CN104096265B - A kind of preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model - Google Patents
A kind of preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model, belonging to vascular pattern technical field.The method comprises the preparation of collencyte mixed solution, the crosslinked of collencyte mixed solution solidifies preparation three steps with three-dimensional shrinkage model, obtained three-dimensional shrinkage model is as a kind of contracting model of improvement, select myofibroblast IMR-90, growth is faster, and the analogue formation cycle is shorter; Meanwhile, add auxiliary contraction factor U0126 and TGF-β irritation cell, make it express contractive protein, there is better shrinkage, and then there is stronger physical strength; The three-dimensional shrinkage model that the present invention obtains can provide better contracting model more efficiently for scientific research, and is applied to after can be and clinically provides reference.
Description
Technical field
The invention belongs to vascular pattern technical field, being specifically related to a kind of preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model.
Background technology
Current three-dimensional shrinkage model is used in the structure artificial blood vessel model in scientific research or biotechnology more, and vascular applications is in clinical even as an alternative.Clinically, although had more ripe biomaterial to substitute for compared with great vessels, for the arteriole such as coronary artery of heart, desirable alternative goods are but still can not find.Although native blood vessel can be selected as one, there are vascular disease in many sufferers, and as atherosclerosis, this makes to find suitable biological substitution goods becomes possible a kind of methods for the treatment of., there is the cell cultures time long in the three-dimensional shrinkage model production method of current utilizable scientific research, the shortcomings such as physical strength is inadequate, make Minitype manual vascular applications be restricted.So the three-dimensional shrinkage models applying providing a kind of scientific and reasonable manufacturing artificial blood vessel similar is extremely urgent in scientific research.
A kind of three-dimensional shrinkage model is refer in the documents such as Small-DiameterArtificialArteriesEngineeredInVitro and PhenotypeMod μ lationinVasc μ larTissueEngineeringUsingBiochemicalandMechanicalStim μ lation, i.e. a kind of cylinder shape belt inner core model, inner core is fixed on columniform mold center, the vascular smooth muscle cell suspended digestion and type i collagen/scleroproein mix the three-dimensional hollow model injecting this, within after putting into 37 DEG C 30 minutes to 1 hour, wait for collagen cross-linking.For increasing the mechanical force of model, somebody also did applied mechanical power to be stimulated, and as pressurization etc., and biotic factor stimulates, as TGF-β, PDGF etc.But it is long that they all exist the cell cultures time, propagation is slow, the shortcomings such as the three-dimensional model physical strength built is not high.Therefore, how overcoming the deficiencies in the prior art is problems that current vascular pattern technical field needs solution badly.
Summary of the invention
The cell proliferation that the present invention is directed to the existence of current three-dimensional shrinkage Modelling process is slow, and the shortcoming that physical strength is low, provides a kind of preparation method of the three-dimensional shrinkage model for building 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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 19 ~ 21*10 by 3.9 ~ 4.1gI Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.5 ~ 10.9 μ l concentration
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, mixes, to obtain final product;
Step (2), the crosslinked of collencyte mixed solution solidifies: the built-in Glass tubing inner core of cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; Collencyte mixed solution step (1) obtained injects cylindrical model device or Tissue Culture Plate, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked cylindrical collencyte mixed solution that solidified or be cross-linked the disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: the crosslinked cylindrical collencyte mixed solution solidified step (2) obtained is squeezed in centrifuge tube, then in centrifuge tube or the Tissue Culture Plate containing the crosslinked disc collencyte mixed solution solidified, foetal calf serum is added and DMEM substratum is that 1: 9 mixed solution mixed was not to have collencyte mixed solution by volume, change DMEM substratum after incubated overnight and continue cultivation 12 hours, the add-on of described DMEM substratum was limited there not to be collencyte mixed solution, then in DMEM substratum, U0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 30-60min, the concentration of TGF-β in substratum is made to be 200pM-400pM, continue cultivation 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 plastics tubing.
Described plastics tubing is Teflon pipe.
Rubber plug described in step (2) is the rubber plug in syringe.
The mode adopted in centrifuge tube squeezed into by the crosslinked cylindrical collencyte mixed solution solidified step (2) obtained is enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball to squeeze in centrifuge tube.
The preparation method of the above-mentioned three-dimensional shrinkage model for building artificial blood vessel's model, comprises the steps:
Step (1), the preparation of collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 19 ~ 21*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, mixes, to obtain final product;
Step (2), the crosslinked of collencyte mixed solution solidifies: the built-in Glass tubing inner core of Teflon cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; Collencyte mixed solution 2ml step (1) obtained injects cylindrical model device, then puts it into 37.After incubator vertically cultivates half an hour, obtain the crosslinked cylindrical collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball and squeeze in centrifuge tube, then in centrifuge tube, add the mixed solution that 8ml foetal calf serum and DMEM substratum are 1: 9 mixing by volume, change 9mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 9 μ lU0126 are added, the concentration of U0126 in substratum is made to be 10 μMs, after 45min, TGF-β is added again after 30-60min, the concentration of TGF-β in substratum is made to be 300pM, continue cultivation 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 collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 20*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, mixes, to obtain final product;
Step (2), the crosslinked of collencyte mixed solution solidifies: collencyte mixed solution 0.5ml step (1) obtained injects 24 porocyte culture plates of low adhesion, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: add 1ml foetal calf serum in 24 porocyte culture plates of the low adhesion containing the crosslinked disc collencyte mixed solution solidified and DMEM substratum is 1: 9 mixed solution mixed by volume, change 1mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 1 μ lU0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, add again after 50min and add TGF-β again, the concentration of TGF-β in substratum is made to be 280pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage disk model invented.
The present invention compared with prior art, its beneficial effect is: the three-dimensional shrinkage model that the preparation method of (1) a kind of three-dimensional shrinkage model for building artificial blood vessel's model provided by the invention obtains is as a kind of contracting model of improvement, select myofibroblast IMR-90, growth is faster, and 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 physical strength; (3) the three-dimensional shrinkage model that obtains of the present invention can provide better contracting model more efficiently for scientific research, and is applied to after can be and clinically provides reference.
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 plug;
Fig. 2 is the comparison schematic diagram adding the various factor in disc mould unit structure of the present invention; Wherein, 21-cultivates half an hour; The non-dosing of 22-cultivates 12 hours; 23-drug treating 3 days;
Fig. 3 is three-dimensional shrinkage model protein immune-blotting method collection of illustrative plates of the present invention;
Fig. 4 is three-dimensional shrinkage model protein immune-blotting method collection of illustrative plates of the present invention.
Specific embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Cell prepares, and the present invention selects myofibroblast (IMR-90) purchased from American DSMZ (ATCC).Myofibroblast cell can express smooth muscle contractility albumen Smooth muscle α-actin (α-actin), calcium conditioning albumen (calponin) and Smoothing Probablities (SM22 α).Meanwhile, IMR-90 vitro growth rates comparatively vascular smooth muscle cell is faster, and incubation time is shorter.A cylindrical model uses cell quantity to be approximately 20*10
6individual; A disc model uses the about 5*10 of cell count
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 collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 20*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: as shown in Figure 1, the built-in Glass tubing inner core of Teflon cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; The collencyte mixed solution 2ml that step (1) obtained injects cylindrical model device, then puts it into after 37 DEG C of incubators vertically cultivate half an hour, obtains the crosslinked cylindrical collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball and squeeze in centrifuge tube, then in centrifuge tube, add the mixed solution that 8ml foetal calf serum and DMEM substratum are 1: 9 mixing by volume, change 9mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 9 μ lU0126 are added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 45min, the concentration of TGF-β in substratum is made to be 300pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the 5cm of Teflon cylindrical tube height described in step (2), outside diameter 1.1cm, interior diameter 0.9cm Glass tubing inner core diameter 0.2cm, long 4cm, rubber plug is the rubber plug 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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 19*10 by 3.9I Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.5 μ l concentration
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: as shown in Figure 1, the built-in Glass tubing inner core of Teflon cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; The collencyte mixed solution 2ml that step (1) obtained injects cylindrical model device, then puts it into after 37 DEG C of incubators vertically cultivate half an hour, obtains the crosslinked cylindrical collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball and squeeze in centrifuge tube, then in centrifuge tube, add the mixed solution that 8ml foetal calf serum and DMEM substratum are 1: 9 mixing by volume, change 8mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 8 μ lU0126 are added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 30min, the concentration of TGF-β in substratum is made to be 200pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the 5cm of Teflon cylindrical tube height described in step (2), outside diameter 1.1cm, interior diameter 0.9cm, Glass tubing inner core diameter 0.2cm, long 4cm, rubber plug is the rubber plug 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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 21*10 by 4.1gI Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.9 μ l concentration
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: as shown in Figure 1, the built-in Glass tubing inner core of Teflon cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; The collencyte mixed solution 2ml that step (1) obtained injects cylindrical model device, then puts it into after 37 DEG C of incubators vertically cultivate half an hour, obtains the crosslinked cylindrical collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball and squeeze in centrifuge tube, then in centrifuge tube, add the mixed solution that 8ml foetal calf serum and DMEM substratum are 1: 9 mixing by volume, change 10mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 10 μ lU0126 are added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 60min, the concentration of TGF-β in substratum is made to be 400pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage cylinder model of the present embodiment, wherein, the 5cm of Teflon cylindrical tube height described in step (2), outside diameter 1.1cm, interior diameter 0.9cm Glass tubing inner core diameter 0.2cm, long 4cm, rubber plug is the rubber plug 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 collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 20*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: collencyte mixed solution 0.5ml step (1) obtained injects 24 porocyte culture plates of low adhesion, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: add 1ml foetal calf serum in 24 porocyte culture plates of the low adhesion containing the crosslinked disc collencyte mixed solution solidified and DMEM substratum is 1: 9 mixed solution mixed by volume, change 1mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 1 μ lU0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 50min, the concentration of TGF-β in substratum is made to be 280pM, continue cultivation 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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 19*10 by 3.9I Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.5 μ l concentration
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: collencyte mixed solution 0.5ml step (1) obtained injects 24 porocyte culture plates of low adhesion, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: add 0.8ml foetal calf serum in 24 porocyte culture plates of the low adhesion containing the crosslinked disc collencyte mixed solution solidified and DMEM substratum is 1: 9 mixed solution mixed by volume, change 0.8mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, U0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, add again after 30min and add TGF-β again, the concentration of TGF-β in substratum is made to be 200pM, continue cultivation 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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 21*10 by 4.1gI Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.9 μ l concentration
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, blowing gently to mixing, obtaining final product with liquid-transfering gun;
Step (2), the crosslinked of collencyte mixed solution solidifies: the 24 porocyte culture plates collencyte mixed solution that step (1) obtains being injected low adhesion, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: add 1.2ml foetal calf serum in 24 porocyte culture plates of the low adhesion containing the crosslinked disc collencyte mixed solution solidified and DMEM substratum is 1: 9 mixed solution mixed by volume, change 1.2mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, U0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 60min, the concentration of TGF-β in substratum is made to be 400pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage disk model of the present embodiment.
The three-dimensional shrinkage model that embodiment 1 ~ 6 obtains all has certain contraction intensity and physical strength.Detected by protein immunoblot 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 is the comparison schematic diagram adding the various factor in disc mould unit structure of the present invention; Fig. 3 is three-dimensional shrinkage model protein immune-blotting method collection of illustrative plates of the present invention, and when being used for illustrating that collagen cultivates three days, contractile protein expression amount is the highest; Fig. 4 is used to add TGF-β after explanation adds U0126 pre-treatment again and expresses more obvious than adding merely TGF-β contractile protein.
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 collencyte mixed solution: simultaneously the compound method of 2ml collencyte mixed solution is, after the sodium hydroxide of 1M mixes, add foetal calf serum, DMEM substratum and 19 ~ 21*10 by 3.9 ~ 4.1gI Collagen Type VI, 200 μ l10 × phosphate buffered saline buffers, 10.5 ~ 10.9 μ l concentration
6it is 2ml that the mixed solution of individual myofibroblast is mixed to cumulative volume, mixes, to obtain final product; The volume ratio of described foetal calf serum and DMEM substratum is 1: 9;
Step (2), the crosslinked of collencyte mixed solution solidifies: the built-in Glass tubing inner core of cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; Collencyte mixed solution step (1) obtained injects cylindrical model device Tissue Culture Plate, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked cylindrical collencyte mixed solution that solidified or be cross-linked the disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: the crosslinked cylindrical collencyte mixed solution solidified step (2) obtained is squeezed in centrifuge tube, then in centrifuge tube or the Tissue Culture Plate containing the crosslinked disc collencyte mixed solution solidified, foetal calf serum is added and DMEM substratum is that 1: 9 mixed solution mixed was not to have collencyte mixed solution by volume, change DMEM substratum after incubated overnight and continue cultivation 12 hours, the add-on of described DMEM substratum was limited there not to be collencyte mixed solution, then in DMEM substratum, U0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, TGF-β is added again after 30-60min, the concentration of TGF-β in substratum is made to be 200pM-400pM, continue cultivation 96 hours, obtain three-dimensional shrinkage cylinder model or three-dimensional shrinkage disk model.
2. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, is characterized in that described Tissue Culture Plate is 24 porocyte culture plates of low adhesion.
3. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, is characterized in that the cylindrical tube described in step (2) is plastics tubing.
4. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 3, is characterized in that described plastics tubing is Teflon pipe.
5. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, is characterized in that rubber plug described in step (2) is the rubber plug in syringe.
6. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, it is characterized in that the mode adopted in centrifuge tube squeezed into by the crosslinked cylindrical collencyte mixed solution solidified step (2) obtained is enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball to squeeze in centrifuge tube.
7. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, is characterized in that comprising the steps:
Step (1), the preparation of collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 19 ~ 21*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, mixes, to obtain final product;
Step (2), the crosslinked of collencyte mixed solution solidifies: the built-in Glass tubing inner core of Teflon cylindrical tube, and two ends adopt rubber plug to close up and down, obtained cylindrical model device; The collencyte mixed solution 2ml that step (1) obtained injects cylindrical model device, then puts it into after 37 DEG C of incubators vertically cultivate half an hour, obtains the crosslinked cylindrical collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: enforce with syringe core rod the crosslinked cylindrical collencyte mixed solution solidified that step (2) obtains by soft rubber ball and squeeze in centrifuge tube, then in centrifuge tube, add the mixed solution that 8ml foetal calf serum and DMEM substratum are 1: 9 mixing by volume, change 9mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 9 μ lU0126 are added, the concentration of U0126 in substratum is made to be 10 μMs, after 45min, TGF-β is added again after 30-60min, the concentration of TGF-β in substratum is made to be 300pM, continue cultivation 96 hours, obtain three-dimensional shrinkage cylinder model.
8. the preparation method of the three-dimensional shrinkage model for building artificial blood vessel's model according to claim 1, is characterized in that comprising the steps:
Step (1), the preparation of collencyte mixed solution: after the sodium hydroxide that the compound method of 2ml collencyte mixed solution is is the type i collagen of 8.56mg/ml by 467 μ l concentration, 200 μ l10 × phosphate buffered saline buffers, 10.7 μ l concentration are 1M mixes, simultaneously add foetal calf serum, DMEM substratum and 20*10
6mixed solution to the cumulative volume of individual myofibroblast is 2ml, and the volume ratio of described foetal calf serum, DMEM substratum is 1: 9, mixes, to obtain final product;
Step (2), the crosslinked of collencyte mixed solution solidifies: collencyte mixed solution 0.5ml step (1) obtained injects 24 porocyte culture plates of low adhesion, then put it into after 37 DEG C of incubators vertically cultivate half an hour, obtain the crosslinked disc collencyte mixed solution solidified;
Step (3), the preparation of three-dimensional shrinkage model: add 1ml foetal calf serum and DMEM substratum is 1: 9 mixed solution mixed by volume in 24 porocyte culture plates of the low adhesion containing the crosslinked disc collencyte mixed solution solidified, change 1mlDMEM substratum after incubated overnight into and continue cultivation 12 hours, then in DMEM substratum, 1 μ lU0126 is added, the concentration of U0126 in substratum is made to be 10 μMs, add again after 50min and add TGF-β again, the concentration of TGF-β in substratum is made to be 280pM, continue cultivation 96 hours, obtain the three-dimensional shrinkage disk model invented.
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| CN101284148A (en) * | 2008-05-29 | 2008-10-15 | 东华大学 | Preparation method of artificial blood vessel and its application |
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