CN104096265A - Preparation method of three-dimensional contraction model for constructing artificial blood vessel model - Google Patents
Preparation method of three-dimensional contraction model for constructing artificial blood vessel model Download PDFInfo
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Abstract
The invention relates to a preparation method of a three-dimensional contraction model for constructing an artificial blood vessel model, and belongs to the technical field of blood vessel models. The method comprises the steps of preparing a collencyte mixed solution, crosslinking and solidifying the collencyte mixed solution, and preparing the three-dimensional contraction model. The prepared three-dimensional contraction model serves as an improved contraction model, adopts myofibroblast IMR-90, grows faster, and is shorter in preparation cycle; auxiliary contraction factors U0126 and TGF-beta are added to stimulate the myofibroblast, so that the three-dimensional contraction model expresses contractive protein, has better contractility, and further has higher mechanical strength; and the prepared three-dimensional contraction model can provide a better and faster contraction model for scientific research, and can provide reference for later clinical application.
Description
Technical field
The invention belongs to vascular pattern technical field, be specifically related to a kind of for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model.
Background technology
Three-dimensional shrinkage model is used in the structure artificial blood vessel model in scientific research or biological engineering more at present, and vascular applications is in clinical even as an alternative.Clinically, although for there has been more ripe biomaterial to substitute compared with trunk, for the small artery such as coronary artery of heart, but still can not find desirable alternative goods.Although native blood vessel can be used as a kind of selection, there is angiopathy in many sufferers, and as atherosclerosis, this makes to find suitable biological substitution goods becomes possible a kind of Therapeutic Method., there is the shortcomings such as the cell culture time is long, and mechanical strength is inadequate in the three-dimensional shrinkage model production method of current utilizable scientific research, Minitype manual vascular applications is restricted.So it is extremely urgent to provide the similar three-dimensional shrinkage model of a kind of scientific and reasonable manufacturing artificial blood vessel to be applied to scientific research.
In the documents such as Small-Diameter Artificial Arteries Engineered In Vitro and Phenotype Mod μ lation in Vasc μ lar Tissue Engineering Using Biochemical and Mechanical Stim μ lation, mentioned a kind of three-dimensional shrinkage model, it is a kind of cylinder shape belt inner core model, inner core is fixed on columniform mold center, the vascular smooth muscle cell that suspended of digestion and type i collagen/fibrin are mixed to this three-dimensional hollow model of injection, put into 37 ℃ after 30 minutes to 1 hour wait collagen cross-linkings.For increasing the mechanical force of model, somebody also did additional mechanical force to be stimulated, and as pressurization etc., and biotic factor stimulates, as TGF-β, PDGF etc.The shortcomings such as but they all exist the cell culture time long, propagation is slow, and the threedimensional model mechanical strength that builds is not high.Therefore, how overcoming the deficiencies in the prior art is problems that current vascular pattern technical field is needed solution badly.
Summary of the invention
The cell proliferation that the present invention is directed to current three-dimensional shrinkage modelling process existence is slow, and the shortcoming that mechanical strength is low, provides a kind of for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model.
The technical solution used in the present invention is as follows:
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 3.9～4.1g type i collagen, 200 μ l10 * phosphate buffers, 10.5～10.9 μ l concentration, simultaneously add hyclone, DMEM culture medium and 19～21*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, and mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the built-in glass tubing inner core of cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor that step (1) is obtained injects cylindrical model device or Tissue Culture Plate, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked cylindrical collagenocyte mixed liquor having solidified or the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: the crosslinked cylindrical collagenocyte mixed liquor having solidified that step (2) is obtained is squeezed in centrifuge tube, then to adding hyclone and DMEM culture medium in centrifuge tube or the Tissue Culture Plate that contains the crosslinked disc collagenocyte mixed liquor having solidified, be that the mixed liquor of mixing in 1: 9 was not to have collagenocyte mixed liquor by volume, after incubated overnight, changing DMEM culture medium continues to cultivate 12 hours, the addition of described DMEM culture medium was not there to be collagenocyte mixed liquor to be limited, then in DMEM culture medium, add U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 30-60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 200pM-400pM, continue to cultivate 96 hours, obtain three-dimensional shrinkage cylinder model or three-dimensional shrinkage disk model.
Described Tissue Culture Plate is 24 porocyte culture plates of low adhesion.
Cylindrical tube described in step (2) is plastic tube.
Described plastic tube is Teflon pipe.
Described in step (2), rubber stopper is the rubber stopper in syringe.
It is with syringe core rod, to enforce the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) to squeeze in centrifuge tube that the crosslinked cylindrical collagenocyte mixed liquor having solidified that step (2) is obtained is squeezed into the mode adopting in centrifuge tube.
Above-mentioned for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 19～21*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, and mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the built-in glass tubing inner core of Teflon cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor 2ml that step (1) is obtained injects cylindrical model device, then puts it into 37.Incubator was vertically cultivated after half an hour, obtained the crosslinked cylindrical collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) and squeeze in centrifuge tube, then to adding 8ml hyclone and DMEM culture medium in centrifuge tube, be the mixed liquor mixing at 1: 9 by volume, after incubated overnight, changing 9mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 9 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 45min, after 30-60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 300pM, continue to cultivate 96 hours, obtain three-dimensional shrinkage cylinder model.
The preparation method of the three-dimensional shrinkage model of above-mentioned structure artificial blood vessel model, comprises the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 20*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, and mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the collagenocyte mixed liquor 0.5ml that step (1) is obtained injects 24 porocyte culture plates of low adhesion, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: be the mixed liquor of mixing in 1: 9 by volume to adding 1ml hyclone and DMEM culture medium in 24 porocyte culture plates of the low adhesion that contains the crosslinked disc collagenocyte mixed liquor having solidified, after incubated overnight, changing 1mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 1 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 50min, add again and add again TGF-β, the concentration that makes TGF-β in culture medium is 280pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage disk model of invention.
The present invention compared with prior art, its beneficial effect is: (1) is provided by the invention a kind of for building three-dimensional shrinkage model that the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model makes as a kind of improved contracting model, select myofibroblast IMR-90, grow faster, the analogue formation cycle is shorter; (2) simultaneously, add auxiliary contraction factor U0126 and TGF-β irritation cell, make it express contractive protein, there is better shrinkage, and then there is stronger mechanical strength; (3) the three-dimensional shrinkage model that the present invention makes can provide contracting model better more efficiently for scientific research, and the clinical reference that provides is provided after can be.
Accompanying drawing explanation
Fig. 1 is cylindrical model apparatus structure schematic diagram of the present invention;
1-cylindrical tube, 2-glass tubing inner core, 3-rubber stopper;
Fig. 2 adds the comparison schematic diagram of the various factors in disc model equipment structure of the present invention; Wherein, 21-cultivates half an hour; 22-not dosing cultivates 12 hours; 23-drug treating 3 days;
Fig. 3 is that three-dimensional shrinkage model protein immunoblotting of the present invention detects collection of illustrative plates;
Fig. 4 is that three-dimensional shrinkage model protein immunoblotting of the present invention detects collection of illustrative plates.
Specific embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Cell is prepared, and the present invention selects myofibroblast (IMR-90) purchased from American Type Culture Collecti (ATCC).Myofibroblast cell can be expressed smooth muscle contraction albumen Smooth muscle α-actin (α-actin), calcium conditioning albumen (calponin) and smooth muscle 22 α (SM22 α).Meanwhile, IMR-90 Growth of Cells speed is faster compared with vascular smooth muscle cell, and incubation time is shorter.It is approximately 20*10 that a cylindrical model is used cell quantity
6individual; A disc model is used the about 5*10 of cell number
6individual.
U0126 is p-ERK specific inhibitor.
Type i collagen is purchased from BD company, and concentration is 8.56mg/ml.
NAC, is N-acetylcysteine, i.e. N-acetylcystein, and concentration is 4mM,
Embodiment 1
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 20*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: as shown in Figure 1, the built-in glass tubing inner core of Teflon cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor 2ml that step (1) is obtained injects cylindrical model device, then puts it into 37 ℃ of incubators and vertically cultivates after half an hour, obtains the crosslinked cylindrical collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) and squeeze in centrifuge tube, then to adding 8ml hyclone and DMEM culture medium in centrifuge tube, be the mixed liquor mixing at 1: 9 by volume, after incubated overnight, changing 9mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 9 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 45min, add again TGF-β, the concentration that makes TGF-β in culture medium is 300pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the high 5cm of Teflon cylindrical tube described in step (2), overall diameter 1.1cm, interior diameter 0.9cm glass tubing inner core diameter 0.2cm, long 4cm, rubber stopper is the rubber stopper in syringe, and its diameter is 4cm, and long is 1cm, centrifuge tube described in step (3) is 15mL centrifuge tube.
Embodiment 2
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 3.9I Collagen Type VI, 200 μ l10 * phosphate buffers, 10.5 μ l concentration, simultaneously add hyclone, DMEM culture medium and 19*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: as shown in Figure 1, the built-in glass tubing inner core of Teflon cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor 2ml that step (1) is obtained injects cylindrical model device, then puts it into 37 ℃ of incubators and vertically cultivates after half an hour, obtains the crosslinked cylindrical collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) and squeeze in centrifuge tube, then to adding 8ml hyclone and DMEM culture medium in centrifuge tube, be the mixed liquor mixing at 1: 9 by volume, after incubated overnight, changing 8mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 8 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 30min, add again TGF-β, the concentration that makes TGF-β in culture medium is 200pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the high 5cm of Teflon cylindrical tube described in step (2), overall diameter 1.1cm, interior diameter 0.9cm, glass tubing inner core diameter 0.2cm, long 4cm, rubber stopper is the rubber stopper in syringe, and its diameter is 4cm, and long is 1cm, centrifuge tube described in step (3) is 15mL centrifuge tube.
Embodiment 3
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 4.1g type i collagen, 200 μ l10 * phosphate buffers, 10.9 μ l concentration, simultaneously add hyclone, DMEM culture medium and 21*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: as shown in Figure 1, the built-in glass tubing inner core of Teflon cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor 2ml that step (1) is obtained injects cylindrical model device, then puts it into 37 ℃ of incubators and vertically cultivates after half an hour, obtains the crosslinked cylindrical collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) and squeeze in centrifuge tube, then to adding 8ml hyclone and DMEM culture medium in centrifuge tube, be the mixed liquor mixing at 1: 9 by volume, after incubated overnight, changing 10mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 10 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 400pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the high 5cm of Teflon cylindrical tube described in step (2), overall diameter 1.1cm, interior diameter 0.9cm glass tubing inner core diameter 0.2cm, long 4cm, rubber stopper is the rubber stopper in syringe, and its diameter is 4cm, and long is 1cm, centrifuge tube described in step (3) is 15mL centrifuge tube.
Embodiment 4
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 20*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the collagenocyte mixed liquor 0.5ml that step (1) is obtained injects 24 porocyte culture plates of low adhesion, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: be the mixed liquor of mixing in 1: 9 by volume to adding 1ml hyclone and DMEM culture medium in 24 porocyte culture plates of the low adhesion that contains the crosslinked disc collagenocyte mixed liquor having solidified, after incubated overnight, changing 1mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 1 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 50min, add again TGF-β, the concentration that makes TGF-β in culture medium is 280pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage disk model of the present embodiment.
Embodiment 5
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 3.9I Collagen Type VI, 200 μ l10 * phosphate buffers, 10.5 μ l concentration, simultaneously add hyclone, DMEM culture medium and 19*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the collagenocyte mixed liquor 0.5ml that step (1) is obtained injects 24 porocyte culture plates of low adhesion, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: be the mixed liquor of mixing in 1: 9 by volume to adding 0.8ml hyclone and DMEM culture medium in 24 porocyte culture plates of the low adhesion that contains the crosslinked disc collagenocyte mixed liquor having solidified, after incubated overnight, changing 0.8mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 30min, add again and add again TGF-β, the concentration that makes TGF-β in culture medium is 200pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage disk model of the present embodiment.
Embodiment 6
For building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, comprise the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 4.1g type i collagen, 200 μ l10 * phosphate buffers, 10.9 μ l concentration, simultaneously add hyclone, DMEM culture medium and 21*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, with liquid-transfering gun, blows gently to mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the collagenocyte mixed liquor that step (1) is obtained injects 24 porocyte culture plates of low adhesion, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: be the mixed liquor of mixing in 1: 9 by volume to adding 1.2ml hyclone and DMEM culture medium in 24 porocyte culture plates of the low adhesion that contains the crosslinked disc collagenocyte mixed liquor having solidified, after incubated overnight, changing 1.2mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 400pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage disk model of the present embodiment.
The three-dimensional shrinkage model that embodiment 1～6 makes all has certain contraction intensity and mechanical strength.By protein immunoblot, detect and find that TGF-β stimulates three kinds of contractive proteins to express and increases, the most obvious at the 3rd day, as shown in Figure 3; After adding U0126, three kinds of protein expression increases are more obvious, and as shown in Figure 4, side light IMR-90 cell, to the differentiation of smooth muscle cell direction, has more powerful contractility.Wherein, Fig. 2 adds the comparison schematic diagram of the various factors in disc model equipment structure of the present invention; Fig. 3 is that three-dimensional shrinkage model protein immunoblotting of the present invention detects collection of illustrative plates, is used for illustrating that when collagen is cultivated three days, contractile protein expression is the highest; Fig. 4 adds and adds TGF-β after U0126 pretreatment again and express more obvious than adding merely TGF-β contractile protein for explanation.
Claims (8)
1. for building a preparation method for the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that comprising the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the compound method of 2ml collagenocyte mixed liquor is is 1M by 3.9～4.1g type i collagen, 200 μ l10 * phosphate buffers, 10.5～10.9 μ l concentration, simultaneously add hyclone, DMEM culture medium and 19～21*10
6it is 2ml that the mixed liquor of individual myofibroblast is mixed to cumulative volume, and mix homogeneously, obtains; The volume ratio of described hyclone and DMEM culture medium is 1: 9;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the built-in glass tubing inner core of cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor that step (1) is obtained injects cylindrical model device Tissue Culture Plate, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked cylindrical collagenocyte mixed liquor having solidified or the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: the crosslinked cylindrical collagenocyte mixed liquor having solidified that step (2) is obtained is squeezed in centrifuge tube, then to adding hyclone and DMEM culture medium in centrifuge tube or the Tissue Culture Plate that contains the crosslinked disc collagenocyte mixed liquor having solidified, be that the mixed liquor of mixing in 1: 9 was not to have collagenocyte mixed liquor by volume, after incubated overnight, changing DMEM culture medium continues to cultivate 12 hours, the addition of described DMEM culture medium was not there to be collagenocyte mixed liquor to be limited, then in DMEM culture medium, add U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 30-60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 200pM-400pM, continue to cultivate 96 hours, obtain three-dimensional shrinkage cylinder model or three-dimensional shrinkage disk model.
2. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that the 24 porocyte culture plates that described Tissue Culture Plate is low adhesion.
3. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that the cylindrical tube described in step (2) is plastic tube.
4. according to claim 3 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that described plastic tube is Teflon pipe.
5. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that described in step (2), rubber stopper is the rubber stopper in syringe.
6. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that it is with syringe core rod, to enforce the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) to squeeze in centrifuge tube that the crosslinked cylindrical collagenocyte mixed liquor having solidified that step (2) is obtained is squeezed into the mode adopting in centrifuge tube.
7. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that comprising the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 19～21*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, and mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the built-in glass tubing inner core of Teflon cylindrical tube, and the sealing of upper and lower two ends employing rubber stopper, make cylindrical model device; The collagenocyte mixed liquor 2ml that step (1) is obtained injects cylindrical model device, then puts it into 37 ℃ of incubators and vertically cultivates after half an hour, obtains the crosslinked cylindrical collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collagenocyte mixed liquor having solidified that rubber closure obtains step (2) and squeeze in centrifuge tube, then to adding 8ml hyclone and DMEM culture medium in centrifuge tube, be the mixed liquor mixing at 1: 9 by volume, after incubated overnight, changing 9mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 9 μ lU0126, the concentration that makes U0126 in culture medium is 10 μ M, after 45min, after 30-60min, add again TGF-β, the concentration that makes TGF-β in culture medium is 300pM, continue to cultivate 96 hours, obtain three-dimensional shrinkage cylinder model.
8. according to claim 1 for building the preparation method of the three-dimensional shrinkage model of artificial blood vessel's model, it is characterized in that comprising the steps:
Step (1), the preparation of collagenocyte mixed liquor: after the sodium hydroxide mix homogeneously that the type i collagen that the compound method of 2ml collagenocyte mixed liquor is is 8.56mg/ml by 467 μ l concentration, 200 μ l10 * phosphate buffers, 10.7 μ l concentration are 1M, simultaneously add hyclone, DMEM culture medium and 20*10
6the mixed liquor of individual myofibroblast to cumulative volume is 2ml, and the volume ratio of described hyclone, DMEM culture medium is 1: 9, and mix homogeneously, obtains;
Step (2), the crosslinked of collagenocyte mixed liquor solidifies: the collagenocyte mixed liquor 0.5ml that step (1) is obtained injects 24 porocyte culture plates of low adhesion, then put it into 37 ℃ of incubators and vertically cultivate after half an hour, obtain the crosslinked disc collagenocyte mixed liquor having solidified;
Step (3), the preparation of three-dimensional shrinkage model: be the mixed liquor of mixing in 1: 9 by volume to adding 1ml hyclone and DMEM culture medium in 24 porocyte culture plates of the low adhesion that contains the crosslinked disc collagenocyte mixed liquor having solidified, after incubated overnight, changing 1mlDMEM culture medium into continues to cultivate 12 hours, then in DMEM culture medium, add 1 μ l U0126, the concentration that makes U0126 in culture medium is 10 μ M, after 50min, add again and add again TGF-β, the concentration that makes TGF-β in culture medium is 280pM, continue to cultivate 96 hours, obtain the three-dimensional shrinkage disk model of invention.
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| CN104096265B (en) | 2016-03-23 |
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