CN104013998B - Preparation method of artificial esophagus with histological structure - Google Patents
Preparation method of artificial esophagus with histological structure Download PDFInfo
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- CN104013998B CN104013998B CN201410227418.6A CN201410227418A CN104013998B CN 104013998 B CN104013998 B CN 104013998B CN 201410227418 A CN201410227418 A CN 201410227418A CN 104013998 B CN104013998 B CN 104013998B
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Abstract
The invention discloses a preparation method of an artificial esophagus with histological structure. The method is characterized by comprising the steps of: extracting a basilemma membrane protein solution from animal esophageal mucosa tissue; then preparing electrospinning / acellular matrix bracket; then preparing a polyurethane sheet with a double-sided microgroove; and finally coating the polyurethane sheet on the electrospinning / acellular matrix bracket and splicing into a tube shape longitudinally, so as to obtain the artificial esophagus with histological structure. The invention has the advantage that the method can prepare the artificial esophagus simulating histological structure of normal human esophageal, and utilizes the principles of tissue engineering to construct in vitro the artificial esophagus with similar microstructure and function as human esophagus; and the artificial esophagus is seamed with residual esophagus after resection of lesion esophagus layer by layer, so that the artificial esophagus gradually grows into a real esophagus and integrates with the original esophagus in the human body. Compared with the traditional alternatives, the artificial esophagus has structure and function similar to those of natural esophagus, and can alleviate the suffering of patients and prolong the patient's life.
Description
Technical field
The present invention relates to tissue engineering field, particularly relate to a kind of preparation method with the artificial esophagus of histological structure.
Background technology
Normal human body esophagus is the hollow pipeline of one about 20 ~ 25 centimeter length, diameter about 1.9 centimetres (different because of individual), connects throat and stomach, is responsible for function food being transported to stomach from throat.From histological angle, esophagus is made up of mucosa, tela submucosa, muscle layer and adventitia etc. four layers, and wherein mucosa and muscle layer are most important functional components.If mucosa is a permeable barrier layer, protection and buffer action are played to esophagus interior tissue; Containing tubular gland in tela submucosa, make mucomembranous surface remain lubrication, with reduce food by time friction, make food can be transported to stomach under the prerequisite not damaging esophagus; And muscle layer contains significantly and goes in ring and outer stringer multilayer structure, realized the delivery functions of food by the contraction of myocyte and wriggling; And the bottom of mucosa is basement membrane, this is the extracellular matrix that skim contains somatomedin, some Special Proteins and other nutrient substance, has impact and regulates the functions such as epithelial adhesion, propagation, migration, differentiation and death.After testing, the thick about 50 ~ 150nm of basement membrane, be the network structure that the fiber of 28 ~ 166nm and the hole of size inequality form by diameter.Containing functional proteins such as IV Collagen Type VI, laminin，LN, nestin and Dan Baiduotang proteoglycan PG in basement membrane.
Human body esophagus pathological changes happens occasionally, and has make a variation the congenital day after tomorrow that also has brought.Especially esophageal carcinoma has become the fifth-largest malignant tumor in the world.And investigation finds in esophageal cancer patients, the predilection site of esophageal carcinoma is hypomere in esophagus, accounts for 90% nearly.These esophagus diseases cause esophageal obstruction and can not take food, and serious threat human life is healthy.Clinically the treatment of early esophageal cancer is coincide based on excision, stomach-cervical part of esophagus esophagus, for long section esophagus pathological changes, also rebuild with excision and be replaced by master.Conventional substitute has two classes: a class is its hetero-organization autologous or organ, as stomach, jejunum, colon etc., wherein, it is the most frequently used succedaneum that domestic esophagus substitutes stomach in operation, but these substitutes damage with injury repairing, simultaneously therapeutic effect is also undesirable, and the mortality as substituted pathological changes esophagus with stomach reaches 7 ~ 20%, postoperative patient syndrome up to more than 50%; Another kind of substitute is plastics, rubber, glass, polyethylene, Teflon, wire loop etc., and its shortcoming of the substitute of these non-degradable, biocompatible is also apparent.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of histological structure of simulating normal human's esophagus, utilize tissue engineering principle, build in vitro and the preparation method with the artificial esophagus of histological structure that human body esophagus has similar microtexture and function, can alleviate sufferer misery, extend sufferer life.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of preparation method with the artificial esophagus of histological structure, specifically comprises the following steps:
(1), get the mucosa in animal esophagus and tela submucosa, clean, shred, and mucosa and submucosa tissue are immersed by NaCl, Tris-HCl(Tris), in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 3.4mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is immersed by NaCl again, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 0.5mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is then immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 2.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is finally immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 4.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, by the supernatant mixing acquired by four times, obtain laminin liquid, for support bag quilt,
(2), get the mucosa in animal esophagus and tela submucosa, clear water is rinsed well, and sterilize with 75% alcohol-pickled 1 ~ 4h, clean with PBS wash buffer after sterilization, dual anti-and the TritonX be made up of penicillin and streptomycin is mixed in PBS buffer and forms PBS mixed liquor, wherein: TritonX is 1% at the percentage by weight of PBS mixed liquor, penicillin and the concentration of streptomycin in PBS mixed liquor are 200U/L, again above-mentioned mucosa and tela submucosa are placed in this PBS mixed liquor, vibrate and continue vortex, every 12h changes PBS mixed liquor, persistent oscillation vortex is used containing dual anti-PBS buffer solution for cleaning mucosa and tela submucosa for 3 days afterwards, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, then mucosa and tela submucosa are immersed at the temperature of 37 DEG C 2h in the PBS buffer containing DNA enzymatic, wherein: the concentration of DNA enzymatic in PBS buffer is 1000 ~ 5000U/L, again with containing dual anti-PBS buffer solution for cleaning, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, obtain esophageal mucosa membrane injury acellular matrix,
(3), dissolve polycaprolactone with trifluoroethanol and obtain polycaprolactone solution, dissolve fibroin albumen with formic acid and obtain silk fibroin protein solution, and polycaprolactone solution and silk fibroin protein solution are mixed to get mixed liquor in the ratio that solute weight ratio is 4:1, then be the concentration of solvent adjustment mixed liquor with hexafluoroisopropanol be 0.125 μ g/ml, and as Electrospun solution, get esophageal mucosa membrane injury acellular matrix prepared by step (2) as substrate, by electrostatic spinning apparatus, Electrospun solution is carried out electrostatic spinning at substrate surface of internal cavity, acquisition fibre diameter is 50 ~ 500nm, thickness is the porous fiber film of 100 ~ 200nm, then this porous fiber film is immersed in the laminin liquid prepared by step (1) together with substrate, take out soak 24 h at 4 DEG C after, and remove unnecessary laminin with PBS wash buffer, obtain bag by the Electrospun of laminin/acellular matrix support, for subsequent use,
(4), degradable polyester type polyurethane is dissolved in 1, in 4-dioxane, be mixed with the polyurethane solutions that weight concentration is 5 ~ 25%, this polyurethane solutions is poured into breaking on the mould of micro groove structure with point continuously, be prepared into and be widely 5 ~ 10 centimetres and with the rectangular sheet of polyurethane of two-sided microflute, wherein the one side of sheet of polyurethane is continuous microflute, and another side is a disconnected microflute, and the structure of continuous microflute is: groove width 200 μm, groove depth 30 μm, separation 30 μm; The structure of the disconnected microflute of point is: groove width 100 ~ 200 μm, groove depth 30 μm, long 100 ~ 200 μm of each partition, cut off wide 30 μm, with in a line adjacent two cut off spacing be 30 μm, described continuous microflute and the described some microflute that breaks is orthogonal, and the bearing of trend of microflute is the length direction of sheet of polyurethane continuously, the bearing of trend of the disconnected microflute of point is the width of sheet of polyurethane;
(5), fibroin albumen is extracted from silkworm silk or Bombyx bombycis, and the free amino that can supply reaction is chemically connected at the surfaces externally and internally of sheet of polyurethane, then the sheet of polyurethane being connected to free amino being immersed in weight concentration is room temperature reaction 1 ~ 5h in the glutaraldehyde solution of 1 ~ 3%, the unreacted glutaraldehyde in sheet of polyurethane surface is removed afterwards with deionized water rinsing, sheet of polyurethane being immersed concentration is in the silk fibroin protein solution of 1mg/ml again, 12 ~ 24h is soaked at the temperature of 4 DEG C, the responseless fibroin albumen in sheet of polyurethane surface is removed with PBS wash buffer after taking out,
(6), by sheet of polyurethane with put disconnected microflute for an inner surface-disconnected microflute be hoop, continuously microflute for the microflute of outer surface-continuously in the bag be longitudinally wrapped in obtained by step (3) by outside the Electrospun of laminin/acellular matrix support, and sheet of polyurethane longitudinal seam is connected into tubulose, obtain the artificial esophagus with histological structure;
(7), by obtained artificial esophagus with 75% alcohol-pickled sterilization 30 minutes, then with sterilizing PBS wash buffer, Preservation in sterile condition;
(8), when obtained artificial esophagus is docked with human body esophagus, by whole for the diseased region in human body esophagus section of excision, by the method for layer/layer docking, artificial esophagus and residue esophageal tissue are sewed up, namely the tela submucosa of Electrospun/acellular matrix and esophagus sews up, and the some section microflute layer in sheet of polyurethane docks with the annular muscle layer in human body esophagus, and the continuous microflute layer in sheet of polyurethane docks with the vertical shape muscle layer in human body esophagus, layer-by-layer suture wound, cultivates 6 ~ 18 months in body.
The PBS buffer used in this method is phosphate buffer.
Compared with prior art, advantage of the present invention is the artificial esophagus of the histological structure can being prepared simulation normal human esophagus by the method, make use of tissue engineering principle, construct the artificial esophagus to human body esophagus with similar microtexture and function in vitro, then the residue esophagus after successively excising with pathological changes esophagus sews up mutually, artificial esophagus is made to grow up to for real esophagus gradually, and merge with original esophagus in human body, solve the weakness of the function shortage that current artificial esophagus brings because structure is single, compared with traditional substitute, the more natural esophagus of convergence on structure and fuction, the misery of sufferer can be alleviated, extend the life of sufferer.
Accompanying drawing explanation
The structural representation of the artificial esophagus of Fig. 1 obtained by the present invention;
The structural representation with a little disconnected microflute one side of the PAUR thin slice that Fig. 2 produces for step of the present invention (4);
The structural representation with continuous microflute one side of the PAUR thin slice that Fig. 3 produces for step of the present invention (4).
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment one: a kind of preparation method with the artificial esophagus of histological structure, specifically comprises the following steps:
(1), get animal esophagus (as pig, Canis familiaris L., the animals such as sheep) in mucosa and tela submucosa, clean, shred, and mucosa and submucosa tissue are immersed by NaCl, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 3.4mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is immersed by NaCl again, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 0.5mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 10000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is then immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 2.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is finally immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 4.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 12000r/min, get centrifugal after supernatant, by the supernatant mixing acquired by four times, obtain laminin liquid, for support bag quilt,
(2), get the mucosa in animal esophagus and tela submucosa, clear water is rinsed well, and sterilize with 75% alcohol-pickled 2h, clean with PBS wash buffer after sterilization, dual anti-and the TritonX be made up of penicillin and streptomycin is mixed in PBS buffer and forms PBS mixed liquor, wherein: TritonX is 1% at the percentage by weight of PBS mixed liquor, penicillin and the concentration of streptomycin in PBS mixed liquor are 200U/L, again above-mentioned mucosa and tela submucosa are placed in this PBS mixed liquor, vibrate and continue vortex, every 12h changes PBS mixed liquor, persistent oscillation vortex is used containing dual anti-PBS buffer solution for cleaning mucosa and tela submucosa for 3 days afterwards, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, then mucosa and tela submucosa are immersed at the temperature of 37 DEG C 2h in the PBS buffer containing DNA enzymatic, wherein: the concentration of DNA enzymatic in PBS buffer is 2000U/L, again with containing dual anti-PBS buffer solution for cleaning, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, obtain esophageal mucosa membrane injury acellular matrix,
(3), dissolve polycaprolactone with trifluoroethanol and obtain polycaprolactone solution, dissolve fibroin albumen with formic acid and obtain silk fibroin protein solution, and polycaprolactone solution and silk fibroin protein solution are mixed to get mixed liquor in the ratio that solute weight ratio is 4:1, then be the concentration of solvent adjustment mixed liquor with hexafluoroisopropanol be 0.125 μ g/ml, and as Electrospun solution, get esophageal mucosa membrane injury acellular matrix prepared by step (2) as substrate, by electrostatic spinning apparatus, Electrospun solution is carried out electrostatic spinning at substrate surface of internal cavity, acquisition fibre diameter is 100nm, thickness is the porous fiber film of 100nm, then this porous fiber film is immersed in the laminin liquid prepared by step (1) together with substrate, take out soak 24 h at 4 DEG C after, and remove unnecessary laminin with PBS wash buffer, obtain bag by the Electrospun of laminin/acellular matrix support, for subsequent use,
(4), degradable polyester type polyurethane is dissolved in 1, in 4-dioxane, be mixed with the polyurethane solutions that weight concentration is 10%, this polyurethane solutions is poured into breaking on the mould of micro groove structure with point continuously, be prepared into and be widely 5 centimetres and with the rectangular sheet of polyurethane of two-sided microflute, wherein the one side of sheet of polyurethane is continuous microflute, and another side is a disconnected microflute, and the structure of continuous microflute is: groove width W
1be 200 μm, groove depth 30 μm, separation D
1it is 30 μm; The structure of the disconnected microflute of point is: groove width W
2be 100 μm, groove depth 30 μm, the long L of each partition is 150 μm, cut off wide W to be 30 μm, to be 30 μm with two space D cut off adjacent in a line, continuous microflute is orthogonal with the disconnected microflute of point, and the bearing of trend of microflute is the length direction of sheet of polyurethane continuously, the bearing of trend of the disconnected microflute of point is the width of sheet of polyurethane;
(5), from silkworm silk or Bombyx bombycis, fibroin albumen is extracted, and the free amino that can supply reaction is chemically connected at the surfaces externally and internally of sheet of polyurethane, then the sheet of polyurethane being connected to free amino being immersed in weight concentration is room temperature reaction 5h in the glutaraldehyde solution of 1%, the unreacted glutaraldehyde in sheet of polyurethane surface is removed afterwards with deionized water rinsing, sheet of polyurethane being immersed concentration is in the silk fibroin protein solution of 1mg/ml again, at the temperature of 4 DEG C, soak 24h, after taking out, remove the responseless fibroin albumen in sheet of polyurethane surface with PBS wash buffer;
(6), by sheet of polyurethane with put disconnected microflute for an inner surface-disconnected microflute be hoop, continuously microflute for the microflute of outer surface-continuously in the bag be longitudinally wrapped in obtained by step (3) by outside the Electrospun of laminin/acellular matrix support, and sheet of polyurethane longitudinal seam is connected into tubulose, obtain the artificial esophagus with histological structure;
(7), by obtained artificial esophagus with 75% alcohol-pickled sterilization 30 minutes, then with sterilizing PBS wash buffer, Preservation in sterile condition.
Embodiment two: a kind of preparation method with the artificial esophagus of histological structure, specifically comprises the following steps:
(1), get animal esophagus (as pig, Canis familiaris L., the animals such as sheep) in mucosa and tela submucosa, clean, shred, and mucosa and submucosa tissue are immersed by NaCl, Tris-HCl(Tris), in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 3.4mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is immersed by NaCl again, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 0.5mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is then immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 2.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is finally immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 4.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000r/min, get centrifugal after supernatant, by the supernatant mixing acquired by four times, obtain laminin liquid, for support bag quilt,
(2), get the mucosa in animal esophagus and tela submucosa, clear water is rinsed well, and sterilize with 75% alcohol-pickled 4h, clean with PBS wash buffer after sterilization, dual anti-and the TritonX be made up of penicillin and streptomycin is mixed in PBS buffer and forms PBS mixed liquor, wherein: TritonX is 1% at the percentage by weight of PBS mixed liquor, penicillin and the concentration of streptomycin in PBS mixed liquor are 200U/L, again above-mentioned mucosa and tela submucosa are placed in this PBS mixed liquor, vibrate and continue vortex, every 12h changes PBS mixed liquor, persistent oscillation vortex is used containing dual anti-PBS buffer solution for cleaning mucosa and tela submucosa for 3 days afterwards, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, then mucosa and tela submucosa are immersed at the temperature of 37 DEG C 2h in the PBS buffer containing DNA enzymatic, wherein: the concentration of DNA enzymatic in PBS buffer is 4500U/L, again with containing dual anti-PBS buffer solution for cleaning, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, obtain esophageal mucosa membrane injury acellular matrix,
(3), dissolve polycaprolactone with trifluoroethanol and obtain polycaprolactone solution, dissolve fibroin albumen with formic acid and obtain silk fibroin protein solution, and polycaprolactone solution and silk fibroin protein solution are mixed to get mixed liquor in the ratio that solute weight ratio is 4:1, then be the concentration of solvent adjustment mixed liquor with hexafluoroisopropanol be 0.125 μ g/ml, and as Electrospun solution, get esophageal mucosa membrane injury acellular matrix prepared by step (2) as substrate, by electrostatic spinning apparatus, Electrospun solution is carried out electrostatic spinning at substrate surface of internal cavity, acquisition fibre diameter is 400nm, thickness is the porous fiber film of 200nm, then this porous fiber film is immersed in the laminin liquid prepared by step (1) together with substrate, take out soak 24 h at 4 DEG C after, and remove unnecessary laminin with PBS wash buffer, obtain bag by the Electrospun of laminin/acellular matrix support, for subsequent use,
(4), degradable polyester type polyurethane is dissolved in 1, in 4-dioxane, be mixed with the polyurethane solutions that weight concentration is 20%, this polyurethane solutions is poured into breaking on the mould of micro groove structure with point continuously, be prepared into and be widely 8 centimetres and with the rectangular sheet of polyurethane of two-sided microflute, wherein the one side of sheet of polyurethane is continuous microflute, and another side is a disconnected microflute, and the structure of continuous microflute is: groove width W
1be 200 μm, groove depth 30 μm, separation D
1it is 30 μm; The structure of the disconnected microflute of point is: groove width W
2be 200 μm, groove depth 30 μm, the long L of each partition is 200 μm, cut off wide W to be 30 μm, to be 30 μm with two space D cut off adjacent in a line, continuous microflute is orthogonal with the disconnected microflute of point, and the bearing of trend of microflute is the length direction of sheet of polyurethane continuously, the bearing of trend of the disconnected microflute of point is the width of sheet of polyurethane;
(5), from silkworm silk or Bombyx bombycis, fibroin albumen is extracted, and the free amino that can supply reaction is chemically connected at the surfaces externally and internally of sheet of polyurethane, then the sheet of polyurethane being connected to free amino being immersed in weight concentration is room temperature reaction 2h in the glutaraldehyde solution of 3%, the unreacted glutaraldehyde in sheet of polyurethane surface is removed afterwards with deionized water rinsing, sheet of polyurethane being immersed concentration is in the silk fibroin protein solution of 1mg/ml again, at the temperature of 4 DEG C, soak 12h, after taking out, remove the responseless fibroin albumen in sheet of polyurethane surface with PBS wash buffer;
(6), by sheet of polyurethane with put disconnected microflute for an inner surface-disconnected microflute be hoop, continuously microflute for the microflute of outer surface-continuously in the bag be longitudinally wrapped in obtained by step (3) by outside the Electrospun of laminin/acellular matrix support, and sheet of polyurethane longitudinal seam is connected into tubulose, obtain the artificial esophagus with histological structure;
(7), by obtained artificial esophagus with 75% alcohol-pickled sterilization 30 minutes, then with sterilizing PBS wash buffer, Preservation in sterile condition.
By the method that the artificial esophagus obtained by this method is docked with human body esophagus be: whole for the diseased region in human body esophagus section is excised, by the method for layer/layer docking, artificial esophagus and residue esophageal tissue are sewed up, namely the tela submucosa of Electrospun/acellular matrix and esophagus sews up, point section microflute layer in sheet of polyurethane docks with the annular muscle layer in human body esophagus, continuous microflute layer in sheet of polyurethane docks with the vertical shape muscle layer in human body esophagus, layer-by-layer suture wound, cultivates 6 ~ 18 months in body.
In step (4) described in above-described embodiment, degradable polyester type polyurethane can have elastic degradable polymeric material with other and substitute, and is dissolved in the solvent suitable with such degradable polymer for the preparation of the polymer flake being with micro groove structure.
Claims (1)
1. there is the artificial esophagus's of a histological structure preparation method, it is characterized in that specifically comprising the following steps:
(1), get the mucosa in animal esophagus and tela submucosa, clean, shred, and mucosa and submucosa tissue are immersed by NaCl, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 3.4mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is immersed by NaCl again, Tris-HCl, in the solution that Phenylmethanesulfonyl fluoride and N-methylmaleimido mix, wherein: NaCl concentration is in the solution 0.5mol/L, Tris-HCl concentration is in the solution 0.05mol/L, Phenylmethanesulfonyl fluoride concentration is in the solution 2mmol/L, N-methylmaleimido concentration is in the solution 1mmol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is then immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 2.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, and centrifugal rear remaining precipitate is finally immersed by guanidine hydrochloride, in the solution that dithiothreitol, DTT and Tris-HCl mix, wherein: guanidine hydrochloride concentration is in the solution 4.0mol/L, dithiothreitol, DTT concentration is in the solution 2mmol/L, Tris-HCl concentration is in the solution 0.05mol/L, after homogenate by homogenate under the low temperature of 4 DEG C with the centrifugal 20min of the rotating speed of 8000 ~ 12000r/min, get centrifugal after supernatant, by the supernatant mixing acquired by four times, obtain laminin liquid, for support bag quilt,
(2), get the mucosa in animal esophagus and tela submucosa, clear water is rinsed well, and sterilize with 75% alcohol-pickled 1 ~ 4h, clean with PBS wash buffer after sterilization, dual anti-and the TritonX be made up of penicillin and streptomycin is mixed in PBS buffer and forms PBS mixed liquor, wherein: TritonX is 1% at the percentage by weight of PBS mixed liquor, penicillin and the concentration of streptomycin in PBS mixed liquor are 200U/L, again above-mentioned mucosa and tela submucosa are placed in this PBS mixed liquor, vibrate and continue vortex, every 12h changes PBS mixed liquor, persistent oscillation vortex is used containing dual anti-PBS buffer solution for cleaning mucosa and tela submucosa for 3 days afterwards, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, then mucosa and tela submucosa are immersed at the temperature of 37 DEG C 2h in the PBS buffer containing DNA enzymatic, wherein: the concentration of DNA enzymatic in PBS buffer is 1000 ~ 5000U/L, again with containing dual anti-PBS buffer solution for cleaning, wherein: form dual anti-penicillin and the concentration of streptomycin in PBS buffer is 200U/L, obtain esophageal mucosa membrane injury acellular matrix,
(3), dissolve polycaprolactone with trifluoroethanol and obtain polycaprolactone solution, dissolve fibroin albumen with formic acid and obtain silk fibroin protein solution, and polycaprolactone solution and silk fibroin protein solution are mixed to get mixed liquor in the ratio that solute weight ratio is 4:1, then be the concentration of solvent adjustment mixed liquor with hexafluoroisopropanol be 0.125 μ g/ml, and as Electrospun solution, get esophageal mucosa membrane injury acellular matrix prepared by step (2) as substrate, by electrostatic spinning apparatus, Electrospun solution is carried out electrostatic spinning at substrate surface of internal cavity, acquisition fibre diameter is 50 ~ 500nm, thickness is the porous fiber film of 100 ~ 200nm, then this porous fiber film is immersed in the laminin liquid prepared by step (1) together with substrate, take out soak 24h at 4 DEG C after, and remove unnecessary laminin with PBS wash buffer, obtain bag by the Electrospun of laminin/acellular matrix support, for subsequent use,
(4), degradable polyester type polyurethane is dissolved in 1, in 4-dioxane, be mixed with the polyurethane solutions that weight concentration is 5 ~ 25%, this polyurethane solutions is poured into breaking on the mould of micro groove structure with point continuously, be prepared into and be widely 5 ~ 10 centimetres and with the rectangular sheet of polyurethane of two-sided microflute, wherein the one side of sheet of polyurethane is continuous microflute, and another side is a disconnected microflute, and the structure of continuous microflute is: groove width 200 μm, groove depth 30 μm, separation 30 μm; The structure of the disconnected microflute of point is: groove width 100 ~ 200 μm, groove depth 30 μm, long 100 ~ 200 μm of each partition, cut off wide 30 μm, with in a line adjacent two cut off spacing be 30 μm, described continuous microflute and the described some microflute that breaks is orthogonal, and the bearing of trend of microflute is the length direction of sheet of polyurethane continuously, the bearing of trend of the disconnected microflute of point is the width of sheet of polyurethane;
(5), fibroin albumen is extracted from silkworm silk or Bombyx bombycis, and the free amino that can supply reaction is chemically connected at the surfaces externally and internally of sheet of polyurethane, then the sheet of polyurethane being connected to free amino being immersed in weight concentration is room temperature reaction 1 ~ 5h in the glutaraldehyde solution of 1 ~ 3%, the unreacted glutaraldehyde in sheet of polyurethane surface is removed afterwards with deionized water rinsing, sheet of polyurethane being immersed concentration is in the silk fibroin protein solution of 1mg/ml again, 12 ~ 24h is soaked at the temperature of 4 DEG C, the responseless fibroin albumen in sheet of polyurethane surface is removed with PBS wash buffer after taking out,
(6), by sheet of polyurethane with put disconnected microflute for an inner surface-disconnected microflute be hoop, continuously microflute for the microflute of outer surface-continuously in the bag be longitudinally wrapped in obtained by step (3) by outside the Electrospun of laminin/acellular matrix support, and sheet of polyurethane longitudinal seam is connected into tubulose, obtain the artificial esophagus with histological structure;
(7), by obtained artificial esophagus with 75% alcohol-pickled sterilization 30 minutes, then with sterilizing PBS wash buffer, Preservation in sterile condition.
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