CN103705542A - Decellularized blood vessel matrix gel, preparation method therefor and applications thereof - Google Patents
Decellularized blood vessel matrix gel, preparation method therefor and applications thereof Download PDFInfo
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Abstract
The invention relates to a kind of decellularized blood vessel matrix gel, a preparation method therefor and applications thereof. The preparation method is as follows: first, after blood vessel outer membranes and anadesma are removed, the blood vessels are cut into blood vessel pieces, the blood vessel pieces are subjected to decellularized treatment in a TritonX-100 solution, then subjected to freeze drying, smashed into powder, and filtered with a 60-mesh screen, and decellularized blood vessel matrix powder is obtained; second, the decellularized blood vessel matrix powder is placed in a hydrochloric acid solution containing pepsin with a concentration of 0.09-0.11%, and subjected to digestion for 66-78h, a sodium hydroxide solution is added to neutralize hydrochloric acid, then PBS is added to balance ion concentration, finally, the gel solution is placed in an incubator with a temperature of 37 DEG C for gel forming. The decellularized blood vessel matrix gel has good molding capability and mechanical properties, the interior is loose, the gel has certain poriness, and the collagen fiber has appropriate diameters and good stability. The gel can meet requirements of preparation of medicines treating ischemic diseases, support materials of tissue engineering and/or cell culture.
Description
Technical field
The present invention relates to engineered gel technique field, specifically, is a kind of de-cell vascular stroma gel and its preparation method and application.
Background technology
Fibrin gel is a kind of good timbering material, is one of emphasis of Tissue Engineering Study always.Fibrin gel mainly consists of natural extracellular matrix components, has good biocompatibility, effective biological activity and biodegradability, also has three-dimensional porous structure and good plasticity simultaneously.In recent years, fibrin gel as timbering material increasingly extensive be applied in Tissue Engineering Study, its formation to muscular tissue, bone and cartilaginous tissue, epithelial tissue etc. has important function, but simultaneously can not and cartilage tissue engineeredly provide enough mechanical strengths for bone.
De-cell vascular stroma gel is a kind of engineered gel, and it can be applicable to following field: 1) for injecting direct treatment ischemic diseases: such as lower limb ischemia, myocardial ischemia etc.; 2) as the carrier of cell therapy, after gel cell mixing, local injection treatment, plays the effect that partial restriction cell flows and runs off; 3), as laying use in endotheliocyte and the outer incubation of level and smooth muscular cell body, thereby play, help cell adhesion, maintain the effects such as cellular morphology and function, promotion propagation; 4) can be used as the carrier that cell three-dimensional is cultivated, Cell differentiation inducing activity etc.
Chinese periodical < < Chinese Medical Journal > >, 09 phase in 2004, the paper of publishing " take liquid collagen build the experimentation of engineered cardiac muscular tissue for support ", the liquid type i collagen that has added the substrate factor is mixed with neonatal cardiac myocytes separated and that cultivate, in cannelure, cast annular myocardial cell/collagen band.Chinese periodical < < China's Tissue Engineering Study and clinical rehabilitation > >, 49 phases in 2008, the paper of publishing " extracellular matrix gel stent mixing human umbilical vein source endotheliocyte builds engineered endotheliocyte lamella ", first it get newborn SD rat Mus tail and prepare I type liquid glue original solution, then it is mixed with Human umbilical vein endothelial cells, observe growth course and the developmental state of endotheliocyte in extracellular matrix gel.Chinese patent literature CN200310118986.4, open day on November 17th, 2004, denomination of invention is " a kind of method that builds tissue engineering bone/cartilage with bone matrix gel ", it first with chondrocyte isolation, is cultivated or the induction of stroma stem cell separation and Culture is that chondrocyte obtains seed cell, then get new zealand rabbit or people's fetal long bone or metaphysis spongy bone and build bone matrix gel BMG, finally seed cell is inoculated in and on cortical bone or spongy bone BMG, carries out In vitro culture and build tissue engineering bone/cartilage.Also having some reports is that the blood plasma in autologous source is obtained respectively to Fibrinogen and thrombin by steps such as frozen, centrifugal, precipitation, ion exchange, dialysis, and Fibrinogen is mixed with thrombin, make it under thrombin action, produce polyreaction, be transformed into fibrin, be cross-linked into three-dimensional network structure, form fibrin gel.
But about de-cell vascular stroma gel and preparation method thereof, have not been reported at present.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of de-cell vascular stroma gel is provided.
One object more of the present invention is that a kind of preparation method of de-cell vascular stroma gel is provided.
Another object of the present invention is that the purposes of above-mentioned de-cell vascular stroma gel is provided.
For achieving the above object, the technical scheme that the present invention takes is:
A de-cell vascular stroma gel, it is prepared by following methods:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 0.9-1.1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 0.9-1.1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.009-0.011mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.09-0.11%, magnetic agitation digestion 66-78 hour, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:(850-950) mol/mL.
Preferably, described de-cell vascular stroma gel is prepared by following methods:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.01mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.1%, magnetic agitation digestion 72 hours, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, and in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:900mol/mL.
Preferably, the de-cell of described concussion is processed specifically at 37 ℃ of shaking tables, under the condition of 200rpm, processes 6-7 days.
Preferably, described 37 ℃ of incubator plastics that gel solution is put into are specifically put into gel solution 37 ℃ of incubators at least 1 hour.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
A preparation method for de-cell vascular stroma gel, it comprises the following steps:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 0.9-1.1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 0.9-1.1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.009-0.011mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.09-0.11%, magnetic agitation digestion 66-78 hour, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:(850-950) mol/mL.
Preferably, the preparation method of described de-cell vascular stroma gel comprises the following steps:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.01mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.1%, magnetic agitation digestion 72 hours, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, and in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:900mol/mL.
Preferably, the de-cell of described concussion is processed specifically at 37 ℃ of shaking tables, under the condition of 200rpm, processes 6-7 days.
Preferably, described 37 ℃ of incubator plastics that gel solution is put into are specifically put into gel solution 37 ℃ of incubators at least 1 hour.
For realizing above-mentioned the 3rd object, the technical scheme that the present invention takes is:
The purposes of de-cell vascular stroma gel as above, described purposes is selected from:
A) medicine of preparation treatment ischemic diseases,
B) as the timbering material of organizational project, and/or
C) prepare the culture medium that cell culture is used.
Described ischemic diseases refers to the disease of organ or tissue's ischemia aspect, as lower limb ischemia, myocardial ischemia etc.; The described timbering material as organizational project refers to the carrier as cell therapy, will after its cell mixing, for local injection, treat; It is described that to prepare the culture medium that cell culture uses can be to use as laying in endotheliocyte and the outer incubation of level and smooth muscular cell body, thereby play and help cell adhesion, maintain the effects such as cellular morphology and function, promotion propagation, it can also be the carrier of cultivating as cell three-dimensional, Cell differentiation inducing activity etc., but be not limited only to this.
It should be noted that, above-mentioned " until blood vessel sheet desiccation is hard ", its method is preferably placed in-80 ℃ of refrigerator freezings by the blood vessel small pieces after de-cell spend the night after, then be placed in vacuum freezing drying oven under ℃ left and right, temperature-50 about lyophilization 46-50 hour.Above-mentioned pepsin digestion process, preferably at 37 ℃, carries out magnetic agitation under 340-360 rev/min of condition.
The invention has the advantages that:
The present invention successfully prepares de-cell vascular stroma gel, this preparation method is used Triton X-100 solution to take off cell, removal effect is thorough, can obtain pure substrate composition, pepsin concn, processing time are reasonable, can guarantee that the protein ingredient in de-cell vascular stroma powder is fully digested, guarantee the homogeneity of gel, equilibrium ion concentration is proper, can guarantee can form fast gel after 1 hour.Preparation-obtained de-cell vascular stroma gel has good moulding ability, kept certain mechanical property, guaranteed again internal defect, there is certain porosity, collagen fiber diameter is suitable, plastic good stability, can meet the medicine of preparation treatment ischemic diseases, as the requirement of timbering material and/or the cell culture of organizational project.
Accompanying drawing explanation
Accompanying drawing 1 is the preparation mode figure of de-cell vascular stroma gel.
Accompanying drawing 2 is states of de-cell vascular stroma powder in de-cell vascular stroma gel preparation course.
Accompanying drawing 3 is evaluations of de-cell vascular stroma gel (5mg/ml).
Accompanying drawing 4 is evaluations of de-cell vascular stroma gel (10mg/ml).
Accompanying drawing 5 is evaluations of de-cell vascular stroma gel (15mg/ml).
Accompanying drawing 6 is collagen fiber diameter analysis results of de-cell vascular stroma gel.
Accompanying drawing 7 is the OD value situations of change in gel 60 minutes.
The specific embodiment
Below in conjunction with accompanying drawing, the specific embodiment provided by the invention is elaborated.
Herein, described its pH of PBS buffer is 7.2～7.4, comprises following material and forms: NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
410mmol/L, KH
2pO4 2mmol/L.10 * PBS buffer is exactly the PBS of 0.1M.
preparation and the detection () of embodiment 1 de-cell vascular stroma gel
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 1% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 6.5 days, during wash every other day and change 1% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 48 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
Take respectively 50,100, the de-cell vascular stroma powder of preparation is positioned over the pepsin (P7012-1G that 10ml is 0.1% containing mass body volume concentrations in 150mg above-mentioned steps 1, Sigma) in 0.01mol/L hydrochloric acid solution, 37 ℃, 350 revs/min of magnetic agitation digest in the 0.1mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 72 hours and hydrochloric acid, then add 10 * PBS equilibrium ion concentration of 1/9 volume (being 1.111ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubators and after 1 hour, can form de-cell vascular stroma gel.
Two, the detection of de-cell vascular stroma gel
1, HE dyeing
By the de-cell vascular stroma gel forming in the blood vessel small pieces after de-cell and above-mentioned 2ml syringe paraffin embedding after 4% paraformaldehyde is fixing, after section dewaxing aquation, haematoxylin dyeing is 5 minutes, the hydrochloride alcohol differentiation several seconds after rinsing, washing is from the beginning returned blue 30 minutes, Yihong dyeing spend the night after ethanol dehydration, the transparent rear resinene mounting of dimethylbenzene.
2, scanning electron microscope
The de-cell vascular stroma gel forming in above-mentioned 2ml syringe is fixed to 24 hours with 2.5% cold glutaraldehyde, and then 1 * PBS rinsing is three times, each 30 minutes.Pass through again gradient ethanol dehydration (30,50,70,90,100), each 30 minutes, be finally positioned in 100% ethanol 4 ℃ and spend the night, second day is used 100% ethanol rinsing 3 times again, each 30 minutes.With the dry rear ion of vacuum drying oven, spray instrument metal spraying afterwards, then carry out scanning electron microscope (FEI QUANTA 250) and observe and take pictures.
3, gel collagen fiber diameter is analyzed
While calculating the collagen fiber diameter in de-cell vascular stroma gel, 100 fibers of random choose on the scanning electron microscope (SEM) photograph of de-cell vascular stroma gel, by its diameter of Image software measurement, finally ask its meansigma methods and standard deviation, and do statistical analysis.
4, gel absorbance test
The de-cell vascular stroma gelled specimen being placed on ice (is comprised to three kinds of de-cell vascular stroma gels prepared by above-mentioned steps, de-cell vascular stroma powder and hydrochloric acid solution ratio are respectively 5mg/ml, 10mg/ml, 15mg/ml) respectively draw 10 μ l and put into 96 orifice plates, each sample repeats 6 holes, put in the multi-functional microplate reader that is preheating to 37 ℃ and detect (SYNERGY/2 MICROPLATE READER, BioTek), detect the data under 405nm absorbance, within every 2 minutes, read once, continue 60 minutes.
Three, experimental result
1, the preparation of de-cell vascular stroma powder
According to the de-cell vascular stroma powder of method preparation in the preparation mode figure (Fig. 1) of de-cell vascular stroma gel, (Fig. 2 A) takes on a red color after fresh pig aorta shreds, remove after cell outward appearance be white in color (Fig. 2 B), HE dyeing showed cell removes totally, remaining substrate composition (Fig. 2 C), after vacuum lyophilization, blood vessel becomes hard and is dried (Fig. 2 D), is uniform white powder (Fig. 2 E) with micromill after smashing.
2, the formation of de-cell vascular stroma gel
After the above-mentioned de-cell vascular stroma powder of smashing is smashed, be dissolved in and in pepsin solution, digest 72 be as a child creamy white afterwards thick (Fig. 2 F), sodium hydroxide neutralization, regulate ion concentration and put into the visible translucent gelatin semi-solid formation after 1 hour of 37 ℃ of incubators, there is good moulding ability (Fig. 3 A, 3B, 4A, 4B, 5A, 5B).
3, gel HE dyeing
The visible de-cell vascular stroma gel of HE dyeing inside has formed the network-like connection that extracellular matrix is Main Ingredients and Appearance, and such structure had both kept certain mechanical property, had guaranteed again internal defect, had certain porosity (Fig. 3 C, 4C, 5C).
4, gel image scanning Electronic Speculum
Scanning electron microscope result shows that de-cell vascular stroma gel surface has good collagen fiber structure, is unordered latticed cross arrangement, and all the other substrate compositions that remove collagen distribute wherein or are attached to collagen fiber surface (Fig. 3 D, 4D, 5D).
5, gel collagen fiber diameter is analyzed
By statistics, the de-cell vascular stroma gel collagen fiber diameter of 5mg/ml is 5.82037nm ± 1.39185nm, 10mg/ml gel collagen fiber diameter is 5.89368nm ± 1.25852nm, the obvious significant difference (P=0.3532) of nothing between the two, and the collagen fiber diameter of 15mg/ml gel is 96.27796nm ± 19.78365nm, there is obvious significant difference (P=4.15593E-68 and 1.48086E-68) (Fig. 6) with the above two.
6, gel 405nm light absorption value detects
At 37 ℃, de-cell vascular stroma gel is in the light absorption value result at 405nm place, and acellular matrix becomes gradually muddy and is gluey, about 20-30 minute, becomes stable, to its OD value maintenance inconvenience (Fig. 7) in 60 minutes, plastic good stability is described.
preparation and the detection (two) of embodiment 2 de-cell vascular stroma gels
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 0.9% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 7 days, during wash every other day and change 0.9% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 46 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
Take respectively 50,100, the de-cell vascular stroma powder of preparation is positioned over the pepsin (P7012-1G that 10ml is 0.09% containing mass body volume concentrations in 150mg above-mentioned steps 1, Sigma) in 0.009mol/L hydrochloric acid solution, 37 ℃, 360 revs/min of magnetic agitation digest in the 0.09mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 78 hours and hydrochloric acid, then add 10 * PBS equilibrium ion concentration of 1/8.5 volume (being 1.176ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubators and after 1 hour, can form de-cell vascular stroma gel.
Two, the detection of de-cell vascular stroma gel
Detection method is with embodiment 1.
Three, experimental result
1, the preparation of de-cell vascular stroma powder
After fresh pig aorta shreds, take on a red color, remove cell after outward appearance be white in color, HE dyeing showed cell removes totally, remaining substrate composition, after vacuum lyophilization, blood vessel becomes firmly and is dried, and after smashing, is uniform white powder with micromill.
2, the formation of de-cell vascular stroma gel
After the above-mentioned de-cell vascular stroma powder of smashing is smashed, be dissolved in pepsin solution, digest 78 be as a child creamy white afterwards thick, sodium hydroxide neutralization, regulate ion concentration and put into the visible translucent gelatin semi-solid formation after 1 hour of 37 ℃ of incubators, there is good moulding ability.
3, gel HE dyeing
The visible de-cell vascular stroma gel of HE dyeing inside has formed the network-like connection that extracellular matrix is Main Ingredients and Appearance, has certain porosity.
4, gel image scanning Electronic Speculum
Scanning electron microscope result shows that de-cell vascular stroma gel surface has good collagen fiber structure, is unordered latticed cross arrangement, and all the other substrate compositions that remove collagen distribute wherein or are attached to collagen fiber surface.
5, gel collagen fiber diameter is analyzed
By statistics, the de-cell vascular stroma gel collagen fiber diameter of 5mg/ml is 5.85068nm ± 1.27134nm, 10mg/ml gel collagen fiber diameter is 5.91672nm ± 1.30861nm, the obvious significant difference of nothing between the two, and the collagen fiber diameter of 15mg/ml gel is 95.87289nm ± 20.34718nm, there is obvious significant difference with the above two.
6, gel 405nm light absorption value detects
At 37 ℃, de-cell vascular stroma gel is in the light absorption value result at 405nm place, and acellular matrix becomes gradually muddy and is gluey, about 20-30 minute, becomes stable, to its OD value maintenance inconvenience in 60 minutes, plastic good stability is described.
preparation and the detection (three) of embodiment 3 de-cell vascular stroma gels
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 1.1% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 6 days, during wash every other day and change 1.1% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 50 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
Take respectively 50,100, the de-cell vascular stroma powder of preparation is positioned over the pepsin (P7012-1G that 10ml is 0.11% containing mass body volume concentrations in 150mg above-mentioned steps 1, Sigma) in 0.011mol/L hydrochloric acid solution, 37 ℃, 340 revs/min of magnetic agitation digest in the 0.11mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 66 hours and hydrochloric acid, then add 10 * PBS equilibrium ion concentration of 1/9.5 volume (being 1.053ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubators and after 1.1 hours, can form de-cell vascular stroma gel.
Two, the detection of de-cell vascular stroma gel
Detection method is with embodiment 1.
Three, experimental result
1, the preparation of de-cell vascular stroma powder
After fresh pig aorta shreds, take on a red color, remove cell after outward appearance be white in color, HE dyeing showed cell removes totally, remaining substrate composition, after vacuum lyophilization, blood vessel becomes firmly and is dried, and after smashing, is uniform white powder with micromill.
2, the formation of de-cell vascular stroma gel
After the above-mentioned de-cell vascular stroma powder of smashing is smashed, be dissolved in pepsin solution, digest 66 be as a child creamy white afterwards thick, sodium hydroxide neutralization, regulate ion concentration and put into the visible translucent gelatin semi-solid formation after 1.1 hours of 37 ℃ of incubators, there is good moulding ability.
3, gel HE dyeing
The visible de-cell vascular stroma gel of HE dyeing inside has formed the network-like connection that extracellular matrix is Main Ingredients and Appearance, has certain porosity.
4, gel image scanning Electronic Speculum
Scanning electron microscope result shows that de-cell vascular stroma gel surface has good collagen fiber structure, is unordered latticed cross arrangement, and all the other substrate compositions that remove collagen distribute wherein or are attached to collagen fiber surface.
5, gel collagen fiber diameter is analyzed
By statistics, the de-cell vascular stroma gel collagen fiber diameter of 5mg/ml is 5.97253nm ± 1.28240nm, 10mg/ml gel collagen fiber diameter is 5.86259nm ± 1.29851nm, the obvious significant difference of nothing between the two, and the collagen fiber diameter of 15mg/ml gel is 94.99804nm ± 21.01755nm, there is obvious significant difference with the above two.
6, gel 405nm light absorption value detects
At 37 ℃, de-cell vascular stroma gel is in the light absorption value result at 405nm place, and acellular matrix becomes gradually muddy and is gluey, about 20-30 minute, becomes stable, to its OD value maintenance inconvenience in 60 minutes, plastic good stability is described.
comparative example 1
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 0.85% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 7 days, during wash every other day and change 0.85% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 46 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
Take respectively 50,100, the de-cell vascular stroma powder of preparation is positioned over the pepsin (P7012-1G that 10ml is 0.09% containing mass body volume concentrations in 150mg above-mentioned steps 1, Sigma) in 0.0009mol/L hydrochloric acid solution, 37 ℃, 360 revs/min of magnetic agitation digest in the 0.009mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 78 hours and hydrochloric acid, then add 10 * PBS equilibrium ion concentration of 1/8.5 volume (being 1.176ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubators and after 1 hour, can form de-cell vascular stroma gel.
Two, the detection of de-cell vascular stroma gel
Detection method is with embodiment 1.
Three, experimental result
1, the preparation of de-cell vascular stroma powder
HE dyeing showed cell removes not thorough, in substrate composition, is mingled with cell.
comparative example 2
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 1% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 6.5 days, during wash every other day and change 1% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 48 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
The de-cell vascular stroma powder that takes respectively preparation in 45mg, 155mg above-mentioned steps 1 is positioned over the pepsin (P7012-1G that 10ml is 0.1% containing mass body volume concentrations, Sigma) in 0.001mol/L hydrochloric acid solution, 37 ℃, 350 revs/min of magnetic agitation digest in the 0.01mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 72 hours and hydrochloric acid, then add 10 * PBS equilibrium ion concentration of 1/9 volume (being 1.111ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubator plastics.
Two, the detection of de-cell vascular stroma gel
Detection method is with embodiment 1.
Three, experimental result
1, the formation of de-cell vascular stroma gel
After the above-mentioned de-cell vascular stroma powder of smashing is smashed, be dissolved in and in pepsin solution, digest 72 and be as a child creamy white afterwards thickly, sodium hydroxide neutralization, regulates ion concentration and puts into 37 ℃ of incubator plastics.The processing that after 5 hours, concentration is 4.5mg/ml still can not form gelatin semisolid; After 1 hour, visible translucent gelatin semi-solid formation of the processing of 15.5mg/ml, has good moulding ability.
2, gel HE dyeing
The dye de-cell vascular stroma gel inner dispersion of visible 15.5mg/ml of HE has a large amount of granules, shows de-cell vascular stroma powder energy catapepsis.
comparative example 3
One, the preparation of de-cell vascular stroma gel
1, the de-cell vascular stroma powder of preparation
From slaughterhouse, buy Adult Pig (about 120kg) aorta, after rejecting adventitia and fascia composition, shred into irregular blood vessel small pieces, be positioned in 1.1% Triton X-100 solution, 37 ℃ of shaking tables, the de-cell of 200rpm concussion is processed 6 days, during wash every other day and change 1.1% Triton X-100 solution until blood vessel sheet turns white.Blood vessel sheet after de-cell is processed is positioned over to-80 ℃ of refrigerator freezings and spends the night and be placed on vacuum freezing drying oven (Labconco Triad 2.5L, the U.S.) lyophilization, ℃ left and right, temperature-50, about 50 hours time, until blood vessel sheet desiccation is hard.Then with micromill, be broken into powder, through 60 eye mesh screens, filtered.
2, the de-cell vascular stroma gel of preparation
Take respectively 50,100, the de-cell vascular stroma powder of preparation is positioned over the pepsin (P7012-1G that 10ml is 0.11% containing mass body volume concentrations in 150mg above-mentioned steps 1, Sigma) in 0.011mol/L hydrochloric acid solution, 37 ℃, 340 revs/min of magnetic agitation digest in the 0.11mol/L sodium hydroxide solution that adds 1/10 volume (being 1ml) after 66 hours and hydrochloric acid, then add respectively 10 * PBS equilibrium ion concentration of 1/8.4 volume (being 1.190ml), 1/9.7 volume (being 1.031ml).Draw 500 μ l gel solutions and put into the 2ml syringe that reams head, put into 37 ℃ of incubator plastics.
Two, the detection of de-cell vascular stroma gel
Detection method is with embodiment 1.
Three, experimental result
1, the formation of de-cell vascular stroma gel
Above-mentioned processing is put into 37 ℃ of incubators and after 3 hours, is had not yet to see translucent gelatin semi-solid formation, and its moulding ability is poor as seen.
2, gel image scanning Electronic Speculum
Scanning electron microscope result shows that de-cell vascular stroma gel surface collagen fiber structure is cross arrangement, but has clustering phenomena, and all the other substrate compositions that remove collagen distribute and also occur clustering phenomena.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
Claims (9)
1. a de-cell vascular stroma gel, is characterized in that, it is prepared by following methods:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 0.9-1.1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 0.9-1.1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.009-0.011mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.09-0.11%, magnetic agitation digestion 66-78 hour, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:(850-950) mol/mL.
2. de-cell vascular stroma gel according to claim 1, is characterized in that, it is prepared by following methods:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.01mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.1%, magnetic agitation digestion 72 hours, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, and in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:900mol/mL.
3. de-cell vascular stroma gel according to claim 1 and 2, is characterized in that, the de-cell of described concussion is processed specifically at 37 ℃ of shaking tables, under the condition of 200rpm, processes 6-7 days.
4. de-cell vascular stroma gel according to claim 1 and 2, is characterized in that, described 37 ℃ of incubator plastics that gel solution is put into are specifically put into gel solution 37 ℃ of incubators at least 1 hour.
5. a preparation method for de-cell vascular stroma gel, is characterized in that, it comprises the following steps:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 0.9-1.1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 0.9-1.1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.009-0.011mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.09-0.11%, magnetic agitation digestion 66-78 hour, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:(850-950) mol/mL.
6. preparation method according to claim 5, is characterized in that, it comprises the following steps:
A) the de-cell vascular stroma powder of preparation: shred into blood vessel small pieces after rejecting tunica adventitia and fascia composition, be positioned in 1% Triton X-100 solution, the de-cell of concussion is processed, during wash every other day and change 1% Triton X-100 solution; The blood vessel small pieces after de-cell being placed in to-80 ℃ of refrigerator freezings spends the night and is placed on vacuum freezing drying oven lyophilization until blood vessel sheet desiccation is hard again; Be broken into powder, 60 eye mesh screens filter;
B) get in de-cell vascular stroma powder prepared by the step a) pepsic 0.01mol/L hydrochloric acid solution that to be positioned over containing concentration be 0.1%, magnetic agitation digestion 72 hours, add in sodium hydroxide solution and hydrochloric acid, then add PBS equilibrium ion concentration, finally gel solution is put into 37 ℃ of incubator plastics; Wherein, described de-cell vascular stroma powder and the ratio of hydrochloric acid solution are 5-15mg/ml, and in described sodium hydroxide solution, sodium hydroxide mole equates with hydrochloric acid mole in hydrochloric acid solution, and described PBS and the ratio of hydrochloric acid solution are 1:900mol/mL.
According to described in claim 5 or 6 preparation method, it is characterized in that, the de-cell of described concussion is processed specifically at 37 ℃ of shaking tables, under the condition of 200rpm, processes 6-7 days.
According to described in claim 5 or 6 preparation method, it is characterized in that, described 37 ℃ of incubator plastics that gel solution is put into are specifically put into gel solution 37 ℃ of incubators at least 1 hour.
9. the purposes of the de-cell vascular stroma gel described in claim 1 or 2, is characterized in that, described purposes is selected from:
A) medicine of preparation treatment ischemic diseases,
B) as the timbering material of organizational project, and/or
C) prepare the culture medium that cell culture is used.
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