CN109008958B

CN109008958B - Intestinal flora research method based on fecal filtration and transplantation

Info

Publication number: CN109008958B
Application number: CN201810612443.4A
Authority: CN
Inventors: 郭莹; 谭志荣; 唐翠; 周婷; 马冲; 刘昭前; 张伟; 周宏灏
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2018-06-14
Filing date: 2018-06-14
Publication date: 2021-10-26
Anticipated expiration: 2038-06-14
Also published as: CN109008958A

Abstract

The invention discloses a research method for interaction between a medicament based on feces filtration and transplantation and intestinal flora, which comprises the following steps: 1 preparation of fecal bacteria transplant recipient experimental animals: the experimental animal of the fecal strain transplanting receptor selects a pseudo-aseptic animal or an aseptic animal, so that an environment with aseptic or low-bacteria intestinal tract is created, and the field planting of external microorganisms is facilitated. 2, preparing a donor experimental animal for the coprophilous fungi transplantation, wherein the donor animal is subjected to disease model molding and drug gavage treatment, and positive and negative experimental controls of drug effect are set. 3 the recipient animal is transplanted with filtered and non-filtered fecal bacteria from the donor animal. The method is based on a classic intestinal flora research method of coprophilous fungus transplantation, is simple and easy to implement, eliminates the direct influence of drugs on the intestinal tract, focuses on the drug action mediated by flora, and provides a simple method for exploring the interaction between the flora regulated by drugs and a host.

Description

Intestinal flora research method based on fecal filtration and transplantation

Technical Field

The invention relates to a step processing method for animal experiments in medical intestinal flora research, in particular to an intestinal flora research method based on fecal filtration and transplantation.

Background

The intestinal flora is a complex and large population, and the total weight of the intestinal flora in a healthy adult can reach about 1-2 kg, wherein the number of contained cells is as high as 10¹³~10¹⁴Is aboutIs 10 times the number of human cells, and the number of the coding genome of the enteric microorganism is about 100 times the number of the coding genome of the human cell, and thus is also referred to as "second genome".

The difference among intestinal flora individuals is as high as 80-90%, mainly the difference on a strain level. In addition, the structure of the intestinal flora is influenced by various factors, including host genetic factors, geographical conditions, dietary habits, health conditions, drug use, and the like; the complexity of the research on the intestinal flora is increased, and a good opportunity is brought to the development of the research on the intestinal flora in the times of precise medical treatment and personalized medical treatment.

In recent years, studies on diseases associated with the intestinal flora have been rapidly progressing. Research has shown that the intestinal flora is not only related to the occurrence and development of various diseases, such as colorectal cancer, rheumatoid arthritis, autism, depression, senile dementia and the like; the treatment effect of the drug on diseases can also be influenced, for example, a study in 2017 finds that the glucose-reducing mechanism of the metformin is related to the metabolism of the drug by intestinal flora. The study on the intestinal flora is one of the hot spots in the life science research field, and the research method on the intestinal flora is also widely concerned.

The current research methods for the intestinal flora mainly include the following 4 methods:

(1) the method based on separation and culture comprises the following steps: the method generally adopts various selective culture media to culture bacteria, separates various bacteria, identifies the bacteria according to methods such as dyeing, biochemical reaction, serological experiment and the like, and can simultaneously carry out multiple dilution and colony counting to determine the number of viable bacteria. Although this method has a certain degree of maturity and is used by many intestinal researchers, 90% -99% of microorganisms in nature cannot be cultured by the traditional method, so the method can only analyze partial flora and is time-consuming. For intestinal microecological systems with huge types and quantities, analysis of only a small part of flora is obviously not comprehensive enough, the relationship between the whole microecological system and the occurrence and development of diseases cannot be reflected, and the analysis result and conclusion have certain limitations.

(2) Feeding in the same cage: when the animals are raised in the same cage, the transplantation of intestinal flora can be realized by the manure habit of the animals. Taking the example of studying the association between autism and intestinal flora, offspring of pregnancy animals fed with high fat tend to show the phenotype of autism, and their composition of intestinal flora is different from that of offspring of pregnancy animals fed with normal diet. And then, the offspring of the pregnant animals fed with high fat and fed with normal diet are fed in the same cage, so that the flora transfer of the animals in different diet modes is realized, and the result shows that the intestinal flora of the offspring of the high fat pregnant animals fed in the same cage is changed, and the flora composition is more similar to that of the offspring of the normal diet pregnant animals. However, the experimental method of the same-cage feeding depends on the independent food and manure habits of animals and the transmission of microorganisms, and is only feasible in animals with the habits, and the phenotypic difference is large, so that positive results are not easy to obtain.

(3) And (3) flora transplantation: functional flora in the feces of healthy animals is transplanted into the gastrointestinal tract of animals with certain diseases, and new intestinal flora is reconstructed to realize the treatment of intestinal and parenteral diseases, namely classical Fecal flora transplantation (FMT). FMT has many applications in the field of medical hygiene, including clinical treatment of diseases (such as clostridium difficile infection, pseudomembranous enteritis, crohn's disease, etc.) and flora-related disease pathogenesis studies. The recipient of the flora transplantation commonly used in the laboratory is a sterile animal or a pseudo-sterile animal treated by antibiotics, namely an SPF-grade experimental animal without specific pathogens is treated by broad-spectrum antibiotics to ensure that the intestinal tract of the experimental animal forms a pseudo-sterile environment, which is beneficial to the colonization of external donor flora, thereby researching the functions of the external donor flora. At present, the use of the sterile animal model requires a very strict sterile animal breeding and feeding environment, is extremely difficult to obtain, and most domestic research institutions cannot achieve the conditions for breeding and feeding the sterile animals.

(4) Correlation analysis of phenotypic localization covariant microorganisms based on molecular biology and biochip analysis methods: the detection of microorganisms is mainly based on an analysis method of DNA fingerprint and an associated analysis method based on DNA sequencing technology. The principle of the DNA fingerprinting technology is mainly that DNA molecular markers representing species in a microbial community are separated in gel by an electrophoresis method according to the characteristics of molecular size, nucleic acid sequence and the like, so that the molecular markers representing different species are migrated to different positions of gel, and the composition structure of the community is displayed by an electrophoresis map, the method has the greatest advantages of convenience, rapidness and intuition, is usually used for detecting dynamic change of the microbial community structure or comparing the structural difference between different communities, mainly comprises Denaturing Gradient Gel Electrophoresis (DGGE) and Terminal fragment length polymorphism (T-RFLP) and the like, can detect more than ten kinds of dominant bacteria in an environmental sample, but cannot detect trace microorganisms, and an electrophoresis strip comprises more than one kind of 16S rDNA sequence and needs to obtain specific strain information, cloning and sequencing are also needed, and the experimental operation is complicated; furthermore, the abundance of the microorganisms cannot be reflected with this method. The DNA sequencing technology directly obtains the nucleic acid sequence information to judge the evolution status of each species in the community, and before, a 16S R RNA gene clone library based on monoclonal plasmid, transformed cell construction and Sanger (Sanger) dideoxy sequencing is widely used for a long time in a method for researching the microbial composition in the community, is applied to diversity analysis of human intestinal flora, the result of the method is far superior to that of a DNA fingerprint map technology in the aspects of species detection depth and species identification level, and with the rapid development of the high-throughput sequencing technology, 1) MWAS (metagenome-wide association analysis) is mainly used at present, wherein the metagenome is a microbial genome in an environmental sample as a research object, functional gene screening and/or sequencing analysis as research means, and the microbial diversity, population structure and structure are related analysis, The evolution relation, the functional activity, the mutual cooperation relation and the relation with the environment are novel microorganism research methods for research purposes. The influence of genetic factors on the intestinal flora can be researched through correlation analysis of the metagenome and the human genome; the role of the intestinal flora in the expression of relevant proteins or metabolic pathways of the human body is researched through the correlation analysis of metagenome and proteomics or metabonomics. The cost of metagenome research sequencing is high. 2) 16S amplicon sequencing correlation analysis, which is to classify microorganisms of different genera and species by sequencing through utilizing the difference of microorganism variable region sequences and perform correlation analysis with phenotypes. When the relation between a certain disease and intestinal flora is not clear, the preliminary screening can be carried out by 16SrDNA sequencing, so that a basis is provided for further follow-up research. However, due to the limitation of the current technology, most microorganisms can not be identified by the identification of 16SrDNA alone, the research on the genus level is more accurate, the pollution is easy, and a false positive result is obtained. In addition, Fluorescence In Situ Hybridization (FISH) and real-time quantitative fluorescent quantitative PCR (real time quantitative PCR) are also commonly used as a means for detecting microorganisms. The biochip can only verify known species, information of microbial diversity is obtained by detecting probes fixed on the biochip, and the abundance of microbes is judged according to the signal intensity, so that the result is inaccurate. Correlation analysis generally needs to follow the research idea of the koch law, and single or several microbial populations are returned to an animal model to prove that correlation is reliable, and the possibility exists that microorganisms are not easy to separate and culture and the influence of the overall change of the flora on the model cannot be observed.

In addition, the above-mentioned research methods can only be used for the research of one or several intestinal bacteria, or belong to the relevance analysis, and cannot directly elucidate the effect and mechanism of the combined action of multiple bacterial colonies actually occurring in vivo.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a simple and convenient intestinal flora research method based on the excrement filtration and transplantation of SPF (specific pathogen free) experimental animals, and provides a basis for exploring the influence of the integral change of the intestinal flora regulated by medicaments on diseases and mechanism research.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the intestinal flora research method based on fecal filtration and transplantation comprises the following steps:

(1) preparation of fecal bacteria transplant recipient experimental animal

The experimental animal of the fecal strain transplant recipient selects a pseudo-aseptic animal or an aseptic animal to form an environment with aseptic or low-bacteria intestinal tract, which is beneficial to the field planting of external microorganisms; the pseudo-sterile animal model needs to use the concentration of fecal bacteria nucleic acid to determine whether the model is successfully made or not through experimental test (for example, as shown in figure 1, the intestinal flora of the animal is obviously reduced after the antibiotic treatment, and pseudo-sterile receptor experimental animals are formed);

(2) preparation of experimental animal for fecal bacteria transplantation donor

The donor animal receives disease model modeling and drug gavage treatment, and positive and negative experimental control of drug effect is established;

(3) recipient animal experimental animal receiving filtration and non-filtration fecal strain transplantation of donor animal experimental animal

Firstly, animal disease molding and drug treatment are carried out, model animal feces for study drug are collected regularly every day, the collected feces are mixed with neutral cold phosphate buffer solution, and then centrifugation is carried out to obtain supernatant. Dividing the supernatant into two parts, wherein one part is the supernatant A, and the other part is the supernatant B;

filtering the supernatant A by using a sterile needle type filter, and then perfusing the supernatant A to receptor experimental animals of the same disease model to form a group A of experimental model animals, and meanwhile, directly and continuously perfusing the supernatant B to receptor experimental animals of the same disease model to form a group B of experimental model animals; the influence of intestinal flora on animal models is researched by judging the disease process difference of two groups of experimental model animals which are controlled in parallel.

Wherein the pore size of the sterile needle filter is 0.22 +/-0.05 mu m, and preferably the pore size of the sterile needle filter is 0.22 mu m. Model animals experimental animals or sterile animals of SPF class, preferably SPF class animals or sterile animals.

In the invention, the aseptic needle type filter can filter out intestinal flora, and the two groups of filters are in parallel control, so that other factors can be eliminated, thereby researching the influence of the integral change of the intestinal flora on the animal model. On the basis, the invention also cultures the filtered supernatant and the unfiltered supernatant respectively (as shown in figure 2), and the number of colonies (a plurality of clones) of the unfiltered supernatant is found to be obviously more than that of the filtered supernatant (without clone formation), thereby further illustrating that the filtering of the invention can effectively eliminate the colonies in the supernatant.

The comparison of the filtered versus non-filtered fecal transplantation of the present invention can be used as a method to study the effect of the overall change in flora on the experimental animals (as shown in fig. 3-6), i.e. the flora-mediated drug effect, while excluding the direct effect of the drug on the intestinal tract. The method is simple and easy to implement, and provides a simpler method for researchers of the intestinal flora.

Drawings

FIG. 1 comparison of total DNA of intestinal flora in mice treated with antibiotics and not treated with antibiotics;

FIG. 2 shows the A supernatant after filtration (left panel) and the B supernatant without filtration (right panel) for anaerobic culture;

figure 3 difference in intestinal flora between FFMT and FMT groups (disease model 1);

figure 4 difference in intestinal flora between FFMT and FMT groups (disease model 2);

figure 5 FFMT group and FMT group differences in disease progression (disease model 1);

figure 6 difference in disease progression between FFMT and FMT groups (disease model 2).

Detailed Description

Animal ethics and labeling

The experiment is audited by an ethical committee, accords with the principles of animal protection, animal welfare and animal ethics, and accords with the related regulation of national experimental animal welfare ethics.

25-40 SPF mice of the appropriate age were selected according to the disease model and numbered for differentiation. The experiment adopts a needle punching method, a small amount of carbon ink is dipped by an eight-gauge needle head, the eight-gauge needle head penetrates into the subcutaneous part at the ear part, the front limb, the rear limb, the tail part and the like, and a black mark is left at the punctured part, so that the purpose of marking is achieved.

Two, random grouping

Mice are adapted in an experimental barrier for one week and then are divided into 5 groups by adopting a random distribution mode, wherein the 5 groups comprise a 1 blank control group, a 2 research drug group, a 3 positive control group, a 4 antibiotic + bacteria liquid filtering group (Filtered microbial transplantation, called FFMT group for short) and a 5 antibiotic + bacteria liquid group (Filtered microbial transplantation, called FMT group for short).

Preparation of experimental animal for fecal bacteria transplantation acceptor

The experimental animal of the fecal strain transplanting receptor can select a pseudo-aseptic mouse or an aseptic mouse, so that an environment with aseptic or low-bacteria intestinal tracts is created, and the field planting of external microorganisms is facilitated. This example employs a readily available pseudo-sterile animal model.

(1) Pseudo sterile mouse model

Experiments formally started in the first week solvent controls were administered to

groups

1, 2 and 3 mice, and groups 4 and 5 mice were antibiotic-producing pseudo-sterile animal models. The specific antibiotic administration method comprises the following steps: after each mouse is perfused with 100mg streptomycin, mixed antibiotic drinking water is immediately given for 7 days, and the formula is as follows: 1g/L ampicillin, 1g/L metronidazole and 1g/L neomycin dissolve 0.5g/L vancomycin.

The specific steps of the intragastric administration are as follows: lifting the tail of the mouse by the right hand, placing the tail on a mouse cage or a rough plane, pinching the skin of the back of the neck of the two ears of the mouse by the thumb and the forefinger of the left hand when the mouse struggles forwards, overturning the mouse to place the mouse in the palm center, straightening the hind limb, and pressing the tail of the mouse by the little finger. In the process of fixing the mouse, the mouse does not need to be over-stressed and does not need to be held on the neck of the mouse, so that the mouse is prevented from suffocating and dying. The head of the stomach irrigation needle is lightly pressed to enable the oral cavity and the esophagus to form a straight line, then the stomach irrigation needle lightly enters the esophagus along the palate wall, and feeding can be started after an elbow of the stomach irrigation needle enters a neck curve. If the position of the gastric perfusion needle is inserted correctly, the mouse can swallow the medicine by himself, the position of the gastric perfusion needle is inserted incorrectly, the mouse struggles violently, the gastric perfusion needle must be pulled out for reinsertion, otherwise, the medicine can be infused into the trachea, and the mouse dies. After the liquid medicine is injected, the intragastric perfusion needle is slightly drawn out.

(2) Molding effect detection

Pseudo-sterile mice were modeled on days 5 and 7 of antibiotic treatment, and feces naturally excreted by the mice were collected for detection of intestinal flora DNA.

The steps of extracting and detecting the DNA of the intestinal flora are as follows:

(1) the feces 180 mg and 220mg are taken and placed in a 2ml centrifuge tube and placed on an ice box.

(2) Add 1ml of InhibitEx Buffer and vortex for 1min until mixed.

(3) Centrifuging at 15-25 deg.c and 20000g for 1 min.

(4) Aspirate 25ul of Proteinase K into a 2ml centrifuge tube (EP).

(5) 600ul of supernatant from step 3 was pipetted into the EP tube in step 4.

(6) 600ul of Buffer AL was added to the above EP, vortexed for 15 s.

(7) Water bath at 70 deg.C for 10 min.

(8) Add 600ul of absolute ethanol and vortex.

(9) Sucking 600ul of the above solution into adsorption column, covering with cover, centrifuging at 15-25 deg.C for 1min at 20000g, and discarding the lower collection tube; the above steps were repeated until all the solutions from step 8 were centrifuged.

(10) The adsorption column was carefully opened and 500ul of Buffer AW1 was added, the mixture was centrifuged at 15 ℃ to 25 ℃ and 20000g for 1min and placed in a new 2ml collection tube, and the original one was discarded.

(11) The adsorption column was opened, 500ul of Buffer AW2 was added, the mixture was centrifuged at 15 ℃ to 25 ℃ and 20000g for 3 min.

(12) Placing the adsorption column into a new collection tube, centrifuging at 15-25 deg.C and 20000g for 3 min.

(13) The adsorption column was placed in a 2ml collection tube, 200ul of Buffer ATE was added, incubated at room temperature for 1min, 15-25 deg.C, 20000g, and centrifuged for 1 min.

(14) The lower layer of liquid was transferred to a labeled 1.5ml EP tube, which was the DNA solution of the intestinal flora.

(15) The DNA concentration of each sample was determined using a Nanodrop 2000 nucleic acid analyzer.

The results are shown in fig. 1, and on the 5 th and 7 th days of antibiotic treatment, the number of intestinal flora was greatly reduced, the flora DNA concentration was significantly reduced, and the pseudo-sterile model mouse model was successful in the antibiotic treatment group compared to the non-antibiotic treatment group.

Preparation of experimental animal for transplanting fecal bacteria to donor

(1) Disease modeling and drug therapy

In the second week all 5 groups of mice were started for disease model modeling and drug gavage (1/day). Group 1 SPF mice were given solvent control (negative control group), group 2 SPF mice were given study drug (gut flora donor group), and group 3 SPF mice were given positive drug of disease model (positive control group).

(2) Preparation of supernatant for fecal bacteria transplantation

The specific steps of treating the supernatant A and B are as follows: feces of SPF class 2 mice (administered with study drug) were collected at a regular time of day at 500mg, resuspended in 10ml of cold phosphate buffer, and centrifuged at 800g at 4 ℃ for 3min to obtain supernatant. And (3) dividing the supernatant into two parts, wherein one part is the supernatant A, and the other part is the supernatant B.

Fifthly, the recipient experimental animal receives the filtered and non-filtered fecal bacteria supernatant transplantation of the donor experimental animal

And filtering the supernatant A by using a 0.22-micron sterile needle type filter, and then performing intragastric administration on the model animals to form a 4 th group of experimental animals, namely an FFMT group, and directly performing intragastric administration on the supernatant B to form a 5 th group of experimental animals, namely an FMT group. The gavage and the disease model molding are carried out simultaneously, and the gavage amount is 0.4 ml/patient.

(1) Culture of filtered and non-filtered supernatant flora

Anaerobic culture is carried out on the filtered supernatant and the unfiltered supernatant, and the influence of filtration on viable bacteria in the supernatant is observed, which comprises the following steps:

1. taking 100mg of intestinal flora feces naturally excreted by the mouse, and suspending in cold PBS solution;

2. 800g, centrifugating for 3min at 4 ℃, and taking the supernatant.

3. The supernatant is divided into two parts, and one part is not processed; one part is filtered by a sterile needle filter (pore size is 0.22 micron), and the filtrate is taken and stored in an ice box.

4. Putting the sample into a refrigerator at 4 ℃ to prepare an aseptic operation table;

5. and diluting the supernatant liquid and the filtrate of the supernatant liquid into a detection range by PBS.

6. And (3) taking 1ml of diluted supernatant liquid and 1ml of filtrate of the supernatant liquid by a liquid transfer device, respectively placing the diluted supernatant liquid and the filtrate of the supernatant liquid into a 3M Petrifilm bacterial colony total number test piece, pressing by a pressing plate, placing the mixture into an anaerobic culture bag, culturing for 1-2 days in an incubator at 37 ℃, and counting bacterial colonies (the test piece automatically develops color).

As shown in fig. 2, most of the microorganisms had been removed after filtration, and no cultured colonies were seen (left); the upper layer of the unfiltered bacterial liquid has live bacteria, namely colonies are formed (right).

Fifth, analysis of differences

After the modeling of the disease model is finished, 16s amplicon sequencing detects the flora distribution difference of FFMT and FMT experimental mice, and whether the flora in the gastric lavage supernatant affects the distribution of intestinal flora of the disease model animal or not is judged. The results show that there is indeed a difference in the population distribution between FFMT and FMT groups of mice in the two disease models (disease model 1: figure 3 and disease model 2: figure 4). The disease progression degree of the mice of the FFMT group and the FMT group was observed, and the results showed that there was indeed a difference in the effects of the FFMT group and the FMT group experimental mice in the two disease models (disease model 1: fig. 5 and disease model 2: fig. 6).

Claims

1. A supernatant combination for use in studying the effect of a pharmacoregulated gut flora on disease model animals, wherein the supernatant combination is a combination of a B supernatant and a C supernatant; the preparation process comprises the following steps:

(1) preparation of fecal bacteria transplant recipient experimental animals: the experimental animal of the fecal strain transplant recipient selects a pseudo-aseptic animal or an aseptic animal to form an environment with aseptic or low-bacteria intestinal tract, which is beneficial to the field planting of external microorganisms; antibiotic was used to make a pseudo-sterile animal model, and specific antibiotic administration methods were: after each mouse is perfused with 100mg streptomycin, mixed antibiotic drinking water is immediately given for 7 days, and the formula is as follows: 1g/L ampicillin, 1g/L metronidazole and 1g/L neomycin dissolve 0.5g/L vancomycin;

(2) preparation of experimental animal of coprophila transplantation donor: the donor animal receives disease model modeling and drug gavage treatment, and positive and negative experimental control of drug effect is established;

(3) recipient experimental animals received filtered and non-filtered fecal transplantation from donor experimental animals:

firstly, molding the disease of an animal and treating the disease by a drug, regularly collecting 500mg of excrement of a model animal for the study drug every day, suspending the collected excrement in 10ml of cold phosphate buffer solution, and centrifuging the solution at the temperature of 800g for 3min at the temperature of 4 ℃ to obtain supernatant; dividing the supernatant into two parts, wherein one part is the supernatant A, and the other part is the supernatant B; filtering the supernatant A by using a sterile needle type filter to obtain a supernatant C; the aperture of the sterile needle type filter is 0.22 +/-0.05 mu m;

filtering the supernatant A by using a sterile needle type filter to obtain supernatant C, performing intragastric administration on the pseudo-aseptic animal subjected to the antibiotic treatment, performing intragastric administration and disease model modeling at the same time to form a group A of experimental model animals, simultaneously, directly performing intragastric administration on the supernatant B, performing intragastric administration on the pseudo-aseptic animal subjected to the antibiotic treatment, and performing intragastric administration and disease model modeling at the same time to form a group B of experimental model animals; the treatment difference between the experimental animals in the group A and the experimental animals in the group B is only whether the lavage supernatant is filtered or not; after the disease model modeling is finished, the influence of the intestinal flora regulated by the medicine on the disease model animal is researched by judging the disease process difference of the two groups of model animals which are parallelly contrasted;

the model animal is a specific pathogen free experimental animal grade animal or a sterile animal.