阅读说明：本技术 一种脱细胞血管基质及其制备方法 (Acellular vascular matrix and preparation method thereof ) 是由 赵子建 王建英 于 2020-04-13 设计创作，主要内容包括：本发明涉及组织工程领域,具体涉及一种脱细胞血管基质及其制备方法,在脱细胞处理步骤中,使用含有[9,9’-二-9H-芴]-9,9’-二甲醛的工作液孵育待用血管组织器官,以脱除细胞,在减少脱细胞所需的时间中有巨大的应用潜力,并且可以在完全去除血管外膜、中膜及内膜中的所有细胞时,并保留完整的三维结构及细胞外基质组分,具有低免疫原性、低细胞毒性和良好的力学性能。(The invention relates to the field of tissue engineering, in particular to a decellularized vascular matrix and a preparation method thereof, wherein in the step of decellularization treatment, working solution containing [9,9 '-bi-9H-fluorene ] -9,9' -dicarboxaldehyde is used for incubating a to-be-used vascular tissue organ to remove cells, so that the decellularized vascular matrix has huge application potential in reducing the time required by decellularization, can retain complete three-dimensional structures and extracellular matrix components when all cells in vascular adventitia, media and intima are completely removed, and has low immunogenicity, low cytotoxicity and good mechanical properties.)

1. A preparation method of a decellularized vascular matrix is characterized in that in the decellularization treatment step, a working solution containing [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde is used for incubating a vascular tissue organ to be used so as to remove cells.

2. The production method according to claim 1, wherein the concentration of [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde in the working solution is 10 μmol/L to 20 μmol/L.

3. The method according to claim 1 or 2, wherein the working solution further comprises a cell culture medium.

4. The method according to claim 3, wherein the working fluid is prepared by a method comprising: dissolving [9,9 '-bi-9H-fluorene ] -9,9' -diformaldehyde in an organic solvent to obtain a mother solution; to the resulting mother liquor, a cell culture medium was added, and [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde was diluted to a set concentration.

5. The method according to claim 1 or 2, wherein the incubation is performed at room temperature for 24 to 48 hours in the decellularization step.

6. The method of manufacturing according to claim 1 or 2, comprising:

pretreating a vascular tissue organ to be used;

performing cell removal treatment;

cleaning;

performing high-low permeability treatment, cleaning, and repeating the high-low permeability treatment at least once;

processing nucleic acid;

cleaning;

and (5) performing disinfection treatment.

7. The method according to claim 6, wherein in the nucleic acid treatment step, the blood vessel tissue/organ to be used is incubated in the DNase I solution for 6 to 12 hours at 37 ℃; the DNase I solution is 50mM Tris-HCl solution containing 160U/ml-320U/ml of DNase I, 10mM-20mM magnesium chloride and pH 7.5.

8. The method according to claim 6 or 7, wherein the step of sterilizing comprises immersing the vascular tissue organ to be treated in 75 vol.% ethanol for 20-40 minutes, followed by washing with PBS for 10-14 hours at 37 ℃ for 3-5 cycles.

9. The preparation method according to claim 6 or 7, wherein in the washing step, the vascular tissue organ to be used is ultrasonically washed 1 to 3 times for 5 to 10 minutes each time under the ultrasonic condition of a frequency of 25KHZ to 45KHZ and a temperature of 37 ℃ to 45 ℃.

10. A xenogenic acellular matrix prepared by the method of any one of claims 1-9.

Technical Field

The invention relates to the field of tissue engineering, in particular to an acellular vascular matrix and a preparation method thereof.

Background

Surveys have shown that cardiovascular disease is the leading cause of death in patients worldwide. At present, the main treatment means of cardiovascular diseases mainly include the following 4 methods, namely, drug treatment, living habit adjustment, interventional therapy and coronary artery bypass transplantation. Among them, the bridge vessels used in coronary artery bypass grafting include autologous vessels and artificial vessels. The autologous blood vessels are the gold standard of blood vessel bypass graft materials, the commonly used autologous blood vessels mainly comprise an internal mammary artery, a radial artery and a great saphenous vein, but the autologous blood vessels have limited sources and cause secondary trauma to patients, and in addition, many patients have no normal blood vessels for autologous transplantation due to the existence of other vascular diseases. There are three main types of artificial blood vessels: heterogeneous acellular matrices, natural polymeric materials (collagen, gelatin, chitosan, etc.), and synthetic polymeric materials (nylon, dacron, and polytetrafluoroethylene). The artificial blood vessel prepared by the high polymer material has good clinical effect on high-flow low-resistance blood vessels such as great vessels like thoraco-abdominal aorta and the like. However, small diameter vessels made of polymeric materials have poor elasticity and compliance and lack an anti-thrombotic surface, resulting in constructed small diameter vessels (<6mm) that continue to suffer from problems such as intimal hyperplasia and thrombosis. The xenogenic tissue is decellularized by physical or chemical methods to remove cell components causing rejection reaction in the tissue transplantation process, and the xenogenic acellular matrix retaining extracellular matrix (ECM) components and a complete scaffold structure has been widely used in biological tissues such as dermis, heart valve and the like, and has achieved good clinical effects.

ECM components include collagen, elastin, fibronectin, laminin, glycosaminoglycans, and growth factors. The ECM ultrastructure and the components are kept to the maximum extent, so that the mechanical property and the biological property of the acellular matrix are ensured. The existing acellular method mainly comprises a chemical method and a physical method. Chemical methods use reagents to remove cells, such as acid-base reagents, hypotonic hypertonic solutions, detergents; biological agents, such as enzymes, non-enzyme factors. Physical methods such as repeated freeze thawing and mechanical treatment. For example, in chinese patent document CN108904884A, a method for preparing a pig aorta acellular matrix mainly uses repeated freezing and thawing and a detergent in combination to remove cells. For another example, in chinese patent document CN109529121A, a acellular vascular matrix and a preparation method thereof use hypotonic treatment, trypsin, and detergent to perform combined treatment for acellular. The method can achieve the cell removing effect of different degrees, has low toxicity and maintains good mechanical property to a certain extent. However, the use of the above chemical or physical methods or the combination of both still alters ECM, destroys ECM ultrastructure, leads to degradation of extracellular matrix components such as collagen, glycosaminoglycan, growth factors, etc., and mechanical and biological properties of acellular matrix are difficult to satisfy clinical therapeutic effects.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to overcome the defect that the mechanical and biological properties of the acellular matrix in the prior art are difficult to satisfy the clinical treatment effect, so as to provide an acellular vascular matrix and a preparation method thereof, wherein when all cells in the adventitia, the media and the intima of a blood vessel are completely removed, the complete three-dimensional structure and extracellular matrix components are retained, and the acellular vascular matrix has low immunogenicity, low cytotoxicity and good mechanical properties.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the present invention provides a method for preparing a acellular vascular matrix, wherein in the step of acellular treatment, a working solution containing [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde (abbreviated as Raptinal) is used for incubating a vascular tissue organ to be used to remove cells.

Preferably, in the working solution, the concentration of [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde is 10 to 20. mu. mol/L.

Preferably, a cell culture medium, such as MEM medium, is also included in the working solution.

The preparation method of the working solution comprises the steps of dissolving [9,9 '-di-9H-fluorene ] -9,9' -diformaldehyde in an organic solvent to obtain a mother solution; to the resulting mother liquor, a cell culture medium was added, and [9,9 '-di-9H-fluorene ] -9,9' -dicarboxaldehyde was diluted to a set concentration. Preferably, the organic solvent may be dimethyl sulfoxide (DMSO).

In the cell removing step, the incubation condition is incubation for 24h-48h at room temperature.

Preferably, the method for preparing the acellular vascular matrix comprises the following steps: pretreating a vascular tissue organ to be used;

performing cell removal treatment;

cleaning;

performing high-low permeability treatment, cleaning, and repeating the high-low permeability treatment at least once;

processing nucleic acid;

cleaning;

and (5) performing disinfection treatment.

Preferably, in the nucleic acid treatment step, the vascular tissue organ to be used is incubated in a DNase I (DNase I) solution for 6 to 12 hours at 37 ℃; the DNase I solution is 50mM Tris-HCl solution containing 160U/ml-320U/ml of DNase I, 10mM-20mM magnesium chloride and pH 7.5.

Preferably, the step of disinfection treatment is to soak the vascular tissue organ to be used in alcohol with the volume percentage of 75% for 30 minutes, and then wash the vascular tissue organ in PBS for 12 hours under vibration at the temperature of 37 ℃ for 3-5 times in a circulating way.

Preferably, in the cleaning step, the vascular tissue organ to be used is cleaned by ultrasonic for 1 to 3 times, each time for 5 to 10 minutes, and the ultrasonic condition is that the frequency is 25KHZ to 45KHZ and the temperature is 37 ℃ to 45 ℃.

Preferably, the hypertonic-hypotonic treatment is to shake (210-230rpm) the blood vessel tissue to be used in the step (3) with 45-55mM Tris-HCL (hypertonic solution) for 10-14h, and then shake (210-230rpm) the blood vessel tissue to be used with 5-15mM Tris-HCL (hypotonic solution) for 10-14 h.

In a second aspect, the present invention provides a xenogenic acellular matrix prepared according to the preparation method.

The technical scheme of the invention has the following advantages:

1. the invention provides a preparation method of acellular vascular matrix, and the applicant researches and discovers that a raptine medicament can induce apoptosis more rapidly compared with other apoptosis-promoting medicaments, the action mechanism of the raptine medicament is to induce apoptosis by a mitochondrial pathway, and the applicant evaluates a plurality of apoptosis-inducing medicaments such as kinase inhibitors (staurosporine and rapamycin), tubulin inhibitors (taxol, vincristine and colchicine), topoisomerase inhibitors (camptothecin, etoposide and doxorubicin), DNA alkylating agents (mitomycin C, cisplatin and MNNG), endoplasmic reticulum/proteasome inhibitors (geldana-mycin, thapsigargin and tunicamycin), ROS inducers (rotenone and antimycin A) and medicaments directly targeting components of the apoptosis pathway such as Bcl-2 inhibitor (gossypol) and XIAP inhibitor (SM-164), apoptosis body promoter (AA2) and direct caspase-3 activator (PAC-1 and 1541/1541B), all of which are treated with 10uM concentration for 1 hour on U-937 cells, and the results show that Raptinal completely activates procaspasen-3 and caspase-3, 1541 (which is a pro-apoptotic drug) and partially activates procaspasen-3 and caspase-3, while other agents do not activate procaspase-3, indicating that Raptinal induces apoptosis at an abnormally rapid rate, has great application potential in reducing the time required for decellularization, and that Raptinal can target and destroy the cellular components in vascular tissues without destroying the vascular extracellular matrix, and retain the complete three-dimensional structure and cellular components when all cells in the vascular adventitia, media and intima are completely removed, the acellular vascular matrix prepared by the invention has the advantages of low immunogenicity, low cytotoxicity and good mechanical property, and is an ideal vascular graft.

2. The invention provides a preparation method of an acellular vascular matrix, which comprises the following steps: pretreating a vascular tissue organ to be used; performing cell removal treatment; cleaning; performing high-low permeability treatment, cleaning, and repeating the high-low permeability treatment at least once; processing nucleic acid; cleaning; sterilizing; in the method, firstly raptinil solution is used for inducing apoptosis and rupture of cell components in tissues, then hypertonic solution and hypotonic solution are used for alternately washing to remove cell protein and DNA, and finally DNaseI solution is used for cracking nucleic acid sequences and removing nucleic acid substances after cell cracking so as to achieve better cell removing effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is the result of HE staining in the experimental example of the present invention; in the figure, the A picture is a natural blood vessel, and the B picture is a xenogenic acellular vascular matrix (the scale in the figure is 100 μm);

FIG. 2 shows the result of DAPI staining in the experimental examples of the present invention; in the figure, the A picture is a natural blood vessel, and the B picture is a heterogenous acellular vascular matrix;

FIG. 3 shows the result of detection of DNA residue in the experimental example of the present invention;

FIG. 4 shows Masson trichrome staining results in an experimental example of the present invention; in the figure, the A picture is a natural blood vessel, and the B picture is a xenogenic acellular vascular matrix (the scale in the figure is 100 μm);

FIG. 5 shows the result of EVG staining in the experimental example of the present invention; in the figure, the A picture is a natural blood vessel, and the B picture is a xenogenic acellular vascular matrix (the scale in the figure is 100 μm);

FIG. 6 shows the results of Alsinoblue staining in the experimental examples of the present invention; in the figure, the A picture is a natural blood vessel, and the B picture is a xenogenic acellular vascular matrix (the scale in the figure is 100 μm).

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The vascular tissue in the following examples is adult healthy porcine carotid artery, the lumen is flushed with Phosphate Buffered Saline (PBS) (PH 7.4) containing antibiotic (penicilin-Streptomycin, Gibco, usa, 2% -5% by volume), the blood in the lumen is cleared, and the outer loose connective tissue is gently stripped off, thus obtaining the vascular tissue to be used.

Raptin is available from sigma aldrich, usa;

DMSO, AR (analytical grade), alatin;

minimum Eagle's Medium (MEM) Medium was purchased from Gibco, USA;

complete medium is MEM medium containing 10% Fetal Bovine Serum (FBS) (Gibco, usa);

the room temperature range of the invention is 10-30 ℃;

DNase I solution was purchased from sigma aldrich, usa;

2% ferric chloride solution (EVG staining kit purchased (google biosciences, cat # G1042) contains 10% ferric chloride, diluted to 2% with distilled water).

EXAMPLE 1 preparation of a xenogenic acellular vascular matrix

The embodiment provides a preparation method of a heterogenous acellular vascular matrix, which comprises the following steps:

(1) pretreating the vascular tissue to be used, adding the vascular tissue into ultrapure water, and cleaning for 2 times, 5 minutes each time, under the ultrasonic condition that the frequency is 35KHZ and the temperature is 41 ℃;

(2) dissolving Raptinal in DMSO to prepare a mother solution containing 20mmol/L Raptinal, diluting the mother solution containing 20mmol/L Raptinal into a working solution containing 10 mu mol/L Raptinal by using a Minimum Eagle's Medium (MEM) culture Medium, and incubating the vascular tissue to be used in the working solution containing 10 mu mol/L Raptinal for 24 hours at room temperature (25 ℃);

(3) then adding the vascular tissue to be used in the step (2) into ultrapure water, and cleaning for 2 times, 5 minutes each time, wherein the ultrasonic condition is 35KHZ in frequency and 41 ℃;

(4) washing the vascular tissue to be used in the step (3) with 50mM Tris-HCL (hypertonic solution) for 12h by shaking (220rpm), and then washing the vascular tissue to be used with 10mM Tris-HCL (hypotonic solution) for 12h by shaking (220 rpm);

(5) adding the vascular tissue to be used in the step (4) into ultrapure water for ultrasonic cleaning for 2 times, wherein each time is 5 minutes, and the ultrasonic condition is that the frequency is 35KHZ and the temperature is 41 ℃;

(6) washing the blood vessel tissue to be used in the step (5) with 50mM Tris-HCL (hypertonic solution) for 12h by shaking (220rpm), and washing the blood vessel tissue with 10mM Tris-HCL (hypotonic solution) for 12h by shaking (220 rpm);

(7) incubating the vascular tissue to be used in step (6) in 160U/ml DNase I solution for 6 hours at 37 ℃, wherein the DNase I solution is 50mM Tris-HCl solution containing 160U/ml DNase I, 10mM magnesium chloride and pH 7.5;

(8) adding the vascular tissue to be used in the step (7) into ultrapure water for ultrasonic cleaning for 2 times, wherein each time is 5 minutes, and the ultrasonic condition is that the frequency is 35KHZ and the temperature is 41 ℃;

(9) soaking the blood vessel tissue to be used in the step (8) in 75% alcohol by volume percentage for 30 minutes, then cleaning the blood vessel tissue in PBS (PH is 7.4) in a shaking way (100rpm) for 12 hours at the temperature of 37 ℃, and circulating for 3 times to obtain the heterogenous acellular blood vessel matrix;

(10) and (4) storing the heterogenous acellular vascular matrix prepared in the step (9) in a refrigerator at the temperature of-20 ℃ for later use.

EXAMPLE 2 preparation of a xenogenic acellular vascular matrix

The embodiment provides a preparation method of a heterogenous acellular vascular matrix, which comprises the following steps:

(1) pretreating the vascular tissue to be used, adding the vascular tissue into ultrapure water, and cleaning for 2 times, 5 minutes each time, under the ultrasonic condition that the frequency is 25KHZ and the temperature is 45 ℃;

(2) dissolving Raptinal in DMSO to prepare a mother solution containing 20mmol/L Raptinal, diluting the mother solution containing 20mmol/L Raptinal into a working solution containing 10 mu mol/L Raptinal by using a Minimum Eagle's Medium (MEM) culture Medium, and incubating the vascular tissue to be used in the working solution containing 10 mu mol/L Raptinal for 26 hours at room temperature (25 ℃);

(3) then adding the vascular tissue to be used in the step (2) into ultrapure water, and cleaning for 1 time for 5 minutes each time under the ultrasonic condition that the frequency is 25KHZ and the temperature is 45 ℃;

(4) washing the vascular tissue to be used in the step (3) with 45mM Tris-HCL (hypertonic solution) for 12h by shaking (220rpm), and then washing the vascular tissue to be used with 5mM Tris-HCL (hypotonic solution) for 12h by shaking (230 rpm);

(5) adding the vascular tissue to be used in the step (4) into ultrapure water for ultrasonic cleaning for 1 time and 10 minutes, wherein the ultrasonic condition is that the frequency is 25KHZ and the temperature is 45 ℃;

(6) washing the blood vessel tissue to be used in the step (5) with 45mM Tris-HCL (hypertonic solution) for 14h by shaking (210rpm), and washing the blood vessel tissue with 5mM Tris-HCL (hypotonic solution) for 14h by shaking (220 rpm);

(7) incubating the vascular tissue to be used in step (6) in a DNase I solution of 320U/ml for 12 hours at 37 ℃ in a 50mM Tris-HCl solution containing 320U/ml DNase I, 20mM magnesium chloride and having a pH of 7.5.

(8) Adding the vascular tissue to be used in the step (7) into ultrapure water for ultrasonic cleaning for 1 time and 10 minutes, wherein the ultrasonic condition is that the frequency is 25KHZ and the temperature is 45 ℃;

(9) soaking the blood vessel tissue to be used in the step (8) in 75% alcohol by volume percentage for 20 minutes, then washing the blood vessel tissue in PBS (PH is 7.4) for 10 hours by oscillating (110rpm), circulating for 5 times at 37 ℃, and obtaining the heterogenous acellular blood vessel matrix;

(10) and (4) storing the heterogenous acellular vascular matrix prepared in the step (9) in a refrigerator at the temperature of-20 ℃ for later use.

EXAMPLE 3 preparation of a heterogeneous acellular vascular matrix

The embodiment provides a preparation method of a heterogenous acellular vascular matrix, which comprises the following steps:

(1) pretreating the vascular tissue to be used, adding the vascular tissue into ultrapure water, and cleaning for 3 times, 5 minutes each time, under the ultrasonic condition that the frequency is 45KHZ and the temperature is 37 ℃;

(2) dissolving Raptinal in DMSO to prepare a mother solution containing 20mmol/L Raptinal, diluting the mother solution containing 20mmol/L Raptinal into a working solution containing 10 mu mol/L Raptinal by using a Minimum Eagle's Medium (MEM) culture Medium, and incubating the vascular tissue to be used in the working solution containing 10 mu mol/L Raptinal for 24 hours at room temperature (25 ℃);

(3) then adding the vascular tissue to be used in the step (2) into ultrapure water, and cleaning for 3 times, 7 minutes each time, under the ultrasonic condition that the frequency is 45KHZ and the temperature is 37 ℃;

(4) washing the vascular tissue to be used in the step (3) with 55mM Tris-HCL (hypertonic solution) for 10h by oscillating (230rpm), and then washing the vascular tissue to be used with 15mM Tris-HCL (hypotonic solution) for 10h by oscillating (220 rpm);

(5) adding the vascular tissue to be used in the step (4) into ultrapure water for ultrasonic cleaning for 1 time, wherein each time is 10 minutes, the frequency is 45KHZ, and the temperature is 37 ℃;

(6) washing the blood vessel tissue to be used in the step (5) with 55mM Tris-HCL (hypertonic solution) for 10h by oscillating (210rpm), and washing the blood vessel tissue with 15mM Tris-HCL (hypotonic solution) for 10h by oscillating (220 rpm);

(7) incubating the vascular tissue to be used in step (6) in 240U/ml DNase I solution at 37 ℃ for 6 hours, the DNase I solution being 50mM Tris-HCl solution containing 240U/ml DNase I, 15mM magnesium chloride and having a pH of 7.5.

(8) Adding the vascular tissue to be used in the step (7) into ultrapure water for ultrasonic cleaning for 2 times, wherein each time is 5 minutes, the frequency is 45KHZ, and the temperature is 37 ℃;

(9) soaking the blood vessel tissue to be used in the step (8) in 75% alcohol by volume percentage for 40 minutes, then washing the blood vessel tissue in PBS (PH is 7.4) for 14 hours by oscillating (90rpm), circulating for 4 times at 37 ℃, and obtaining the heterogenous acellular blood vessel matrix;

(10) and (4) storing the heterogenous acellular vascular matrix prepared in the step (9) in a refrigerator at the temperature of-20 ℃ for later use.

Experimental examples biological evaluation of different acellular vascular matrices

1 method

1.1 hematoxylin-eosin (HE)

(1) Paraffin sections (the sections each containing natural vascular tissue of adult healthy porcine carotid artery to be used and xenogenic acellular vascular matrix prepared in example 1) were deparaffinized in xylene for 10min, deparaffinized 2 times, sequentially added with absolute ethanol, 95% ethanol by volume, 85% ethanol by volume and 75% ethanol by volume each for 3min, and rinsed with tap water for 5 min;

(2) staining with hematoxylin solution for 5min, and washing with tap water for 5 min;

(3) differentiating with 1% hydrochloric acid alcohol for several seconds, and then washing with water for 1 min;

(4) and dyeing for 1min in eosin dyeing solution.

(5) Anhydrous ethanol, 95% ethanol by volume, 85% ethanol by volume, and 75% ethanol by volume, dehydrating for 3min, respectively, and allowing xylene to be transparent for 3min and 2 times. Neutral gum was mounted and examined under a microscope (leica upright microscope, DM 500).

1.24, 6-diamidino-2-phenylindole (DAPI) staining

(1) Paraffin sections (the sections each containing native vascular tissue of adult healthy porcine carotid artery to be used and xenogenic acellular vascular matrix prepared in example 1) were deparaffinized in xylene for 10min, deparaffinized 2 times, sequentially with absolute ethanol, 95% ethanol by volume, 85% ethanol by volume and 75% ethanol by volume each for 3min, and rinsed with tap water for 5 min.

(2) DAPI working solution was added, incubated for 10min at room temperature in the dark, and then rinsed three times with PBS for 5min each.

(3) After mounting with a water-soluble mounting agent, observation was carried out under a fluorescence microscope (model LEICA, DM 2500LED type upright fluorescence microscope)).

1.3 DNA residue detection (Prep for DNA extraction kit)Residual DNA Sample Preparation Kit, Thermo Fisher, USA)

(1) Test sample preparation: taking the heterogenous acellular vascular matrix prepared in example 1 as a test sample, taking natural blood vessels (untreated blood vessel tissues to be used) as a control, taking 3 parallel samples of each group of test samples, shearing and filling the samples into a 1.5ml sterile centrifugal tube, taking 3 parallel samples with the same mass, taking 3 parts for experiments, and taking 3 parts for freeze drying and weighing;

(2) DNA extraction:

1) and (3) protease K digestion: mu.l proteinase K and 60. mu.l proteinase K buffer (10X) were mixed to prepare a digestion solution, and 70. mu.l digestion solution was added to each test sample and digested in a 56 ℃ water bath until the test sample was completely digested and no particle sample was visible to the naked eye.

2) Add 400. mu.l lysate to each digested group, vortex for 10s, and let stand for 10min until completely dissolved.

3) Add 30. mu.l of magnetic beads (preheated in 37 ℃ water bath) and shake for 10 min.

4) Mu.l of binding solution (reagent contained in the purchased DNA extraction kit, Thermo Fisher, USA) was added and incubated with shaking at room temperature for 10 min.

5) Centrifuging (12000rpm/min) for 5s, placing on a magnetic frame, and standing for 5 min.

6) Discarding the supernatant, adding 300. mu.l of washing solution, vortexing for 10s, rapidly centrifuging (12000rpm/min) for 15s, placing in a magnetic frame, and standing for 1 min.

7) Repeating the steps 3) -5), rinsing the DNA for at least 2 times, quickly centrifuging (12000rpm/min) for 25s for the last time, and collecting the magnetic beads.

8) After discarding the supernatant, the tube is left open to dry at room temperature for no more than 5 min.

9) Add 100. mu.l of the eluate, vortex and shake for 5min, and water bath at 70 ℃ for 5min (vortex midway).

10) The mixture was centrifuged rapidly (12000rpm/min) for 25s, placed in a magnetic rack for 2min, and the original groups were transferred to a new group of centrifuge tubes.

11) Add 50. mu.l of lysis solution (reagents included in the purchased DNA kit, Thermo Fisher, USA), vortex for 5min, and water bath at 70 ℃ for 5min (vortex halfway).

12) Repeating the step 10), obtaining 150 mu l of dissolution liquid in a new group of centrifuge tubes, mixing the dissolution liquid in a vortex mode, and quickly centrifuging for 25s to obtain the DNA after final purification. (-20 ℃ C. storage)

(3) Determination of DNA content (DNA detection kit is Quant-iT)^TM PicodsDNAargent and Kits, Thermo Fisher, USA)

1) Drawing a standard curve: lambda DNA (100 ng/. mu.l) was diluted 50-fold with 1 XTE buffer to a concentration of 2000ng/ml, and then the lambda DNA at a concentration of 2000ng/ml was diluted with 1 XTE buffer to a concentration of 0ng/ml,50ng/ml,100ng/ml,200ng/ml,400ng/ml,800ng/ml, and the diluted standard solutions were added to a 96-well plate in an amount of 125. mu.l per well, 3 wells per portion.

2) And (3) measuring the concentration of the sample DNA obtained in the step (2) by using a spectrophotometer, diluting according to corresponding times, and controlling the concentration range to be in a standard curve.

The diluted sample DNA was added to a 96-well plate in an amount of 125. mu.l per well, 3 wells each.

3) Fluorescent staining: diluting the Reagent fluorescent staining solution (Reagent contained in the purchased DNA detection kit, Thermo Fisher, USA) by 200 times for use, respectively adding 125 mul of the diluted Reagent fluorescent staining solution (mixed with the sample in equal volume) into the 96-well plate, shaking and mixing uniformly, incubating for 5min at room temperature in a dark place, and measuring by using a fluorescent microplate reader under the measuring conditions: the data obtained were plotted and analyzed by using 480nm as the excitation wavelength, 520nm as the emission wavelength and 530nm as the cut-off wavelength. The Relative Fluorescence Intensity (RFI) of each well measured was measured and recorded with the fluorescence intensity measured using 1 XTE buffer as a sample as a base value.

4) And (4) calculating a result: the relative fluorescence intensity values were first determined for the standard and test sample groups, averaged, and subtracted with the background (RFI values measured in the absence of DNA but 1 × TE buffer). And drawing a standard curve by using the RFI value of the standard product as an ordinate (Y) and the addition amount (ng/ml) as an abscissa (X) to obtain a regression equation: x ═ y-a₁)/b₁，R₁ ². Substituting the RFI value of the sample into a standard curve to obtain a concentration value, multiplying the concentration value by the total volume and dilution times of the extracted DNA to obtain the total DNA content of the test sample with the unit of ng, and dividing the total DNA content of the test sample by the total dry weight of the sample to obtain the residual amount of DNA (ng/mg) of the test sample

1.4 Masson trichrome dyeing

(1) Paraffin sections (the sections each containing natural vascular tissue of adult healthy porcine carotid artery to be used and xenogenic acellular vascular matrix prepared in example 1) were deparaffinized in xylene for 10min, deparaffinized 2 times, sequentially with absolute ethanol, 95% ethanol by volume, 85% ethanol by volume and 75% ethanol by volume each for 3min, and rinsed with tap water for 5 min.

(2) The sections were mounted in bouin solution (Masson trichrome kit containing reagents, Solebao, cat # G1345), mordanted at 37 ℃ in an incubator for 2h, and washed with running water until the yellow color on the sections disappeared.

(3) And (5) slightly washing the azure blue by water after 2-3min of drop dyeing.

(4) Mayer hematoxylin drop staining for 8min and washing with water slightly.

(5) An acidic ethanol differentiation solution (reagent contained in Masson trichrome kit, 1% hydrochloric alcohol) was differentiated for several seconds, and washed with running water for 10min (reverse blue).

(6) Ponceau fuchsin was stained for 8min and washed slightly with water.

(7) And (3) treating with phosphomolybdic acid for 10 min.

(8) The supernatant was decanted off, and the slices were directly dropped into aniline blue dye solution for 5min without washing with water.

(9) Treating with weak acid solution for 2 min.

(10) The ethanol with the volume percentage of 95 percent is dehydrated quickly, and the absolute ethanol is dehydrated twice, each time for 5 to 10 seconds.

(11) Transparent sealing sheet

1.5 Verhoeff' S VAN GIESON (EVG) staining

(1) Paraffin sections (the sections each containing native vascular tissue of adult healthy porcine carotid artery to be used and xenogenic acellular vascular matrix prepared in example 1) were deparaffinized in xylene for 10min, deparaffinized 2 times, sequentially with absolute ethanol, 95% ethanol by volume, 85% ethanol by volume and 75% ethanol by volume each for 3min, and rinsed with tap water for 5 min.

(2) Dyeing with prepared EVG dye solution (Google biology, Cat G1042) in greenhouse for 30min until the color is dark black.

(3) Washing with tap water, differentiating with 2% ferric trichloride solution for 10-20s, and observing with microscope that elastic fiber is black and background is gray.

(4) Washing with tap water and slightly washing with distilled water; van Gieseon's liquid (Google organism, cat # G1042) was counterstained for 3-5 min.

(5) Dehydrated for 3min respectively by absolute ethyl alcohol, 95% by volume of ethyl alcohol, 85% by volume of ethyl alcohol and 75% by volume of ethyl alcohol, and the xylene is transparent for 3min and 2 times. And sealing the neutral gum into pieces, and performing microscopic examination.

1.6 cytotoxicity assays

(1) And (3) sterilizing the vascular tissue: the xenogenic acellular vascular matrix prepared in example 1 and native blood vessels were treated with 5% peroxyacetic acid for 3 hours, respectively, and then washed with sterile PBS for 12 hours.

(2) Preparing the leaching solution by sterilizing blood vessel tissue with a length of 6cm²The cells were incubated at a density of one ml/ml in complete medium at 37 ℃ and 100rpm for 24h, complete medium being used as a control.

(3) Cell culture: mouse fibroblasts (L929) were cultured in 5% CO₂Incubate at 37 ℃ to near confluency (80% plating); making into 3 × 10⁴The cell suspension was seeded in 96-well plates at 100. mu.l/well, 3X 10³Cells, inoculation hole 6 x 3; discarding the culture medium when the cells grow to be in a nearly confluent state;

(4) inoculating a leaching solution: adding 100 μ l of control leaching solution into each well of 96-well cell-grown plate, wherein the control group is complete culture medium and 5% CO₂The culture was continued in the 37 ℃ incubator for 24h, 48h,72h.

(5) And (3) detecting cytotoxicity: and (3) detecting cell proliferation toxicity by using a CCK-8 kit: cells were cultured in the extract for 24h, 48h,72h, respectively, and then the extract was discarded, and 100. mu.l of a new culture medium (MEM complete medium) and 10. mu.l of CCK-8 solution were added to each well, followed by incubation in an incubator for 2h and then OD450 was measured.

(6) The OD value of the leaching liquor/the OD value of the control group is the relative proliferation rate.

1.7 Alsinoblue staining

(1) Paraffin sections of vascular tissue (the sections all contain natural vascular tissue of adult healthy pig carotid artery to be used and xenogenic acellular vascular matrix prepared in example 1) were dewaxed in xylene for 10min, 2 times, then 3min each were sequentially added with absolute ethanol, 95% ethanol by volume, 85% ethanol by volume and 75% ethanol by volume, and tap water was washed for 5 min.

(2) Alxin blue staining solution (Google biology, Cat. G1027) is used for staining for 10min and is washed with water slightly.

(3) Dehydrated for 3min respectively by anhydrous ethanol, 95% ethanol by volume, 85% ethanol by volume, and 75% ethanol by volume, and transparent for 2 times and 3 min. And sealing the neutral gum into pieces, and performing microscopic examination. (leica upright microscope, DM500)

2. Results

2.1 Decellularization Effect

(1) HE staining and DAPI staining results

When the tissue sections of the natural blood vessels and the acellular vascular matrices prepared in example 1 were stained with hematoxylin-eosin staining solution, the nuclei were stained with hematoxylin in a vivid bluish-purple color (dark gray spots in black and white), and the cytoplasm and extracellular matrices were stained in a red color (gray spots in black and white), as shown in fig. 1, a in fig. 1 was the natural blood vessels, and B in fig. 1 was the acellular vascular matrices, and as seen from the graphs, HE was stained with natural blood vessels to see a large amount of blue (dark gray spots in black and white) nuclear structures, while HE was stained with acellular vascular matrices to see only extracellular matrices stained in a red color (gray spots in black and white).

Tissue sections of native vessels and the xenogenic acellular vascular matrix prepared in example 1 were DAPI stained, and then normal nuclei showed bright blue fluorescence and no cytoplasm fluorescence, as shown in FIG. 2, in which graph A shows native vessels and graph B shows xenogenic acellular vascular matrix, and as can be seen from the graph, the native vascular DAPI staining showed a large amount of blue fluorescence (white spots in the graph) and the acellular vascular matrix DAPI staining showed no fluorescence (white spots in the graph).

The results show that the heterogenous acellular vascular matrixes prepared by the invention are completely removed, and only the extracellular matrix structure is reserved.

(2) DNA residue detection result

As shown in FIG. 3, it can be seen that the DNA content of natural blood vessel is 2717 + -161.935 ng/mg and the DNA content of xenogenic acellular vascular matrix is 41.064 + -2.209 ng/mg.

2.2 extracellular matrix component Retention

The Masson trichrome results are shown in fig. 4, in which the a-picture is the native blood vessel, the B-picture is the xenogenic acellular vascular matrix, and the collagen fibers are dark blue (dark grey in black and white pictures), from which the collagen fibers are visible, indicating that collagen is better retained in the xenogenic acellular vascular matrix.

The EVG staining results are shown in fig. 5, in which a is a natural blood vessel and B is a xenogenic acellular vascular matrix, and the EVG staining results show that the elastic fibers are black, and the elastic fibers are visible from B, indicating that elastin is well retained in the xenogenic acellular vascular matrix.

The results of alcian blue staining are shown in fig. 6, wherein a is a natural blood vessel, B is a xenogenic acellular vascular matrix, and alcian blue staining shows that the glycosaminoglycan is light gray, from which the light gray glycosaminoglycan can be seen, indicating that the glycosaminoglycan is better retained in the xenogenic acellular vascular matrix.

2.3 cytotoxicity assays

Culturing L929 cells by using the acellular vascular matrix leaching liquor, and calculating the relative cell proliferation rate (RGR), wherein the relative cell proliferation rate (RGR) is equal to (OD mean value of each detection group/OD mean value of a control group) 100%, the toxicity of the material is evaluated according to the response grading standard of cytotoxicity experiments in ISO and GB/T, the RGR (100%) > 100 is positioned on a grade 0, the RGR (100%) > 75-99 is positioned on a grade 1, the RGR (100%) > 50-74 is positioned on a grade 2, the RGR (100%) > 25-49 is positioned on a grade 3, the RGR (100%) > 1-24 is positioned on a grade 4, and the RGR (100%) > 0 is positioned on a grade 5. Wherein the grade of 0-1(RGR is more than or equal to 75%) toxic reaction is qualified medical material. The results are shown in Table 1 below.

TABLE 1 cytotoxicity assay results

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.