CN106148180A - A kind of for simulated environment pollutant in the apparatus and method of the digested absorption of digestive system - Google Patents
A kind of for simulated environment pollutant in the apparatus and method of the digested absorption of digestive system Download PDFInfo
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- CN106148180A CN106148180A CN201610529834.0A CN201610529834A CN106148180A CN 106148180 A CN106148180 A CN 106148180A CN 201610529834 A CN201610529834 A CN 201610529834A CN 106148180 A CN106148180 A CN 106148180A
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- 239000003344 environmental pollutant Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000010521 absorption reaction Methods 0.000 title claims abstract description 30
- 231100000719 pollutant Toxicity 0.000 title claims abstract description 26
- 210000002249 digestive system Anatomy 0.000 title claims description 20
- 230000000968 intestinal effect Effects 0.000 claims abstract description 67
- 230000002496 gastric effect Effects 0.000 claims abstract description 23
- 230000008569 process Effects 0.000 claims abstract description 13
- 239000000356 contaminant Substances 0.000 claims abstract description 9
- 230000007613 environmental effect Effects 0.000 claims abstract description 9
- 239000011521 glass Substances 0.000 claims description 153
- 238000004088 simulation Methods 0.000 claims description 93
- 210000001815 ascending colon Anatomy 0.000 claims description 66
- 210000003384 transverse colon Anatomy 0.000 claims description 65
- 210000001072 colon Anatomy 0.000 claims description 49
- 210000002784 stomach Anatomy 0.000 claims description 43
- 239000007788 liquid Substances 0.000 claims description 31
- 239000002609 medium Substances 0.000 claims description 30
- 235000015097 nutrients Nutrition 0.000 claims description 30
- 210000004027 cell Anatomy 0.000 claims description 29
- 210000001731 descending colon Anatomy 0.000 claims description 24
- 239000004816 latex Substances 0.000 claims description 24
- 229920000126 latex Polymers 0.000 claims description 24
- 230000002572 peristaltic effect Effects 0.000 claims description 23
- 210000001819 pancreatic juice Anatomy 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 13
- 201000010897 colon adenocarcinoma Diseases 0.000 claims description 11
- 208000029742 colonic neoplasm Diseases 0.000 claims description 11
- 230000029087 digestion Effects 0.000 claims description 11
- 244000005700 microbiome Species 0.000 claims description 11
- 238000003760 magnetic stirring Methods 0.000 claims description 10
- 239000000523 sample Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 8
- 102000001621 Mucoproteins Human genes 0.000 claims description 8
- 108010093825 Mucoproteins Proteins 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 210000000170 cell membrane Anatomy 0.000 claims description 8
- 210000003608 fece Anatomy 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000005374 membrane filtration Methods 0.000 claims description 8
- 229920001277 pectin Polymers 0.000 claims description 8
- 239000001814 pectin Substances 0.000 claims description 8
- 235000010987 pectin Nutrition 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- SATHPVQTSSUFFW-UHFFFAOYSA-N 4-[6-[(3,5-dihydroxy-4-methoxyoxan-2-yl)oxymethyl]-3,5-dihydroxy-4-methoxyoxan-2-yl]oxy-2-(hydroxymethyl)-6-methyloxane-3,5-diol Chemical compound OC1C(OC)C(O)COC1OCC1C(O)C(OC)C(O)C(OC2C(C(CO)OC(C)C2O)O)O1 SATHPVQTSSUFFW-UHFFFAOYSA-N 0.000 claims description 7
- 229920000189 Arabinogalactan Polymers 0.000 claims description 7
- 239000001904 Arabinogalactan Substances 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 7
- 235000019312 arabinogalactan Nutrition 0.000 claims description 7
- 235000013305 food Nutrition 0.000 claims description 7
- 238000005070 sampling Methods 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 6
- 235000011890 sandwich Nutrition 0.000 claims description 6
- 235000020188 drinking water Nutrition 0.000 claims description 5
- 239000003651 drinking water Substances 0.000 claims description 5
- 238000004064 recycling Methods 0.000 claims description 5
- 210000000813 small intestine Anatomy 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 4
- 239000003833 bile salt Substances 0.000 claims description 4
- 229940093761 bile salts Drugs 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 230000036760 body temperature Effects 0.000 claims description 4
- 239000008366 buffered solution Substances 0.000 claims description 4
- 210000002615 epidermis Anatomy 0.000 claims description 4
- DUYAAUVXQSMXQP-UHFFFAOYSA-N ethanethioic S-acid Chemical compound CC(S)=O DUYAAUVXQSMXQP-UHFFFAOYSA-N 0.000 claims description 4
- 239000011229 interlayer Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 210000000496 pancreas Anatomy 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 239000012930 cell culture fluid Substances 0.000 claims description 2
- 239000006481 glucose medium Substances 0.000 claims description 2
- 229940055695 pancreatin Drugs 0.000 claims description 2
- 239000002699 waste material Substances 0.000 claims description 2
- 229920001221 xylan Polymers 0.000 claims description 2
- 150000004823 xylans Chemical class 0.000 claims description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims 1
- 239000012091 fetal bovine serum Substances 0.000 claims 1
- 235000008935 nutritious Nutrition 0.000 claims 1
- 239000003643 water by type Substances 0.000 claims 1
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 5
- 231100000440 toxicity profile Toxicity 0.000 abstract description 4
- 238000012502 risk assessment Methods 0.000 abstract description 2
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 13
- 239000004695 Polyether sulfone Substances 0.000 description 8
- 229920006393 polyether sulfone Polymers 0.000 description 8
- 229940106691 bisphenol a Drugs 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000000605 extraction Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000192142 Proteobacteria Species 0.000 description 2
- 229910052785 arsenic Inorganic materials 0.000 description 2
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- 241000605059 Bacteroidetes Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 238000001927 high performance liquid chromatography-inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
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- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention discloses a kind of absorption plant of being simulated with Transwell by external gastrointestinal analog and combine the apparatus and method digested and assimilated by intestinal after research environment pollutant enter human gastrointestinal tract, assembly of the invention and method intactly simulate the process digested and assimilated by intestinal after environmental contaminants enter human gastrointestinal tract, being more nearly process of digesting and assimilating really, toxicity profile research and environmental contaminants after being absorbed by the body subsequent environments pollutant are significant to human healthy risk assessment.
Description
Technical field
The present invention relates to the external gastrointestinal model of a kind of employing and Transwell model carrys out research environment pollutant at Digestive
The situation of digested absorption in system, belongs to biological technology field.
Background technology
External model has the shortest compared to zoopery, relatively accurate, will not cause the advantages such as ethical issues.And mesh
The external model of front application mainly have Manual Suture simplify extraction method (SBET), biological away from extraction method (PBET),
Rodriguez external gastrointestinal method (IVG) etc., these methods have been widely used for Metals in Environments ion, organic pollution etc.
The research of Manual Suture in human body.But this several method only simulates the environmental contaminants digestion feelings at harmonization of the stomach small intestine site
Condition, it is impossible to the digestion situation at reaction large intestine position and enteric microorganism effect wherein.Ghent, Belgium university research is sent out
Bright SHIME device (Simulator of the Human Intestinal Microbial Ecosystem) increases and finishes
Simulation that intestines is divided and be vaccinated with enteric microorganism, but it is merely able to simulated environment pollutant at stomach, small intestinal and colon portion
Manual Suture and the process of metabolism, enter human consumption to environmental pollutants and absorb and the poison that causes after digesting and assimilating
Property change can not carry out complete simulation, therefore present invention improves over SHIME model, it combined with Transwell model
The complete metabolism simulated after environmental contaminants are orally ingested in human gastrointestinal tract and absorption process and through digesting and assimilating
After toxicity profile.
Summary of the invention
Goal of the invention: the technical problem to be solved is to provide one for simulated environment pollutant at Digestive
Uniting the device of digested absorption, external gastrointestinal model and Transwell models coupling are got up by this device, it is possible to complete simulation
Environmental contaminants be orally ingested after metabolism in human gastrointestinal tract and absorbing state.
The present invention also to solve the technical problem that be to provide above-mentioned digested at digestive system for simulated environment pollutant
The method absorbed.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is:
A kind of for simulated environment pollutant at the device of the digested absorption of digestive system, including external gastrointestinal model and
Transwell cell；
Wherein, described external gastrointestinal model includes the glass jar I of five sandwichs, and five glass jars 1 simulate stomach, little respectively
Intestinal, ascending colon, transverse colon and descending colon；Also include the glass jar II equipped with Nutrient medium and the glass jar III equipped with pancreatic juice；
It is connected by latex tubing, equipped with the glass jar III of pancreatic juice with the glass jar I of simulation stomach equipped with the glass jar II of Nutrient medium
It is connected by latex tubing with the glass jar I of simulation small intestinal, simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass
Glass tank I passes sequentially through latex tubing and is connected, and each latex tubing is respectively provided with peristaltic pump；Simulation stomach, small intestinal, ascending colon, transverse colon
It is respectively provided with magnetic stirring apparatus with in colon descendens glass jar I；On simulation small intestinal, ascending colon, transverse colon and colon descendens glass jar I
Also setting up thief hatch, arrange PES filter membrane outside each thief hatch, the sample solution that thief hatch flows out is through PES membrane filtration；Simulation rises
Colon, transverse colon and colon descendens glass jar I arrange pH-meter；
The thief hatch of described simulation small intestinal, ascending colon, transverse colon and descending colon glass jar I is connected respectively one
Transwell cell, described Transwell cell includes room and lower room, arranges filter membrane bottom described upper room, and filter membrane is inoculated
Human colon adenocarcinoma cell line Caco-2, Human colon adenocarcinoma cell line Caco-2 forms a cell membrane, and the sample solution that thief hatch flows out is through PES
Cultivate in upper room after membrane filtration.
Wherein, described PES filter membrane is polyethersulfone millipore filter, and its aperture is 0.45 μm.
Wherein, the cell culture fluid in described Transwell cell is 500mL incomplete high glucose medium+50mL embryo
Ox blood serum.
A kind of for simulated environment pollutant in the method for the digested absorption of digestive system, comprise the steps:
Step 1, the outer gastrointestinal model of construct: external gastrointestinal model includes the glass jar I of five sandwichs, five glass jars
I simulates stomach, small intestinal, ascending colon, transverse colon and descending colon respectively；Also include the glass jar II equipped with Nutrient medium and equipped with pancreas
The glass jar III of liquid；It is connected by latex tubing with the glass jar I of simulation stomach equipped with the glass jar II of Nutrient medium, equipped with
The glass jar III of pancreatic juice is connected by latex tubing with the glass jar I of simulation small intestinal, simulates stomach, small intestinal, ascending colon, transverse colon
Pass sequentially through latex tubing with colon descendens five glass jar I to be connected, each latex tubing is respectively provided with peristaltic pump；Simulation stomach, little
Intestinal, ascending colon, transverse colon and colon descendens glass jar I are respectively provided with magnetic stirring apparatus；Simulation small intestinal, ascending colon, transverse colon and
Thief hatch is also set up on colon descendens glass jar I；Simulation ascending colon, transverse colon and colon descendens glass jar I arrange pH regulator
Device；
Step 2, logical 37 DEG C of perseverances in simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass jar I interlayers
Warm water simulation body temperature；Starting the magnetic stirring apparatus in five glass jar I, mixing speed is 150rpm；By micro-for the intestinal of people
Bacterization is to simulating in ascending colon, transverse colon and colon descendens glass jar I；
Step 3, the operation of device: with peristaltic pump, 200ml Nutrient medium is added in the glass jar I simulating stomach, simulation
Food is in the digestion situation of stomach, and liquid is transferred completely into the glass jar I of simulation small intestinal after stomach position stops 2h, simultaneously with compacted
Dynamic pump adds pancreatic juice in the glass jar I of simulation small intestinal, after small intestine site digestion 4h, is fully transferred to simulation with peristaltic pump and rises
The glass jar I of colon, is finally transferred to residual liquid with the speed of 0.625mL/min from ascending colon glass jar I continuously with peristaltic pump
In transverse colon glass jar I, descending colon glass jar I and waste liquid cylinder；
Step 4, after external gastrointestinal model runs three weeks by the stable operation of step 3, in the Nutrient medium of step 3
Adding environmental contaminants, simulated environment pollutant enter human body by food or drinking-water, in simulation small intestinal, ascending colon, transverse colon
The thief hatch sampling arranged with colon descendens glass jar I, it is little that the thief hatch correspondence of each glass jar I connects a Transwell
Room, obtains Manual Suture part after the PES membrane filtration of sample solution sampled mouth outer surface；
Step 5, carries out certain pretreatment by the Manual Suture part obtained, and after pretreatment, adds on Transwell
Room, inoculates Human colon adenocarcinoma cell line Caco-2, Human colon adenocarcinoma cell line Caco-2 growth formation one on filter membrane bottom the upper room of Transwell
Opening cell membrane, Human colon adenocarcinoma cell line Caco-2 cell membrane is used for the suction to environmental pollutants of simulating human intestinal epidermis mucomembranous cell
Receipts situation.
Wherein, in step 2, described being inoculated into by the enteric microorganism of people simulates ascending colon, transverse colon and colon descendens glass
In glass tank I, concrete seeded process is the fresh adult feces taking 6 months and not taking in antibiotic, adds 80mLpH and be in feces
7.0, concentration is the phosphate buffered solution of 0.7M and 1g thiacetic acid. is received, and is homogenized by sample, centrifugal under 3000rpm rotating speed
3min, takes 20mL supernatant and transfers to successively simulate in ascending colon, transverse colon and colon descendens glass jar I after being centrifuged；Toward simulation
Ascending colon, transverse colon and colon descendens glass jar I are separately added into SHIME special culture media, until simulation ascending colon, transverse colon
It is respectively 800ml, 1200ml and 800ml with the volume of liquid in descending colon glass jar I, stands a night；Recycling pH controller
The pH of liquid in simulation ascending colon, transverse colon and descending colon glass jar I is controlled respectively 5.5～5.9,6.0～6.4 and 6.5
～6.9；
Wherein, in step 3, the pH value of described Nutrient medium is 2, and the formula of described Nutrient medium is: 1.0g/L Ah
Draw primary galactan, 2.0g/L pectin, 1.0g/L xylan, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L glucose,
1.0g/L yeast extract, 4.0g/L be mucoprotein and 1.0g/L peptone.
Wherein, after described Nutrient medium prepares, it is placed at 121 DEG C and is cooled to room temperature after sterilizing.
Wherein, in step 3, the formula of described pancreatic juice is: 12.5g/L sodium bicarbonate, 6.0g/L bile salts and 0.9g/L
Pancreatin.
Wherein, in described pH controller, acid regulation liquid is the hydrochloric acid of 0.01M, and alkali regulation liquid is 0.5M sodium bicarbonate.
The formula of described SHIME special culture media is: 1.0g/L arabinogalactan, 2.0g/L pectin, 4.0g/L form sediment
Powder, 0.5g/L cysteine, 0.4g/L glucose, 1.0g/L yeast extract, 4.0g/L be mucoprotein and 1.0g/L peptone.
According to apparatus of the present invention absorbing state to environmental pollutants, recycling cell toxicity test is carried out research environment and is polluted
Toxicity profile after the digested absorption of thing.
Compared to existing simulated environment pollutant at the device and method of the digested absorption of digestive system, the present invention is used for
Simulated environment pollutant have the beneficial effect that at the device and method of the digested absorption of digestive system
Assembly of the invention and method intactly simulate after environmental contaminants enter human gastrointestinal tract is inhaled by intestinal digestion
The process received, is more nearly process of digesting and assimilating really, and the toxicity profile after being absorbed by the body subsequent environments pollutant grinds
Study carefully and environmental contaminants are significant to human healthy risk assessment.
Accompanying drawing explanation
Fig. 1 is the present invention for simulated environment pollutant at the structural representation of the device of the digested absorption of digestive system；
Fig. 2 is biological community structure figure in analog.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, technical scheme is described further.
Embodiment 1
By external gastrointestinal analog with Transwell simulation absorption plant binding Arsenic in Drinking Water at digestive system
In the situation of digested absorption:
Step 1, the outer gastrointestinal model of construct: external gastrointestinal model includes the glass jar I of five sandwichs, five glass jars
1 simulates stomach, small intestinal, ascending colon, transverse colon and descending colon respectively；Also include the glass jar II equipped with Nutrient medium and equipped with pancreas
The glass jar III of liquid；It is connected by latex tubing with the glass jar I of simulation stomach equipped with the glass jar II of Nutrient medium, equipped with
The glass jar III of pancreatic juice is connected by latex tubing with the glass jar I of simulation small intestinal, simulates stomach, small intestinal, ascending colon, transverse colon
Passing sequentially through latex tubing with colon descendens five glass jar I to be connected, each latex tubing is respectively provided with peristaltic pump, peristaltic pump is used for
Realize the connection between different tank body；Simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens glass jar I are respectively provided with magnetic force
Agitator；Thief hatch is also set up on simulation small intestinal, ascending colon, transverse colon and colon descendens glass jar I；Simulation ascending colon, horizontal knot
Intestinal and colon descendens glass jar I arrange pH-meter；
Step 2, logical 37 DEG C of perseverances in simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass jar I interlayers
Warm water simulation body temperature；Start the magnetic stirring apparatus in five glass jar I, by magnetic stirring apparatus simulation human gastrointestinal tract
Wriggling, mixing speed is 150rpm；The enteric microorganism of people is inoculated into simulation ascending colon, transverse colon and colon descendens glass jar
In I；Concrete seeded process is the fresh adult feces taking 6 months and not taking in antibiotic, toward feces in addition 80mLpH be 7.0,
Concentration is the phosphate buffered solution of 0.7M, adds 1g thiacetic acid. and receives as reducing agent, is homogenized by sample, in 3000rpm
Centrifugal 3min under rotating speed, takes 20mL supernatant and transfers to successively simulate ascending colon, transverse colon and colon descendens glass jar I after being centrifuged
In；It is separately added into (the special training of SHIME of SHIME special culture media in simulation ascending colon, transverse colon and colon descendens glass jar I
The formula supporting base is: 1.0g/L arabinogalactan, 2.0g/L pectin, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L
Glucose, 1.0g/L yeast extract, 4.0g/L be mucoprotein and 1.0g/L peptone), until simulation ascending colon, transverse colon and
In descending colon glass jar I, the volume of liquid is respectively 800ml, 1200ml and 800ml, stablizes overnight；Recycling pH controller leads to
Cross 0.01M hydrochloric acid and 0.5M sodium bicarbonate to be controlled respectively by the pH value of liquid in simulation ascending colon, transverse colon and descending colon glass jar I
System is 5.5～5.9,6.0～6.4 and 6.5～6.9:
Step 3, the operation of device: with peristaltic pump by (nutrition in the glass jar I of 200ml Nutrient medium addition simulation stomach
The pH value of culture medium is 2, and the formula of Nutrient medium is: 1.0g/L arabinogalactan, 2.0g/L pectin, 1.0g/L wood are poly-
Sugar, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L glucose, 1.0g/L yeast extract, 4.0g/L mucoprotein and
1.0g/L peptone；The Nutrient medium prepared is placed at 121 DEG C and after sterilizing, is cooled to room temperature；), simulation food is at stomach
Digestion situation, liquid stops the glass jar I being transferred completely into simulation small intestinal after 2h in stomach position, simultaneously with peristaltic pump toward simulation
(pancreatic juice formula is 12.5g/L sodium bicarbonate, 6.0g/L bile salts and 0.9g/L pancreatic juice to add pancreatic juice in the glass jar I of small intestinal
Element), after small intestine site digestion 4h, it is fully transferred to simulate colon ascendens glass jar I with peristaltic pump, finally with peristaltic pump by residual
Liquid is transferred to transverse colon glass jar I, descending colon glass jar I and useless from ascending colon glass jar I continuously with the speed of 0.625mL/min
In fluid cylinder；External gastrointestinal model stable operation three weeks, in simulation ascending colon glass jar I, simulation transverse colon glass jar I and simulation
The thief hatch sampling of descending colon glass jar I, carries out the extraction of STb gene, and Miseq high-flux sequence, the group obtained to sample
The structure that falls contrasts with the human intestinal microorganisms's structure of community having been reported, and its main door is rear wall bacterium door, Bacteroidetes
And Proteobacteria, consistent with human intestinal microorganisms's structure of community, as shown in Figure 2；
Step 4, when after external gastrointestinal model stable operation three weeks, is separately added into 100ppb in step 3 Nutrient medium
With 600ppb arsenic trioxide solution, simulation As enters human body by drinking water, after exposing one week respectively, at simulation small intestinal glass
The thief hatch sampling of tank I, simulation ascending colon glass jar I, simulation transverse colon glass jar I and simulation descending colon glass jar I, sampling
Liquid, after the PES membrane filtration of thief hatch outer surface, obtains the As of Manual Suture part；
Step 5, carries out certain pretreatment by the As of the Manual Suture part obtained, and pre-treatment step is: will obtain
As solution lyophilizing to no liquid, add overall height sugar culture-medium, more degerming with the filtering with microporous membrane that aperture is 0.22 μm；Pre-place
Solution adds after reason the upper room of Transwell cell, and room and the contact position of upper room under Transwell, i.e. go up room in advance
Human colon adenocarcinoma cell line Caco-2 is inoculated so that it is grow up to a cell membrane, simulate on bottom filter membrane (filter membrane is polycarbonate membrane)
Human intestine's epidermis mucomembranous cell absorption process to environmental pollutants.
Sample in the lower room of Transwell cell every 30min, after utilizing HPLC-ICP-MS to measure digested absorption
The form of arsenic and concentration.
Embodiment 2
By external gastrointestinal analog with Transwell simulation absorption plant binding bisphenol-A (BPA) at digestive system
In the situation of digested absorption:
Step 1, the outer gastrointestinal model of construct: external gastrointestinal model includes the glass jar I of five sandwichs, five glass jars
1 simulates stomach, small intestinal, ascending colon, transverse colon and descending colon respectively；Also include the glass jar II equipped with Nutrient medium and equipped with pancreas
The glass jar III of liquid；It is connected by latex tubing with the glass jar I of simulation stomach equipped with the glass jar II of Nutrient medium, equipped with
The glass jar III of pancreatic juice is connected by latex tubing with the glass jar I of simulation small intestinal, simulates stomach, small intestinal, ascending colon, transverse colon
Passing sequentially through latex tubing with colon descendens five glass jar I to be connected, each latex tubing is respectively provided with peristaltic pump, peristaltic pump is used for
Realize the connection between different tank body；Simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens glass jar I are respectively provided with magnetic force
Agitator；Thief hatch is also set up on simulation small intestinal, ascending colon, transverse colon and colon descendens glass jar I；Simulation ascending colon, horizontal knot
Intestinal and colon descendens glass jar I arrange pH-meter；
Step 2, logical 37 DEG C of perseverances in simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass jar I interlayers
Warm water simulation body temperature；Start the magnetic stirring apparatus in five glass jar I, by magnetic stirring apparatus simulation human gastrointestinal tract
Wriggling, mixing speed is 150rpm；The enteric microorganism of people is inoculated into respectively simulation ascending colon, transverse colon and colon descendens glass
In glass tank I；Concrete seeded process is the fresh adult feces taking 6 months and not taking in antibiotic, adds 80mLpH and be in feces
7.0, concentration is the phosphate buffered solution of 0.7M, adds 1g thiacetic acid. and receives as reducing agent, is homogenized by sample, in
Centrifugal 3min under 3000rpm rotating speed, centrifugal after take 20mL supernatant and transfer to simulate ascending colon, transverse colon and colon descendens successively
In glass jar I；It is separately added into SHIME special culture media (SHIME in simulation ascending colon, transverse colon and colon descendens glass jar I
The formula of special culture media is: 1.0g/L arabinogalactan, 2.0g/L pectin, 4.0g/L starch, 0.5g/L cysteine,
0.4g/L glucose, 1.0g/L yeast extract, 4.0g/L be mucoprotein and 1.0g/L peptone), until simulation ascending colon, horizontal stroke
In colon and descending colon glass jar I, the volume of liquid is respectively 800ml, 1200ml and 800ml, stablizes overnight；Recycling pH control
Device processed will simulate the pH value of liquid in ascending colon, transverse colon and descending colon glass jar I by 0.01M hydrochloric acid and 0.5M sodium bicarbonate
Control respectively 5.5～5.9,6.0～6.4 and 6.5～6.9；
Step 3, the operation of device: with peristaltic pump, 200ml Nutrient medium is added in the glass jar I simulating stomach, (nutrition
The pH value of culture medium is 2, and the formula of Nutrient medium is: 1.0g/L arabinogalactan, 2.0g/L pectin, 1.0g/L wood are poly-
Sugar, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L glucose, 1.0g/L yeast extract, 4.0g/L mucoprotein and
1.0g/L peptone；The Nutrient medium prepared is placed at 121 DEG C and after sterilizing, is cooled to room temperature；) simulate food at stomach
Digestion situation, liquid stops the glass jar I being transferred completely into simulation small intestinal after 2h in stomach position, simultaneously with peristaltic pump toward simulation
(pancreatic juice formula is 12.5g/L sodium bicarbonate, 6.0g/L bile salts and 0.9g/L pancreatic juice to add pancreatic juice in the glass jar I of small intestinal
Element), after small intestine site digestion 4h, it is fully transferred to simulate colon ascendens glass jar I with peristaltic pump, finally with peristaltic pump by residual
Liquid is transferred to transverse colon glass jar I, descending colon glass jar I and useless from ascending colon glass jar I continuously with the speed of 0.625mL/min
In fluid cylinder；External gastrointestinal analog stable operation three weeks, simulation ascending colon glass jar I, simulation transverse colon glass jar I and
The thief hatch sampling of simulation descending colon glass jar I, carries out the extraction of STb gene, and Miseq high-flux sequence, obtains sample
Structure of community contrast with the human intestinal microorganisms's structure of community having been reported, its main door be rear wall bacterium door, intend bar
Bacterium door and Proteobacteria, consistent with human intestinal microorganisms's structure of community, see accompanying drawing 2；
Step 4, when after external gastrointestinal model stable operation three weeks, is separately added into BPA solution in step 3 Nutrient medium
(concentration is respectively 25mg/L, 250mg/L and 2500mg/L), simulation BPA enters human body by food or drinking-water, exposes one respectively
Zhou Hou, at simulation small intestinal glass jar I, simulation ascending colon glass jar I, simulation transverse colon glass jar I and simulation descending colon glass
The thief hatch sampling of tank I, sample solution, after the PES membrane filtration of thief hatch outer surface, obtains Manual Suture part
BPA；
Step 5, carries out certain pretreatment by the BPA of the Manual Suture part obtained, and pre-treatment step is: will obtain
BPA solution lyophilizing to no liquid, add overall height sugar culture-medium, more degerming with the filtering with microporous membrane that aperture is 0.22 μm；In advance
After process, dye poison liquid is added the upper room of Transwell cell, in Transwell cell, inoculates human colon carcinoma in advance thin
Born of the same parents Caco-2 so that it is grow up to a cell membrane, carrys out simulating human intestinal epidermis mucomembranous cell absorption process to environmental pollutants.
Sample in the lower room of Transwell cell every 30min, utilize the material after GC-MS analysis BPA metabolism with dense
Degree.
Obviously, above-described embodiment is only for clearly demonstrating example of the present invention, and not to the present invention
The restriction of embodiment.For those of ordinary skill in the field, can also be made it on the basis of the above description
The change of its multi-form or variation.Here without also cannot all of embodiment be given exhaustive.And these belong to this
What bright spirit was extended out obviously changes or changes among still in protection scope of the present invention.
Claims (10)
1. one kind is used for the simulated environment pollutant device in the digested absorption of digestive system, it is characterised in that: include external stomach
Intestinal model and Transwell cell；
Wherein, described external gastrointestinal model includes the glass jar I of five sandwichs, five glass jar I simulate respectively stomach, small intestinal,
Ascending colon, transverse colon and descending colon；Also include the glass jar II equipped with Nutrient medium and the glass jar III equipped with pancreatic juice；Dress
The glass jar II of nutritious culture medium and simulation stomach glass jar I be connected by latex tubing, equipped with pancreatic juice glass jar III with
The glass jar I of simulation small intestinal is connected by latex tubing, simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass
Tank I passes sequentially through latex tubing and is connected, and each latex tubing is respectively provided with peristaltic pump；Simulation stomach, small intestinal, ascending colon, transverse colon and
Colon descendens glass jar I is respectively provided with magnetic stirring apparatus；On simulation small intestinal, ascending colon, transverse colon and colon descendens glass jar I also
Arranging thief hatch, arrange PES filter membrane outside each thief hatch, the sample solution that thief hatch flows out is through PES membrane filtration；Simulation rises knot
Intestinal, transverse colon and colon descendens glass jar I arrange pH-meter；
The thief hatch of described simulation small intestinal, ascending colon, transverse colon and descending colon glass jar I is connected respectively one
Transwell cell, described Transwell cell includes room and lower room, arranges filter membrane bottom described upper room, and filter membrane is inoculated
Human colon adenocarcinoma cell line Caco-2, Human colon adenocarcinoma cell line Caco-2 forms a cell membrane, and the sample solution that thief hatch flows out is through PES
Cultivate in upper room after membrane filtration.
The most according to claim 1, be used for the simulated environment pollutant device in the digested absorption of digestive system, its feature exists
In: the aperture of described PES filter membrane is 0.45 μm.
The most according to claim 1, be used for the simulated environment pollutant device in the digested absorption of digestive system, its feature exists
In: the cell culture fluid in described Transwell cell is 500mL incomplete high glucose medium+50mL fetal bovine serum.
4. one kind is used for the simulated environment pollutant method in the digested absorption of digestive system, it is characterised in that include walking as follows
Rapid:
Step 1, the outer gastrointestinal model of construct: external gastrointestinal model includes the glass jar I of five sandwichs, five glass jars 1 point
Do not simulate stomach, small intestinal, ascending colon, transverse colon and descending colon；Also include the glass jar II equipped with Nutrient medium and equipped with pancreatic juice
Glass jar III；It is connected by latex tubing, equipped with pancreas with the glass jar I of simulation stomach equipped with the glass jar II of Nutrient medium
The glass jar III of liquid and simulation small intestinal glass jar I be connected by latex tubing, simulate stomach, small intestinal, ascending colon, transverse colon and
Colon descendens five glass jar I pass sequentially through latex tubing and are connected, and each latex tubing is respectively provided with peristaltic pump；Simulation stomach, small intestinal,
Ascending colon, transverse colon and colon descendens glass jar I are respectively provided with magnetic stirring apparatus；Simulation small intestinal, ascending colon, transverse colon and fall knot
Thief hatch is also set up on the glass jar I of intestinal；Simulation ascending colon, transverse colon and colon descendens glass jar I arrange pH-meter；
Step 2, logical 37 DEG C of thermostatted waters in simulation stomach, small intestinal, ascending colon, transverse colon and colon descendens five glass jar I interlayers
Simulation body temperature；Starting the magnetic stirring apparatus in five glass jar I, mixing speed is 150rpm；By the enteric microorganism of people
It is inoculated in simulation ascending colon, transverse colon and colon descendens glass jar I；
Step 3, the operation of device: with peristaltic pump, 200ml Nutrient medium is added in the glass jar I simulating stomach, simulate food
In the digestion situation of stomach, liquid is transferred completely into the glass jar I of simulation small intestinal after stomach position stops 2h, uses peristaltic pump simultaneously
In the glass jar I of simulation small intestinal, add pancreatic juice, after small intestine site digestion 4h, be fully transferred to simulate ascending colon with peristaltic pump
Glass jar I, finally with peristaltic pump, residual liquid is transferred to horizontal knot from ascending colon glass jar I continuously with the speed of 0.625mL/min
In intestinal glass jar I, descending colon glass jar I and waste liquid cylinder；
Step 4, after external gastrointestinal model runs three weeks by the stable operation of step 3, adds in the Nutrient medium of step 3
Environmental contaminants, simulated environment pollutant enter human body by food or drinking-water, in simulation small intestinal, ascending colon, transverse colon and fall
The thief hatch sampling that the glass jar I of colon is arranged, the thief hatch correspondence of each glass jar I connects a Transwell cell, adopts
Manual Suture part is obtained after the PES membrane filtration of sample liquid sampled mouth outer surface；
Step 5, carries out certain pretreatment by the Manual Suture part obtained, and after pretreatment, adds room on Transwell,
Inoculating Human colon adenocarcinoma cell line Caco-2 bottom the upper room of Transwell on filter membrane, Human colon adenocarcinoma cell line Caco-2 growth forms one
Cell membrane, Human colon adenocarcinoma cell line Caco-2 cell membrane is used for the absorption to environmental pollutants of simulating human intestinal epidermis mucomembranous cell
Situation.
The most according to claim 4, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: in step 2, described being inoculated into by the enteric microorganism of people is simulated in ascending colon, transverse colon and colon descendens glass jar I, tool
Body seeded process is the fresh adult feces taking 6 months and not taking in antibiotic, in feces add 80mLpH be 7.0, concentration be
Phosphate buffered solution and the 1g thiacetic acid. of 0.7M are received, and are homogenized by sample, and under 3000rpm rotating speed, centrifugal 3min, centrifugal
After take 20mL supernatant and transfer to successively simulate in ascending colon, transverse colon and colon descendens glass jar I；Toward simulation ascending colon, horizontal stroke
Colon and colon descendens glass jar I are separately added into SHIME special culture media, until simulation ascending colon, transverse colon and descending colon
In glass jar I, the volume of liquid is respectively 800ml, 1200ml and 800ml, stands a night；Simulation is risen by recycling pH controller
In colon, transverse colon and descending colon glass jar I, the pH of liquid controls respectively 5.5～5.9,6.0～6.4 and 6.5～6.9；
The most according to claim 4, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: in step 3, the pH value of described Nutrient medium is 2, and the formula of described Nutrient medium is: 1.0g/L arabinogalactan
Sugar, 2.0g/L pectin, 1.0g/L xylan, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L glucose, 1.0g/L yeast
Extract, 4.0g/L be mucoprotein and 1.0g/L peptone.
The most according to claim 6, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: after described Nutrient medium prepares, it is placed at 121 DEG C and is cooled to room temperature after sterilizing.
The most according to claim 4, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: in step 3, the formula of described pancreatic juice is: 12.5g/L sodium bicarbonate, 6.0g/L bile salts and 0.9g/L pancreatin.
The most according to claim 5, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: in described pH controller, acid regulation liquid is the hydrochloric acid of 0.01M, and alkali regulation liquid is 0.5M sodium bicarbonate.
The most according to claim 5, be used for the simulated environment pollutant method in the digested absorption of digestive system, its feature exists
In: described SHIME special culture media is consistent with the formula of Nutrient medium, and the formula of described SHIME special culture media is:
1.0g/L arabinogalactan, 2.0g/L pectin, 4.0g/L starch, 0.5g/L cysteine, 0.4g/L glucose, 1.0g/L
Yeast extract, 4.0g/L be mucoprotein and 1.0g/L peptone.
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