CN109008958A - A kind of study on intestinal flora method for filtering and transplanting based on excrement - Google Patents
A kind of study on intestinal flora method for filtering and transplanting based on excrement Download PDFInfo
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Abstract
The research method of drug and intestinal flora interaction that the invention discloses a kind of to be filtered and be transplanted based on excrement, this method comprises the following steps: the preparation of 1 caprophyl transplant recipient experimental animal: caprophyl transplant recipient experimental animal selects pseudo- germfree animal or germfree animal, it causes a kind of enteron aisle sterile or the environment of few bacterium, is conducive to the field planting of external microorganism.The preparation of 2 caprophyl transplantation donor experimental animals, donor animal receive disease model modeling and drug stomach-filling processing, set up the positive and negative experiment contrast of drug effect.3 receptor assay animals receive the filtering of donor assay animal and non-filtered caprophyl is transplanted.Study on intestinal flora method of this method based on classical caprophyl transplanting, and it is simple, easy to implement, it excludes drug to directly affect enteron aisle, focuses the drug effect that flora mediates, provide a kind of succinct method for the flora of exploration medicament adjusting and the interaction of host.
Description
Technical field
The present invention relates to a kind of step process methods of zoopery in medicine study on intestinal flora, and in particular to Yi Zhongji
In the study on intestinal flora method that excrement is filtered and transplanted.
Background technique
Intestinal flora is a complexity and huge group, in a healthy adult human body, intestinal flora total weight
Can be of about 1 ~ 2 kilogram, cell quantity wherein included is up to 1013~1014It is a, it is approximately 10 times of human body cell number, enteron aisle is micro-
Biological encoding gene group number is about 100 times of human body encoding gene number, therefore also referred to as " the second genome ".
Intestinal flora inter-individual difference is up to 80% ~ 90%, the mainly difference on species level.In addition, intestinal flora
Structure will receive the influence of many factors, including host genetic factor, region situation, eating habit, health status, drug use
Etc.；This not only adds the complexity of study on intestinal flora, are also study on intestinal flora in accurate medical treatment and personalized medicine
The development in epoch brings good chance.
Disease research rapid development relevant to intestinal flora in recent years.Existing research show intestinal flora not only with it is various
The occurrence and development of disease are related, such as colorectal cancer, rheumatoid arthritis, self-closing disease, depression, senile dementia etc.；It can also shadow
Drug is rung to the therapeutic effect of disease, and the hypoglycemic mechanism and intestinal flora of a such as 2017 research discovery melbine are to medicine
The metabolism of object is related.One of the hot spot that current life science field is just like had become about the research of intestinal flora, about
The research method of intestinal flora is also widely paid close attention to.
Mainly there are following 4 kinds about the research method of intestinal flora at present:
(1) method based on separation, culture: various selective medium culture bacteriums are usually used in this method, will be various thin
Bacterium separates and is identified according to the methods of dyeing, biochemical reaction and Serologic test bacterium, while can carry out doubling dilution
Number of viable is measured with bacterium colony counting.Although this method has certain maturity, and is utilized by many enteron aisle researchers,
There is 90%~99% microorganism that can not be turned out with conventional method come therefore this method can only be to the flora of part in nature
It is analyzed, and time-consuming.For intestinal microecology system so huge for type, quantity, only fraction flora is carried out
Analysis is obviously not comprehensive enough, can not reflect the relationship of entire microecosystem and disease development, the result and conclusion of analysis
There is certain limitation.
(2) raise with cage: when raising animal with cage, the transplanting of intestinal flora is may be implemented in the coprophagy habit of animal.To grind
Study carefully self-closing disease it is related to intestinal flora for, the pregnancy period animal offspring of feeding high in fat is easy to show the phenotype of self-closing disease, and
The composition of its intestinal flora is different from the pregnancy period animal offspring that normal diet is fed.Then feeding high in fat, normal diet are fed
Pregnancy period animal offspring use mode raise with cage, realize that the flora of different eating pattern animals shifts, as a result, it has been found that same cage
The intestinal flora of the pregnancy period animal offspring high in fat of raising is changed, and flora composition and normal diet pregnancy period animal offspring are more
It is close.But only there is this habit by the propagated of animal autonomous coprophagy habit and microorganism with the experimental method of cage raising
The animal of property is feasible, and phenotypic difference is big, is not easy to obtain positive findings.
(3) flora is transplanted: by the functional flora in healthy animal excrement, being transplanted to the animal gastrointestinal tract with certain disease
It is interior, new intestinal flora is rebuild, realizes the treatment of enteron aisle and parenterally disease, as classical caprophyl transplants (Fecal
Microbiota transplantation, FMT).FMT has many applications in medicine and hygiene fields, and the clinic including disease is controlled
Treat (such as clostridium difficile infection, pseudomembranous enteritis, Crohn disease) and the relevant disease incidence Mechanism Study of flora.Laboratory
Common flora transplant recipient is the pseudo- germfree animal of germfree animal or antibiotic treatment, the i.e. SPF of no-special pathogen
Grade experimental animal is handled with broad-spectrum antibiotic makes its enteron aisle form a kind of pseudo- gnotobasis, and it is fixed to be conducive to external donor flora
It plants, to study the function of foreign donor bacterium.Currently, the use of Aseptic forceps need it is very strict sterile dynamic
Object breeding and feeding environment, pole are not easy to obtain, and most of the country research institution is unable to reach breeding and raises germfree animal
Condition, in comparison, pseudo- germfree animal is using more convenient.
(4) the phenotype location co-variation microorganism association analysis based on molecular biology and analyzing biochips method: micro- life
The detection of object is based primarily upon the analysis method of DNA finger-print and the association analysis method based on DNA sequencing technologies.DNA refers to
Line graphical spectrum technology principle will represent the DNA of species in microbiologic population predominantly according to features such as molecular size, nucleic acid sequences
Molecular marked compound is separated in gel with the method for electrophoresis, and the molecular labeling for representing different plant species is made to move to the difference of glue
Position shows that the composed structure of group, great advantage are easily and fast, intuitively, to be usually used in detecting microorganism with electrophorogram
Architectural difference between the dynamic change of structure of community or the different groups of comparison mainly includes denaturing gradient gel electrophoresis
(Denatured gradient gel electrophoresis, DGGE) and terminal fragment length polymorphism (Terminal
Restriction fragment length polymorphism, T-RFLP) etc., it can detect in environmental sample that ten is several
Dominant bacteria, but trace microorganism can not but be detected, also, include more than one 16S rDNA sequence in electrophoretic band, it
It learns specific strain information, also needs to be cloned, be sequenced, experimental implementation is cumbersome；In addition, cannot reflect in this way micro-
The abundance situation of biology.DNA sequencing technologies are by directly acquiring nucleic acid sequence information, and then the evolution to species each in group
Status judges, before this, the 16S based on monoclonal plasmid, transformed cells building and the sequencing of mulberry lattice (Sanger) dideoxy
R RNA gene cloning library be widely used in for a long time research group in microorganism group at method, be applied to human enteric bacteria
The diversity analysis of group, result is detected in species is far superior to DNA finger-print skill in depth and species identification level
Art, it is main with 1) MWAS(metagenome- at this stage with the rapid development of high throughput sequencing technologies
Wideassociationstudies, macro genome association analysis), macro genome is a kind of micropopulation in environmental sample
Body genome is research object, using functional gene screening and/or sequencing analysis as research means, with microbial diversity, population
Structure, functional activity, cooperates relationship and relationship between environment is that the new microorganism of research purpose is ground at evolutionary relationship
Study carefully method.Influence of the inherent cause to intestinal flora can be studied by macro genome and human genome association analysis；By macro
Genome and proteomics or metabolism group association analysis research intestinal flora are in human body correlative protein expression or metabolic pathway
In effect.Macro genome research sequencing cost is higher.2) association analysis is sequenced in 16S amplicon, refers to micro- with being utilized by sequencing
The difference of biological variable region sequences is associated analysis, the party with phenotype to classify to the microorganism for not belonging to and planting
Method is easy to operate, cheap.In indefinite a certain disease and the relationship of intestinal flora, 16SrDNA sequencing can be first passed through
Carry out preliminary screening, for it is subsequent deeper into research foundation is provided.But due to current technical restriction, most microorganism is independent
Kind cannot be identified by 16SrDNA identification, it is more accurate in the research for belonging to horizontal, and it is easy pollution, obtain false positive results.
In addition, fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH) and real-time quantitative is glimmering
Fluorescent Quantitative PCR (Real time quantitative PCR) is also common microorganism detection means.Biochip is able to validate only
Known species obtain the information of microbial diversity by detecting the probe being fixed on chip, are judged by signal strength or weakness micro-
The abundance of biology is as a result inaccurate.Association analysis usually requires to defer to kock rule Research Thinking, will be single or several micro-
Biotic population is fed back to animal model to prove the reliable of correlation, is not readily separated culture there are microorganism and can not observe flora
A possibility that overall variation influences model.
In addition, above-mentioned research method can only study one or more of enterobacteriaceaes, or belong to association analysis, nothing
Method directly illustrates the coefficient effect of a variety of floras and mechanism that reality occurs in vivo.
Summary of the invention
It is real the present invention is directed to overcome the deficiencies of the prior art and provide a kind of easy, based on no-special pathogen SPF grade
The study on intestinal flora method of the excrement filtering and transplanting of animal is tested, to explore the intestinal flora overall variation of medicament adjusting to disease
The influence of disease and Mechanism Study provide basis.
In order to achieve the above object, technical solution provided by the invention are as follows:
The study on intestinal flora method for being filtered and being transplanted based on excrement includes the following steps:
(1) preparation of caprophyl transplant recipient experimental animal
Caprophyl transplant recipient experimental animal selects pseudo- germfree animal or germfree animal, causes a kind of enteron aisle sterile or the ring of few bacterium
Border is conducive to the field planting of external microorganism；Pseudo- Aseptic forceps need to check whether modeling with caprophyl nucleic acid concentration measurement experiment
Success (animal itself intestinal flora significantly reduces after such as Fig. 1 antibiotic treatment, and pseudo- sterile receptor assay animal is formed)；
(2) preparation of caprophyl transplantation donor experimental animal
Donor animal receives disease model modeling and drug stomach-filling processing, sets up the positive and negative experiment contrast of drug effect；
(3) receptor assay animal receives the filtering of donor assay animal and non-filtered caprophyl is transplanted
1. carrying out Disease model to animal and giving drug therapy, daily timed collection gives the animal pattern excrement of research drug
Just, the excrement of collection and neutral cold phosphate buffer are mixed, then are centrifuged, obtain supernatant.Supernatant is divided into two, one
Part is A supernatant, another is B supernatant；
2. it is real to be formed A group for the receptor assay animal of A supernatant same disease model of stomach-filling after the filtering of sterile syringe filter
Animal pattern is tested, meanwhile, by the receptor assay animal of the direct continuous gavage same model of B supernatant, it is dynamic to form B group experimental model
Object；By judging that the disease process difference of two groups of experimental model animals of parallel control studies intestinal flora to animal model
It influences.
Wherein, sterile pin type filter pore size is 0.22 ± 0.05 μm, it is preferable that sterile pin type filter pore size is 0.22
μm.SPF grades of experimental animals of animal pattern or germfree animal, preferably SPF grades of animal or germfree animal.
In the present invention, sterile syringe filter can filter out intestinal flora, two groups of parallel controls, can eliminate other because
Element, to study influence of the intestinal flora overall variation to animal model.On this basis, the present invention is also by filtered supernatant
(as shown in Figure 2) is cultivated respectively with unfiltered supernatant, it is found that unfiltered supernatant Microflora (several clones) is significantly more than
Thus the supernatant of (no Clone formation) after filtering further illustrates that present invention filtering can effectively eliminate the flora in supernatant.
The comparison that filtering of the invention is transplanted with non-filtered caprophyl can be used as a kind of research flora overall variation to reality
The method (as shown in Fig. 3-Fig. 6) of the influence of animal, the i.e. drug effect of flora mediation are tested, and excludes drug to the straight of enteron aisle
Connect effect.This method is simple, easy to implement, provides more succinct method for the researcher of intestinal flora.
Detailed description of the invention
Fig. 1 antibiotic treatment group is compared with non-antibiotic processing group mouse intestinal flora total DNA；
Fig. 2 is A supernatant (left figure) and the culture of B supernatant (right figure) Anaerobic culturel not filtered after filtering；
Fig. 3 FFMT group and FMT group intestinal flora difference (disease model 1)；
Fig. 4 FFMT group and FMT group intestinal flora difference (disease model 2)；
Fig. 5 FFMT group and FMT group progression of disease difference (disease model 1)；
Fig. 6 FFMT group and FMT group progression of disease difference (disease model 2).
Specific embodiment
One, animal welfare and label
Experiment is audited by Ethics Committee, meets animal protection, animal welfare and animal welfare principle, and it is dynamic to meet National Laboratory
The relevant regulations of object welfare ethics.
25-40 SPF grades of of the right age mouse are chosen according to disease model, and label is numbered to show difference.Experiment is taken
Needle point method dips a small amount of carbon black ink with No. eight syringe needles, is pierced into ear, front and back limb and tail portion etc. subcutaneously, by thorn portion
There are a density bullets for position, to achieve the purpose that label.
Two, random grouping
Mouse adapts to that the mode being randomly assigned is taken to be divided into 5 groups, including 1 blank control group, 2 researchs after a week in experiment barrier
Medicine group, 3 positive controls, 4 antibiotic+bacterium solution filtering group (Filtered fecal microbiota
Transplantation, filtering rear caprophyl transplanting, abbreviation FFMT group), 5 antibiotic+bacterium solution group (Fecal
Microbiota transplantation, caprophyl transplanting, abbreviation FMT group).
Three, the preparation of caprophyl transplant recipient experimental animal
Caprophyl transplant recipient experimental animal can choose pseudo-germfree mice either germfree mouse, cause a kind of enteron aisle sterile or
The environment of few bacterium, is conducive to the field planting of external microorganism.The present embodiment is using the pseudo- Aseptic forceps easily obtained.
(1) pseudo-germfree mice modeling
Experiment is formal to be started to give 1,2 and 3 group of mouse solvent control in first week, and the 4th and 5 group of mouse antibiotic manufacture is pseudo- sterile
Animal model.Specific antibiotic administration way are as follows: after every intragastric administration on mice 100mg streptomysin, give mixing antibiotic drink at once
With water 7 days, formula are as follows: the ampicillin of 1g/L, 1g/L metronidazole, 1g/L neomycin dissolve the vancomycin of 0.5g/L.
Specific step is as follows for stomach-filling: lifting mousetail with the right hand, is placed in small mouse cage or coarse plane, when small
When mouse struggles forward, left-handed thumb and index finger pinch two ear posterior neck butt skin of mouse, and overturning mouse is placed in the centre of the palm, after being straightened
Limb pushes down mousetail with little finger of toe., should not be firmly excessive during fixed mouse, its neck is not held, in order to avoid death by suffocation
It dies.Its head is gently pressed with gastric perfusion needle, keeps oral cavity in alignment with esophagus, then gastric perfusion needle is gently entered into esophagus along palate wall,
It can start to feed after the elbow of gastric perfusion needle enters neck bend.If the position insertion of gastric perfusion needle is correct, mouse can be voluntarily
Medicine is swallowed, gastric perfusion needle insertion position is incorrect, and mouse can struggle strongly, it is necessary to which extraction is inserted again, otherwise drug may be poured into gas
Pipe, causes dead mouse.Gastric perfusion needle is gently extracted out after having infused medical fluid.
(2) modeling effect detection
Pseudo-germfree mice modeling collected the excrement that mouse drains naturally and carries out enterobacteriaceae the 5th day of antibiotic treatment and the 7th day
The detection of group DNA.
Intestinal flora DNA is extracted and detecting step is as follows:
(1) excrement 180-220mg is taken, is placed in 2ml centrifuge tube, is placed on ice chest.
(2) 1ml InhibitEx Buffer, vortex 1min is added, until mixing.
(3) 15 DEG C -25 DEG C, 20000g, it is centrifuged 1min.
(4) the Proteinase K of 25ul is drawn into 2ml centrifuge tube (EP).
(5) 600ul is drawn from the supernatant in 3 steps to add into the EP pipe in 4 steps.
(6) it is added in the Buffer AL to above-mentioned EP of 600ul, vortex 15s.
(7) 70 DEG C of water-bath 10min.
(8) 600ul dehydrated alcohol is added, is vortexed.
(9) the above solution 600ul is drawn into adsorption column, is closed the lid, and 15 DEG C -25 DEG C, 20000g, is centrifuged 1min, is abandoned
Collective low pipe；Repeat above step, it is known that finish solution all in 8 steps centrifugation.
(10) the Buffer AW1 that 500ul is added in adsorption column is carefully opened, 15 DEG C -25 DEG C, 20000g, 1min is centrifuged, puts
Enter in new 2ml collecting pipe, abandons original.
(11) adsorption column is opened, the Buffer AW2 of 500ul is added, 15 DEG C -25 DEG C, 20000g, is centrifuged 3min.
(12) adsorption column is put into new collecting pipe, 15 DEG C -25 DEG C, 20000g, is centrifuged 3min.
(13) adsorption column is put into 2ml collecting pipe, is added the Buffer ATE of 200ul, be incubated at room temperature 1min, 15 DEG C-
25 DEG C, 20000g, it is centrifuged 1min.
(14) lower liquid is transferred in markd 1.5mlEP pipe, this liquid is the DNA solution of intestinal flora.
(15) each sample DNA concentration is measured with 2000 nucleic acids instrument of Nanodrop.
As a result as shown in Figure 1, antibiotic treatment the 5th day and the 7th day, antibiotic treatment group and non-antibiotic processing group phase
Than the quantity of intestinal flora greatly reduces, and flora DNA concentration is remarkably decreased, pseudo- sterile model mice model success.
Four, the preparation of caprophyl transplantation donor experimental animal
(1) Disease model and drug therapy
Second week starts to carry out disease model modeling and drug stomach-filling processing (1 times/day) to all 5 groups of mouse.1st group SPF grades
Mouse is given solvent control (negative control group), and the 2nd group of SPF grades of mouse, which are given, studies drug (intestinal flora donor group), and the 3rd group
SPF grades of mouse give the positive drug (positive controls) of disease model.
(2) caprophyl transplanting supernatant prepares
The processing specific steps of A and B supernatant are as follows: the excrement of the 2nd group of SPF grades of mouse of daily timed collection (giving research drug)
The excrement of collection is resuspended in the cold phosphate buffer of 10ml, 800g by 500mg, and 4 DEG C of centrifugation 3min obtain supernatant.It will be upper
Clear liquid is divided into two, and portion is A supernatant, another is B supernatant.
Five, receptor assay animal receives the filtering of donor assay animal and non-filtered caprophyl supernatant transplanting
Stomach-filling animal pattern after the 0.22 micron of sterile syringe filter filtering of A supernatant, forms the 4th group of experimental animal, i.e.,
FFMT group, the direct stomach-filling animal pattern of B supernatant form the 5th group of experimental animal, i.e. FMT group.Stomach-filling and the disease model modeling same period
It carries out, stomach-filling amount is 0.4 milliliter/.
(1) filtering and non-filtered supernatant bacteria group culture
Anaerobic culturel is carried out to the unfiltered supernatant of filtered supernatant, the influence to viable bacteria in supernatant is filtered in observation, specifically
Steps are as follows:
1, the intestinal flora excrement 100mg for taking mouse to drain naturally, is resuspended in cold PBS solution；
2,800g, 4 DEG C of centrifugation 3min, takes supernatant.
3, supernatant is divided into two parts, and portion is not handled；It is a to be filtered with sterile syringe filter (0.22 micron of aperture),
Filtrate is taken, ice chest saves.
4, sample is put into 4 DEG C of refrigerators, prepares aseptic operating platform；
5, the filtered fluid of supernatant bacterium solution and supernatant bacterium solution, PBS are diluted in detection range.
6, pipettor takes the filtered fluid 1ml of supernatant bacterium solution and supernatant bacterium solution after dilution to be respectively placed in 3M Petrifilm bacterium
It falls in total testing piece, pressing plate pressing, is put in anaerobism training bag, is cultivated 1-2 days in 37 DEG C of incubators, carry out bacterium colony counting
(testing piece develops the color automatically).
As shown in Fig. 2, most microorganism has been removed after filtering, culture bacterium colony (left side) is had no；Unfiltered upper layer
Bacterium solution has bacterium colony to form (right side) with the presence of viable bacteria.
Five, variance analysis
After disease model modeling, the Flora distribution difference of 16s amplicon sequencing detection FFMT and FMT group experiment mice is sentenced
Whether there is or not whether influence the distribution of disease model animals intestinal flora for flora in disconnected stomach-filling supernatant.The results show that in two diseases
FFMT and FMT group mouse is implicitly present in Flora distribution difference (disease model 1: Fig. 3 and disease model 2: Fig. 4) in disease model.It sees
The progression of disease degree of FFMT group and FMT group mouse is examined, the results show that FFMT group and the experiment of FMT group in two disease models
Mouse is implicitly present in the difference (disease model 1: Fig. 5 and disease model 2: Fig. 6) of effect.
Claims (4)
1. one kind is filtered and is transplanted intestinal flora research method based on excrement, which is characterized in that described method includes following steps:
(1) preparation of caprophyl transplant recipient experimental animal: caprophyl transplant recipient experimental animal selects pseudo- germfree animal or sterile dynamic
Object causes a kind of enteron aisle sterile or the environment of few bacterium, is conducive to the field planting of external microorganism；
(2) preparation of caprophyl transplantation donor experimental animal: donor animal receives disease model modeling and drug stomach-filling processing, sets up
The positive and negative experiment contrast of drug effect；
(3) receptor assay animal receives the filtering of donor assay animal and non-filtered caprophyl is transplanted:
1. carrying out Disease model to animal and giving drug therapy, daily timed collection gives the animal pattern excrement of drug, will
The excrement and phosphate buffer of collection mix, then are centrifuged, and obtain supernatant, and supernatant is divided into two, and portion is A supernatant,
Another is B supernatant；
2. by A supernatant after the filtering of sterile syringe filter, pseudo- germfree animal after stomach-filling antibiotic treatment, stomach-filling and disease
The model modeling same period carries out, and forms A group experimental model animal, meanwhile, by puppet of the direct stomach-filling of B supernatant after antibiotic treatment
Germfree animal, stomach-filling and the disease model modeling same period carry out, and form B group experimental model animal；A group experimental animal and B group are tested
Whether the processing difference of animal is only the filtering of stomach-filling supernatant；After disease model modeling, by judge parallel control two
The disease process difference of group model animal studies influence of the intestinal flora to disease model animals of medicament adjusting.
2. the method as described in claim 1, which is characterized in that the filter pore size is 0.22 ± 0.05 μm.
3. method according to claim 2, which is characterized in that the filter pore size is 0.22 ± 0.05 μm.
4. method as described in any one of claims 1 to 3, which is characterized in that the animal pattern is no-special pathogen grade
Experimental animal grade animal or germfree animal.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111642457A (en) * | 2020-05-25 | 2020-09-11 | 西南大学 | Rat model for methamphetamine intervention pseudo-aseptic, construction method and application |
| CN114586897A (en) * | 2022-05-09 | 2022-06-07 | 斯贝福（北京）生物技术有限公司 | Composition, application thereof and sterile-like mouse model constructed by using composition |
| CN115097044A (en) * | 2022-06-30 | 2022-09-23 | 山西中医药大学 | Construction method for coprophilous bacteria transplantation through intervention of intestinal flora in rheumatoid arthritis model |
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| CN115097044A (en) * | 2022-06-30 | 2022-09-23 | 山西中医药大学 | Construction method for coprophilous bacteria transplantation through intervention of intestinal flora in rheumatoid arthritis model |
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