CN105963784A

CN105963784A - Method for recovering residual stress and residual strain of decellurarized blood vessel and reagent adopted by method

Info

Publication number: CN105963784A
Application number: CN201610291908.1A
Authority: CN
Inventors: 叶志义; 王贵学; 骆青松; 潘君
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2016-05-05
Filing date: 2016-05-05
Publication date: 2016-09-28
Anticipated expiration: 2036-05-05
Also published as: CN105963784B

Abstract

The invention provides a method for recovering the residual stress and residual strain of the decellurarized blood vessel. The method comprises the following steps: (1) preparing aqueous solution of methylene blue with the volume percentage being 0.01-0.05 %; (2) placing the decellurarized blood vessel into a transparent container filled with the aqueous solution, and placing the transparent container under UV-irradiation for 1-4 h; and (3) cleaning the decellurarized blood vessel for removing residues. With the adoption of the method, the residual stress and the residual strain of the decellurarized blood vessel can be obviously recovered, no hemolysis effect exists, and the requirements for national medical materials are satisfied.

Description

A kind of recovery takes off cellular vascular residual stress and the method for overstrain and reagent thereof

Technical field

The present invention relates to small-caliber artificial blood vessel field, particularly relate to a kind of recovery and take off cellular vascular residual stress and remnants The method of strain and reagent thereof.

Background technology

Numerous studies show that cardiovascular disease disease is sent out rate and increased with the growth at age, along with the aging of Chinese population Seriously, will appear from increasing cardio-cerebral vascular disease patient, wherein quite a few cardio-cerebral vascular disease patient there will be blood vessel blockage and Angionecrosis, is accomplished by blood vessel transplantation in this case.At present can be by other people normal blood vessels transplant, but the method donor Quantity is few, expensive, and has strong rejection；Self other part blood vessel transplantation the most useful, such as: great saphenous vein, breast is dynamic Arteries and veins etc., but the method can damage other site tissue, therefore develops artificial blood vessel in terms for the treatment of condition particularly important.

The comparative maturity that heavy caliber artificial blood vessel has applied at present, but compare the long-term patency rate of autogenous vessel graft still Differ greatly, the most only the half of autologous vein patency rate；And the material used in the preparation of small-caliber vascular is except greatly Outside the macromolecular material that calibre is conventional, biomaterial, composite are each applied in preparation, although occur in that and much exist Breakthrough in small-caliber vascular research, but application clinically is still extremely limited, effect extreme difference, and long-term patency rate is bad.Study carefully Its reason, it may be possible to cause owing to vascular grafts performance and former blood vessel differ greatly, when blood contacts with artificial blood vessel's tube wall Time, owing to blood vessel wall roughness is high, elastic poor, it is impossible to as normal blood vessels, the pulsation according to blood is shunk, easily Formation stress is concentrated, and has caused plaque rupture, has assembled and form thrombosis and cause angiostenosis, and then has affected the patency rate of blood vessel, And have been reported that and show that extracellular matrix framework has material impact to cardiovascular disease such as atherosclerosiss.Therefore development and people Body vascular grafts is similar to, and the similar artificial blood vessel of mechanical property is particularly important.

In recent years, de-cellular vascular was once becoming the focus of research, the preparation principle of de-cellular vascular be utilize physics, The method such as chemical, biological, by the cell clearance in allogeneic blood vessels, retains the epimatrix of cell, and extracellular matrix contribute to blood vessel Major part mechanical property, therefore takes off cellular vascular and retains most of mechanical property of original blood vessel, and this contributes to the adhesion of cell With growth.In recent years, many to the research report of de-cellular vascular mechanical property, but de-cellular vascular mechanics is ground by the overwhelming majority Study carefully mainly conventional at blood vessel mechanical property research, the such as change of elastic modelling quantity, burst pressure, suture before and after vascular digest The Testing index of the mechanical performances such as tolerance, and their research conclusion is more consistent, the de-burst pressure of cellular vascular, suture Tolerance test does not has significant difference, elastic modelling quantity to increase with untreated blood vessel.Outwardly, the blood after digestion While rejection removed by pipe, the most too many change of mechanical performance, but the present invention studies discovery digestion process and destroys The connection each other of smooth muscle cell, endotheliocyte, elastic fiber, collagen fiber makes the residual stress of de-cellular vascular subtract Little, and residual stress is significant to keeping the original physiological status of blood vessel.

Summary of the invention

For overcoming defect of the prior art, the present invention provides a kind of and recovers de-cellular vascular residual stress and overstrain Method, including: 1) dose volume percent is the aqueous solution of 0.01-0.05% methylene blue；2) de-cellular vascular is put into dress Have in the transparent vessel of above-mentioned aqueous solution, be placed in ultraviolet light and irradiate 2-4 hour；3) clean de-cellular vascular and remove removal of residue.

As preferably, the present invention recovers the method for de-cellular vascular mechanical property, including: 1) prepare containing 50-200u/ml Heparin, percentage by volume is the aqueous solution of 0.01-0.03% methylene blue and hydrogen peroxide that percentage by volume is 0.125%；2) will De-cellular vascular is put in the transparent vessel equipped with above-mentioned aqueous solution, is placed in ultraviolet light and irradiates 2-4 hour；3) de-cell blood is cleaned Pipe removes removal of residue.

As further preferably, step 1) also include in aqueous solution, be passed through oxygen.

The present invention also provides for a kind of for recovering de-cellular vascular residual stress and the reagent of overstrain, described reagent bag Include the aqueous solution that percentage by volume is 0.01-0.03% methylene blue.

As preferably, the reagent of the present invention also includes anticoagulant and/or antibacterial.

Further preferably, anticoagulant includes but not limited to heparin, EDTA, Disodium oxalate., one or more in citrate. It is further preferred that 50-200u/ml heparin.

Further preferably, antibacterial includes but not limited to hydrogen peroxide.It is further preferred that percentage by volume is the dioxygen of 0.125% Water.

It is that 0.01-0.03% aqueous solution of methylene blue is for recovering de-cellular vascular that the present invention also provides for a kind of percentage by volume Residual stress and the purposes of overstrain.

Accompanying drawing explanation

Fig. 1 glutaraldehyde cross-linking is with de-cellular vascular open-angle average ratio relatively

Photooxidation open-angle average ratio under Fig. 2 different condition is relatively

Fig. 3 normal blood vessels, de-cellular vascular compare with optimal crosslinking vessel expansion angle

Fig. 4 normal blood vessels, de-cellular vascular, crosslinking Ink vessel transfusing outer wall strain average ratio are relatively

Detailed description of the invention

By particular specific embodiment, embodiments of the present invention being described below, those skilled in the art can be by this explanation Content disclosed by book understands other advantages and effect of the present invention easily.

Preparation example 1: the preparation of de-cellular vascular

The acquisition of 1.1 rat left common carotid artery

All rats are all from Military Medical Univ No.3, P.L.A's Experimental Animal Center, and Animal Sex is male, Body weight 300g ± 25, use disconnected cervical approach to be put to death by rat.After rats death, rat is fixed on cystosepiment, then uses The hair of rat neck is cut by aseptic nipper and shears, breaks cervical region and searches out rat left common carotid artery, finds the left neck of rat total After tremulous pulse, progressively will adhere to along left common carotid artery lower end (connect heart at) to upper end (at aortic bifurcation) nerve thereon with Muscular tissue is removed and is made its only artery-sparing.After the nerve on left common carotid artery surface to be removed and muscle, take at body with camera Manage and measure its length, cut blood vessel from left common carotid artery lower end (connecting at heart), upper end (at aortic bifurcation) and use afterwards Camera takes its in vitro photo and measures its length.

The pretreatment of 1.2 rat left common carotid artery

After taking off rat left common carotid artery, it is immediately placed in equipped with in the container of PBS solution, takes tweezers and will remain in blood Nerve fiber on tube wall is torn with muscular tissue, will be trapped in internal blood vessel with syringe toward intravascular space perfusion PBS solution Blood rinse well, rinse 5-8 time, then carry out pretreatment, pre-treatment step is as follows:

1) the rat left common carotid artery obtained is put in the EP equipped with tri-distilled water and manages interior, and Hypotonic treatment 24 hours on shaking table To make vascular endothelial cell and smooth muscle cell rupture, temperature is 37 DEG C, rotating speed 120r/min；

2) after Hypotonic treatment, the distilled water in EP pipe is outwelled, load PBS solution, be then put in-80 DEG C of refrigerators, 37 DEG C water-bath alternate treatment 3 times, processes time 2 h every time ,-80 DEG C, 37 DEG C of water-baths process 1 hour respectively, it is intended to enter one Step destroys cells of vascular wall；

3), after multigelation, the PBS solution in EP is outwelled, then toward the ethanol of addition 70% in EP pipe, and with injecting Device makes it hydraulically full toward intravascular space perfusion ethanol, the most at room temperature processes and within 24 hours, carries out fat and process, and the 2nd, 4, within 8,14 hours, change liquid, change PBS solution for the last time into.

The digestion process of 1.3 rat left common carotid artery

In rat left common carotid artery after pretreatment, the most just carry out digestion process, the concrete steps of digestion process As follows:

1) pretreated rat left common carotid artery puts into the EP pipe of PBS solution, irrigates to internal blood vessel with syringe Solution is carried out, and cleans 5-8 time, is subsequently placed on shaking table process 2 hours, changes liquid every half an hour, change during liquid same every time Being rinsed with syringe, temperature is 37 DEG C, rotating speed 120r/min；

2), after treating that pretreated blood vessel cleans, put it in 0.125% trypsin solution and process 1.5 hours, and It is placed on shaking table, rotating speed 120r/min, temperature 37 DEG C, puts into trypsin solution before trypsin solution with syringe to Endovascular Perfusion so that it is intracavity is full of solution, and irrigates once with syringe every half an hour；

3) blood vessel after trypsinization processes is put into the EP pipe of PBS solution, is placed on bed processing 2 hours, Change liquid every half an hour, period uses irrigation with syringe every time, and temperature is 37 DEG C, rotating speed 120r/min.

Preparation example 2: the glutaraldehyde cross-linking-matched group of de-cellular vascular

Glutaraldehyde cross-linking is usually acid catalysis crosslinking, and the aldehyde radical on glutaraldehyde can react with hydroxyl or amino, can Monomer, linear macromolecule are transformed into the material of tridimensional network, thus improve the hot strength of collagen fiber and anti-degraded Ability, and its bio-mechanical property is had a certain impact, therefore the present invention select glutaraldehyde as one of which cross-linking reagent, Method is as follows:

1. with PBS, the glutaraldehyde solution of 50% is diluted to 0.25%；

2. with syringe, the glutaraldehyde solution of 0.25% is injected in de-cellular vascular tube chamber, in making tube chamber, be full of liquid Body, the most whole is soaked in solution 30 minutes, in environment is 4 DEG C of refrigerators；

3. take out blood vessel after having cross-linked to put in PBS, and repeatedly rinse inside with syringe, be subsequently placed on shaking table 6 Hour, often change liquid every other hour.

Preparation example 3-5 (of the present invention group of 1-3)

Experimental procedure is as follows:

1. 0.01% methylene blue+ultra-vioket radiation (2h)

1) configuration is containing 100u/ml heparin, the deionized water of 0.01% methylene blue；

2) logical oxygen in the solution of configuration, and add hydrogen peroxide solution and make its concentration be 0.125%；

3) de-cellular vascular is put into the transparent EP pipe equipped with configuration solution, and with syringe to de-cellular vascular tube chamber Primer solution, is subsequently placed under the ultraviolet light of medium wavelength irradiation 2 hours；

4) until de-cellular vascular through UV-crosslinked complete after, put it into PBS solution EP pipe in, be placed on shaking table place Managing 2 hours, change liquid every half an hour, rotating speed is 120r/min, and temperature is 37 DEG C.

2. 0.03% methylene blue+ultra-vioket radiation (4h)

1) configuration is containing 100u/ml heparin, the deionized water of 0.03% methylene blue；

3) de-cellular vascular is put into the transparent EP pipe equipped with configuration solution, and with syringe to de-cellular vascular tube chamber Primer solution, is subsequently placed under the ultraviolet light of medium wavelength irradiation 4 hours；

3. 0.005% methylene blue+ultra-vioket radiation 1h

1) configuration is containing 100u/ml heparin, the deionized water of 0.005% methylene blue；

3) the de-cellular vascular through pretreatment is put into the transparent EP pipe equipped with configuration solution, and with syringe to de- Cellular vascular tube chamber primer solution, is subsequently placed under the ultraviolet light of medium wavelength irradiation 1 hour；

Effect example 1: open-angle measures

Packet: normal blood vessels group, de-cellular vascular group, cross-linking agent matched group, of the present invention group of 1-3, often organizes each 3 blood vessels It is measured, selects the every blood vessel open-angle at point 0%, 20%, 40%, 60%, 80%, 100%, and try to achieve at each point Meansigma methods, the meansigma methods of every blood vessel can be tried to achieve by the meansigma methods of each point, the most again the meansigma methods of every blood vessel is asked again Meansigma methods, in the hope of normal blood vessels group, takes off cellular vascular group, the open-angle meansigma methods of crosslinking blood vessel group.

The metering system of open-angle is as follows:

1. take out fresh rat left common carotid artery blood vessel (or de-cellular vascular, crosslinking blood vessel), be immersed in PBS In；

Observe the most under the microscope, shoot photo；

3. with scalpel, blood vessel is cut into vertically annulus, vascular ring width 1mm；

The most horizontal (vessel lumen is towards the level) and vertical (vessel lumen is upward) of each ring is carried out Observe, take pictures；

5. vascular ring is cut off, static a period of time (about 1 minute), observe and shoot picture；

6. with the picture of image tools processing screened, measure open-angle, and be analyzed.

Measurement result such as Fig. 1-3

Measurement result shows:

1) the open-angle average of de-cellular vascular is less than the open-angle average of normal blood vessels, and has significant difference, table The bright vascular wall residual stress through digestion process reduces.

2) the de-cellular vascular open-angle through glutaraldehyde cross-linking do not increase the most significantly reduce and have the most notable Sex differernce, shows that the blood vessel wall residual stress after glutaraldehyde cross-linking reduces.

3) before of the present invention group of 1-3 is processed relative to methylene blue by the de-cellular vascular after methylene blue process+ultra-vioket radiation De-cellular vascular, residual stress all has recovery.By 0.01% methylene blue+ultra-vioket radiation crosslinking method of two hours to de-thin The residual stress of born of the same parents' blood vessel recovers the most obvious.Make T inspection by SPSS to find, 0.005% methylene blue+ultra-vioket radiation 1h crosslinking side Method has significant difference with the often group open-angle average of 0.01% methylene blue+ultra-vioket radiation 2h；0.03% methylene blue+ultraviolet is shone The open-angle average that 4h compares 0.01% methylene blue+ultra-vioket radiation 2h is smaller, but does not have significant difference.

Effect example 2

Overstrain measures: reflection Ink vessel transfusing outer wall mechanics situation

Packet: normal blood vessels group, de-cellular vascular group, cross-linking agent matched group, of the present invention group 1, often organize each 3 blood vessels and enter Row measures, and selects what every blood vessel was put at each at the strain value trying to achieve of point 0%, 20%, 40%, 60%, 80%, 100% Meansigma methods, can be tried to achieve the meansigma methods of every blood vessel, the most again the meansigma methods of every blood vessel be asked flat again by the meansigma methods of each point Average in the hope of normal blood vessels group, de-cellular vascular group, the inside and outside wall overstrain meansigma methods of crosslinking blood vessel group.

The present invention calculate blood vessel overstrain time in order to make result closer to blood vessel practical situation use image Too l is real

Survey Ink vessel transfusing outer wall girth.

Concrete operation step is as follows:

1) take out fresh rat left common carotid artery blood vessel (or de-cellular vascular, crosslinking blood vessel), be immersed in PBS In；

2) observe under the microscope, shoot photo；

3) with scalpel, blood vessel is cut into vertically annulus, vascular ring width 1mm；

4) under the microscope horizontal (vessel lumen is towards the level) and vertical (vessel lumen is upward) of each ring is carried out Observe, take pictures；

5) vascular ring is cut off, static a period of time (about 1 minute), observe and shoot picture；

6) with the picture of image tools processing screened, whole length of vessel, vascular ring lateral length, blood vessel are measured Ring cuts off front inside and outside wall girth, vascular ring cuts off rear inside and outside wall girth, then tries to achieve according to the strain tensor formula of Ge Xi-Green Overstrain, formula is:L_unload ^iθRepresent and cut off front vascular ring Inwall girth, L_unload ^eθRepresent the outer wall girth cutting off front vascular ring, L_0-stress ^eθRepresent the inwall girth cutting off rear vascular ring, L_0-stress ^eθRepresent the outer wall girth cutting off rear vascular ring.

Measurement result such as Fig. 4

Test result indicate that:

By 3 groups of normal blood vessels of comparison, 3 groups take off cellular vascular and 3 groups of crosslinking Ink vessel transfusing outer wall overstrain averages, and make Corresponding T inspection, it can be deduced that conclusion:

1) absolute value of the outer wall overstrain average of normal blood vessels is more than the absolute value of inwall overstrain average, and deposits In pole significant difference；The absolute value of the inwall overstrain average of de-cellular vascular and the absolute value of outer wall overstrain average There is not significant difference.

2) there is not significance with the inwall overstrain average of de-cellular vascular in normal blood vessels inwall overstrain average Difference；The outer wall overstrain average of normal blood vessels is more than taking off the outer wall overstrain average of cellular vascular and there is pole significance Difference.

3) finding de-cellular vascular and the overstrain average ratio of crosslinking blood vessel are relatively middle, de-cellular vascular and crosslinking blood The change of inside pipe wall overstrain is little, there is not significant difference, and cross-links vessel outer wall overstrain average ratio and take off cell blood The outer wall overstrain average of pipe is big, there is significant difference.

Effect example 3: the blood compatibility experiment of crosslinking blood vessel

Choose of the present invention group 1 and cross-link blood vessel 3 groups, negative control 3 groups, positive control 3 groups, record number by spectrophotometer It is worth as follows:

Table 1 takes off cellular vascular blood compatibility correlation values

According to erythrocyte hemolysis rate (Erythrocyte hemolysis rate, EHR) computing formula:

Can obtain crosslinked after de-cell erythrocyte hemolysis rate be 2.8%.Therefore can be determined that crosslinked after de-cell Blood vessel does not has

Haemolysis.

Effect example 4: the cytotoxicity experiment of crosslinking blood vessel

Cell relative to the rate of increase ((Relative growth rate, RGR) computing formula is as follows:

Wherein toxic reaction 0-1 level is qualified medical material.

The reaction grade scale of table 2 cytotoxicity experiment

Table 3 cell proliferation rate table

Cytotoxicity experiment shows that this crosslinking blood vessel meets the requirement of medical material.

Above-described embodiment all can be carried out by any person skilled in the art under the spirit and the scope of the present invention Modify or change.Therefore, such as art has usually intellectual without departing from disclosed spirit with All equivalences completed under technological thought are modified or change, and must be contained by the claim of the present invention.

Claims

1. one kind is recovered de-cellular vascular residual stress and the method for overstrain, it is characterised in that comprise the steps: 1) join System comprises the aqueous solution that percentage by volume is 0.01-0.03% methylene blue；2) de-cellular vascular is put into equipped with above-mentioned aqueous solution Transparent vessel in, be placed in ultraviolet light irradiate 2-4 hour；3) clean de-cellular vascular and remove removal of residue.

2. recover as claimed in claim 1 de-cellular vascular residual stress and the method for overstrain, it is characterised in that include as Lower step: 1) preparation is containing 50-200u/ml heparin, and percentage by volume is 0.01-0.03% methylene blue and percentage by volume is The aqueous solution of the hydrogen peroxide of 0.125%；2) de-cellular vascular is put in the transparent vessel equipped with above-mentioned aqueous solution, be placed in ultraviolet Light irradiates 2-4 hour；3) clean de-cellular vascular and remove removal of residue.

3. recover de-cellular vascular residual stress and the method for overstrain as claimed in claim 1 or 2, it is characterised in that step Rapid 1) also include in aqueous solution, be passed through oxygen.

4., for recovering the de-de-cellular vascular residual stress of cellular vascular recovery and a reagent for overstrain, its feature exists In, it is 0.01-0.03% aqueous solution of methylene blue including percentage by volume.

5. reagent as claimed in claim 4, it is characterised in that also include anticoagulant and/or antibacterial in described aqueous solution.

6. as claimed in claim 5 reagent, it is characterised in that described anticoagulant includes but not limited to heparin, EDTA, Disodium oxalate. or One or more in citrate.

7. reagent as claimed in claim 5, it is characterised in that described anticoagulant is 50-200u/ml heparin.

8. reagent as claimed in claim 5, it is characterised in that described antibacterial is hydrogen peroxide.

9. reagent as claimed in claim 8, it is characterised in that the percentage by volume of described hydrogen peroxide is 0.125%.

10. reagent as described in any one in claim 4 to 9 is used for recovering de-cellular vascular residual stress and overstrain Purposes.