CN107583110A - A kind of preparation method of the artificial esophagus with biological structure and function - Google Patents
A kind of preparation method of the artificial esophagus with biological structure and function Download PDFInfo
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- 210000003238 esophagus Anatomy 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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- 210000004876 tela submucosa Anatomy 0.000 claims abstract description 32
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- 210000004379 membrane Anatomy 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 8
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- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 210000000981 epithelium Anatomy 0.000 claims abstract description 7
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 3
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 23
- 102000019280 Pancreatic lipases Human genes 0.000 claims description 18
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- 229930182555 Penicillin Natural products 0.000 claims description 17
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 17
- 229940049954 penicillin Drugs 0.000 claims description 17
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- 229940116369 pancreatic lipase Drugs 0.000 claims description 15
- 230000009514 concussion Effects 0.000 claims description 10
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
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- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 8
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 4
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 4
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a kind of preparation method of the artificial esophagus with biological structure and function, feature is to take fresh mammalian gut to carry out de- cell processing, and lyophilized preservation after leaving mucous membrane and submucosa；Then fresh placenta amnion is taken to leave comprising de- cell amnion is made after epithelium layer, basilar memebrane and intact amniotic tissue without vascular stroma, de- cell amnion is laid on lyophilized esophageal mucosa membrane again, dry at room temperature, obtain the artificial esophagus with biological structure and function；Advantage is that the artificial esophagus obtained by this method simulates human normal esophageal tissue on macroscopic form and microstructure, and because it can successfully realize epithelialization, the biological function of esophageal tissue functionally can be also played to greatest extent so that the artificial esophagus parts of lesions esophagus preferably for substituting patient or infected animal.
Description
Technical field
The invention belongs to biomedical engineering field, more particularly to a kind of artificial esophagus with biological structure and function
Preparation method.
Background technology
Normal human's esophagus is the hollow pipeline of 25 cms length, connects throat and stomach, it is main rise conveying food and
The effect of water.Esophagus is made up of mucous membrane, submucosa, muscle layer and outer membrane, mucous membrane by non-angling pavement epithelium cells group
Into, be grown on basement membrane, basement membrane provide cell growth required for Special Proteins, growth factor and other nutriments, with adjust
Propagation, differentiation, migration and death of ganglion cell etc..Muscle layer is made up of myocyte, based on smooth muscle cell, under mucous membrane and mucous membrane
Layer is common to be played secreting mucus and prevents food and water seepage, is that esophagus can bring into normal play the most important part of function.
At present, the death that esophagus lesion is triggered increasingly has caused the concern of people, this kind of lesion refer mainly to cancer of the esophagus,
Chronic reflux esophagitis and Barret esophaguses etc..Wherein cancer of the esophagus is one of most common malignant tumour, it has also become the world
The fifth-largest malignant tumour.China is even more the district occurred frequently of cancer of the esophagus, and about 250,000 people, which are diagnosed, every year suffers from cancer of the esophagus, is nearly accounted for complete
The half of world patient.Therefore, the structure of tissue engineered esophageal has increasingly caused the concern of scientists from all over the world.
Patent No. ZL01118316.0 Chinese invention patent disclose " cell scaffold with composite structure for tissue engineering and
Its preparation method and purposes ", degradable polymer such as lactic acid/ethanol copolymer and poly (l-lactic acid), is dissolved in dichloromethane by it respectively
It is well mixed afterwards with salt grain, pours and cast from mould, solvent flashing is dried in vacuo 24 hours after 24 hours at room temperature, is then immersed in
Desalination in ionized water, bonded after the porous support for having taken off salt is dried 24 hours with dichloromethane, then be dried in vacuo 48 hours and make
Into.Cytoskeleton is made from the method for above-mentioned casting desalination, not only complex process, speed are slow, it is often more important that it is difficult to will envelope
Salinity in closed pore leaches completely.In addition, with dichloromethane bonding the pore structure of whole support can be caused uneven, and influence bonding
Locate the growth of cell.
Patent No. ZL02145461.2 Chinese invention patent discloses tissue engineered esophageal, and it is with cell free pig
Skin be used as support (containing epidermis and skin corium), is implanted into endothelial cell to build blood vessel in support and fibroblast to copy
Skin corium constructs, it is therefore an objective to for substituting the One-stage reconstruction of impaired esophagus.But simply proposing one kind prepares suitable a variety of devices for this
The conventional method of official's support, do not made according to the actual microstructure of human body esophagus made from support there is institute of esophageal tissue
Distinctive biological function.
The content of the invention
The technical problems to be solved by the invention are to provide one kind and simulate human normal on macroscopic form and microstructure
Esophageal tissue and the preparation method with the artificial esophagus of human body esophageal tissue biological structure and function.
Technical scheme is used by the present invention solves above-mentioned technical problem：A kind of people with biological structure and function
The preparation method of work esophagus, including step in detail below：
（1）, take fresh mammalian gut to clean up, peel off muscle layer, leave mucous membrane and submucosa；
（2）, mucous membrane and submucosa be placed in the alcohol that volumetric concentration is 75~95% and rinse 3~10 minutes, then use clear water
Wash away alcohol；
（3）, mucous membrane and submucosa be placed in the Triton X-100 that mass concentration is 1~5% and embathed 1~5 day, during which every 12 is small
When change liquid once, finally use PBS（Phosphate buffer）Cleaning；
（4）, mucous membrane and submucosa immersed in pancreatic lipase/PBS solution that temperature is 30~40 DEG C 5~20 hours, wherein：Pancreas
The concentration of lipase in the solution is 1000~5000U/L, then takes out and is rinsed in the alcohol that volumetric concentration is 75~95%, then
Rinsed with PBS；
（5）, mucous membrane and submucosa are immersed in DNA enzymatic/PBS solution that temperature is 30~40 DEG C and soaked 2~10 hours, its
In：The concentration of DNA enzymatic in the solution is 1000~5000U/L, then takes out and is rinsed with PBS；
（6）, mucous membrane and submucosa soaked 1~5 hour in the alcohol that volumetric concentration is 75~95%, then take out and use PBS
Rinsing, and lyophilized preservation；
（7）, take fresh placenta amnion to remove clot and chorion, leave comprising epithelium layer, basilar memebrane and without blood vessel base
The intact amniotic tissue of matter, and with PBS, obtain clean translucent amnion；
（8）, with volumetric concentration be 75% alcohol by translucent amnion rinse 1~2 time, then with PBS rinse 2~4 times, then will
Dual anti-be mixed into PBS being made up of penicillin and streptomysin forms dual anti-/PBS solution, wherein：Penicillin and streptomysin are double
Content in anti-/PBS solution is 100~500U/L, then by translucent amnion is 1 by volume：1~3 is placed in dual anti-/PBS
Concussion is soaked in solution, changes liquid once within every 12 hours, altogether twice；
（9）, penicillin, streptomysin, Triton X-100 are mixed into PBS form comprehensive PBS solution, wherein：Penicillin and strepto-
Content of the element in comprehensive PBS solution is 100~500U/L, weight ratio of the Triton X-100 in comprehensive PBS solution for 1~
5%, then by translucent amnion by volume be 1：1~3 ratio, which is placed in comprehensive PBS solution, soaks concussion, changes within every 6~8 hours
Liquid once, altogether twice, then takes out translucent amnion, is rinsed 1~2 time with PBS；
（10）, by translucent amnion press 1：1~3 volume ratio is immersed in pancreatic lipase/PBS solution, wherein：Pancreatic lipase is in pancreas fat
Content in enzyme/PBS solution is 500~5000U/L, and is handled 5~20 hours at being 30~40 DEG C in solution temperature, then will
Translucent amnion takes out to be rinsed 1~2 time with PBS, then translucent amnion is pressed into 1：It is molten that 1~3 volume ratio is immersed in DNA enzymatic/PBS
In liquid, wherein：Content of the DNA enzymatic in DNA enzymatic/PBS solution is 1000~5000U/L, and is 30~40 DEG C in solution temperature
Lower processing 2~5 hours；
（11）, translucent amnion taken out from DNA enzymatic/PBS solution, be placed in dual anti-/PBS solution and shake rinsing, wherein：It is blue or green
The content of mycin and streptomysin in dual anti-/PBS solution is 100~500U/L, changes liquid once within every 12 hours, altogether twice, is obtained
It is put into PBS, is preserved at a temperature of 4 DEG C stand-by to de- cell amnion, and by de- cell amnion；
（12）, de- cell amnion is laid in step（6）On the lyophilized esophageal mucosa membrane being prepared, dry, had at room temperature
There is the artificial esophagus of biological structure and function.
Further, described PBS pH value is 7.4.
Further, described mammalian gut is pig esophagus, dog food road, monkey esophagus, rabbit food road or sheep esophagus etc..
Further, described placenta amnion behaviour placenta amnion or animal placenta amnion.
Compared with prior art, it is an advantage of the invention that this method is used as people by the use of animal esophageal mucosa membrane after de- cell processing
The support of work esophagus, close with human body esophagus on macroscopic form and structure, esophageal cells epimatrix composition contained therein is non-
Often be advantageous to growth and the Function of neonatal cell, and after the de- cell amnion of its surface covering, take off contained in cell amnion
Proteins and growth factor be advantageous to the growth and differentiation of esophageal mucosa membrane cell, can be quick after being implanted into animal body or human body
Promoting the epithelialization of esophageal mucosal layer, the regeneration and reparation to esophagus are highly beneficial, therefore, the artificial esophagus obtained by this method
Human normal esophageal tissue is simulated on macroscopic form and microstructure, and because it can successfully realize epithelialization, in work(
Can on can also play the biological function of esophageal tissue to greatest extent so that the artificial esophagus is preferably to substitute patient or illness
The parts of lesions esophagus of animal.
Embodiment
The present invention is described in further detail with reference to embodiments.
Embodiment one：A kind of preparation method of the artificial esophagus with biological structure and function, including walk in detail below
Suddenly：
（1）, take fresh pig esophagus to clean up, peel off muscle layer, leave mucous membrane and submucosa；
（2）, mucous membrane and submucosa be placed in the alcohol that volumetric concentration is 75% and rinse 10 minutes, then wash away wine with clear water
Essence；
（3）, mucous membrane and submucosa be placed in the Triton X-100 that mass concentration is 5% and embathed 1 day, change liquid within during which every 12 hours
Once, PBS is finally used；
（4）, mucous membrane and submucosa immersed in pancreatic lipase/PBS solution that temperature is 30 DEG C 20 hours, wherein：Pancreatic lipase exists
Concentration in solution is 1000U/L, then takes out and is rinsed in the alcohol that volumetric concentration is 75%, then is rinsed with PBS；
（5）, mucous membrane and submucosa are immersed in DNA enzymatic/PBS solution that temperature is 40 DEG C and soaked 2 hours, wherein：DNA enzymatic exists
Concentration in solution is 5000U/L, then takes out and is rinsed with PBS；
（6）, mucous membrane and submucosa soaked 1 hour in the alcohol that volumetric concentration is 95%, then take out and rinsed with PBS, and
It is lyophilized to preserve；
（7）, take fresh Human plactnta amnion to remove clot and chorion, leave comprising epithelium layer, basilar memebrane and without blood vessel
The intact amniotic tissue of matrix, and with PBS, obtain clean translucent amnion；
（8）, with volumetric concentration be 75% alcohol by translucent amnion rinse 1 time, then with PBS rinse 2 times, then will be by mould
Dual anti-be mixed into PBS of element and streptomysin composition forms dual anti-/PBS solution, wherein：Penicillin and streptomysin are in dual anti-/PBS
Content in solution is 500U/L, then by translucent amnion by volume be 1：1 is placed in immersion concussion in dual anti-/PBS solution,
Change liquid once within every 12 hours, altogether twice；
（9）, penicillin, streptomysin, Triton X-100 are mixed into PBS form comprehensive PBS solution, wherein：Penicillin and strepto-
The plain content in comprehensive PBS solution is 100U/L, and weight ratio of the Triton X-100 in comprehensive PBS solution is 1%, then will be partly
Transparent amnion is 1 by volume：3 ratio, which is placed in comprehensive PBS solution, soaks concussion, changes liquid once within every 6 hours, altogether twice,
Then translucent amnion is taken out, rinsed 1 time with PBS；
（10）, by translucent amnion press 1：1 volume ratio is immersed in pancreatic lipase/PBS solution, wherein：Pancreatic lipase pancreatic lipase/
Content in PBS solution is 4000U/L, and is handled 6 hours at being 30 DEG C in solution temperature, then takes out translucent amnion and uses
PBS is rinsed 2 times, then translucent amnion is pressed into 1：1 volume ratio is immersed in DNA enzymatic/PBS solution, wherein：DNA enzymatic is in DNA
Content in enzyme/PBS solution is 5000U/L, and is handled 2 hours at being 30 DEG C in solution temperature；
（11）, translucent amnion taken out from DNA enzymatic/PBS solution, be placed in dual anti-/PBS solution and shake rinsing, wherein：It is blue or green
The content of mycin and streptomysin in dual anti-/PBS solution is 100U/L, changes liquid once within every 12 hours, altogether twice, is obtained de- thin
Born of the same parents' amnion, and de- cell amnion is put into PBS, preserved at a temperature of 4 DEG C stand-by；
（12）, de- cell amnion is laid in step（6）On the lyophilized esophageal mucosa membrane being prepared, dry, had at room temperature
There is the artificial esophagus of biological structure and function.
Embodiment two：A kind of preparation method of the artificial esophagus with biological structure and function, including walk in detail below
Suddenly：
（1）, take fresh rabbit food road to clean up, peel off muscle layer, leave mucous membrane and submucosa；
（2）, mucous membrane and submucosa be placed in the alcohol that volumetric concentration is 85% and rinse 7 minutes, then wash away wine with clear water
Essence；
（3）, mucous membrane and submucosa be placed in the Triton X-100 that mass concentration is 1% and embathed 5 days, change liquid within during which every 12 hours
Once, PBS is finally used；
（4）, mucous membrane and submucosa immersed in pancreatic lipase/PBS solution that temperature is 40 DEG C 7 hours, wherein：Pancreatic lipase is molten
Concentration in liquid is 3000U/L, then takes out and is rinsed in the alcohol that volumetric concentration is 75%, then is rinsed with PBS；
（5）, mucous membrane and submucosa are immersed in DNA enzymatic/PBS solution that temperature is 35 DEG C and soaked 8 hours, wherein：DNA enzymatic exists
Concentration in solution is 3000U/L, then takes out and is rinsed with PBS；
（6）, mucous membrane and submucosa soaked 3 hours in the alcohol that volumetric concentration is 85%, then take out and rinsed with PBS, and
It is lyophilized to preserve；
（7）, take fresh animal placenta amnion to remove clot and chorion, leave comprising epithelium layer, basilar memebrane and without blood
The intact amniotic tissue of pipe matrix, and with PBS, obtain clean translucent amnion；
（8）, with volumetric concentration be 75% alcohol by translucent amnion rinse 2 times, then with PBS rinse 2 times, then will be by mould
Dual anti-be mixed into PBS of element and streptomysin composition forms dual anti-/PBS solution, wherein：Penicillin and streptomysin are in dual anti-/PBS
Content in solution is 100U/L, then by translucent amnion by volume be 1：3 are placed in immersion concussion in dual anti-/PBS solution,
Change liquid once within every 12 hours, altogether twice；
（9）, penicillin, streptomysin, Triton X-100 are mixed into PBS form comprehensive PBS solution, wherein：Penicillin and strepto-
The plain content in comprehensive PBS solution is 300U/L, and weight ratio of the Triton X-100 in comprehensive PBS solution is 3%, then will be partly
Transparent amnion is 1 by volume：2 ratio, which is placed in comprehensive PBS solution, soaks concussion, changes liquid for every eight hours once, altogether twice,
Then translucent amnion is taken out, rinsed 2 times with PBS；
（10）, by translucent amnion press 1：3 volume ratio is immersed in pancreatic lipase/PBS solution, wherein：Pancreatic lipase pancreatic lipase/
Content in PBS solution is 1000U/L, and is handled 12 hours at being 40 DEG C in solution temperature, then takes out translucent amnion
Rinsed 2 times with PBS, then translucent amnion is pressed 1：3 volume ratio is immersed in DNA enzymatic/PBS solution, wherein：DNA enzymatic is in DNA
Content in enzyme/PBS solution is 1000U/L, and is handled 5 hours at being 40 DEG C in solution temperature；
（11）, translucent amnion taken out from DNA enzymatic/PBS solution, be placed in dual anti-/PBS solution and shake rinsing, wherein：It is blue or green
The content of mycin and streptomysin in dual anti-/PBS solution is 500U/L, changes liquid once within every 12 hours, altogether twice, is obtained de- thin
Born of the same parents' amnion, and de- cell amnion is put into PBS, preserved at a temperature of 4 DEG C stand-by；
（12）, de- cell amnion is laid in step（6）On the lyophilized esophageal mucosa membrane being prepared, dry, had at room temperature
There is the artificial esophagus of biological structure and function.
Embodiment three：A kind of preparation method of the artificial esophagus with biological structure and function, including walk in detail below
Suddenly：
（1）, take fresh sheep esophagus to clean up, peel off muscle layer, leave mucous membrane and submucosa；
（2）, mucous membrane and submucosa be placed in the alcohol that volumetric concentration is 90% and rinse 5 minutes, then wash away wine with clear water
Essence；
（3）, mucous membrane and submucosa be placed in the Triton X-100 that mass concentration is 3% and embathed 3 days, change liquid within during which every 12 hours
Once, PBS is finally used；
（4）, mucous membrane and submucosa immersed in pancreatic lipase/PBS solution that temperature is 35 DEG C 12 hours, wherein：Pancreatic lipase exists
Concentration in solution is 5000U/L, then takes out and is rinsed in the alcohol that volumetric concentration is 85%, then is rinsed with PBS；
（5）, mucous membrane and submucosa are immersed in DNA enzymatic/PBS solution that temperature is 30 DEG C and soaked 10 hours, wherein：DNA enzymatic
Concentration in the solution is 1000U/L, then takes out and is rinsed with PBS；
（6）, mucous membrane and submucosa soaked 5 hours in the alcohol that volumetric concentration is 75%, then take out and rinsed with PBS, and
It is lyophilized to preserve；
（7）, take fresh Human plactnta amnion to remove clot and chorion, leave comprising epithelium layer, basilar memebrane and without blood vessel
The intact amniotic tissue of matrix, and with PBS, obtain clean translucent amnion；
（8）, with volumetric concentration be 75% alcohol by translucent amnion rinse 2 times, then with PBS rinse 4 times, then will be by mould
Dual anti-be mixed into PBS of element and streptomysin composition forms dual anti-/PBS solution, wherein：Penicillin and streptomysin are in dual anti-/PBS
Content in solution is 300U/L, then by translucent amnion by volume be 1：2 are placed in immersion concussion in dual anti-/PBS solution,
Change liquid once within every 12 hours, altogether twice；
（9）, penicillin, streptomysin, Triton X-100 are mixed into PBS form comprehensive PBS solution, wherein：Penicillin and strepto-
The plain content in comprehensive PBS solution is 500U/L, and weight ratio of the Triton X-100 in comprehensive PBS solution is 5%, then will be partly
Transparent amnion is 1 by volume：1 ratio, which is placed in comprehensive PBS solution, soaks concussion, changes liquid once within every 7 hours, altogether twice,
Then translucent amnion is taken out, rinsed 2 times with PBS；
（10）, by translucent amnion press 1：2 volume ratio is immersed in pancreatic lipase/PBS solution, wherein：Pancreatic lipase pancreatic lipase/
Content in PBS solution is 2500U/L, and is handled 20 hours at being 35 DEG C in solution temperature, then takes out translucent amnion
Rinsed 2 times with PBS, then translucent amnion is pressed 1：2 volume ratio is immersed in DNA enzymatic/PBS solution, wherein：DNA enzymatic is in DNA
Content in enzyme/PBS solution is 3000U/L, and is handled 4 hours at being 35 DEG C in solution temperature；
（11）, translucent amnion taken out from DNA enzymatic/PBS solution, be placed in dual anti-/PBS solution and shake rinsing, wherein：It is blue or green
The content of mycin and streptomysin in dual anti-/PBS solution is 300U/L, changes liquid once within every 12 hours, altogether twice, is obtained de- thin
Born of the same parents' amnion, and de- cell amnion is put into PBS, preserved at a temperature of 4 DEG C stand-by；
（12）, de- cell amnion is laid in step（6）On the lyophilized esophageal mucosa membrane being prepared, dry, had at room temperature
There is the artificial esophagus of biological structure and function.
In above-described embodiment, PBS pH value is 7.4.
Claims (4)
1. a kind of preparation method of the artificial esophagus with biological structure and function, it is characterised in that including walking in detail below
Suddenly：
（1）, take fresh mammalian gut to clean up, peel off muscle layer, leave mucous membrane and submucosa；
（2）, mucous membrane and submucosa be placed in the alcohol that volumetric concentration is 75~95% and rinse 3~10 minutes, then use clear water
Wash away alcohol；
（3）, mucous membrane and submucosa be placed in the Triton X-100 that mass concentration is 1~5% and embathed 1~5 day, during which every 12 is small
When change liquid once, finally use PBS；
（4）, mucous membrane and submucosa immersed in pancreatic lipase/PBS solution that temperature is 30~40 DEG C 5~20 hours, wherein：Pancreas
The concentration of lipase in the solution is 1000~5000U/L, then takes out and is rinsed in the alcohol that volumetric concentration is 75~95%, then
Rinsed with PBS；
（5）, mucous membrane and submucosa are immersed in DNA enzymatic/PBS solution that temperature is 30~40 DEG C and soaked 2~10 hours, its
In：The concentration of DNA enzymatic in the solution is 1000~5000U/L, then takes out and is rinsed with PBS；
（6）, mucous membrane and submucosa soaked 1~5 hour in the alcohol that volumetric concentration is 75~95%, then take out and use PBS
Rinsing, and lyophilized preservation；
（7）, take fresh placenta amnion to remove clot and chorion, leave comprising epithelium layer, basilar memebrane and without blood vessel base
The intact amniotic tissue of matter, and with PBS, obtain clean translucent amnion；
（8）, with volumetric concentration be 75% alcohol by translucent amnion rinse 1~2 time, then with PBS rinse 2~4 times, then will
Dual anti-be mixed into PBS being made up of penicillin and streptomysin forms dual anti-/PBS solution, wherein：Penicillin and streptomysin are double
Content in anti-/PBS solution is 100~500U/L, then by translucent amnion is 1 by volume：1~3 is placed in dual anti-/PBS
Concussion is soaked in solution, changes liquid once within every 12 hours, altogether twice；
（9）, penicillin, streptomysin, Triton X-100 are mixed into PBS form comprehensive PBS solution, wherein：Penicillin and strepto-
Content of the element in comprehensive PBS solution is 100~500U/L, weight ratio of the Triton X-100 in comprehensive PBS solution for 1~
5%, then by translucent amnion by volume be 1：1~3 ratio, which is placed in comprehensive PBS solution, soaks concussion, changes within every 6~8 hours
Liquid once, altogether twice, then takes out translucent amnion, is rinsed 1~2 time with PBS；
（10）, by translucent amnion press 1：1~3 volume ratio is immersed in pancreatic lipase/PBS solution, wherein：Pancreatic lipase is in pancreas fat
Content in enzyme/PBS solution is 500~5000U/L, and is handled 5~20 hours at being 30~40 DEG C in solution temperature, then will
Translucent amnion takes out to be rinsed 1~2 time with PBS, then translucent amnion is pressed into 1：It is molten that 1~3 volume ratio is immersed in DNA enzymatic/PBS
In liquid, wherein：Content of the DNA enzymatic in DNA enzymatic/PBS solution is 1000~5000U/L, and is 30~40 DEG C in solution temperature
Lower processing 2~5 hours；
（11）, translucent amnion taken out from DNA enzymatic/PBS solution, be placed in dual anti-/PBS solution and shake rinsing, wherein：It is blue or green
The content of mycin and streptomysin in dual anti-/PBS solution is 100~500U/L, changes liquid once within every 12 hours, altogether twice, is obtained
It is put into PBS, is preserved at a temperature of 4 DEG C stand-by to de- cell amnion, and by de- cell amnion；
（12）, de- cell amnion is laid in step（6）On the lyophilized esophageal mucosa membrane being prepared, dry, had at room temperature
There is the artificial esophagus of biological structure and function.
2. the preparation method of artificial esophagus with biological structure and function as claimed in claim 1 a kind of, its feature exist
In：Described PBS pH value is 7.4.
3. the preparation method of artificial esophagus with biological structure and function as claimed in claim 1 a kind of, its feature exist
In：Described mammalian gut is pig esophagus, dog food road, monkey esophagus, rabbit food road or sheep esophagus.
4. the preparation method of artificial esophagus with biological structure and function as claimed in claim 1 a kind of, its feature exist
In：Described placenta amnion behaviour placenta amnion or animal placenta amnion.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102698318A (en) * | 2012-03-02 | 2012-10-03 | 首都医科大学附属北京朝阳医院 | Biological-material complex patch |
| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| CN104013998A (en) * | 2014-05-26 | 2014-09-03 | 宁波大学 | Preparation method of artificial esophagus with histological structure |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
| CN105979976A (en) * | 2013-12-10 | 2016-09-28 | 法国国家健康医学研究院 | Methods for adhering tissue surfaces and materials and biomedical uses thereof |
| EP2197270B1 (en) * | 2007-09-07 | 2016-12-14 | Surgical Biologics | Placental tissue grafts and improved methods of preparing and using the same |
-
2017
- 2017-10-24 CN CN201710997587.1A patent/CN107583110A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2197270B1 (en) * | 2007-09-07 | 2016-12-14 | Surgical Biologics | Placental tissue grafts and improved methods of preparing and using the same |
| CN102698318A (en) * | 2012-03-02 | 2012-10-03 | 首都医科大学附属北京朝阳医院 | Biological-material complex patch |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| CN105979976A (en) * | 2013-12-10 | 2016-09-28 | 法国国家健康医学研究院 | Methods for adhering tissue surfaces and materials and biomedical uses thereof |
| CN104013998A (en) * | 2014-05-26 | 2014-09-03 | 宁波大学 | Preparation method of artificial esophagus with histological structure |
Non-Patent Citations (1)
| Title |
|---|
| QIUXIANG SHEN等: "Progress on materials and scaffold fabrications applied to esophageal tissue engineering", 《MATERIALS SCIENCE AND ENGINEERING C》 * |
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