from other species from Pancreas Sus domestica, and this has just supported above-mentioned application prospect.For example, the traditional nothing of known use determines that the preparation of forming that contains collagenase is easy to make the island cell dissociation from Pancreas Sus domestica to become less, useless fragment (25).Special property by the island cell aggregation can be made explanations to this, contains in the aggregate much protease-sensitive cell is contacted with intercellular, is having between internal secretion and the exocrine cell and between endocrine cell and the island cell.Thereby the target of improving separation method can only be the aggregate that obtains a large amount of complete island cells, and the disintegration of aggregate＜100 μ m can only be carried out to a small extent.
Being used for from the enzyme mixture of Pancreas Sus domestica detached island cell of using in this Application Example is the lyophilized products of the elastoser of the AZ collagenase of HP collagenase, 5U of 130U and 21U, and it is dissolved in the dissociating buffer of 10ml.In this solution, add routinely deoxyribonuclease (0.4mg/10ml, 440 Kunitz units/mg, Sigma).
The distribution of sizes and other the parameter commonly used of aggregate that can be by resulting intact cell are determined isolating result.
Use a special advantage of enzyme mixture of the present invention can be described as, will be higher than ratio in the traditional preparation process method greater than the ratio of the free island cell of 100 μ m.Embodiment K
The The effects that wound is handled in the body
In the healing of wound, collagenase with remove necrotic tissue effectively, make the conversion of the cellular-restoring of damage location and extracellular matrix relevant.
In the body inner model experiment that the wound of being described by Webster (19) is cleaned, in the rat body, check purifying enzyme organize resolution characteristic.
When being used for third degree burn, the dissociation yield of observing devitalized tissue in initial 16 hours increases, and this depends on the amount of employed purifying enzyme.These enzymes are used separately or be used in combination with other enzyme.When using HP collagenase (3-48 U/cm
2, be preferably 16U/cm
2), AZ collagenase (0.2-3 U/cm
2, be preferably 1U/cm
2) and elastoser (1-12 U/cm
2, 3U/cm preferably
2) mixture the time obtained optimal results.In this case, 8 scab in 15 animals is dissociated fully, and 6 scab in 15 animals is partly dissociated.In the situation of back, compare with control group, remove necrotic tissue with mechanical means and become very easy.
Having used equal amount and after 4 hours treatment time than weak point, having found the softening of downright bad material, under any circumstance can satisfactorily scab be removed (comparing) with control group.
Except this mixture, can also successfully use other mixture, for example HP collagenase (3-48U/cm
2) and elastoser (1-12U/cm
2) mixture.Embodiment L
The The effects that external wound is handled
The resulting result of the necrotic tissue that dissociates (embodiment K) has further obtained sign in an external model.After with HP collagenase, AZ collagenase and/or elastoser the burn scab mentioned among the embodiment K being handled 2,4,6 and 24 hours in the buffered soln of vibration, measure the 4-oxyproline (20) that is discharged.
The vigor of employed purifying enzyme is identical with the relative vigor of the preparation that contains collagenase of available ratio trial of strength.In the simplification of this experiment, ignored the contribution separately that other component discharges oxyproline in the preparation that contains collagenase.
When using purifying enzyme, compare with the preparation that contains collagenase that uses respective numbers, can find the release of the oxyproline of analogous amount in all cases, it is relevant with the time significantly.
According to The above results, purifying enzyme of the present invention and enzyme mixture also can be applicable to the treatment field, for example in wound is handled and in the processing of keloid and fibrous tissue knurl.But their external applications or be used for the injection.
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Claims (9)
1. enzyme that can obtain from clostridium histolyticum, it both can be
A) a kind of collagenase, in with the analysis of synthetic six peptide Z-Gly-Pro-Gly-Gly-Pro-Ala as the Nordwig of substrate and Strauch, its ratio work is 20 U/mg at least, it can only transform the collagen of inactivation to a small extent, for example gelatin and A Zuokao, however it but destroys ox flesh key collagen, measuring its molecular weight by sds gel electrophoresis is 106,000 Da, its iso-electric point is pH5.8 to 6.0, perhaps
B) a kind of collagenase, in with the analysis of A Zuokao (Azokoll) as the people such as Mandl of substrate, its ratio work is 10 U/mg at least, it can transform the collagen of inactivation effectively, for example gelatin and A Zuokao, and can transform ox flesh key collagen, yet it can not destroy the synthetic proteins of short chain, such as 2-furans acryloyl-Leu-Gly-Pro-Ala, 4-phenylazo-benzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg[sic] and Z-Gly-Pro-Gly-Gly-Pro-Ala, measuring its molecular weight by sds gel electrophoresis is 111,000 Da, its iso-electric point is pH5.9 to 6.1, perhaps
C) elastoser, in the analysis of elastin as substrate from the ox ligament of neck, the work of its ratio is 2U/mg at least, is 35,000 Da by its molecular weight of SDS cataphoretic determination,
Also can be the mixture of above-mentioned enzyme, wherein enzyme ratio work a) be at least 20 U/mg, enzyme b) ratio work be at least 10 U/mg, enzyme d) the ratio work of [sic] is at least 2U/mg.
2. according to the enzyme mixture of claim 1, wherein contain to additivity thiol proteinase-clostripain.
In the claim 1 enzyme a) and enzyme c) mixture be used for from the purposes of human or animal tissues isolated cell or fragment of tissue.
In the claim 1 enzyme a), b) and mixture c) be used for from the purposes of human or animal tissues isolated cell or fragment of tissue.
5. the mixture of claim 2 is used for from the purposes of human or animal tissues isolated cell or fragment of tissue.
6. the enzyme of claim 1 separately or be used for wound with the enzyme of form of mixtures or claim 2 and handle or be used to handle the purposes that changes relevant imbalance with the collagen protein metabolism.
7. the HP collagenase of pure state.
8. the AZ collagenase of pure state.
9. the elastoser of pure state.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 96192616 CN1178552A (en) | 1995-03-16 | 1996-03-12 | New difined enzyme mixture for obtaining cells and treating wounds |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19509584.7 | 1995-03-16 | ||
| DE19532906.6 | 1995-09-07 | ||
| CN 96192616 CN1178552A (en) | 1995-03-16 | 1996-03-12 | New difined enzyme mixture for obtaining cells and treating wounds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1178552A true CN1178552A (en) | 1998-04-08 |
Family
ID=5128375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 96192616 Pending CN1178552A (en) | 1995-03-16 | 1996-03-12 | New difined enzyme mixture for obtaining cells and treating wounds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1178552A (en) |
-
1996
- 1996-03-12 CN CN 96192616 patent/CN1178552A/en active Pending
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