CN101125216A - Transgenic cell activation modified chitosan medical dressing and preparation method and application - Google Patents
Transgenic cell activation modified chitosan medical dressing and preparation method and application Download PDFInfo
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Abstract
The present invention discloses a medical dressing and preparation method of chitosan activated and modified by the transgeneic cell and the application. The manufacturing procedures of the medical dressing of chitosan activated and modified by the transgeneic cell and the application includes the two steps as follows:(1) the sulphydrylation chitosan is added with cell complete culture medium with the proportion of 15 to 25 mg/ml to make the culture medium solution;(2) adding the skin fibroblast cell or keratinocyte cell of growth gene which is derivatized by transfected platelet into the culture medium solution of procedure (1) in the thickness of 4 multiplied by 104-4 multiplied by 105/ml to be rotarylis cultured. The present invention applies gel with the performances of hydrophilicity, hygroscopicity, ventilation property and flexility as the cell scaffold, meeting the dressing requirement of modern medical. With the technique of gene recombination, the seed cell in the medical dressing of the present invention provides the partial growth gene of PDGF-BB a great phrasing and high exudation, cooperating with the extracellular matrix to control the micro-environment of canker and reaching the recovery.
Description
Technical field
The invention belongs to genetic engineering, bioengineered tissue field, particularly relate to a kind of transgenic cell activation modified chitosan medical dressing and Preparation method and use.
Background technology
Diabetes have now become global commonly encountered diseases and frequently-occurring disease, are listed in the third-largest disease after cardiovascular disease and tumor in developed country.Show that according to IDF's statistics in 2006 whole world 100,015,000 people suffer from type 2 diabetes mellitus at present, but its sickness rate differs in various places.Nearly nearly 3,000 ten thousand diabeticss of China.By the sickness rate in increasing at present, diabetes will become one of the most general disease in the whole world in many decades.WHO predicts global diabetics in 2025 will reach 3.3 hundred million, 75% diabetics wherein be arranged in developing countries such as India, China.The number of China's diabetics is only second to India and occupies the second place of the world.The harm of diabetes not only is its disease itself, and is its various acute and chronic complication, and diabetes have become the arch-criminal who jeopardizes the human survival quality.
Wherein, diabetic foot ulcers is the main cause that causes the diabetics amputation.Along with global diabetics number increases and the prolongation of the average life span year by year, because of the difficult more ulcer of diabetes foot needs the patient of amputation constantly to increase.In existing 1,600 ten thousand diabeticss of the U.S., 15% patient may foot ulcers occur in its certain stage in life.Investigation shows that the annual foot ulcers neopathy of diabetics rate is at 2%-3%.Annual about 25% diabetics and at least 50% is to be infected by the diabetes foot to cause among the atraumatic lower extremity amputation patient because foot infects and hospitalization.Because diabetes cause foot ulcers sickness rate height, wound healing is slow, and complication is many, is having a strong impact on patient's quality of life.Therefore, early stage understanding and correct this disease of handling have important clinical significance.
In recent years, the wound healing obstacle that causes of diabetes is the emphasis of Chinese scholars research always.Its mechanism relates to many-sides such as cellular metabolism, immunologic function, somatomedin and cell oxygen supply.
This complex process of wound healing has related to following four processes: stable interior environment, inflammatory reaction, cell proliferation reach plastotype again, comprise the mutual adjusting of inflammatory cell, repair cell, cytokine and extracellular matrix.At whole repairing phase, the participation of somatomedin is most important.Somatomedin is one group and has the growth of the cell of promotion, propagation, the protein of anabolic effect or the proper noun of polypeptide.It has the effect that promotes that fibroblast breeds in a large number, collagen fiber obviously accumulate and strengthen the wound surface tension stress, increases endotheliocyte and fibroblastic chemotaxis, makes its migration to damaged part, promotes fibroblast to produce collagen protein; Vascular endothelial cell, keratinocyte and fibroblasts proliferation differentiation be can stimulate, vascularization and re-epithelialization quickened; It is the mitogen and the chemotactic factor of inflammatory cell, and the latter's gathering, threshing then are the important initiating agents of wound repair; In addition, it is synthesizing of scalable extracellular matrix also, promotes ulcer healing jointly.
For some refractory wound surface, there is the scholar to think that somatomedin is dispersed in the extracellular matrix and the hydrolase of proteolysis enhancing causes due to the somatomedin deficiency, so exogenous some somatomedin that replenishes in the wound healing process, or by strengthening the activity of endogenous somatomedin, or the expression of rise growth factor receptors, to reach the effect of initiatively regulating and control wound healing.Can promote wound healing yet somatomedin is applied to wound surface change local microenvironment, but also have the following disadvantages: the somatomedin half-life is shorter; The partial hydrolase of proteolysis of wound surface is stronger, can decompose the raised growth factor; Local heavy dose of somatomedin that uses has side effect; Somatomedin can limit its bioavailability with emulsifiable paste or saline solution as carrier.In order to overcome these deficiencies, will inevitably reuse somatomedin in a large number, cause the waste of medical resource on the one hand, be easy to generate drug resistance on the other hand, thereby cause somatomedin not play one's part to the full.
The somatomedin that with platelet derivation somatomedin (PDGF) is representative has brought breakthrough for the diabetes wound healing in the application of repair in trauma.PDGF was at first found by Ross etc. in 1974, obtained from platelet at first, so be called platelet derivation somatomedin.But found afterwards that many cells beyond the platelet such as endotheliocyte, macrophage, vascular smooth muscle cell etc. can both synthesis secretion PDGF or PDGF like growth factors.PDGF is as a kind of multi-functional cytokine, and is in close relations with wound healing.In wound tissue, the content of PDGF and biological activity thereof change the tissue reconstruction after can influencing repairing quality usually and repairing.PDGF is that important mitogenesis is former, can directly act on tissue repair cells such as fibroblast and keratinocyte, shortens cell cycle, quickens cell proliferation, promotes wound healing.PDGF quicken cell proliferation mechanism may to induce the cell that is in the contact inhibition state to enter cell cycle relevant as starting the factor with PDGF.An akinete (G0 phase cell) enters cell cycle, must and advance two stages through startup.Starting under the effects such as the factor such as PDGF, basic fibroblast and keratinocyte growth factor, relevant promotor gene is expressed, for cell enters S phase accumulated substance and energy, then under the effect that promotes the factor such as epidermal growth factor, transforming growth factor and insulin-like growth factor-i etc., cell enters the S phase very soon, quickens cell proliferation.PDGF is proved with external in vivo with the synergism that promotes the factor.PDGF also can induce fibroblast and keratinocyte excreting insulin like growth factor-1, promotes cell proliferation indirectly.In addition, PDGF can promote the synthetic and secretion of extracellular matrixs such as fibronectin, polydextrose amine and collagen, influences the reconstruction of organizing after the wound repair.
Up to now, PDGF unique proves effectively and by U.S. food and drug administration (FDA) approval and the somatomedin that goes on the market of success clinically in three phases.Becaplermin is that people's recombinant platelet source somatomedin (PDGF-BB) adopts recombinant DNA technology to express production (CA 94608 for U.S.License No.1106, Emeryville) in yeast by U.S. Chiron company; Ortho-McNeil drugmaker successfully is that the Regranex of Becaplermin introduces (U.S.License No.1196 to the market with the effective active composition, San German, Puerto Rico 00683), be used for treating the diabetes foot neurogenic ulcer that subcutaneous deep has sufficient vascularity.Regranex is a kind of carboxymethyl cellulose type ointment, is colourless or pale pink, and 1g contains 100ug Becaplermin, and 2g is arranged at present, and three kinds of specification packings of 7.5g and 15g are limited to external.Ointment formulation is present unique PDGF dosage form.
Chinese patent application numbers 96198773.1 discloses a kind of gel preparation that contains somatomedin, somatomedin is PDGF, gel-type vehicle is a cellulosic polymer, and available electrically charged chemical substance, be selected from comprise lysine, arginine, histidine, protamine, alanine, methionine, proline, serine, aspartic acid, tryptophan, aminoguanidine, the combination of zinc and magnesium, wherein compositions is the at room temperature about 1000-500 of viscosity, the aqueous gel in the 000cps scope.
Two patents of Jinsaishi Biological Pharmaceutical Technology Development Co., Ltd., Beijing's application, application number is respectively 02153958.8: the preparation method of recombinant platelet source somatomedin aqueogel; 02153957.X: the aqueogel of recombinant platelet source somatomedin.They also are the gel preparations of PDGF, the rhPDGF-BB that invention relates to, be to take general technique for gene engineering, make up the hydrogel PDGF-BB bacterial strain of recombined human, amplification culture, fermentation, purification, obtain lipidated protein greater than 97%, protein content is the rhPDGF-BB medicinal liquid sample of 1mg/ml.Hydrogel matrix is Ka Baimu, adds a certain amount of pH regulator agent, isotonic agent, stabilizing agent and antiseptic etc.
Nearest studies show that; extracellular matrix (extracellular matrix; ECM) be the complicated network that constitutes by macromole; not only bring into play physical actions such as support, connection, water conservation, protection statically; for the existence of cell and movablely provide suitable place; and dynamically pair cell produces comprehensive influence, influences shape, metabolism, function, migration, propagation and the differentiation of cell by signal transduction system.Extensively be present in the life deductive procedure in multiple tissue and the organ, extracellular matrix is in the continuous renewal process, and in process of tissue reparation the important especially supporting construction of extracellular matrix.Studies show that extracellular matrix is essential in wound healing.
The research of the mechanism of the chronic difficult healing wound surface of body surface comprises between the change of tissue repair cytoskeleton, the excessive apoptosis of repair cell, somatomedin and receptor in target cell network adjustment " out of control " between the signal transduction " mistake coupling " and the multiple factor.Highlighted somatomedin in the past and promote ulcer healing, and in fact extracellular matrix is bigger to the ulcer healing role, therefore, for the research of ulcer, it is far from being enough studying somatomedin merely, should study in conjunction with extracellular matrix.
Cytoskeleton is selected for use the reason of chitosan derivatives hydrogel to be chitosan aboundresources, low price and is had excellent biological compatibility and biological degradability, catabolite generally has no side effect to human body, do not put aside in vivo, numerous advantages such as non-immunogenicity, apply to ulcer treatment and also show bacteria growing inhibiting, accelerate the advantage of ulcer healing.Yet chitosan itself can not form hydrogel, has only through behind the modification to become glue.
Summary of the invention
The objective of the invention is to overcome prior art, a kind of good hydrophilicity, moisture retention, breathability and pliability and good transgenic cell activation modified chitosan medical dressing are provided.
Second purpose of the present invention provides a kind of preparation method of transgenic cell activation modified chitosan medical dressing.
The 3rd purpose of the present invention provides the application of a kind of transgenic cell activation modified chitosan medical dressing in preparation treatment diabetic ulcer medicine.
Technical scheme of the present invention is summarized as follows:
A kind of transgenic cell activation modified chitosan medical dressing, make with following method: (1) adds cell complete medium solution with the sulfhydrylation chitosan with the 15-25mg/mL ratio, makes culture medium; (2) with the skin flbroblast of transfection platelet derivation growth factor gene or keratinocyte with 4 * 10
4-4 * 10
5The concentration of/mL is inoculated in the culture medium that step (1) makes, with 10-15 rev/min of rotating and culturing 2-5 days.
The molecular weight of described sulfhydrylation chitosan is 20-1000KDa.
The preferred N-acetyl-L-cysteine modification of chitosan of described sulfhydrylation chitosan or 2,3-dimercaptosuccinic acid modification of chitosan can also be selected other for use, as: TGA modification of chitosan, thiacetic acid. alcohol modification of chitosan etc.
Described N-acetyl-L-cysteine modification of chitosan is made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%-3%, stirs until forming clear solution;
(2) be 1 with chitosan and N-acetyl-L-cysteine in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
Described 2,3-dimercaptosuccinic acid modification of chitosan is made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%-3%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1 in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
The preparation of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine aqueous solution is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine to be added to dissolve fully in the entry get final product.
A kind of preparation method of transgenic cell activation modified chitosan medical dressing comprises the steps: that (1) is that the sulfhydrylation chitosan of 20-1000KDa adds cell complete medium solution with the 15-25mg/mL ratio with molecular weight, makes culture medium; (2) skin flbroblast of transfection platelet derivation growth factor gene or keratinocyte are inoculated in the culture medium that step (1) makes, with 10-15 rev/min of rotating and culturing 2-5 days with the concentration of 4 * 104-4 * 105mg/mL.
Described sulfhydrylation chitosan is N-acetyl-L-cysteine modification of chitosan or 2, and 3-dimercaptosuccinic acid modification of chitosan can also be selected other for use, as: TGA modification of chitosan, thiacetic acid. alcohol modification of chitosan etc.
Described N-acetyl-L-cysteine modification of chitosan is made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%-3%, stirs until forming clear solution;
(2) be 1 with chitosan and N-acetyl-L-cysteine in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/LEDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/LNaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2～5 times.
Described 2.3-dimercaptosuccinic acid modification of chitosan is made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%～3%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1 in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after in the aqueous solution of that dropwise add and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2～5 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2～5 times.
The preparation of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine aqueous solution is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine to be added to dissolve fully in the entry get final product.
The application of a kind of transgenic cell activation modified chitosan medical dressing in preparation treatment diabetic ulcer medicine.
Hydrogel adjuvant with this method preparation is used for the treatment of various skin traumas and ulcer, the especially regeneration of diabetic foot neurogenic ulcer and reparation.
Advantage of the present invention:
1. cytoskeleton is selected the natural macromolecular material with antibiotic property for use---and chitosan realizes that by making it form cystine linkage at aqueous solution state after thiolated modified nature glue connection forms gel, this gel has superior hydrophilic, moisture retention, breathability and pliability and good functions such as biological activity, can farthest satisfy the requirement of modern medicine to dressing.
2. seed cell is realized local PDGF-BB somatomedin high expressed, strong secretion by gene recombination technology in the transgenic cell activation modified chitosan medical dressing, regulates and control the ulcer microenvironment jointly with the compound cells epimatrix, reaches the function of reconstruction.
Description of drawings
Fig. 1 is the comparison of different time healing rate between each group.
The specific embodiment
The present invention is further illustrated below by embodiment.
One. the present invention relates to the design and optimization of hydrogel, introduce sulfydryl, realize the hydrogel preparation, measure sulfydryl stability and swelling behavior respectively and it is carried out the physicochemical properties sign by chitosan.With following gained solution shape material, use 0.22 μ m filter to filter and reach aseptic condition, divide be installed on 2-8 ℃ standby.
Embodiment 1
The preparation of N-acetyl-L-cysteine modification of chitosan
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 4 mol ratio, the mass percentage concentration that makes chitosan is 2%, stirs until forming clear solution;
(2) with chitosan and N-acetyl-L-cysteine be 1: 5 ratio in mass ratio, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 5, stirs 3h down in 20 ℃;
(3) take out reactant liquor, dialysed 3 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 3 days under 10 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 3 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 3 times.
Embodiment 2
The preparation of N-acetyl-L-cysteine modification of chitosan
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 1 mol ratio, the mass percentage concentration that makes chitosan is 1%, stirs until forming clear solution;
(2) with chitosan and N-acetyl-L-cysteine be 1: 0.5 ratio in mass ratio, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 4, stirs 4h down in 15 ℃;
(3) take out reactant liquor, dialysed 4 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 4 days under 8 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/LNaCl 5 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 5 times.
Embodiment 3
The preparation of N-acetyl-L-cysteine modification of chitosan
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 8 mol ratio, the mass percentage concentration that makes chitosan is 3%, stirs until forming clear solution;
(2) with chitosan and N-acetyl-L-cysteine be 1: 10 ratio in mass ratio, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 6, stirs 2h down in 25 ℃;
(3) take out reactant liquor, dialysed 2 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 2 days under 15 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2 times.
Embodiment 4
The preparation of N-acetyl-L-cysteine modification of chitosan
N-acetyl-L-cysteine with 500mg chitosan adding 100mg after treating to dissolve fully, dropwise adds the 4mlEDAC aqueous solution, regulates pH to 5 and keeps constant with 1mol/LNaOH, stirs 3h under the room temperature.Take out reactant liquor, under 10 ℃ of lucifuge conditions, in 3d, dialyse once, with the 5mmol/L hydrochloric acid solution dialysis twice that contains 1%NaCl, twice of reuse 1mmol/L hydrochloric acid solution dialysis with the 5mmol/L hydrochloric acid solution that contains 0.2%EDTA.
Embodiment 6
The preparation of TGA modification of chitosan
Take by weighing the 2.5g chitosan, adding 47.5g water, to be made into concentration be 5% chitosan solution.Drip the 2.5g TGA, measure pH value behind the mix homogeneously.Add behind the 1.12mL EDAC stirring reaction 3h under room temperature.Take out mixed solution, under 10 ℃ of lucifuge conditions, use the hydrochloric acid solution of 5mmol/L to dialyse once in the time, 5mmolPL hydrochloric acid/1%NaCl mixed solution dialysis twice, twice of reuse 1mmol/L hydrochloric acid solution dialysis at 3d.
Embodiment 7
The preparation of thiacetic acid. alcohol modification of chitosan
Take by weighing 0.8072g thiacetic acid. alcohol, be made into 8ml solution, add the 4ml acetic anhydride, after stirring, add two concentrated sulphuric acids, be put in the 3h that vibrates in the constant temperature oscillator, placement is spent the night, and water or ammonia are washed till neutrality.
Embodiment 8
2.3-the preparation of dimercaptosuccinic acid modification of chitosan:
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 4 mol ratio, the mass percentage concentration that makes chitosan is 2%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1: 5 ratio in mass ratio, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 5, stirs 3h down in 20 ℃;
(3) take out reactant liquor, dialysed 3 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 3 days under 10 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 3 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 3 times.
Embodiment 9
2.3-the preparation of dimercaptosuccinic acid modification of chitosan:
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 1 mol ratio, the mass percentage concentration that makes chitosan is 1%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1: 0.5 ratio in mass ratio, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 4, stirs 4h down in 15 ℃;
(3) take out reactant liquor, dialysed 2 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 2 days under 8 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2 times.
Embodiment 10
2.3-the preparation of dimercaptosuccinic acid modification of chitosan:
(1) chitosan, I-hydroxybenzotriazole are scattered in the water with 1: 8 mol ratio, the mass percentage concentration that makes chitosan is 3%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1: 10 ratio in mass ratio, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, regulating pH is 6, stirs 2h down in 25 ℃;
(3) take out reactant liquor, dialysed 4 times with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA in 4 days under 15 ℃ of lucifuge conditions, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 5 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 5 times.
The present invention successfully introduces sulfydryl in the chitosan molecule structure after, under solution state, can oxidation form disulfide bond, the mutual glue connection of the latter, the result forms the hydrogel of network interpenetrating.
Two, the modification of engineered seed cell is handled
Embodiment 11
The modification of seed cell: platelet derivation somatomedin (PDGF-BB) genetic modification skin flbroblast or keratinocyte
The total RNA that extracts with Human umbilical vein endothelial cells is a template, synthetic cDNA sequence, regular-PCR amplifying target genes fragment is connected into the pMD-19T carrier, carry out that double digestion is handled and with its product and the pSELECT-GFPzeo-mcs carrier that contains green fluorescent protein and can independently express through T
4Ligase is connected, and successfully makes up the carrier for expression of eukaryon recon pSELECT-GFPzeo-mcs/PDGF-BB of PDGF-BB gene.Further the liposome-mediated rotaring dyeing technology of utilization imports skin flbroblast or keratinocyte with the PDGF-BB gene, to realize the expression of PDGF-BB gene in eukaryotic cell.
Three, hydrogel/cell three-dimensional complex is cultivated in rotation altogether under the microgravity condition
Embodiment 12
A kind of transgenic cell activation modified chitosan medical dressing, make with following method:
(1) molecular weight with embodiment 1 preparation is that 100KDa N-acetyl-L-cysteine modification of chitosan adds cell complete medium solution with the 20mg/mL ratio, makes culture medium; (2) with the skin flbroblast of transfection platelet derivation growth factor gene with 4 * 10
4The concentration of/mL was inoculated in the culture medium that step (1) makes, with 10 rev/mins of rotating and culturing 3 days.(can use 2.3-dimercaptosuccinic acid modification of chitosan or TGA modification of chitosan, thiacetic acid. alcohol modification of chitosan to substitute the N-acetyl-L-cysteine modification of chitosan in the present embodiment, become new embodiment)
Embodiment 13
A kind of transgenic cell activation modified chitosan medical dressing, make with following method:
(1) molecular weight with embodiment 2 preparations is that 20KDa N-acetyl-L-cysteine modification of chitosan adds cell complete medium solution with the 15mg/mL ratio, makes culture medium; (2) with the skin flbroblast of transfection platelet derivation growth factor gene with 4 * 10
4The concentration of/mL was inoculated in the culture medium that step (1) makes, with 10 rev/mins of rotating and culturing 5 days.(can use 2.3-dimercaptosuccinic acid modification of chitosan or TGA modification of chitosan, thiacetic acid. alcohol modification of chitosan to substitute the N-acetyl-L-cysteine modification of chitosan in the present embodiment, become new embodiment)
Embodiment 14
A kind of transgenic cell activation modified chitosan medical dressing, make with following method:
(1) molecular weight with embodiment 3 preparations is that 1000KDa N-acetyl-L-cysteine modification of chitosan adds cell complete medium solution with the 25mg/mL ratio, makes culture medium; (2) with the keratinocyte of transfection platelet derivation growth factor gene with 4 * 10
5The concentration of/mL was inoculated in the culture medium that step (1) makes, with 15 rev/mins of rotating and culturing 2 days.(can use 2.3-dimercaptosuccinic acid modification of chitosan or TGA modification of chitosan, thiacetic acid. alcohol modification of chitosan to substitute the N-acetyl-L-cysteine modification of chitosan in the present embodiment, become new embodiment)
Four, a kind of transgenic cell activation modified chitosan medical dressing (hydrogel/cell three-dimensional complex) treatment diabetic ulcer animal model.
Embodiment 15
Laboratory animal is selected heritability diabetes (db/db) rat for use, adaptability fed for 1 week, experiment fasting on the same day, the clean hair of anesthesia lower back portion, area 9cm * 8cm, back to be sterilized is cut 4 circular wound surface with special card punch altogether in spinal column both sides, back, diameter 2cm, be deep to subcutaneously, after the hemostasis, be divided into 4 groups at random: (1) model control group; (2) medicine (Regranex Gel
*) the treatment group; (3) hydrogel treatment group; (4) a kind of transgenic cell activation modified chitosan medical dressing group (hydrogel/cell three-dimensional complex treatment group).Single cage is fed, and changes aerogel dressing weekly 2 times, until wound healing.Among (see figure 1) Fig. 1, A group: a kind of transgenic cell activation modified chitosan medical dressing group (hydrogel/cell three-dimensional complex group); B group: medicine ((Regranex Gel
*) the treatment group; C group: hydrogel treatment group; D group: model control group.
Treatment back 1,3,7,14,21d in ulcer the instillation isotonic saline solution to liquid level with till edge of wound skin is equal, and note down; Treatment back 1,3,7,14,21d hyaline membrane flap coverage, film is cut in the setting-out of edgewise edge, puts analytical balance and takes by weighing quality and be converted into area, is converted to re-epithelialization speed again.Re-epithelialization speed=(the original wound surface area-wound surface area of not healing)/original wound surface area * 100% (seeing table 1 for details); Variation by digital photographing, computer stored, analysis, comparative observation wound tissue color, form and the speed of growth.
The comparison of table 1 different time wound healing rate
| 3 days (%) | 7 days (%) | 14 days (%) | 21 days (%) | |
| Medical dressing group model matched group of the present invention | 41.89±13.9 56.37±5.07 | 73.71±6.59 61.39±8.2 | 92.71±1.95 84.34±2.71 | 97.61±1.45 89.81±2.91 |
Respectively in treatment 1,3,7,14,21d puts to death 3 animals, the face of whenever forming is left and taken 12 specimen, inserts in the 4% neutral paraformaldehyde solution fixedly 1h, with phosphate buffer eluting 3 times, dewater transparent after, paraffin-embedded tissue is cut into slices, HE dyes.By om observation inflammatory cell (neutrophilic granulocyte, phagocyte etc.) form, infiltration situation and fibroblast, vascular endothelial cell, epithelial cell form and number change, observe its lesion characteristic from tissue and cellular level.Ultrastructure by cell in the electron microscope observation granulation tissue.
By being carried out animal experiment study, a kind of transgenic cell activation modified chitosan medical dressing provided by the invention (hydrogel/cell three-dimensional complex) shows, a kind of transgenic cell activation modified chitosan medical dressing provided by the invention is compared the diabetes rat skin ulcer with Regranex Gel, obviously accelerate the reparation of ulcer, and cost is lower.
The present invention uses (the rotary cell culture system of cell rotating and culturing system of microgravity three-dimensional tissue, RCCS), rat skin fibroblast and keratinocyte are carried out the microgravity three-dimensional histoculture, and the purpose of cell rotating and culturing system of microgravity three-dimensional tissue is to set up the dimensional culture system of zooblast.Through rotation, gravity vector is continued randomization in container for cell and culture fluid, and cell is suspended in the culture medium in the mode of a continuous freely falling body of terminal velocity, thereby is in a kind of simulation free falling body state, is similar to microgravity environment to a certain extent.Cultured cell can produce low-shearing force, low eddy current under microgravity condition, reduces the mechanical damage that the culture fluid pair cell produces, and can increase the transfer function of cytotrophy, quickens the eliminating of metabolite, thereby helps the propagation and the differentiation of cell.In addition, the microgravity rotating and culturing can also be avoided the generation of bubble, can finish gas exchange by filter membrane, and gas, nutritional labeling and metabolic waste can bestly be spread, and these advantages are that static cultural method is beyond one's reach.The present invention successfully turns out a kind of transgenic cell activation modified chitosan medical dressing, can effectively realize somatomedin and extracellular matrix combined effect in ulcer surface to promote its reparation.
The present invention is transfected into skin flbroblast or keratinocyte by the recon that gene recombination technology will contain the PDGF-BB gene, become three-dimensional composite with the chitosan macromolecule hydrogel material co-cultivation in RCCS that contains sulfydryl, be used for the treatment of the diabetic ulcer animal model, realize continuing synthetic consistently and the secretion somatomedin, thereby reach the purpose of using somatomedin effectively and rationally, and with the extracellular matrix combined effect, effectively influence the natural process of tissue repair, shorten wound healing time, function and outward appearance after promoting the healing of difficult more ulcer and effectively improving wound healing.
A kind of transgenic cell activation modified chitosan medical dressing provided by the invention is given full play to the advantage of hydrogel in the Peptic Ulcers treatment, and show: this hydrogel has good barrier function, and water and gas are had two-way permeability; And can effectively resist bacterial invasion, prevent extraneous the infection; Wound surface is in the moistening microenvironment all the time, not only helps the healing of wound, and can soak into the peripheral nerve outside being exposed to, alleviate patient's pain; This hydrogel can effectively absorb wound exudate, avoids adhesion; Hydrogel has good flexibility, plasticity and adhesiveness, with the skin ulcer face contact performance is preferably arranged; Seed cell continues to discharge the effect of somatomedin by gel layer, brings into play therapeutical effect jointly with extracellular matrix; This hydrogel is realized self-crosslinking by the chitosan sulfhydrylation, has excellent biological compatibility, and biodegradable, can not cause the infection of rejection and wound; Its outward appearance is transparence, helps the observation to the wound healing situation; Easy to operate, to wound surface result satisfaction or the like.
Characterize by physicochemical properties a kind of transgenic cell activation modified chitosan medical dressing provided by the invention, the functional examination of contained seed cell and carry out zoopery evaluation model ulcer healing speed, the new vessels number, PDGF-BB and acceptor gene thereof are expressed, the extracellular matrix equal size, result of study shows that a kind of transgenic cell activation modified chitosan medical dressing can reach the process of quickening to organize the nature reparation, effectively shorten wound healing time, function and outward appearance after promoting the healing of difficult more ulcer and effectively improving wound healing, cost performance are better than at present unique by the Regranex Gel of drugs approved by FDA and listing treatment diabetic foot ulcer.
One, cell base culture medium
[constituent]
Composition (mg/L): calcium chloride---200; The L-serine---42; Nine water ferric nitrates---0.1; The L-threonine---95; Potassium chloride---400; The L-tryptophan---16; Anhydrous magnesium sulfate---97.67; L-tyrosine---72; Sodium chloride---6400; The L-valine---94; AMSP---108.7; The D-glucose---1000; The L-arginine monohydrochloride---84; Sodium Pyruvate---110; The L-cystine hydrochloride---63; Phenol red---15; L-glutaminate---584; The D-calcium pantothenate---4; Glycine---30; Choline chloride---4; The L-histidine hydrochloride---42; Folic acid---4; The L-isoleucine---105; The i-inositol---7.2; The L-leucine---105; Nicotiamide---4; L-lysine hydrochloride---146; Pyridoxal hydrochloride---4; The L-methionine---30; Riboflavin---0.4; The L-phenylalanine---66; Thiamine hydrochloride---4
[product index]
Outward appearance: pink solid powder, dissolubility, dissolving fully, solution is clear and bright.
PH value: (1) adds NaHCO
37.50～8.10 (2) do not add NaHCO
36.00～6.60
Moisture (%) :≤5.0
Osmotic pressure (mOsm/kgH
2O): (1) adds NaHCO
3300～332; (2) do not add NaHCO
3238～263
Bacterial endotoxin: (EU/ml)≤10
Microbial check: (CFU/g)≤1000
[storage] 2 ℃ of-8 ℃ of cold preservations, drying, sealing, lucifuge storage.
[specification] 1L/ bag
[effect duration] 2 years
[compound method]
(1) bag culture medium is all poured in the container, residual culture medium in the bag is washed, incorporate into and add injection water (20 ℃-30 ℃ of water temperatures) behind the container, the gentle agitation dissolving to 950mL with a small amount of water for injection;
(2) add the 3.7g sodium bicarbonate;
(3) gentle agitation dissolving adds the injection water to 1L;
(4) transfer pH to desirable value with 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution;
(5) with 0.2 μ m filter membrane malleation filtration sterilization;
(6) solution should keep in Dark Place under 2 ℃-8 ℃.
Two, cell complete medium
[compound method]
(1) gets basal medium 88.5ml after the filtration;
(2) add hyclone 10ml;
(3) add glutamine 1ml;
(4) penicillin of adding mixing and streptomycin (two anti-) solution 0.5ml;
(5) mixing is regulated pH value to 7.2-7.4, keeps in Dark Place under 2 ℃-8 ℃.
Claims (10)
1. transgenic cell activation modified chitosan medical dressing is characterized in that making with following method: (1) adds cell complete medium solution with the sulfhydrylation chitosan with the 15-25mg/mL ratio, makes culture medium; (2) with the skin flbroblast of transfection platelet derivation growth factor gene or keratinocyte with 4 * 10
4-4 * 10
5The concentration of/mL is inoculated in the culture medium that step (1) makes, with 10-15 rev/min of rotating and culturing 2-5 days.
2. a kind of transgenic cell activation modified chitosan medical dressing according to claim 1, the molecular weight that it is characterized in that described sulfhydrylation chitosan is 20-1000KDa.
3. a kind of transgenic cell activation modified chitosan medical dressing according to claim 1 is characterized in that described sulfhydrylation chitosan is N-acetyl-L-cysteine modification of chitosan or 2,3-dimercaptosuccinic acid modification of chitosan.
4. a kind of transgenic cell activation modified chitosan medical dressing according to claim 3 is characterized in that described N-acetyl-L-cysteine modification of chitosan made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%～3%, stirs until forming clear solution;
(2) be 1 with chitosan and N-acetyl-L-cysteine in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
5. a kind of transgenic cell activation modified chitosan medical dressing according to claim 3 is characterized in that describedly 2, and 3-dimercaptosuccinic acid modification of chitosan is made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%～3%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1 in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2 ~ 5 times, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
6. the preparation method of a transgenic cell activation modified chitosan medical dressing, it is characterized in that comprising the steps: that (1) is that the sulfhydrylation chitosan of 20-1000KDa adds cell complete medium solution with the 15-25mg/mL ratio with molecular weight, make culture medium; (2) with the skin flbroblast of transfection platelet derivation growth factor gene or keratinocyte with 4 * 10
4-4 * 10
5The concentration of/mL is inoculated in the culture medium that step (1) makes, with 10-15 rev/min of rotating and culturing 2-5 days.
7. the preparation method of a kind of transgenic cell activation modified chitosan medical dressing according to claim 6 is characterized in that described sulfhydrylation chitosan is N-acetyl-L-cysteine modification of chitosan or 2,3-dimercaptosuccinic acid modification of chitosan.
8. the preparation method of a kind of transgenic cell activation modified chitosan medical dressing according to claim 7 is characterized in that described N-acetyl-L-cysteine modification of chitosan made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%～3%, stirs until forming clear solution;
(2) be 1 with chitosan and N-acetyl-L-cysteine in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add N-acetyl-L-cysteine, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/L NaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
9. the preparation method of a kind of transgenic cell activation modified chitosan medical dressing according to claim 7 is characterized in that described 2.3-dimercaptosuccinic acid modification of chitosan made by following method:
(1) with chitosan, I-hydroxybenzotriazole with 1: the mol ratio of 1-8 is scattered in the water, and the mass percentage concentration that makes chitosan is 1%-3%, stirs until forming clear solution;
(2) with chitosan and 2, the 3-dimercaptosuccinic acid is 1 in mass ratio: the ratio of 0.5-10, in above-mentioned solution, add 2, the 3-dimercaptosuccinic acid, after dropwise add in the aqueous solution with 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine preparation of quality such as chitosan, adjusting pH is 4-6, stirs 2-4h down in 15-25 ℃;
(3) take out reactant liquor, under 8-15 ℃ of lucifuge condition, in 2-4 days, dialyse 2-4 time with the 5mmol/L aqueous hydrochloric acid solution that contains 2 μ mol/L EDTA, with the 5mmol/L aqueous hydrochloric acid solution dialysis that contains 1g/LNaCl 2-5 time, the dialysis of reuse 1mmol/L aqueous hydrochloric acid solution is made for 2-5 time.
10. the application of a kind of transgenic cell activation modified chitosan medical dressing of one of claim 1-5 in preparation treatment diabetic ulcer medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100587383A CN101125216A (en) | 2007-08-14 | 2007-08-14 | Transgenic cell activation modified chitosan medical dressing and preparation method and application |
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|---|---|---|---|
| CNA2007100587383A CN101125216A (en) | 2007-08-14 | 2007-08-14 | Transgenic cell activation modified chitosan medical dressing and preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210733A (en) * | 2010-04-08 | 2011-10-12 | 中国科学院兰州化学物理研究所 | Method for preparing thiolated chitosan coated ginseng nano particles |
| CN114306717A (en) * | 2021-11-30 | 2022-04-12 | 中国科学院南海海洋研究所 | Marine biomedical material for repairing skin wounds and preparation method and application thereof |
-
2007
- 2007-08-14 CN CNA2007100587383A patent/CN101125216A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102210733A (en) * | 2010-04-08 | 2011-10-12 | 中国科学院兰州化学物理研究所 | Method for preparing thiolated chitosan coated ginseng nano particles |
| CN114306717A (en) * | 2021-11-30 | 2022-04-12 | 中国科学院南海海洋研究所 | Marine biomedical material for repairing skin wounds and preparation method and application thereof |
| CN114306717B (en) * | 2021-11-30 | 2022-07-26 | 中国科学院南海海洋研究所 | Marine biomedical material for repairing skin wounds and preparation method and application thereof |
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