CN1654071A - Biological fluid film of recombined human keratinized cell growth factor-2 and its preparing process - Google Patents
Biological fluid film of recombined human keratinized cell growth factor-2 and its preparing process Download PDFInfo
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Abstract
The present invention is recombinant human keratinized cell growth factor-2 (rhKGF-2) biofluid membrane for treating epithelium tissue damage and its preparation process. The rhKGF-2 biofluid membrane includes purified rhKGF-2 liquid and biofluid dressing comprising chitosan, gelatin, glycerin and PVP. The purified rhKGF-2 liquid is prepared through connecting KGF-2 gene onto the expression plasmid pLY4 to constitute expression vector transforming colibacillus and clone engineering bacillus; culturing engineering bacillus and raising temperature to induce expressing rhKGF-2; and separating and purifying the expressed rhKGF-2. The biofluid dressing is prepared is prepared with chitosan as main matrix, and gelatin, glycerin and PVP as supplementary material in optimized compounding proportion.
Description
Technical field
The present invention relates to a kind of recombinant human keratinized cell growth factor-2 biofluid film that is used for the treatment of the epithelial tissue damage, particularly recombinant human keratinized cell growth factor-2 biofluid film and preparation technology's method thereof.
Background technology
Epithelial cell damage body surface shows as the skin ulcer of the burn and scald of skin, traumatic wound and a variety of causes etc., involves a wide range of knowledge.At present, the processing of burn and scald, traumatic wound surface is comprised wound cleaning, dressing application and three aspects of Drug therapy clinically substantially, wherein dressing application and Drug therapy are often carried out simultaneously, help wound healing in solution flap coverage problem.
In the prior art, the cytokine medicine of treatment epithelial tissue damage mainly contains human epidermal growth factor (EGF).Promote the factor because EGF is non-specific horn cell, therefore in the treatment damage, may stay tangible cicatrix.Use human body keratinized cell growth factor-2 treatment epithelial tissue at present and damage great attention and the approval that has caused medical circle, for example, the trade name Inc (HGS) of HUMAN GEMNOME SCIENCES exploitation reorganization KGF-2 had once just reported on May 23rd, 2000 that KGF-2 treated the result of study of chronic venous ulcer IIa clinical trial.HGS has the patient of chronic venous ulcer to give the experiment that KGF-2 estimates its safety and preliminary effectiveness to 94, and the result shows that human body has excellent biological compatibility and notable therapeutic effect to KGF-2.Not only can treat and hinder equivalent damage, also can be used for treating chronic trauma and comprise venous ulcer, diabetic ulcer etc. as burn, scald, otch.Medical circle and then discovery again: recombinant human keratinized cell growth factor-2 is a kind of rho factor that wound healing is had very much the treatment potentiality, not only have obvious promotion wound tissue healing speed and healing quality, and have obvious promotion epidermis cell to divide a word with a hyphen at the end of a line apart from the effect of (P＜0.05) and the area of dividing a word with a hyphen at the end of a line (P＜0.01).Can breed by the differential stimulus horn cell simultaneously, the unnecessary collagen of degrading, because of reducing the formation of cicatrix, application prospect is very good when the damage of treatment large area skin comprises burn and scald, traumatic wound, chronic ulcer for it.
Yet, although recombinant human keratinized cell growth factor-2 is a kind of wound healing to be had the rho factor of treatment potentiality very much, how to be applied to clinically, how about be made into certain dosage form, be that pendulum is in current medical circle urgency problem to be solved.
KGF-2 once made a kind of injection and injectable powder in the U.S., but it is only used for treating a kind of medicine that the epithelial tissue damage is used, and when this medicine of extraction, adopted the method for occlusion body to extract KGF-2, and its shortcoming is that purity is low, complex process.And aspect dressing, though having with chitin derivativ (chitosan) in the prior art is the treatment that the biological dressing film of substrate preparation is used for various wounds, but the chitin biological dressing film of listing is a kind of solid film, and this solid film can not closely touch the various aspects of wound surface when using, and easily produces hydrops and causes infection; The solid film size need increase and decrease according to wound surface simultaneously, and is extremely inconvenient, thereby limited being extensive use of of solid dressing film.
For the advantage that makes full use of KGF-2 overcomes the shortcoming of solid film again, the present invention is exactly in order to provide a kind of recombinant human keratinized cell growth factor-2 that utilizes to inject biological fluid dressing, to be prepared into recombinant human horny cell growth factor-2 biofluid film.Make recombinant human horny cell growth factor-2 and biological fluid dressing have Synergistic and slow releasing function.
Because in the treatment to burn and scald, traumatic wound surface, dressing application and Drug therapy are often carried out simultaneously, and this requires dressing and medicine to have the good compatibility.Biological fluid dressing is to be primary raw material with the chitosan among the present invention, its character gentleness, contain suitability stabilizing agent widely, the reasonable mixture ratio of substrate and adjuvant, can be respectively and multiple medicament mixed, still stable after the long term storage, and the substrate of its preparation is biodegradation material, can be used as pharmaceutical carrier, make medicine have slow releasing function.
Summary of the invention
For overcoming the shortcoming that prior art exists, one of purpose of the present invention provides a kind of biofluid film that contains recombinant human keratinized cell growth factor-2.
Two of purpose of the present invention provides the process for producing method of the biofluid film of recombinant human keratinized cell growth factor-2.
The present invention is achieved by following technical proposals:
The alleged recombinant human keratinized cell growth factor-2 biofluid film of the present invention comprises recombinant human keratinized cell growth factor-2 purification stock solution, and with chitosan, the biofluid adjuvant that gelatin, glycerol, polyvinylpyrrolidone are formed.
First purpose of the present invention is achieved in that
The present invention is injected into recombinant human keratinized cell growth factor-2 purification stock solution in the biological fluid dressing, and this biological fluid dressing is to be main matrix with the chitosan, is that adjuvant adopts ratio optimization to make with gelatin, glycerol, polyvinylpyrrolidone.Described adjuvant filling ratio is chitosan 1.0-2.0%; Gelatin 0.2-1.0%; Polyvinylpyrrolidone 0.02-0.1%; Biofluid adjuvant pH value 5.0-7.0.
Described recombinant human keratinized cell growth factor-2 is a lyophilized powder; Fermentation purification stock solution form.
Second purpose of the present invention is achieved in that
Preparation technology's method of recombinant human keratinized cell growth factor-2 biofluid film of the present invention is as follows: the 1. preparation of purification stock solution; 2. the preparation of biological fluid dressing; 3. mixing of purification stock solution and biological fluid dressing, the i.e. main preparation methods of recombinant human keratinized cell growth factor-2 biofluid film.
1. the preparation method of purification stock solution comprises:
(a) the KGF-2 gene is connected on the expression vector pLY4 transformed into escherichia coli, clone's high expression engineering strain;
(b) culturing engineering bacterial strain is by improving temperature-induced expression rhKGF-2;
(c) go out expressed rhKGF-2 in the step (b) by the chromatographic technique separation and purification;
2. the preparation method of biological fluid dressing comprises:
Deacetylation placed greater than 80% chitosan in 80 ℃ the baking oven 2 hours, the weighing of 1.0-2.0% concentration is pressed in cooling.With the abundant stirring and dissolving of above-mentioned chitosan, mixing speed is 65-296r/min with the acetum of 0.5-1.0%, and 12 hours time, the pH value of solution is 1-5;
In dissolved chitosan solution, add (0.2-1.0%) gelatin, glycerol, mixing respectively.Chitosan is 3.2: 1～5: 1 with the dry weight ratio of gelatin.Sodium bicarbonate solution 2500-4000ml with 0.5-0.7% neutralizes, solution PH 5.0-7.0.Add (0.02-0.1%) polyvinylpyrrolidone, fully stir.Filter froth breaking, sterilization.The viscosity of measuring chitosan solution next day is 500-5000mpa.s, and the product P H-number is 5-7, carries out sterility test.
3. purification stock solution adds the biological fluid dressing process:
Get aseptic recombinant human keratinized cell growth factor-2 purification stock solution water solution, slowly join in the biological fluid dressing, fully stir, filtration, froth breaking, fill is in the medical aluminum pipe of 80 ℃ of baking 4h.The rhKGF-2 final concentration is 50-200 μ g/ml in the fluid film.
The preparation method of high expression engineering strain of the present invention comprises:
Expression plasmid is pLY4, and the host bacterium is escherichia coli BL 21 (D3), and (efficient expression plasmid pLY4, escherichia coli BL 21 (D3) are provided by Liu Xinyuan professor laboratory, and this is the temperature-induced type high-expression vector that Liu Xinyuan professor laboratory makes up.The structure data is consulted " Chinese science " (B collects), 1995,25 (10): 1063-1070 in detail.) with 0 section complementary oligonucleotide of KGF-2 cDNA sequence Synthetic 2, and 5 ' end, 3 ' end introduces special restriction enzyme site EcoR I, BamH I respectively, handled 30 minutes for 37 ℃ with T4 phage polynucleotide kinase, each oligonucleotide fragment of phosphorylation mixes to wait molal quantity, 94 ℃ of degeneration 5 minutes, fade to 65 ℃ of annealing 10 minutes immediately, add the T4 ligase then, 16 ℃ of reactions are spent the night.
The coded sequence that expression vector EcoRI/BamHI has inserted in the site human horny cell growth factor-2 also comprises the degenerate sequence of SEQID NO:1 sequence, should " degenerate sequence " be meant that coding had the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
Fermentation culture stage of the present invention is: temperature: 37 ℃; PH:6.0; OD when inducing
600: 2.0; Cultivated altogether 5 hours; In the fermentation inducement stage, temperature: 42 ℃, pH:6.8, induction time are 2 hours.
Purification phase of the present invention is:
1., fermentation liquid is below 4 ℃, the centrifugal 10min of 4500rpm, thalline.
2., the thalline height crushes bacterium, the centrifugal 30min of 7000rpm, supernatant.
3., chromatography 1 (affinity chromatograph):
Chromatography media: heparin (Heparin) CL-6B
Buffer: solution A: Tris-HCl (PH7.4,20mM)
Solution B: Tris-HCl (PH7.4,20mM)+1M NaCl
Clean: clean chromatographic column with the solution A of 6 column volumes (CV) after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10 column volumes (CV).
4., chromatography 2 (cation-exchange chromatography):
Chromatography media: SP Sepharose FF
Buffer: solution A: Tris-HCl (PH7.4,20mM)
Solution B: Tris-HCl (PH7.4,20mM)+1M NaCl
Clean: clean chromatographic column with the solution A of 6 column volumes (CV) after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10 column volumes (CV).]
Above-mentioned chromatographic technique also comprises gel permeation chromatography, cation-exchange chromatography, anion-exchange chromatography, affinity chromatograph, and gel permeation chromatography is meant: behind the sample ultrafiltration and concentration, can desalt and purification with gel permeation chromatography.
Implement the present invention and can produce following beneficial effect:
The present invention is in making up engineering bacteria, because employing matrix extraction process, so extraction process is simple, rhKGF-2 content height.And do not belong to ointment, colloid with the fluid dressing that process of the present invention is prepared into, its physical behavior is a semifluid, has permeability, and energy and wound surface are combined closely, can form transparent membrane after being applied to the wound surface drying, have physics and chemical double barrier anti-infectious function, exudate can be controlled and absorb to this thin-films Oxygen permeability height, use the back wound surface convergence, exudate significantly reduces, crust face drying, polishing, not pollution clothes, and this fluid dressing room temperature stable storing.By high-load rhKGF-2 the present invention who is produced that combines, have following four aspect advantages with fluid dressing:
(1) expression process is simple.Because the carrier that the present invention has selected suitable rhKGF-2 to express, this carrier helps rhKGF-2 to exist with soluble form, does not need degeneration, renaturation technology; Easy, temperature-induced mode has improved the industrialization value of rhKGF-2 cheaply; Separation and purification is easy.
(2) expression height.By controlling crucial technological condition for fermentation, make expression be higher than the level with the occlusion body formal representation of present report.Experiment shows, stable manufacturing process of the present invention is simple to operate, and the cycle is short, and cost is low, and every liter of fermentation liquid can obtain the pure product 150mg of rhKGF-2.Be fit to industrialization production.
(3) contain the fluid dressing of chitosan; skin wound had excellent biological compatibility and natural antibacterial activity; various biological effects such as wound surface local immunity, epithelial cell growth and healing acceleration can be promoted, after the local use of this dressing drying, the thin film of layer of transparent can be formed; have physics and chemical double barrier protective effect; and exudate can be controlled and absorb to oxygen permeability height, uses the back wound surface convergence; exudate significantly reduces, and crust face drying, polishing be pollution clothes not.
(4) recombinant human keratinized cell growth factor-2 (rhKGF-2) is a kind of rho factor that wound healing is had very much the treatment potentiality, can stimulate horn cell propagation specifically, the unnecessary collagen of degrading, because of reducing the formation of cicatrix, application prospect is very good when the damage of treatment large area skin comprises burn, traumatic wound surface, chronic ulcer for it.Recombinant human keratinized cell growth factor-2 has synergistic function with biological fluid dressing.
The specific embodiment:
Embodiment 1: the preparation of recombinant human keratinized cell growth factor-2 biofluid membrane matrix:
Take by weighing through chitosan 492 grams (final concentration is 1.6%) of 80 ℃ of oven dry and put in the agitator, adding 25 kilograms of purified water stirs evenly, mixing speed is 196r/min, slowly adds the 250ml acetum while stirring, and pH value is 3, process needs 12 hours approximately, after finishing, add the gelatin 150g that is dissolved in advance in the 1500ml water while hot, and 150ml glycerol, mixing, slowly regulate chitosan solution pH value to 6.5 with 0.7% sodium bicarbonate solution 3700ml then, process needs 2-3 hour, adds 24.5 gram polyvinylpyrrolidones again, fully stir, filter heating, the bubble that degass, sterilization, measure the pH value and the viscosity of chitosan solution next day, medical then aluminum pipe potting package.Recording viscosity with rotary viscosimeter is 3570mpa.s.This substrate storage at normal temperature is stable, is the semifluid shape, can directly apply skin wound.
The quality index of chitosan raw material
The clarity of acetum and color: 1% acetate dissolution becomes this sample solution of 1%, should be thickness, visual no granule slightly, and opalescence is arranged slightly, and its color should be not darker than tested liquid.Tested liquid: take by weighing 0.018g iron chloride and be dissolved into 10ml solution.
Viscosity-average molecular weight: it is an amount of to get chitosan, is solvent with 0.1mol/L acetic acid and 0.2mol/L sodium chloride solution, and with the intrinsic viscosity of determination of ubbelohde viscometer chitosan, and the calculating molecular weight ranges is 2 * 10
5～5 * 10
5
Dynamic viscosity: it is an amount of to get this product, becomes the solution of 1% concentration with 1% acetate dissolution, after room temperature is placed 20h, with the range of viscosities 5～100mpa.s of NDJ-1 type rotary viscosity design determining this product.
Deacetylation: this product takes by weighing 0.5g behind 40 ℃ of dry 24h, places the 250ml conical flask, adds 0.1mol/LHCl standard solution 35ml, makes its dissolving.Methyl orange is indicator, with the titration of 0.1mol/L NaOH titer to terminal.Other gets a 0.5g of this product, and 105 ℃ dry to constant weight, and survey its moisture, are not less than 80% in its deacetylation of dry product.
Loss on drying: take by weighing this product 2.0g, 105 ℃ are dried to constant weight, subtract weight loss and must not cross 10% (two appendix VIIIL of Chinese Pharmacopoeia).
Ignition residue (ash): take by weighing this product 5.0g, slowly blazing to carbonization fully, check (two appendix VIIIN of Chinese Pharmacopoeia) in accordance with the law, leave over residue and must not cross 2.0%.
Acid insoluble ash: the residue after the above-mentioned calcination is dissolved in the mixed liquor of 5ml 5% hydrochloric acid and 5ml water, boils, filter, 105 ℃ are dried to constant weight, and acid insoluble ash must not cross 0.5%.
Heavy metal: take by weighing this product 1.0g, put in the crucible of ignition to constant weight, precision weighing, slowly blazing to carbonization fully, blazingly make complete ashing in 550 ℃, check in accordance with the law (95 editions two appendix VIIIH second methods of Chinese Pharmacopoeia) that heavy metal (in the Pb value) content must not be crossed 10/1000000ths (10ppm).
Arsenic salt: take by weighing this product 1.0g, add natrium carbonicum calcinatum 1.0g, mix, add water and stir, after the drying, burn with little baked wheaten cake earlier and make carbonization, blazingly make complete ashing in 550 ℃ again, put coldly, add hydrochloric acid 5ml and water 23ml, check in accordance with the law (95 editions two appendix VIIIL first methods of Chinese Pharmacopoeia) that arsenic content must not be crossed 2/1000000ths (2mg/kg).
Embodiment 2: the preparation of recombinant human keratinized cell growth factor-2 stock solution:
Expression plasmid is pLY4, and the host bacterium is escherichia coli BL 21 (D3).CDNA sequence according to the KGF-2 in the gene library, 0 section complementary oligonucleotide of Synthetic 2, and 5 ' end, 3 ' end introduces special restriction enzyme site EcoR I, BamH I respectively, adopts the conventional method of molecular cloning, at first handled 30 minutes for 37 ℃ with T4 phage polynucleotide kinase.Each oligonucleotide fragment of phosphorylation mixes to wait molal quantity, and 94 ℃ of degeneration 5 minutes fade to 65 ℃ of annealing 10 minutes immediately, add the T4 ligase then, and 16 ℃ of reactions are spent the night.
PLY4 plasmid EcoR I reclaims big segment behind the BamH I double enzymolysis, and be connected with synthetic rhKGF-2 cDNA segment and spend the night, direct transformed into escherichia coli host bacterium BL 21 (D3), the screening of reuse amicillin resistance obtains the positive colony bacterial strain; Prepare plasmid in a small amount, go out to contain the rhKGF-2 full length cDNA sequence with the restricted enzyme evaluation and screening.
The preparation of embodiment 3:rhKGF-2 biofluid film
With recombinant human horny cell growth factor-2 (rhKGF-2) collocation of fluid dressing membrane and debita spissitudo, make rhKGF-2-fluid film novel form, its recipe design is:
Get 1 part of aseptic 0.4% recombinant human keratinized cell growth factor-2 (rhKGF-2) aqueous solution, slowly join in 19 parts of the rhKGF-2 biofluid membrane matrixs, fully stir, filter, froth breaking, fill is in the medical aluminum pipe of 80 ℃ of baking 4h.The rhKGF-2 final concentration is 200 μ g/ml in the fluid film.
The test of embodiment 4:rhKGF-2 biofluid membrane stability
1, long-term experiment (experiment carry out in): under actual storage requirement, carry out, provide foundation so that work out the effect duration of medicine near medicine.3 batch samples are placed in 6 ℃ ± 2 the environment, and the 0th day, the 3rd month, the 6th month, the 9th month, the 12nd month, a year and a half, 2 years, take a sample once for the year ends three, detection index is according to the rules monitored.Detect index: sterility test, character, color and luster, pH value, activity, dynamic viscosity, skin film property.
2, accelerated tests: this test is to carry out under extraordinary condition, and purpose is the stability by chemistry that quickens pharmaceutical preparation or physical change prediction pharmaceutical preparation.
3 batch samples are placed in temperature 25 ℃ ± 2 incubators, placed 6 months under the condition of humidity 60 ± 5%, and in 1st month, the 2nd month, the 3rd month, the 6th sampling at the end of month of experimental session once, detection index is according to the rules monitored.
021001,021002,021,003 3 group of test is carried out in recombinant human keratinized cell growth factor-2 biofluid membrane stability test (for a long time) altogether, and the investigation data that just list wherein a group are as shown in the table:
Lot number: 021001 keeps sample the date: the 2002.10.9 experiment condition: 6 ± 2 ℃ of temperature
| Aseptic | Up to specification | ????- | ?????- | ????- | Up to specification |
| Color and luster | Faint yellow | Faint yellow | Faint yellow | Faint yellow | Faint yellow |
| The skin film property | Well | Well | Well | Well | Well |
| Dynamic viscosity (mpa.s) | ????2800 | ????2700 | ????2730 | ????2650 | ????2600 |
| Active (being labelled amount) | ????100 | ????99.7 | ????99.0 | ????97.2 | ????96.8 |
Through finding out in the accelerated test, the tiring of sample that the three batches of quilts are investigated changes at extraordinary condition end, but all in the 90%-110% of labelled amount.
Calculate that by long-term stable experiment fluid film was respectively 6 ± 2 ℃ of condition following effect duration: 021001 batch 31.1 months, 021002 batch 35.9 months, 021003 batch 34.3 months.Estimate that in view of the above this fluid film effect duration is tentative to be 2 years.
The pharmacodynamics test of embodiment 5:rhKGF-2 fluid film
1, the rhKGF-2 fluid film is to the therapeutical effect of rat skin scald
Experimental technique:
50 of male SD rats, body weight 350～400g.Rat back spinal column both sides are with 8% sodium sulfide (being dissolved in the 1%CMC solution) depilation, the about 6 * 8cm of area
2, the appropriate 40mg/kg tail vein injection of receiving of sulfur benzene was anaesthetized in the 2nd day, and the 50g counterweight (diameter 2cm) of putting into the boiling water preheating is in advance pressed in depilation district, rat back spinal column both sides, scalded for 8 seconds, 2 of every sides, totally 4.(scalding the back the 2nd day) was divided into 5 groups at random with rat, 10 every group in the 3rd day.Earlier trace the wound size with translucent pan paper, analytical balance is weighed, and is used for the preceding wound area of administration and calculates.Respectively organize wound then and smear normal saline 100 μ l/ wounds (matched group), KGF-2 solution 20 μ g/ wounds (positive group), KGF-2 fluid film 20,10 and 5 μ g/ wounds (to contain the KGF-2 amount), the administration volume is 100 μ l/ wounds, administration every day 1 time, continuous 16 days, during traced wound 1 time in per 4 days.Calculate wound healing rate in the medication process.
Experimental result:
The KGF-2 fluid film can obviously be accelerated the healing rate after rat skin is scalded, and dose relationship is clear and definite.Same dose (20 μ g/ wound) is better than the therapeutic effect of KGF-2 solution, and it is faster to heal.
Table 1 KGF-2 fluid film is to burned rats wound area (mg) and healing rate (%)
| Wound area/healing rate | Before the administration | Administration the 4th day | Administration the 8th day | Administration the 12nd day | Administration the 16th day |
| Matched group | 15.2±2.2 | ?14.2±2.5 | ?11.6±1.8 | ?8.8±1.5 | ?5.6±1.7 |
| ?8.1±6.2 | ?24.8±12.3 | ?42.5±10.2 | ?62.7±11.0 | ||
| KGF-2 solution 20 μ g/ wounds | 16.2±2. ????1 | ?14.2±1.7 | ?9.5±1.4** | ?6.8±2.1** | ?4.1±1.7** |
| ?10.6±7.6 | ?41.2±8.0** | ?57.7±12.9* ????* | ?75.7±10.3* ????* | ||
| KGF-2 fluid film 20 μ g/ wounds | 16.6±2. ????5 | ?14.3±2.4 | ?8.5±1.8** | ?6.0±1.9** | ?2.3±2.0** |
| ?13.8±7.5 | ?48.6±13.7 | ?63.7±13.5* ????* | ?86.4±13.1* ????* | ||
| KGF-2 fluid film 10 μ g/ wounds | 15.8±2. ????3 | ?14.1±2.0* | ?10.3±2.1* ????* | ?6.6±1.9** | ?3.1±1.9** |
| ?11.3±8.6** | ?31.4±16.4 ????* | ?58.4±13.0* ????* | ?80.1±13.6* ????* | ||
| KGF-2 fluid film 5 μ g/ wounds | 16.0±3. ????3 | ?14.7±3.6 | ??11.5±2.9 | ?7.6±2.6** | ?4.0±1.7** |
| ?8.1±6.1* | ??28.5±16.6 | ?52.3±14.0* ????* | ?75.4±9.2** |
Annotate: n=40 and matched group compare, * * p＜0.01 * p＜0.05
2, the rat skin holostrome is excised the influence of wound healing
Experimental technique:
50 of female sd inbred rats, body weight 250 ~ 300g.Above-mentioned with quadrat method at rat back about 6 * 8cm that loses hair or feathers
2, the appropriate 40mg/kg dosage tail vein injection of receiving of sulfur benzene was anaesthetized in the 2nd day.Depilation district, back 75% alcohol disinfecting, every Mus is cooked two 1.4 * 5.2cm with scraper in the spinal column both sides
2The holostrome otch, and skin and subcutaneous sheath wiped out through Musclar layer, but muscle injury shin film not.Be divided into 5 groups at random on the 2nd day, and traced the wound size with translucent pan paper, analytical balance is weighed, and is used for the preceding wound area of administration and calculates.Give normal saline 250 μ l/ wounds respectively, KGF-2 solution (0.2mg/ml) 250 μ l/ wounds, KGF-2 fluid film 40,20 and 10 μ g/ wounds (to contain the KGF-2 amount), the administration volume is 250 μ l/ wounds, administration every day 1 time, during traced wound 1 time in per 4 days.Calculate wound healing rate in the medication process.
Table 2 rhKGF-2 fluid film is to the influence (%) of rat skin holostrome excision wound healing rate
| Group/healing rate | Administration the 4th day | Administration the 8th day | Administration the 12nd day |
| Matched group | ??6.3±4.7 | ??16.2±7.5 | 39.8±11.4 |
| KGF-2 solution 40 μ g/ wounds | ??8.6±4.2 | ??28.0±9.2** | 46.0±11.5** |
| KGF-2 fluid film 40 μ g/ wounds | ??9.7±5.4 | ??32.4±11.1** | 58.6±12.4** |
| KGF-2 fluid film 20 μ g/ wounds | ??9.1±5.0 | ??30.2±12.9** | 54.2±15.4** |
| KGF-2 fluid film 10 μ g/ wounds | ??8.9±6.4 | ??23.6±8.8* | 48.5±13.0* |
Annotate: n=20 and matched group compare, * * p＜0.01 * p＜0.05
This experiment does not finish as yet, has only 12 days data.
Result of the test:
The rhKGF-2 fluid film can obviously be accelerated the healing rate of rat skin holostrome excision wound, and dose relationship is clear and definite.Same dose (40 μ g/ wound) is better than the therapeutic effect of KGF-2 solution, and it is faster to heal.
Compare with scalding model, the healing rate of this model wound is lower, may with wound area big relevant (bigger 1.32 times) than the scald wound.But wound healing perusal after the administration, in the skin holostrome excision model, the wound state of appearance is better, may can fully contact with wound with this model Chinese medicine, and in scalding model, that dead crust on the early stage wound has hindered medicine and had a repair function or organize contacts, and has influenced the reparation of wound.
Above-mentioned cited embodiment is only for the usefulness that the present invention is described; and be not to be limitation of the present invention; the those of ordinary skill in relevant technologies field; under the situation that does not break away from the spirit and scope of the present invention; the various variations of having done, all technical schemes that are equal to also should belong to the category of the present invention's protection.
Claims (9)
1 one kinds of recombinant human keratinized cell growth factor-2 biofluid films is characterized in that comprising recombinant human keratinized cell growth factor-2 purification stock solution, and with chitosan, the biological fluid dressing that gelatin, glycerol, polyvinylpyrrolidone are formed.
2, recombinant human keratinized cell growth factor-2 biofluid film according to claim 1 is characterized in that described biological fluid dressing filling ratio is chitosan 1.0-2.0%; Gelatin 0.2-1.0%; Polyvinylpyrrolidone 0.02-0.1%; Biological fluid dressing pH value 5.0-7.0.
3, recombinant human keratinized cell growth factor-2 biofluid film according to claim 1 is characterized in that described recombinant human keratinized cell growth factor-2 is lyophilized powder or fermentation purification stock solution form.
4, a kind of recombinant human keratinized cell growth factor-2 biofluid film preparation process is characterized in that the purification stock solution that will extract joins in the biological fluid dressing as claimed in claim 1;
1. the preparation method of purification stock solution comprises:
(a) the KGF-2 gene is connected on the expression vector transformed into escherichia coli, clone's high expression engineering strain;
(b) culturing engineering bacterial strain is by improving temperature-induced expression rhKGF-2;
(c) go out the rhKGF-2 that expresses in the step (b) by the chromatographic technique separation and purification;
2. the preparation method of biological fluid dressing comprises:
Deacetylation placed greater than 80% chitosan in 80 ℃ the baking oven 2 hours, the weighing of 1.0-2.0% concentration is pressed in cooling, with the acetum of 0.5-1.0% with the abundant stirring and dissolving of above-mentioned chitosan, mixing speed is 65-296r/min, and 12 hours time, the pH value of solution is 1-5;
In dissolved chitosan solution, add 0.2-1.0% gelatin, glycerol respectively, mixing, chitosan is 3.2: 1～5: 1 with the dry weight ratio of gelatin, sodium bicarbonate solution 2500-4000ml with 0.5-0.7% neutralizes, and solution PH 5.0-7.0 adds (0.02-0.1%) polyvinylpyrrolidone, fully stir, filter, froth breaking, sterilization, the viscosity of measuring chitosan solution next day is 500-5000mpa.s, the product P H-number is 5-7, carries out sterility test;
3. purification stock solution adds the biological fluid dressing process:
Get aseptic recombinant human keratinized cell growth factor-2 purification stock solution water solution, slowly join in the biological fluid dressing, fully stir, filtration, froth breaking, fill is in the medical aluminum pipe of 80 ℃ of baking 4h.
5, according to the described recombinant human keratinized cell growth factor-2 biofluid of claim 4 film preparation process, it is characterized in that the preparation of described high expression engineering strain comprises:
Expression plasmid is pLY4, the host bacterium is escherichia coli BL 21 (D3), with 0 section complementary oligonucleotide of KGF-2 cDNA sequence Synthetic 2, and 5 ' end, 3 ' end introduces special restriction enzyme site EcoR I, BamH I respectively, handles 30 minutes for 37 ℃ with T4 phage polynucleotide kinase, and each oligonucleotide fragment of phosphorylation mixes to wait molal quantity, 94 ℃ of degeneration 5 minutes, fade to 65 ℃ of annealing 10 minutes immediately, add the T4 ligase then, 16 ℃ of reactions are spent the night.
6, recombinant human keratinized cell growth factor-2 biofluid film preparation process according to claim 5, the coded sequence that it is characterized in that the human horny cell growth factor-2 that insert in described expression vector EcoRI/BamHI site also comprises the degenerate sequence of SEQ ID NO:1 sequence, should " degenerate sequence " be meant that coding had the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
7, recombinant human keratinized cell growth factor-2 biofluid film preparation process according to claim 4 is characterized in that described fermentation culture stage is temperature: 37 ℃; PH:6.0; OD when inducing
600: 2.0; Cultivated altogether 5 hours; In the fermentation inducement stage, temperature: 42 ℃, pH:6.8, induction time are 2 hours.
8, recombinant human keratinized cell growth factor-2 biofluid film preparation process according to claim 4 is characterized in that described purification phase is:
1., 4 ℃ of centrifugal 10min of following 4500rpm of fermentation liquid, thalline;
2., the thalline height crushes bacterium, the centrifugal 30min of 7000rpm, supernatant;
3., chromatography 1 (affinity chromatograph):
Chromatography media: heparin (Heparin) CL-6B
Buffer: solution A: Tris-HCl (PH7.4,20mM)
Solution B: Tris-HCl (PH7.4,20mM)+1M NaCl
Clean: clean chromatographic column with the solution A of 6 column volumes (CV) after going up sample,
Gradient: clean the back and solution B is risen to 100% from 0% with 10 column volumes (CV),
4., chromatography 2 (cation-exchange chromatography):
Chromatography media: SP Sepharose FF
Buffer: solution A: Tris-HCl (PH7.4,20mM)
Solution B: Tris-HCl (PH7.4,20mM)+1M NaCl
Clean: clean chromatographic column with the solution A of 6 column volumes (CV) after going up sample,
Gradient: clean the back and solution B is risen to 100% from 0% with 10 column volumes (CV),
9, recombinant human keratinized cell growth factor-2 biofluid film preparation process according to claim 4, it is characterized in that described chromatographic technique comprises gel permeation chromatography, cation-exchange chromatography, anion-exchange chromatography, affinity chromatograph, gel permeation chromatography is meant: behind the sample ultrafiltration and concentration, can desalt and purification with gel permeation chromatography.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533722A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump |
| CN106377760A (en) * | 2016-10-26 | 2017-02-08 | 李天学 | Application of preparation containing KGF-2 (keratinocyte growth factor-2) to relieving of diabetic ulcer |
| CN111407717A (en) * | 2020-03-27 | 2020-07-14 | 河北银丰鼎诚生物技术有限公司 | Stem cell factor freeze-dried powder and preparation method and application thereof |
-
2004
- 2004-02-12 CN CNB2004100394353A patent/CN1284600C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533722A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump |
| CN102533722B (en) * | 2010-12-24 | 2014-03-12 | 北京义翘神州生物技术有限公司 | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump |
| CN106377760A (en) * | 2016-10-26 | 2017-02-08 | 李天学 | Application of preparation containing KGF-2 (keratinocyte growth factor-2) to relieving of diabetic ulcer |
| CN111407717A (en) * | 2020-03-27 | 2020-07-14 | 河北银丰鼎诚生物技术有限公司 | Stem cell factor freeze-dried powder and preparation method and application thereof |
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