A kind of organization engineering skin and its construction method based on omentum majus acellular matrixTechnical field
The invention belongs to organizational project and regeneration medicine technology field, and in particular to it is a kind of using omentum majus acellular matrix asThe organization engineering skin and its construction method of timbering material.
Background technology
Skin is the primary barrier for protecting body from external world's invasion and attack, is played extremely in body homeostasis is maintainedImportant effect.Severe trauma, large-area burns, ablation of tumors etc. cause traditional treatment method of defect of skin to be moved for skinPlant, however dermatoplasty at present there are problems that donor source it is not enough and, therefore in the urgent need to one kind reasonThe Graftskin thought repairs injured skin.By Method of Tissue Engineering, it can be built using cell and extracellular matrixGo out the artificial skin with certain function, new method is provided to build Graftskin and skin injury clinical repair.
Organization engineering skin is that the reparing skin defect surface of a wound brings hope, and at present, portion of tissue engineering skin product isIt is latent with huge development applied to clinic, such as Integra, Dermagraft, Alloderm organization engineering skin productPower.But its transplanting survival rate is significantly lower than auto-skin grafting, clinical effectiveness is still not very obvious.Main reason is that tissueEngineering skin lacks enough vascularizations and causes ischemic injuries after being largely limited by transplanting, and the cell in turn resulted in is suppliedSupport deficiency, or even graft failure.
Just because of this, domestic and international researcher has carried out numerous studies in terms of organization engineering skin vascularization.At present, promoteEnter the method for organization engineering skin vascularization mainly the direct application including angiogenic factors, addition vascular endothelial cell,Encode particular growth factor gene transfection seed cell etc..The above method is played in terms of organization engineering skin vascularization is solvedCertain effect, but there is also obvious limitation.For example, adding the exogenous endothelium life of blood vessel when organization engineering skin is builtThe regulatory factors such as the long factor (VEGF), basic fibroblast growth factor (bFGF), although can chemotactic host ECs, rushEnter the vascularization of engineered skin.However, exogenous growth factor is expensive, Half-life in vivo is short, and high dose is when applyingHemorheology may be caused abnormal, and then produce serious side effects.And add vascular endothelial cell when building engineered skinMethod there is also autologous source of endothelial cells is limited and materials process produces the deficiency such as wound to body, not only such asThis, vascular endothelial cell is generally terminally differentiated cells, its ability bred and the equal Shortcomings of cytokine secretion ability, limitationIn the application of organization engineering skin.Coding particular growth factor gene transfection seed cell also be present.CauseThis, needs badly and finds the new method for being capable of promotion organization engineering skin vascularization and strategy.
The appearance of de- cell technology brings new hope for the structure and its vascularization of organization engineering skin.It is thin using taking offNatural biological timbering material and acellular matrix that born of the same parents' technology removes various cell components and inhereditary material in tissue and obtainedMaterial, remains the three-dimensional structure and natural component of extracellular matrix, is conducive to cell adherence, propagation, differentiation and its biologyThe performance of function, in tissue repair with having the incomparable advantage of other timbering materials in regeneration.At present, researcher has beenObtain the acellular matrix timbering material of various different tissue sources, such as small intestine, skin, cornea, the heart, lung, liver, kidney.
Omentum majus (greater omentum) be connect greater curvature to transverse colon peritoneal tissues, between stomach and intestines toPreceding bulging, in the sagging formation pleat in the front of intestines, hides by empty, ileum in apron shape.Omentum majus wide material sources, very vascular,Rich in all kinds of growth factors, the internal transplanting platform of all kinds of grafts is widely used at present.For example, it can be used for surgery weightBuild the reparation to inflammatory or defective tissue, the vascularization and regeneration of promotion organization in operation.However, omentum majus takes off cell at presentMatrix correlative study is at the early-stage, there is not yet using omentum majus acellular matrix as timbering material, building the phase of organization engineering skinClose research report.
The content of the invention
According to an aspect of the invention, there is provided the construction method of organization engineering skin, comprises the following steps：
(1) omentum majus acellular matrix timbering material is prepared, it is including the use of slow containing lauryl sodium sulfate and DNA enzymaticFliud flushing immersion omentum majus carries out the step of de- cell is handled；And
(2) organization engineering skin is built and culture.
There is provided the construction method of organization engineering skin according to another aspect of the present invention, it is characterised in that according to followingOperating procedure is carried out：
(1) omentum majus acellular matrix timbering material is prepared
Fresh big net membrane tissue concussion is soaked in and mixed by methanol, chloroform with ether using volume ratio as 1: 1: 0.2-1Handled into degreasant solution, degreasant solution soak time is 18-30 hours, concussion frequency is 150-250r/min；Then, willThe omentum majus for removing fat is organized in cleaning in PBS, and concussion is soaked in lauryl sodium sulfate/DNA enzymatic content from high to lowGradient de-cell liquid in handle, lauryl sodium sulfate/DNA enzymatic content is respectively 1.5-3.5% weights in gradient de-cell liquidMeasure the lauryl sodium sulfate/3500-4500U/L DNA enzymatic of ratio, 0.6-1.5% weight than lauryl sodium sulfate/2500-3500U/L DNA enzymatic, 0.1-0.6% weight than lauryl sodium sulfate/1500-2500U/L DNA enzymatic, per ladderIt is 4-12h to spend solution soak time, and concussion frequency is 100-200r/min, finally, big after being handled through gradient de-cell liquidThe freeze-dried rear irradiation sterilization of omental organization；And
(2) organization engineering skin is built and culture
By 5 × 105-1×107/ mL skin fibroblasts suspension 1-3mL is inoculated in the omentum majus prepared in step (1)On acellular matrix material, compound construction is obtained；The compound construction is incubated at 37 DEG C, 5%CO21-2 is small in incubatorWhen, DMEM nutrient solution 10-15mL are then added, continue to cultivate to the 7th day, compound construction is then subjected to gas-liquid face culture,A nutrient solution is changed daily, and organization engineering skin can be obtained by terminating culture within the 14-28 days.
There is provided the organization engineering skin obtained according to the above method according to another aspect of the present invention.
Compared with the organization engineering skin that other modes are built, the organization engineering skin built in the present invention is de- with omentum majusCellular matrix is timbering material, and due to being rich in angiogenic growth factor in omentum majus acellular matrix, it can promote capillaryThe organization engineering skin of transplanting is grown into by the surface of a wound, so as to be conducive to the vascularization and wound healing of organization engineering skin, to tissueThe clinical practice of engineering skin is significant.In addition, medium-height trestle material source of the present invention is extensive, with low cost, operative employeeSkill is simple.
Brief description of the drawings
Organization engineering skins of the Fig. 1 based on omentum majus acellular matrix live/dead (Live/Dead) dyeing × 200.
Vegf expression situation (* p ＜ 0.01) in organization engineering skins of the Fig. 2 based on omentum majus acellular matrix.
BFGF expressions (* p ＜ 0.01) in organization engineering skins of the Fig. 3 based on omentum majus acellular matrix.
The HE dyeing × 200 after skin wound is transplanted 7 days of Fig. 4 organization engineering skins.
Embodiment
In this application, the implication of abbreviation is as follows：
DMEM：Dulbecco ' s modified eagle medium nutrient solutions are a kind of containing various amino acid and grapeThe culture medium of sugar；
HEPEs：4- hydroxyethyl piperazineethanesulfonic acids；
PBS：Phosphate buffer；
SDS：Lauryl sodium sulfate；
VEGF：The exogenous endothelial growth factors of blood vessel；
bFGF：Basic fibroblast growth factor；
HE is dyed：Hematoxylin eosin staining；
EthD-III：Ethidium bromide homodimer-III；
calcein AM：Diacetyl methyl esters
DNA enzymatic：Deoxyribonuclease；
min：Minute.
According to an aspect of the invention, there is provided the construction method of organization engineering skin, comprises the following steps：
(1) omentum majus acellular matrix timbering material is prepared, it is including the use of slow containing lauryl sodium sulfate and DNA enzymaticFliud flushing immersion omentum majus carries out the step of de- cell is handled；And
(2) organization engineering skin is built and culture.
In certain embodiments, also shaken in the step of de- cell is handled is carried out.
In certain embodiments, the step of step (1) also includes ungrease treatment.
In preferred embodiments, the step of ungrease treatment is carried out before the step of de- cell is handled.
In preferred embodiments, the step of ungrease treatment uses methanol, chloroform and the second that volume ratio is 1: 1: 0.2-1The mixed solution of ether is carried out.In some further preferred embodiments, the volume ratio of methanol, chloroform and ether is 1: 1: 0.4-0.7.In the most preferred embodiment, the volume ratio of methanol, chloroform and ether is 1: 1: 0.5.
In certain embodiments, the step of de- cell is handled is that point gradient is carried out, and the gradient includes at least firstGradient and the second gradient.
In certain preferred aspects, in first gradient, the concentration of lauryl sodium sulfate is 1.5-3.5% weightsAmount ratio, the concentration of DNA enzymatic is 3500-4500U/L, and pH is 7.2-7.4.In some further preferred embodiments, in the first ladderIn degree, the concentration of lauryl sodium sulfate is 1.5-2.5% weight ratio, and the concentration of DNA enzymatic is 3600-4400U/L.It is some moreIn preferred embodiment, in first gradient, the concentration of lauryl sodium sulfate is 1.8-2.2% weight ratio, DNA enzymatic it is denseSpend for 3800-4200U/L.In the most preferred embodiment, in first gradient, the concentration of lauryl sodium sulfate is2.0% weight ratio, the concentration of DNA enzymatic is 4000U/L.
In certain preferred aspects, in the second gradient, the concentration of lauryl sodium sulfate is 0.6-1.5% weightsAmount ratio, the concentration of DNA enzymatic is 2500-3500U/L, and pH is 7.2-7.4.In some further preferred embodiments, in the second ladderIn degree, the concentration of lauryl sodium sulfate is 0.7-1.3% weight ratio, and the concentration of DNA enzymatic is 2600-3400U/L.It is some moreIn preferred embodiment, in the second gradient, the concentration of lauryl sodium sulfate is 0.8-1.2% weight ratio, DNA enzymatic it is denseSpend for 2800-3200U/L.In the most preferred embodiment, in the second gradient, the concentration of lauryl sodium sulfate is1.0% weight ratio, the concentration of DNA enzymatic is 3000U/L.
In certain preferred aspects, the gradient also includes 3rd gradient, wherein in the 3rd gradient, tenThe concentration of sodium dialkyl sulfate is 0.1-0.6% weight ratio, and the concentration of DNA enzymatic is 1500-2500U/L, and pH is 7.2-7.4.In some further preferred embodiments, in 3rd gradient, the concentration of lauryl sodium sulfate is 0.2-0.6% weight ratio, DNAThe concentration of enzyme is 1600-2400U/L.In some further preferred embodiments, in 3rd gradient, lauryl sodium sulfateConcentration is 0.4-0.6% weight ratio, and the concentration of DNA enzymatic is 1800-2200U/L.In the most preferred embodiment, in the 3rd ladderIn degree, the concentration of lauryl sodium sulfate is 0.5% weight ratio, and the concentration of DNA enzymatic is 2000U/L.
There is provided the construction method of organization engineering skin according to another aspect of the present invention, it is characterised in that according to followingOperating procedure is carried out：
(1) omentum majus acellular matrix timbering material is prepared
Fresh big net membrane tissue concussion is soaked in and mixed by methanol, chloroform with ether using volume ratio as 1: 1: 0.2-1Handled into degreasant solution, degreasant solution soak time is 18-30 hours, concussion frequency is 150-250r/min；Then, willThe omentum majus for removing fat is organized in cleaning in PBS, and concussion is soaked in lauryl sodium sulfate/DNA enzymatic content from high to lowGradient de-cell liquid in handle, lauryl sodium sulfate/DNA enzymatic content is respectively 1.5-3.5% weights in gradient de-cell liquidMeasure the lauryl sodium sulfate/3500-4500U/L DNA enzymatic of ratio, 0.6-1.5% weight than lauryl sodium sulfate/2500-3500U/L DNA enzymatic, 0.1-0.6% weight than lauryl sodium sulfate/1500-2500U/L DNA enzymatic, per ladderIt is 4-12h to spend solution soak time, and concussion frequency is 100-200r/min, finally, big after being handled through gradient de-cell liquidThe freeze-dried rear irradiation sterilization of omental organization；And
(2) organization engineering skin is built and culture
By 5 × 105-1×107/ mL skin fibroblasts suspension 1-3mL is inoculated in the omentum majus prepared in step (1)On acellular matrix material, compound construction is obtained；The compound construction is incubated at 37 DEG C, 5%CO21-2 is small in incubatorWhen, DMEM nutrient solution 10-15mL are then added, continue to cultivate to the 7th day, compound construction is then subjected to gas-liquid face culture,A nutrient solution is changed daily, and organization engineering skin can be obtained by terminating culture within the 14-28 days.
In certain embodiments, the omentum majus in step (2) is from mammals such as pig, ox, sheep.
In this application, omentum majus is commercially available, can be purchased from such as slaughterhouse.
In certain embodiments, DMEM nutrient solutions are containing the DMEM cultures that concentration of volume percent is 10-15% serumBase；Wherein, the compound method of DMEM culture mediums for DMEM culture medium dry powders one are wrapped, NaHCO32.4g, HEPEs 2.383g are moltenSolve and constant volume is in 1L ultra-pure waters.
There is provided the organization engineering skin obtained according to the above method according to another aspect of the present invention.
The present invention is described in detail by following examples, but following examples are merely possible to illustration, it is not rightThe present invention constitutes any limitation.
Reagent matches somebody with somebody needed for the organization engineering skin based on omentum majus acellular matrix used in following examples is builtSystem：
1)PBS：Weigh 8g NaCl, 0.2g KCl, 3.491g Na2HPO4·12H2O, 0.2g KH2PO4, it is dissolved in 1L and surpassesIn pure water, regulation pH value is 7.2-7.4,121 DEG C of high pressure steam sterilization 20min, 4 DEG C of preservations.
2) degreasant solution：400mL methanol, 400mL chloroforms and 200mL ether are mixed.
3) de-cell liquid I：Weigh 20g SDS to be dissolved in PBS, simultaneously constant volume, in 1L, reaches DNA enzymatic concentration to addition DNA enzymaticTo 4000U/L, pH to 7.2-7.4 is adjusted, normal temperature is preserved after filtration sterilization.
4) de-cell liquid II：Weigh 10g SDS to be dissolved in PBS, simultaneously constant volume, in 1L, reaches DNA enzymatic concentration to addition DNA enzymaticTo 3000U/L, pH to 7.2-7.4 is adjusted, normal temperature is preserved after filtration sterilization.
5) de-cell liquid III：Weigh 5g SDS to be dissolved in PBS, simultaneously constant volume, in 1L, reaches DNA enzymatic concentration to addition DNA enzymaticTo 2000U/L, pH to 7.2-7.4 is adjusted, normal temperature is preserved after filtration sterilization.
6) DMEM cell culture mediums：One bag of DMEM dehydrated mediums, 3.7g NaHCO3, 2.383g HEPES are dissolved in 1LIn ultra-pure water, regulation pH value is 7.2-7.4, degerming through 0.22 μm of filtering with microporous membrane, 4 DEG C of preservations.10% tire is added when usingCow's serum (FBS).
7) 4% paraformaldehyde fixer：0.1M PBS solution 1L is heated to seething with excitement, 40g paraformaldehydes is added, stirs moltenSolution, regulation pH value is 7.2-7.4, filtering and impurity removing matter, 4 DEG C of preservations.
Fell skin tissue used is donated by Chinese People's Liberation Army General Hospital's medical science in embodiment.
The culture of the human skin fibroblasts of embodiment 1
By fell skin tissue after PBS, shred that (tissue block size is about 0.1cm3) to be placed in 0.5% clostridiopetidase A moltenIn liquid, digested 2 hours in 37 DEG C of incubators.Then, by 200 mesh sieve net filtrations, PBS to 10mL, 800rpm centrifugation 5min are added,Clostridiopetidase A is washed away, precipitation is skin fibroblasts.Supernatant discarding, adds DMEM nutrient solutions and cell suspension is made, by 1 ×106/ mL is seeded in blake bottle, puts 37 DEG C, 5%CO2Incubator culture.
Build before organization engineering skin, by the cell of culture by Trypsin Induced, be resuspended with DMEM nutrient solutions thinBorn of the same parents, and it is 5 × 10 to prepare cell density6/ mL skin fibroblasts suspension.
The preparation of the omentum majus acellular matrix of embodiment 2
Fresh miniature pig peritonaeum big net membrane tissue (being purchased from slaughterhouse) is taken, is cleaned 3 times in PBS.By the group after cleaningKnit concussion and be soaked in 2L degreasant solutions and handle 24h, concussion frequency is 200r/min.Then, the omentum majus group of fat will be removedIt is woven in PBS and cleans 3 times, and concussion is soaked in 1L de-cell liquids I and handles 8h, concussion frequency is 150r/min.Then, will be throughOmentum majus after de-cell liquid I processing, which is organized in PBS, to be cleaned 3 times, and concussion is soaked in 1L de-cell liquids II and handles 8h, shakesSwing frequency 150r/min.Then, the omentum majus after being handled through de-cell liquid II is organized in PBS and cleaned 3 times, and shake immersion8h is handled in 1L de-cell liquids III, concussion frequency is 150r/min.Finally, by the omentum majus after being handled through de-cell liquid IIIFreeze-dried rear irradiation sterilization is organized, normal temperature is preserved.
The structure of organization engineering skin of the embodiment 3 based on omentum majus acellular matrix and culture
By 5 × 106ML skin fibroblasts suspension 2mL is inoculated on the omentum majus acellular matrix material of preparation,Obtain compound construction.Compound construction is incubated at 37 DEG C, 5%CO21.5h in incubator, then adds DMEM nutrient solutions10mL, continued to cultivate to the 7th day, and compound construction then is carried out into gas-liquid face culture, a nutrient solution, the 21st day are changed dailyTerminate culture and obtain organization engineering skin.
The cytoactive detection of organization engineering skin of the embodiment 4 based on omentum majus acellular matrix
Take the organization engineering skin of culture 21 days in new culture dish, 20 μ l 2mM EthD-III are drawn with liquid-transfering gunSolution is added in 10mL PBS, obtains 4 μM of EthD-III working solutions, then 5 μ l 4mM calcein AM solution are transferred toIn EthD-III working solutions, mixed liquor (2 μM of calcein AM and 4 μM of EthD-III) is configured to.Then add enoughLive/Dead mixed liquors are to cover organization engineering skin, and then in incubation at room temperature 30-45min, PBS solution is cleaned three times.GlimmeringThe cell of viewed under light microscopy fluorescence labeling.
It is dyed, because living cells has esterase active, non-fluorescence calcein AM can be changed into energy green-emitting glimmeringThe calcein of light, and because the cell membrane of dead cell loses selectivity, EthD-III is attached on nucleus into cell, is madeRed fluorescence is presented in dead cell.Live/dead coloration results are visible, a large amount of cell survivals in the organization engineering skin of culture 21 daysWell (green), only a small amount of cell death (red) occurs, and shows the organization engineering skin based on omentum majus acellular matrixWith good activity (Fig. 1).
The rush Angiogenesis detection of expression of organization engineering skin of the embodiment 5 based on omentum majus acellular matrix
Detect and compare based on omentum majus acellular matrix organizational project skin by enzyme linked immunoassay determination method (ELISA)Promote Angiogenesis VEGF and bFGF expression in skin and organization engineering skin based on collagen scaffold.Weigh respectively100mg or so above two engineered skins, take supernatant after homogenate, set blank control group, standard item group, sample sets.In enzymeMark standard items on coating plate and be accurately loaded in 50 μ l, testing sample hole and first add the μ l of sample diluting liquid 40, testing sample is then added again10μl；With the rearmounted 37 DEG C of incubations 30min of shrouding film shrouding；Then, carefully take shrouding film off, discard liquid, drying, filled it up with per holeCleaning solution, stand 30s after discard, so repeatedly 5 times, pat dry.Then added per hole except the μ l of enzyme marking reagent 50, blank well；ThroughAfter incubation, washing, developer A50 μ l are first added per hole, developer B50 μ l are added, gently concussion is mixed, 37 DEG C of lucifuges develop the color10min；Finally, the μ l terminating reactions of terminate liquid 50 are added per hole；With blank air-conditioning zero, 450nm wavelength sequentially measures the suction in each holeLight value.Standard curve is drawn, sample standard concentration is calculated, is multiplied by the actual concentrations of extension rate, as sample.As a result find,Base is all remarkably higher than based on the expression for promoting Angiogenesis VEGF and bFGF in omentum majus acellular matrix organization engineering skinIn the organization engineering skin of collagen scaffold, show that what the present invention built is had based on omentum majus acellular matrix organization engineering skinGood rush blood vessel function (Fig. 2, Fig. 3).
The foundation of the rat wound infection model of embodiment 6 and organization engineering skin xenotransplantation experiment
Rats by intraperitoneal injection 0.5mL 2% Nembutal sodium solution is anaesthetized.Then, 75% is sprayed in rat backAlcohol disinfecting, then shaves its back with scissors and covers hair, with depilatory cream that the hair of operative region is de- clean；Delimited in rat back1.5 × 1.5cm square field of operation, then cuts off its full skin；By organization engineering skin flap coverage, with suture by itself and weekSkin closure is enclosed, aseptic dressing pressure dressing is fixed.The conventional tincture of iodine, alcohol disinfecting, and more change dressings two days later, are removed after 7 daysSuture, the organization engineering skin sample for cutting part transplanting carries out making histological examination.HE coloration results show, based on omentum majusThe organization engineering skin of acellular matrix can effectively facilitate wound healing, and inflammatory cell is significantly reduced, and can significantly increase damageThe vascularization degree (Fig. 4) in region.