CN106497854B - Lactobacillus D8 and its application - Google Patents
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- CN106497854B CN106497854B CN201710039037.9A CN201710039037A CN106497854B CN 106497854 B CN106497854 B CN 106497854B CN 201710039037 A CN201710039037 A CN 201710039037A CN 106497854 B CN106497854 B CN 106497854B
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0635—B lymphocytes
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0679—Cells of the gastro-intestinal tract
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses lactobacillus D8 and its applications.Lactobacillus D8 of the invention, be it is isolated from healthy swinery, classification naming be lactobacillus (Lactobacillus sp.), deposit number is CGMCC No.13112.Pass through the building of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model, it has been found that lactobacillus D8 can effectively facilitate the Proliferation, Differentiation of intestinal stem cell and then safeguard the integrality of gut barrier.Further animal experiment confirms that oral lactobacillus D8 can significantly improve the length of intestinal villi and the depth of crypts of small intestine, reduces enteritis symptom.Therefore, which is expected to exploitation as prevention and/or the probiotics for the treatment of enteritis.
Description
Technical field
The present invention relates to biological Control Technologies, and in particular to lactobacillus D8 and its application, lactobacillus D8 can be effectively facilitated
The Proliferation, Differentiation of intestinal stem cell repairs the damage of TNF-α induction；Oral Lactobacillus animalis D8 can significantly improve intestinal inflammation
Shape.
Background technique
1. the present Research and its immunological role of lactobacillus
Lactobacillus (Lactobacillus) belongs to hard bacterium door (Firmicutes), bacillus guiding principle on taxology
(Bacilli), lactobacillus mesh (lactobacillales), Lactobacillaceae (lactobacillaceae) are a kind of gram sun
Property bacillus, do not formed bud embrace, decompose sugar can generate lactic acid.Lactobacillus is the normal bacterium of humans and animals enteron aisle, with host's nutrition
The absorption of substance, the development of Intestinal Mucosal Immunity system suffer from close relationship.Research shows that lactobacillus is as probiotics,
Inhibit the field planting of enteropahtogenic microganism, promote intestinal health etc. plays an important role；Lactobacillus also can be used as mucosa-immune
Adjuvant promotes reaction of the mucosal immune system to antigen, to improve the function and effect of vaccine.In addition to this, lactobacillus can be with
Live vector as drug and antigen molecule.
The bootable nospecific immunity adjustment effect of lactobacillus stimulates intestine immunity cell, reinforces the immune of enteron aisle itself
System function especially enhances the function of huge saliva cell, and by stimulation specific immune response, increases IgA, IgG in serum
With IgM level, it is mature to promote T lymphocyte and bone-marrow-derived lymphocyte, to enhance cellular immunity, improves intestinal immunity and disease-resistant
Power resists the diseases such as intestinal canal tumour, inflammation.Research confirms that Yue Shi lactobacillus and Bacillus bifidus can enhance in vitro and gulps down saliva cell
The effect of sighing is gulped down to Escherichia coli.The immune rush healthy functions of lactobacillus be unable to do without the receptor on the ligand and enteron aisle on its surface,
After external source lactobacillus enters enteron aisle, only its surface mass is combined with the specific receptors on enterocyte surface, and passes through letter
Breath transduction, could activate the immune response of body.Recent study confirms that lactobacillus surface composition rises in enhancing host immune
Important role, such as rouge dish Teichaic acid (LTA), whole cell peptidoglycan (PG), cell surface protein (S-protein) and some
Unknown cell surface extract.Lactobacillus surface mass is activated immune signal access as ligand after Receptor recognition, generated
Cell factor and chemotactic factor (CF).In relation to lactobacillus surface protein win the Effect study in attached gut epithelium obtain it is more, and for
Effect of the surface protein in immunological regulation is also known little about it.The study found that Yue Shi lactobacillus (L.johnsonii NCC533)
The heat stress proteins of cell surface can promote the bacterium attached to the domestic animal of cell and the withered film of intestines, and can promote gut epithelium and it is huge sigh it is thin
Born of the same parents generate cell factor.It studies while finding, slpA gene can promote the generation of IL-1 β, IL-6, IL-12 and TNF-α.Rouge phosphorus
Teichaic acid (LTA) is prevalent in gram-positive bacterium, and is largely present in the cell surface of lactobacillus, can not only be mediated
Bacterium it is withered attached, also as the ligand of cell surface receptor, in conjunction with receptor after stimulation body generate immune factor, such as TNF-α
With interleukins (IL-1).Studies have shown that in vitro experiment lactobacillus cell wall peptide glycan energy stimulating expression of macrophage at
It is ripe, and can induce the generation of cell factor, generate immunology effect.
2. the present Research and its application of enteron aisle organoid
Intestines organoid culture (organoid culture) is by the single stem cell of enteric epithelium or containing the hidden of stem cell
Nest is separated, and is cultivated in vitro.Necessary growth factor (R-spondin1, Noggin and EGF are added in the medium
Deng), make single Lgr5 stem cell or crypts gradually structure of the Proliferation, Differentiation with similar intestines organ, passes through the life of organoid
Long budding, the Proliferation, Differentiation process of in-vitro simulated enteric epithelium, the structure not only have " enteric cavity ", also with the various function of intestinal mucosa
It can cell.Its more single microenvironment provides one to the development impact research of enteric epithelium for different factors and easily puts down
Platform has been increasingly becoming people and has explored the growth and development of intestines and the research model that enteritis intestinal cancer is the strongest.This organoid tool
There are the structure and function similar with normal bowel epithelium.At present small intestine organoid culture technique oneself be widely used in it is dry thin
The correlative studys such as born of the same parents, disease model W and regenerating medicine.
Currently, it is any about the report for isolating lactobacillus D8 separation identification from healthy swinery not yet, do not have yet
Any report about the foundation of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model, more not about
The immunological role of lactobacillus is probed into using lactobacillus D8 and to the effect of intestinal mucosa.
Summary of the invention
Goal of the invention: there is provided a kind of lactobacillus D8 for first technical problem to be solved by this invention.The lactobacillus
Enteritis can be effectively relieved in D8.The Proliferation, Differentiation of intestinal stem cell can be effectively facilitated, the damage of TNF-α induction is repaired；It is oral
Lactobacillus animalis D8 can significantly improve enteritis symptom.
There is provided a kind of external lactobacillus-enteron aisle organoids-for second technical problem to be solved by this invention inherently
Layer lymphocyte co-culture model.
There is provided external lactobacillus-enteron aisle organoid-lamina propria leaching for third technical problem to be solved by this invention
The construction method of bar cell co-culture model.
There is provided lactobacillus D8 or external lactobacillus-enteron aisle class devices for 4th technical problem to be solved by this invention
Application of the official-lamina propria lymphocyte co-culture model in terms of preparation prevention or treatment intestines problem drug.
For 5th technical problem to be solved by this invention there is provided a kind of probiotics, the probiotics includes described
Lactobacillus D8.
Technical solution: in order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is as follows: a kind of lactobacillus D8,
The classification naming of the lactobacillus D8 is lactobacillus (Lactobacillus sp.), is preserved in China on October 14th, 2016
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.13112；Address: court, Beijing
The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101.
The content of present invention further includes a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model, institute
Stating co-culture model is by the way that isolated lamina propria lymphocyte and enteron aisle organoid to be mixed, then described in addition
Lactobacillus D8 construct the external lactobacillus to be formed-enteron aisle organoid-lamina propria lymphocyte co-culture model.
The content of present invention further includes the building of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model
Method, comprising the following steps:
1) enteron aisle organoid is separately cultured；
2) lamina propria lymphocyte is separately cultured；
3) lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model foundation.
Wherein, the volume ratio of lamina propria lymphocyte and enteron aisle organoid is 3:1~10:1 in above-mentioned step 3).
Preferably, the volume ratio of lamina propria lymphocyte and enteron aisle organoid is 7:1 in step 3) of the invention.
Wherein, the lactobacillus in above-mentioned step 3) is lactobacillus D8, the lactobacillus D8 additional amount be every hole 1 ×
103CFU~1 × 104CFU。
Preferably, the lactobacillus in step 3) of the invention is lactobacillus D8, the lactobacillus D8 additional amount is every hole 1
×104CFU。
Lactobacillus of the invention-enteron aisle organoid-lamina propria lymphocyte co-culture model building specific steps referring to
Embodiment 2~4 in specific embodiment mode.
The content of present invention further includes the lactobacillus D8 or the external lactobacillus-enteron aisle organoid-lamina propria leaching
The application of bar cell co-culture model in terms of preparation prevention or treatment intestines problem drug.
Wherein, above-mentioned intestines problem is enteritis or colitis.
Wherein, said medicine dosage form is tablet, capsule, sustained release tablets, controlled release tablet, oral solution, syrup, dripping pill, injection liquor
One of type or freeze-dried powder dosage form.
The content of present invention further includes a kind of probiotics, and the probiotics includes the lactobacillus D8.
The utility model has the advantages that compared with prior art, the present invention have the advantages that following characteristic and:
1) present invention separation identification for the first time obtains lactobacillus D8；The characteristics of bacterial strain: Gram-positive is in bar
Shape；Oral lactobacillus D8 can significantly improve the length of intestinal villi and the depth of crypts of small intestine, reduce enteritis symptom；
2) present invention constructs a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model for the first time；
The model can be used to probe into the immunological role of lactobacillus and the effect to intestinal mucosa；Lactobacillus D8 can effectively facilitate intestines
It is horizontal to promote Intestinal Mucosal Immunity for the Proliferation, Differentiation of road stem cell and then the integrality for safeguarding gut barrier.
3) first demonstration that lactobacillus D8 can effectively facilitate the Proliferation, Differentiation of intestinal stem cell, TNF-α is repaired
The damage of induction.
4) the isolated lactobacillus D8 Direct-fed animal of the present invention can mitigate animal intestinal inflammation shape, safeguard enteron aisle
Health；The bacterium is expected to develop into the probiotics of prevention enteritis.
Detailed description of the invention
Fig. 1: lactobacillus D8 optical microscopy Gram's staining observation figure；
Fig. 2: external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model optical microphotograph sem observation figure；A:
Lactobacillus (D8)-enteron aisle organoid (intestinal organoids)-lamina propria lymphocyte (LPLs) co-culture model mould
Formula figure；B: lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model photo under optical microscopy；C: lactobacillus-intestines
1 to 6 days growth and development optical microphotograph sem observation figures of enteron aisle organoid in road organoid-lamina propria lymphocyte co-culture model；
Fig. 3: lactobacillus D8 can promote enteron aisle organoid to grow, and improve the damage of the enteron aisle organoid as caused by TNF-α；
Ctrl: negative control, normal incubation medium culture are without any processing；Lactobacillus D8 group: every hole adds 1 × 104CFU lactobacillus D8；
TNF-α damage group: the TNF-α of 30ng is added in every hole；Lactobacillus D8 reparation group (D8+TNF- α): the TNF- of 30ng is first added in every hole
α effect 6h is added 1 × 10 after causing damage4CFU lactobacillus D8；
Fig. 4: lactobacillus D8 can be improved enteron aisle organoid BrdU Positive area；It is grouped same Fig. 3；
Fig. 5: lactobacillus D8 can be improved enteron aisle organoid Ki67 Positive area；It is grouped same Fig. 3；
Fig. 6: lactobacillus D8 can be improved Jejunum Crypt depth and height of naps, improves small intestinal mucosa caused by DSS and damages；
Ctrl: negative control, the PBS of daily stomach-filling 100ul；D8: the daily stomach-filling 1 × 10 of mouse8CFU lactobacillus D8, continuously feeds 27
It；DSS: the dextran sulfate sodium (DSS) of mass percent 5% is added in drinking-water in the 21st day of feeding；D8+DSS: to mouse
Daily stomach-filling 1 × 108CFU lactobacillus D8, continuous feeding 27 days, adds the Portugal of mass percent 5% in the 21st day drinking-water
Glycan sodium sulphate (DSS)；
Fig. 7: the histopathology symptom of Mouse oral lactobacillus D8 postcolon inflammation mitigates；Feed phosphate buffered saline solution
The mouse of (PBS, pH=7.4) DSS inducing colitis, shows apparent histopathologic change；Epithelial cell shedding, mucous membrane
Bleeding in lower and enteric cavity, with lamina propria inflammatory cell infiltration under mucous membrane, intestinal wall hyperplasia thickens；Enteritis tissue after feeding lactobacillus D8
Pathological change is substantially reduced；Without clearly visible blutpunkte and inflammatory cell infiltration, epithelial cell is more complete, and intestinal wall increases without obvious
It is raw；It is grouped same Fig. 6；
Fig. 8: Mouse oral lactobacillus D8 postcolon inflammation shape mitigates.Colon lengths increase is shown as, bleeding mitigates；Grouping
Same Fig. 6；
Fig. 9: lactobacillus D8 can promote co-culture model secreting leukocytes mesonium 22 (IL-22)；Ctrl: negative control；
Lactobacillus D8 group: every hole adds 1 × 104CFU lactobacillus D8；TNF-α damage group: the TNF-α of 30ng is added in every hole；Lactobacillus D8 is repaired
Multiple group (D8+TNF- α): the TNF-α effect 6h that 30ng is first added causes addition 1 × 10 after damage4CFU lactobacillus D8；
Figure 10: lactobacillus D8 can stimulate co-culture model secretion IL-22 to improve the damage of the enteron aisle organoid as caused by TNF-α
Wound；Ctrl: negative control；TNF-α damage group: the TNF-α of 30ng is added in every hole；Lactobacillus D8 reparation group (D8+TNF- α): first
The TNF-α effect 6h that 30ng is added causes addition 1 × 10 after damage4CFU lactobacillus D8 co-incubation；In IL-22 antibody and group:
After first the TNF-α effect 6h of addition 30ng causes damage, while being added 1 × 104CFU lactobacillus D8 and interleukin 22 neutralize
Antibody (anti-IL-22) (Sigma, the U.S.) co-incubation.
Specific embodiment
Below by specific embodiment and attached drawing, the present invention is further described.In following embodiments method therefor for example without
It illustrates, is conventional reagent and conventional method.
The separation of 1 lactobacillus D8 of embodiment is identified
The source of 1.1 samples and the preliminary screening of bacterial strain
Sample comes from 30 age in days Duroc piggys, buys in Jiangsu Province Agriculture Science Institute, and piglet opens abdominal cavity after butchering, and uses
Sterilizing cotton cord ties duodenal both ends, is immediately placed in the physiological saline of pre-cooling, send in 30min to laboratory after cutting
Make strain separating, divides bacterium operation to carry out in superclean bench, longitudinally cut off intestinal segment with sterile scissors, then with sterile PBS
Buffer rinses the content on intestines surface, and intestinal wall mucous membrane about 0.5g or so is then gently scraped with blade, places and bead is housed
Erlenmeyer flask in sufficiently vibrate, after being stored at room temperature 2-3min, take 100 μ l suspensions be coated with MRS agar medium (pH 5.8) table
Face, 37 DEG C of candle cylinder culture 24-36h.The doubtful bacterium colony of Sample selection carries out Gram's staining, the optics of lactobacillus D8 as shown in Figure 1
Micro- sem observation figure.
The identification of 1.2 lactobacillus specific PCRs
The bacterial strain of preliminary screening is selected, MRS culture medium is added, and (Bifidobacterium selective culture medium is bought in Qingdao Hai Bosheng
Object, China) after amplification cultivation, bacterial genomes are extracted with bacterial genomes DNA extraction kit (Tiangen, China), with one
PCR amplification is carried out by the bacterial genomes of extraction to lactobacillus 16s universal primer P1 and P2.
P1:AGAGTTTGATCCTGGCTCAG
P2:GGTTACCTTGTTACGACTT (sequence is referring to the SEQ ID NO:2 and SEQ ID NO:3 in sequence table)
The above primer is synthesized by Nanjing Jin Sirui company, and reaction uses 25 μ l systems (12.5 2 × Master of μ l BU-Taq
PCR mix (Takara, China), 2 μ l template DNAs (bacterial genomes), upstream and downstream primer each 1 μ l, 9.5 μ l ddH2O)
Biometra PCR instrument (Bio-Rad, the U.S.) carries out, response procedures are as follows: 95 DEG C of initial denaturations 3min, 94 DEG C of 30s, 72 DEG C of 1min,
30 circulations, last 72 DEG C of extensions 15min are executed altogether.Reaction product is sequenced resulting SEQ ID NO:1 sequence and passes through Blast ratio
It is right, discovery and Lactobacillus reuteri strain CTI0324-RS-018 16S ribosomal RNA gene,
Partial sequence (GenBank:KU754503.1) similitude reaches 99%, so that it is determined that being lactobacillus D8, the bacterial strain
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.13112.
The foundation of the external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model of embodiment 2
2.1 enteron aisle organoids are separately cultured
2.1.1 in Yangzhou University's Experimental Animal Center, four week old C57 mouse take small intestine about 15cm, longitudinally cut open for mouse purchase
After opening, with pre-cooling added with mass percent be 1% penicillin and mass percent be 1% streptomysin sterile PBS buffering it is molten
Liquid cleans for several times；
2.1.2, clean small intestine is cut into the segment of 3-5mm or so, is transferred in 50ml sterile centrifugation tube, 10mL is added
20mM pre-cooling EDTA, digest 20min on ice, with liquid-transfering gun draw liquid abandon, retain small intestine；
2.1.3 the PBS of pre-cooling is added in small intestine, is softly persistently blown and beaten with liquid-transfering gun, and is aobvious by optics in time
Micro mirror microscopy discards the supernatant containing a large amount of villus, until there is massive small bowel crypts in the visual field, starts to collect supernatant；
2.1.4 70 μM of cell filters of supernatant with crypts of small intestine are filtered, supernatant 800rpm, 4 DEG C of centrifugations
5min collects precipitating；
2.1.5 with pre-cooling PBS be resuspended precipitating, then with 600rpm centrifugation, collect, repeatedly 3-4 time up to clean it is extra
Villus fragment obtains crypts of small intestine；
2.1.6 crypts of small intestine is resuspended with 50 μ l Matrigel matrigels (Corning, the U.S.), is inoculated into after mixing
24 well culture plate culture hole centers；DMEM/F-12 culture medium is added after Matrigel solidification (500 μ l are added in every hole)
(Gibco, the U.S.), be added in every hole culture medium the recombined small-mouse of 0.25 μ l (concentration is 100 μ g/mL) EGF (Peprotech,
The U.S.), be added in every hole culture medium 1 μ l (concentration is 50 μ g/ml) recombined small-mouse Noggin (Peprotech, the U.S.) and
The R-spondin (Peprotech, the U.S.) of the recombined human of 1 μ l (concentration is 250 μ g/ml) is added in every hole culture medium, changes every other day
Liquid；I.e. it is (three-dimensional comprising various types of 3D such as intestinal stem cell to obtain enteron aisle organoid (intestinal organoids) for culture
Culture model).
2.2 lamina propria lymphocytes are separately cultured
2.2.1 in no Ca2+、Mg2+100ml Hanks equilibrium liquid (source training, China) in 5g bovine serum albumin(BSA) is added
(Sigma, the U.S.), 58mg EDTA, 15.4mg dithiothreitol (DTT) (Sigma, the U.S.) prepare separating liquid.Add in 100mlPBS
Enter 5g bovine serum albumin(BSA), VIII Collagenase Type of 0.15g (Sigma, the U.S.), the DNase I (Sigma, the U.S.) of 100U, in 37 DEG C
Lower incubation 5min prepares digestive juice.9:1 is mixed Percoll cell separating liquid (connection section, China) by volume with 10 × PBS, is matched
Make 100% isotonic Percoll mother liquor.With Percoll mother liquor, DMEM high sugar juice and FCS distinguish 8:1:1 and 4:5 by volume:
1 mixing, prepares 80% isotonic Percoll solution and 40% isotonic Percoll solution.
2.2.2 cervical dislocation put to death mouse, take out about 7-8cm colon and ileal tissue immediately, be placed in pre-cooling not calcic,
Magnesium PBS (PBS-/-) in, the aggregated lymphatic follicles of removal fat, mesenterium connective tissue and small intestine；Intestinal tube is indulged along mesenterium side
To splitting, gently rinsed in not calcium-magnesium-containing pre-cooling PBS, until the complete rinsed clean of excrement, is laterally cut into about 0.5-1.0cm's
Intestinal tissue segment.
2.2.3 intestinal tissue segment is moved into 50ml centrifuge tube, 5ml separating liquid is added, is placed in constant temperature oscillation case, in 37
(250r/min) 15min is shaken at DEG C；It is placed on vortex mixer, vortex 30s, then passes through the intestinal tissue segment after vortex
100 μm of nylon leaching net filterings, filtrate is intestinal intraepithelial lymphocytes and enterocyte at this time；By filtered intestinal tissue segment
Again it moves into 50ml centrifuge tube, 5ml separating liquid is added, repeat above-mentioned concussion, vortex and filtration step.
2.2.4 the intestinal tissue segment after filtration treatment is moved into new 50ml centrifuge tube, 5ml digestive juice is added, is placed in
In constant temperature oscillation case, (250r/min) 45min is shaken at 37 DEG C；It is placed on vortex mixer, vortex 30s, after vortex
Intestinal tissue segment collects filtrate in 15ml centrifuge tube, is centrifuged (400g) at 4 DEG C by 100 μm of nylon leaching net filterings
10min discards supernatant liquid, and precipitating includes lamina propria lymphocyte (lamina propria lymphocytes, LPLs) at this time.
2.2.5 80% isotonic Percoll solution 4ml is laid on new 15ml and is centrifuged bottom of the tube；With 40% isotonic Percoll
Above-mentioned precipitating is resuspended in solution 8ml, is sufficiently layered on 80% isotonic Percoll solution after piping and druming uniformly, centrifuge tube is tilted 180 degree
Liquid is added slowly along tube wall, visible clearly interface between two layers of liquid；In 20 DEG C of zero acceleration and deceleration density gradients of downlink
It is centrifuged (500 × g) 20min；Discarding supernatant liquid to residual volume is 7ml, and the opaque cellular layer between two bed boundarys is sucked out, and is moved
Enter in new 15ml centrifuge tube, PBS is added-/-It is 15ml to volume, after mixing well, (400 × g) 8min is centrifuged at 4 DEG C,
Liquid is discarded supernatant, precipitating is resuspended containing the RPMI1640 culture solution that mass percent is 10% fetal calf serum with 5ml, sufficiently piping and druming is equal
The even cell suspension for preparing is up to lamina propria lymphocyte.
The co-cultivation of 2.3 lactobacillus-enteron aisle organoid-lamina propria lymphocyte
This experiment is by isolated lamina propria lymphocyte (lamina propria lymphocytes) and enteron aisle class
Organ (intestinal organoids) is mixed according to the ratio that volume ratio is 7:1, and (Specific amounts is 7 μ l lamina propria lymphs
Cell and 1 μ l enteron aisle organoid), Matrigel matrigel (every 50 μ l of hole) is added and is resuspended, is laid in 24 orifice plates, every hole is added 50
DMEM/F-12 culture medium (Gibco, the beauty of 500 μ l is added in μ l Matrigel matrigel after Matrigel solidification in every hole
State) (the R- of the recombined human of the Noggin and 125ng of the recombined small-mouse of EGF, 50ng of the recombined small-mouse containing 25ng
Spondin), it is placed in cell incubator and is cultivated.The every hole of lactobacillus processing group is needed to be added 1 × 104The lactobacillus of CFU
D8 constructs a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model, probes into the immune of lactobacillus
Effect and the effect to intestinal mucosa.As shown in Figure 2.
The foundation of the external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model of embodiment 3
Almost the same with embodiment 2, different is, lamina propria lymphocyte (lamina propria
Lymphocytes it) is mixed with enteron aisle organoid (intestinal organoids) according to the ratio that volume ratio is 3:1
(Specific amounts is 3 μ l lamina propria lymphocytes and 1 μ l enteron aisle organoid) is added (every 50 μ l of hole) Matrigel and is resuspended, be laid on 24
In orifice plate, 50 μ l Matrigel are added in every hole, and the DMEM/F-12 culture medium of 500 μ l is added in every hole after Matrigel solidification
(Gibco, the U.S.) (recombined human of the Noggin and 125ng of the recombined small-mouse of EGF, 50ng of the recombined small-mouse containing 25ng
R-spondin), it is placed in cell incubator and is cultivated.The every hole of lactobacillus processing group is needed to be added 1 × 103The newborn bar of CFU
Bacterium D8 constructs a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model.
The foundation of the external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model of embodiment 4
Almost the same with embodiment 2, different is, lamina propria lymphocyte (lamina propria
Lymphocytes it) is mixed with enteron aisle organoid (intestinal organoids) according to the ratio that volume ratio is 10:1
(Specific amounts is 10 μ l lamina propria lymphocytes and 1 μ l enteron aisle organoid) is added (every 50 μ l of hole) Matrigel and is resuspended, be laid on 24
In orifice plate, 50 μ l Matrigel are added in every hole, and the DMEM/F-12 culture medium of 500 μ l is added in every hole after Matrigel solidification
(Gibco, the U.S.) (recombined human of the Noggin and 125ng of the recombined small-mouse of EGF, 50ng of the recombined small-mouse containing 25ng
R-spondin), it is placed in cell incubator and is cultivated.The every hole of lactobacillus processing group is needed to be added 1 × 103The newborn bar of CFU
Bacterium D8 constructs a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model.
Embodiment 5 detects effect of the lactobacillus D8 to enteron aisle organoid
Experimental group is divided into four groups.Control group (Ctrl group in Fig. 3), lactobacillus D8 group (D8 group in Fig. 3), TNF-α damage group
(TNF-α group in Fig. 3), TNF-α cause damage that lactobacillus reparation group (D8+TNF- α group in Fig. 3) is added.Lactobacillus D8 group is past
1 × 10 is added in the every hole of co-culture model that embodiment 2 constructs4CFU lactobacillus D8, continuous action for 24 hours more than；TNF-α group is
Into the co-culture model that embodiment 2 constructs, the TNF-α of 30ng is added in every hole, and continuous action is for 24 hours；TNF-α causes damage to be added
After lactobacillus D8 reparation group causes damage toward the co-culture model TNF-α effect 6h that first 30ng is added in every hole that embodiment 2 constructs
It is added 1 × 104CFU lactobacillus D8, continuous action for 24 hours more than；Control group is the co-culture model that embodiment 2 constructs, normal to train
Base culture is supported, it is without any processing.Ordinary optical microscope observes the surface area of each group enteron aisle organoid, germination percentage and damage
Degree.The increment situation of enteron aisle organoid is observed by BrdU and Ki67 immunofluorescence dyeing respectively.
5.1 simple microscopes observe the variation of enteron aisle organoid morphological indices
Enteron aisle organoid is placed directly in the surface area of microscope microscopic observation enteron aisle organoid, germination percentage and damage
Degree.It was found that lactobacillus D8 can promote enteron aisle organoid to grow, the surface area and germination percentage of enteron aisle organoid are promoted, is improved
The damage of the enteron aisle organoid as caused by TNF-α.As shown in Figure 3.
The increment situation of 5.2 BrdU and Ki67 immunofluorescence dyeings observation enteron aisle organoid
The BrdU dyestuff (final concentration of 30 μ g/L) that 300 μ l are added in the every hole 2h before BrdU is dyed acts on 2h.It discards supernatant,
4% 4 DEG C of fixations of formaldehyde are overnight.1 × PBS is washed 3 times, each 10min.0.4%Triton X-100 permeabilization 5 minutes；1×PBS
It washes 3 times, each 10min；Mass percent is that 5%BSA room temperature closes 30min；It is separately added into primary antibody (Arigo, the China of Brdu
Taiwan) or Ki-67 antibody (Arigo, TaiWan, China) (with 1%BSA dilute 1:100) be placed in wet box, 4 spend night；1×
PBS is washed 3 times, each 10min；It is separately added into the Brdu secondary antibody (diluting 1:200 with 1%BSA) or 100 μ l of 100 μ l fluorescent markers
The Ki-67 secondary antibody (connection section, China) (diluting 1:200 with 1%BSA) of fluorescent marker 30 minutes, black out；1 × PBS washes 3 times, every time
10min.Enteron aisle organoid staining conditions are observed with Laser Scanning Confocal Microscope (Zeiss).It was found that lactobacillus D8 can be improved enteron aisle class
Organ BrdU and Ki67 Positive area.As shown in Figure 4 and Figure 5.Bacillus acidi lactici group is compared to control group, enteron aisle organoid
BrdU and Ki67 Positive area promotes 50.8% and 34.4% respectively.Bacillus acidi lactici reparation group is compared to damage group, enteron aisle
Organoid BrdU and Ki67 Positive area promotes 118.2% and 146.3% respectively.
Influence after the oral lactobacillus D8 of the detection of embodiment 6 to mouse small intestine and colitis
To the daily stomach-filling of mouse 1 × 108CFU lactobacillus D8, continuous feeding added mass percent after 21 days in drinking-water
For 5% dextran sulfate sodium (DSS) inducing colitis.Cervical dislocation puts to death mouse observation colon lengths variation after 7 days, and to sky
Field, colon carry out HE dyeing, evaluate Crypt depth height of naps and histopathologic change.
It was found that lactobacillus D8 can be improved Jejunum Crypt depth and height of naps, improves small intestinal mucosa caused by DSS and damage.
Ctrl: negative control, the PBS of daily 100 μ l of stomach-filling；D8: the daily stomach-filling 1 × 10 of mouse8CFU lactobacillus D8, continuously feeds 27
It；DSS: the dextran sulfate sodium (DSS) of mass percent 5% is added in drinking-water in the 21st day of feeding；D8+DSS: to mouse
Daily stomach-filling 1 × 108CFU lactobacillus D8, continuous feeding 27 days, adds the Portugal of mass percent 5% in the 21st day drinking-water
Glycan sodium sulphate (DSS)；As shown in Figure 6.For lactobacillus D8 group compared to PBS control group, it is left that small intestinal villous height promotes 35.7%
The right side, Crypt depth promote 33.4% or so.Lactobacillus D8 reparation group is promoted compared to DSS damage group, small intestinal villous height
83.2% or so, Crypt depth increases by 21.4% or so.As shown in fig. 6, the tissue disease of Mouse oral lactobacillus D8 postcolon inflammation
Symptom is managed to mitigate.After damage group mouse feeds DSS, apparent enteritis histopathologic change: epithelial cell shedding, mucous membrane is shown
Bleeding in lower and enteric cavity, with lamina propria inflammatory cell infiltration under mucous membrane, intestinal wall hyperplasia thickens.Bacillus acidi lactici reparation group feeds newborn bar
Enteritis histopathologic change is substantially reduced after bacterium D8: without visible blutpunkte and inflammatory cell infiltration, epithelial cell is more complete, intestines
Wall is without obvious hyperplasia.As shown in Figure 7.Mouse oral lactobacillus D8 postcolon inflammation shape mitigates: showing as colon lengths increase, out
Blood mitigates.As shown in Figure 8.For Bacillus acidi lactici group compared to control group, colon lengths are unchanged.Bacillus acidi lactici reparation group is compared to damage
Hurt group, colon lengths promote 33.3% or so.
7 lactobacillus D8 of embodiment, which stimulates co-culture model to secrete IL-22, improves enteritis symptom
7.1 lactobacillus D8 can promote co-culture model to secrete IL-22
Experimental group is divided into four groups.Control group (Ctrl group in Fig. 9), lactobacillus D8 group (D8 group in Fig. 9), TNF-α damage group
(TNF-α group in Fig. 9), TNF-α cause damage that lactobacillus reparation group (D8+TNF- α group in Fig. 9) is added.Lactobacillus D8 group is past
1 × 10 is added in the every hole of co-culture model that embodiment 2 constructs4CFU lactobacillus D8, continuous action for 24 hours more than；TNF-α damage
Group is the TNF-α that 30ng is added in every hole into the co-culture model that embodiment 2 constructs, and continuous action is for 24 hours；TNF-α causes to damage
Lactobacillus D8 reparation group is added to cause to damage toward the co-culture model TNF-α effect 6h that first 30ng is added in every hole that embodiment 2 constructs
1 × 10 is added after wound4CFU lactobacillus D8, continuous action for 24 hours more than；Control group is without any processing, normal incubation medium culture.
Supernatant is collected, using IL-22 concentration in ELISA kit (eBioscience, the U.S.) detection culture solution.As a result, it has been found that newborn bar
Bacterium D8 can significantly improve IL-22 concentration in intestines organoid supernatant.It is 198pg/ml, cream relative to IL-22 concentration in control group
IL-22 concentration is 2253pg/ml in bacillus D8 group, dramatically increases 10.3 times.It is relative to IL-22 concentration in TNF-α damage group
980pg/ml, IL-22 concentration is 1230pg/ml in lactobacillus reparation group, significantly improves 25.5%.
7.2 lactobacillus D8 can stimulate co-culture model secretion IL-22 to improve the damage of enteron aisle organoid
Experimental group is divided into four groups.Control group (Ctrl group in Figure 10), TNF-α damage group (TNF-α group in Figure 10): every hole adds
Enter the TNF-α of 30ng；Lactobacillus D8 reparation group (the D8+TNF- α in Figure 10): the TNF-α effect 6h that 30ng is first added in every hole draws
1 × 10 is added after playing damage4CFU lactobacillus D8 co-incubation；In IL-22 antibody and the group (D8+TNF- α+anti-in Figure 10
IL-22): after the TNF-α effect 6h that 30ng is first added in every hole causes damage, while being added 1 × 104CFU lactobacillus D8 and white thin
22 neutralizing antibody of born of the same parents' interleukin (every hole adds 2.5ng) (anti-IL-22) (Sigma, the U.S.) co-incubation；Control group does not do any
Processing, normal incubation medium culture.It was found that the intestines organoid that TNF-α can induce 78.1% is dead, intestines class in IL-22 antibody and in group
Organic death rate is 80.8%.Relative to TNF-α damage group, the intestines organoid death rate is only in lactobacillus D8 reparation group
20.2%, the intestines organoid death rate declines up to 57.9%.As a result the co-cultivation that lactobacillus D8 can stimulate embodiment 2 to construct is confirmed
Model secretes IL-22, safeguards the integrality of intestines organoid.
It above are only the preferred embodiment of the invention, there is no need and unable to illustrate to all embodiments.It is right
For those skilled in the art, without departing from the principle of the present invention, other different forms can also be made
Variation or variation, these also should belong to protection scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of Nanjing
<120>lactobacillus D8 and its application
<130> SG2017001
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1424
<212> DNA
<213> Lactobacillus
<400> 1
aatggttagg ccaccgactt tgggcgttac aaactcccat ggtgtgacgg gcggtgtgta 60
caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc gattccgact 120
tcgtgtaggc gagttgcagc ctacagtccg aactgagaac ggctttaaga gattagctta 180
ctctcgcgag cttgcgactc gttgtaccgt ccattgtagc acgtgtgtag cccaggtcat 240
aaggggcatg atgatctgac gtcgtcccca ccttcctccg gtttgtcacc ggcagtctca 300
ctagagtgcc caacttaatg ctggcaacta gtaacaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga cgaccatgca ccacctgtca ttgcgtcccc 420
gaagggaacg ccttatctct aaggttagcg caagatgtca agacctggta aggttcttcg 480
cgtagcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc aattcctttg 540
agtttcaacc ttgcggtcgt actccccagg cggagtgctt aatgcgttag ctccggcact 600
gaagggcgga aaccctccaa cacctagcac tcatcgttta cggcatggac taccagggta 660
tctaatcctg ttcgctaccc atgctttcga gcctcagcgt cagttgcaga ccagacagcc 720
gccttcgcca ctggtgttct tccatatatc tacgcattcc accgctacac atggagttcc 780
actgtcctct tctgcactca agtcgcccgg tttccgatgc acttcttcgg ttaagccgaa 840
ggctttcaca tcagacctaa gcaaccgcct gcgctcgctt tacgcccaat aaatccggat 900
aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt gactttctgg 960
ttggataccg tcactgcgtg aacagttact ctcacgcacg ttcttctcca acaacagagc 1020
tttacgagcc gaaacccttc ttcactcacg cggtgttgct ccatcaggct tgcgcccatt 1080
gtggaagatt ccctactgct gcctcccgta ggagtatgga ccgtgtctca gttccattgt 1140
ggccgatcag tctctcaact cggctatgca tcatcgcctt ggtaagccgt taccttacca 1200
actagctaat gcaccgcagg tccatcccag agtgatagcc aaagccatct ttcaaacaaa 1260
agccatgtgg cttttgttgt tatgcggtat tagcatctgt ttccaaatgt tatcccccgc 1320
tccggggcag gttgcctacg tgttactcac ccgtccgcca ctcactggtg atccatcgtc 1380
aatcaggtgc aagcaccatc aatcagttgg gccagtgcgt acga 1424
<210> 2
<211> 20
<212> DNA
<213>upstream primer P1
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213>downstream primer P2
<400> 3
ggttaccttg ttacgactt 19
Claims (8)
1. a kind of lactobacillus D8, which is characterized in that the classification naming of the lactobacillus D8 be lactobacillus (Lactobacillus sp.), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on October 14th, 2016, preservation is compiled
Number be CGMCC No. 13112.
2. a kind of external lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-culture model, which is characterized in that the total training
Feeding model is by the way that claim 1 is then added by isolated lamina propria lymphocyte and enteron aisle organoid mixed culture
The lactobacillus D8 constructs the external lactobacillus to be formed-enteron aisle organoid-lamina propria lymphocyte co-culture model.
3. external lactobacillus as claimed in claim 2-enteron aisle organoid-lamina propria lymphocyte co-culture model building side
Method, which comprises the following steps:
1) enteron aisle organoid is separately cultured；
2) lamina propria lymphocyte is separately cultured；
3) lactobacillus D8 building lactobacillus-enteron aisle organoid-lamina propria lymphocyte co-cultivation mould of claim 1 institute is added
Type.
4. the building of external lactobacillus according to claim 3-enteron aisle organoid-lamina propria lymphocyte co-culture model
Method, which is characterized in that the volume ratio of lamina propria lymphocyte and enteron aisle organoid is 3:1 ~ 10:1 in the step 3).
5. the building of external lactobacillus according to claim 3-enteron aisle organoid-lamina propria lymphocyte co-culture model
Method, which is characterized in that the lactobacillus D8 additional amount in the step 3) is every hole 1 × 103CFU~ 1×104 CFU。
6. the external lactobacillus of lactobacillus D8 or as claimed in claim 2-enteron aisle organoid described in claim 1-lamina propria leaching
The application of bar cell co-culture model in terms of preparation prevention or treatment intestines problem drug, the intestines problem is enteritis or knot
Enteritis.
7. application according to claim 6, which is characterized in that the pharmaceutical dosage form is tablet, capsule, sustained release tablets, controlled release
One of piece, oral solution, syrup, dripping pill, parenteral solution formulation or freeze-dried powder dosage form.
8. a kind of probiotics, which is characterized in that the probiotics includes lactobacillus D8 described in claim 1.
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