CN115316343A - Kit for constructing humanized flora mouse model - Google Patents
Kit for constructing humanized flora mouse model Download PDFInfo
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- CN115316343A CN115316343A CN202211103776.7A CN202211103776A CN115316343A CN 115316343 A CN115316343 A CN 115316343A CN 202211103776 A CN202211103776 A CN 202211103776A CN 115316343 A CN115316343 A CN 115316343A
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit which comprises a broad-spectrum antibiotic composition, human intestinal flora, dietary fiber pectin and a gastric lavage device. The humanized flora mouse model can be constructed by adopting the kit, and a tumor model is constructed on the basis of the humanized flora mouse model and is used for screening or evaluating an immune checkpoint inhibitor.
Description
Technical Field
The invention belongs to the field of medicine, and particularly relates to a kit for constructing a humanized flora mouse model, application of the kit and application of the constructed mouse model in screening or evaluation of an immune checkpoint inhibitor.
Background
The tumor treatment method mainly comprises operations, chemotherapy, radiotherapy, targeted therapy and the like, and the initial clinical treatment effect of many tumors is good, but the tumors often generate drug resistance and easily relapse along with the prolonging of the treatment time of the drugs, thereby bringing serious challenges to clinical treatment.
The human intestinal microorganisms have complex compositions and diversity, have important influence on human health and disease processes, and are widely considered as an overlooked 'important organ', 'second genome' and 'second brain' in the body. Researches show that the specific intestinal flora is used as a cancer promotion or inhibition factor to influence the generation, development and metastasis of tumors. Meanwhile, the compound can affect the pharmacokinetics, anticancer activity and toxicity of the medicine from multiple aspects, and has important influence on antitumor treatment. The difference among intestinal flora individuals is as high as 80% -90%, mainly the difference on the 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 conditions and the like; the complexity of flora research is increased, and good opportunities are brought to the development of the intestinal flora research in the times of precise medical treatment and personalized medical treatment.
In recent years, with the progress of research on correlation between intestinal flora and human diseases, the demand for establishment and application of intestinal microorganism transplantation mouse models is also increasing. At present, the mouse model is still the first choice for the study of intestinal flora. The intestinal microorganism transplantation mouse model has become the most effective choice for studying the causal relationship between microorganisms and human-related diseases and the preclinical proof of concept of possible effective intervention treatment methods. Because the composition of intestinal microorganisms of a mouse is obviously different from that of a human, the method for deducing the experimental result of a mouse model to the human body still needs to be carefully considered in the research field of the intestinal flora and the human related diseases. At present, the application of sterile mice to establish a human-derived intestinal microorganism transplantation mouse model to research the influence of intestinal flora on human health and related diseases has become an important research strategy in the field at present. However, the use of sterile animal models requires very strict sterile animal breeding and feeding environments, is not easy to obtain, and most domestic research institutions cannot achieve the conditions for breeding and feeding sterile animals. In addition, the sterile mouse model for the coprophilous fungus transplantation experiment is not only complicated to construct, but also poor in model effect and difficult to truly react.
Disclosure of Invention
In view of the important clinical research value of the human intestinal microorganism transplantation mouse model, the inventor provides a system and a method for constructing a humanized flora mouse model, in particular a humanized flora tumor-bearing mouse model in order to overcome the defects in the prior art, so that the construction method of the experimental mouse model is simplified, the obtained mouse model can truly and reliably reflect the microenvironment state of the human intestinal tract, and a foundation is laid for researching the action mechanism of the intestinal flora and discovering key prognosis targets and formulating individualized accurate medication schemes. Specifically, the present invention includes the following technical solutions.
A kit for constructing a humanized flora mouse model, comprising: a broad spectrum antibiotic composition for use in eliminating indigenous intestinal flora in mice; human intestinal flora used for intragastric mice to implant new flora, the intestinal flora derived from feces of patients with predetermined diseases, namely fecal flora, wherein the predetermined diseases refer to diseases of a pre-constructed mouse model; the dietary fiber is used for promoting the colonization and growth of human donor bacteria in the intestinal tract of the mouse; an intragastric administration device.
In one embodiment, the human intestinal flora is derived from stool from a cancer patient. Accordingly, the mouse model is a cancer mouse model.
For this purpose, the kit further comprises: mouse cancer cells for constructing cancer mouse model.
For example, the mouse cancer cell is a mouse tumor cell such as a mouse colon cancer cell (MC 38 cell) used to construct a tumor mouse model such as a colon cancer mouse model.
Preferably, the kit further comprises: a tool for separating intestinal bacteria from stool. The tool comprises: a sampling spoon, a filter such as a sieve, a centrifuge tube, a buffer/buffer solution suitable for holding live bacteria.
The buffer solution includes, but is not limited to, phosphate buffered saline PBS, PBS containing 20% glycerol, carbonate buffered saline CBS, borate buffered saline, amino acid buffered saline, citrate buffered saline, disodium hydrogen phosphate-citric acid buffered saline, tris buffered saline. The preferred buffer solution for cryopreserving feces is PBS containing 20% glycerol.
The PBS buffer solution is a solvent medium, and the concentration of each component is as follows: 137mM sodium chloride, 2.7mM potassium chloride, 10mM sodium dihydrogen phosphate, 2mM potassium dihydrogen phosphate. The pH value of the combined stool sample is preferably 7.0-7.5.
20% glycerol in PBS buffer was added to reduce cell death due to ice crystal formation during cryopreservation of the fecal samples.
Preferably, the broad spectrum antibiotic composition includes, but is not limited to: vancomycin, neomycin sulfate, metronidazole and ampicillin.
The administration amount of each component of the broad-spectrum antibiotic composition can be as follows: vancomycin 50-200mg/kg mouse body weight, preferably about 100mg/kg mouse body weight; neomycin sulfate 100-300mg/kg mouse body weight, preferably about 200mg/kg mouse body weight; metronidazole 100-300mg/kg mouse body weight, preferably about 200mg/kg mouse body weight; ampicillin is 100-300mg/kg of mouse body weight, preferably about 200mg/kg of mouse body weight. The administration was 1 time per day for 3 consecutive days. Such an amount of administration has no significant influence on the physiological state of the mouse. The components of the broad spectrum antibiotic composition may be dissolved in sterile physiological saline for administration.
The dietary fiber may be pectin. Pectin is a soluble fiber existing in primary cell wall and mesocyte layer of higher plant, and its main component is D-galacturonic acid polymer with molecular mass above 100 kDa. Pectin retains its colloidal activity without being digested after entering the digestive tract, and provides an environment suitable for the growth of beneficial bacterial flora, thereby promoting the colonization efficiency and growth rate of donor bacterial flora in the intestinal tract of a recipient.
The inventor finds that the lavage of pectin has an obvious promoting effect on the colonization and growth of exogenous fecal bacteria in the intestinal tract of the mouse, so that the colonization success rate of the exogenous fecal bacteria in the intestinal tract of the mouse can be improved by at least 50%, the construction speed of the model mouse is greatly shortened, the success rate is improved, and the cost for constructing the mouse model is obviously reduced.
The dietary fiber pectin is derived from fruits, such as pericarp or pomace of fruits such as fructus Citri Grandis, fructus Citri Limoniae, mandarin orange, fructus Mali Pumilae, and fructus Musae, by self-extracting and purifying; it can also be purchased commercially, for example, commercial pectin having a galacturonic acid content (dry basis) of > 74.0% (Cat. No: P112756, manufactured by Aladdin reagent (Shanghai) Co., ltd.).
The amount of pectin to be administered is 5-20mg/kg of mouse body weight, preferably about 10mg/kg of mouse body weight. 2 times daily for 4 days.
It should be understood that when numerical features are expressed herein, the terms "about" or "approximately" mean that the number indicated may have a margin of error or variance of ± 10%, ± 8%, ± 6%, ± 4% or ± 2%.
In one embodiment, the mouse is a Specific Pathogen Free (SPF) mouse.
The second aspect of the present invention provides a method for constructing the humanized flora mouse model, which is implemented by using the kit, and comprises the following steps:
1) Isolating human intestinal flora: separating fecal bacteria from human fresh or frozen fecal samples;
2) The broad-spectrum antibiotic composition is used for gastric perfusion of a mouse to remove endogenous flora of the mouse, and has no obvious influence on the physiological state of the mouse, so that a sterile animal model is obtained;
3) Perfusing human intestinal flora into a gastric mouse, transplanting about 200 mu l of fecal bacteria liquid into a sterile animal model in a gastric perfusion mode, 1 time per day for 3 consecutive days;
4) Smearing fecal bacteria solution on the skin and hair of the mouse (preventing mouse intestinal bacteria from breeding or cross infection of mouse intestinal bacteria by habitual scratching of the mouse, and preventing skin allergy and other pathological changes of the mouse caused by using antibiotics. The bacteria which are habitually scratched on the skin and ingested through the oral cavity of the mouse are also human intestinal bacteria, so that the human intestinal bacteria are planted in the intestinal tract of the mouse. ) Approximately 100. Mu.l of fecal inoculum was applied to the fur of each mouse;
5) The fecal strain-transplanted mice were fed pectin by gavage at a gavage of approximately 10mg/kg of mouse body weight. 2 times daily for 4 consecutive days;
6) Evaluation of transplantation efficiency: sequencing intestinal flora of the mice by adopting a high-throughput sequencing method, carrying out bioinformatics analysis, detecting the structure and diversity of fecal flora, and evaluating the transplanting efficiency to obtain a humanized flora mouse model.
Preferably, the above method further comprises the steps of:
7) Constructing a tumor model: culturing sufficient tumor cells such as MC38 cells in vitro, and injecting the cells into mice subcutaneously to cause the mice to form tumors in vivo to obtain humanized flora tumor-bearing mouse model.
Correspondingly, the kit also comprises a cell injection device.
In the above method, the operation of step 1) is, for example: collecting fresh human fecal specimen, weighing, mixing in equal volume (w/v) of PBS containing 20% glycerol, and freezing; before the coprophilous fungi is transplanted, a coprophilous fungi sample is unfrozen and suspended in PBS with the same volume, after uniform mixing, the coprophilous fungi sample is filtered by a 100-mesh filter screen, such as a 100-mesh filter screen, filtrate is collected by a centrifugal tube, the volume of the filtrate is recorded, centrifugation is carried out, supernatant is discarded, normal saline is added to the original volume, uniform mixing is carried out, the steps are repeated for a plurality of times, lower-layer precipitates are diluted by the normal saline, and coprophilous fungi is obtained after uniform mixing and used for coprophilous fungi transplantation.
In a second aspect, the invention provides the use of the above-described tumor mouse model in screening and evaluating immune checkpoint inhibitors.
For example, the immune checkpoint inhibitor may be a PD-1 inhibitor.
The experimental mouse model disclosed by the invention is simple in construction method and low in cost, the construction power is obviously improved, the obtained mouse model can truly and reliably reflect the micro-environment state of the intestinal tract of a human body, and a foundation is laid for researching the action mechanism of intestinal flora and finding a key prognosis target point and formulating an individualized accurate medication scheme.
Drawings
FIG. 1 shows the shape characterization of the construction and validation of humanized flora tumor-bearing mice. Wherein, A: cecal photographs of mice; b: a sparse curve displayed by sequencing of the intestinal flora 16s rDNA gene; C. d, E: the Chao index, shannon index and ACE index of intestinal flora 16s rDNA gene sequencing. In the figure, control is the original mouse without the construction procedure and ABX cocktail is the mouse fed with the broad-spectrum antibiotic composition.
FIG. 2 shows a graph of species composition analysis in mouse model intestinal flora. Wherein, A: phylogenetic level-based species composition analysis in intestinal flora; b: species composition analysis based on genus level. In the figure, control is the original mouse without performing the construction operation, and ABX cocktail is the mouse fed with the broad-spectrum antibiotic composition.
Figure 3 shows validation of humanized intestinal flora tumor-bearing mice. Wherein, A: clustering analysis of intestinal microbial evolution of individual human donors and recipient mice, an evolutionary tree of intestinal flora was constructed using dissimilarity coefficients based on the Bray-Curtis distance. B: the ratio of the intestinal flora of recipient mice derived from human donor feces was estimated using Source Tracker analysis.
FIG. 4 shows the results of testing the effect of humanized intestinal flora tumor-bearing mouse model on the treatment of PD-1 inhibitor (anti-PD-1). Wherein, A: tumor growth curves of each humanized intestinal flora tumor-bearing mouse model, in which the abscissa is the number of days after tumor inoculation (days after tumor inoculation) and the ordinate is the tumor volume; b: survival curves of each group of humanized intestinal flora tumor-bearing mice, wherein the survival time is expressed in days, the abscissa of the graph is the days (days after tumor inoculation) after tumor inoculation, and the ordinate is the survival rate of the mice; c: the tumor mass of each group of humanized intestinal flora tumor-bearing mice is summarized, the abscissa in the graph is the grouping of the mice, and the ordinate is the tumor mass. Control (Control) was the original mouse without construction, ABX cocktail was the mouse fed with the broad-spectrum antibiotic composition, HV was the tumor-bearing mouse receiving fecal bacteria from a healthy volunteer (health volunteer), COL was the tumor-bearing mouse receiving fecal bacteria from a colon cancer patient (colon cancer).
Figure 5 shows tumor immune microenvironment assessment and analysis of a humanized intestinal flora tumor-bearing mouse model. At the end of the experiment, immunohistochemical staining images (a) and statistical analysis results (B) of CD4, CD8 in tumor tissues of each group of humanized intestinal flora tumor-bearing mouse models. In panel B, the abscissa is the mouse cohort, the left ordinate is the IHC score of CD4 in the tumor, and the right ordinate is the IHC score of CD8 in the tumor, where IHC is immunohistochemical staining.
Detailed Description
According to the invention, after a broad-spectrum antibiotic composition is adopted to treat a mouse, the mouse is transplanted with fecal bacteria, dietary fiber pectin treatment is adopted to promote the colonization and growth of donor fecal bacteria in a receptor intestinal tract, a humanized flora mouse model is obtained, and a tumor model is constructed on the basis.
In the clinical field, sterile mice are often used as animal models for fecal transplantation. However, germ free mice have the following disadvantages in constructing the model: 1. sterile animals do not exist in nature and can only depend on artificial breeding. Therefore, the requirement of the feeding environment is strict, and the feeding cost is high; 2. the breeding rate is low, the pollution rate is high, the survival rate is low, and some special genotype mice can not be bred and raised; 3. the existing method has the defects of innate immunity and nerves, poor life and high mortality rate after disease modeling, so that the research result of related diseases is unreliable. According to the invention, after endogenous flora of SPF mice is completely eliminated by using the broad-spectrum antibiotic composition, sterile mice can be obtained, and a coprophilous transplantation model is constructed by using the sterile mice, so that the experimental steps can be greatly simplified, the experimental animal samples can be reduced, and the experimental cost can be reduced. More importantly, the SPF level mouse is similar to a healthy and normal mouse, and compared with a sterile mouse, the SPF level mouse can reflect the physiological state of the simulated body more truly.
It is understood that the mouse model constructed by the present invention may be a humanized flora mouse model simulating a healthy person, i.e. the fecal bacteria are derived from a healthy person individual; the strain may be a humanized flora mouse model simulating a certain disease, i.e., the fecal strain is derived from a patient with a certain disease. In order to guarantee the typicality of the humanized flora mouse model, an individual as a fecal flora donor should satisfy the following conditions: 1) No combined infectious diseases exist within 2 months before the collection of the fecal sample; no serious dysfunction of heart, liver and hematopoietic system, no diarrhea or constipation and serious malnutrition, etc.; 2) At least 3 months prior to stool sample collection, without any antibiotics, probiotics, or antimicrobial agents; 3) Non-autoimmune diseases and metabolic diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic scleroderma, gout, diabetes, obesity, etc.; 4) No history of receiving fecal bacteria transplantation; 5) No diet or strict vegetarian diet; 6) There is no history of serious cardiovascular and cerebrovascular diseases.
In a preferred embodiment, the fecal bacteria for the mice after intragastric administration is mixed fecal bacteria from a plurality of donors of the same type, for example, fecal suspensions of a plurality of patient samples are mixed in equal amount, and fecal bacteria difference caused by individual difference of the samples is eliminated, so that representativeness and typicality of the constructed humanized flora mouse model are guaranteed, the microenvironment state of the intestinal tract of a human body is truly and reliably reflected, and accurate information is provided for researching the action mechanism of the intestinal flora and finding of key prognosis targets.
In an exemplary embodiment, the method for constructing the humanized flora tumor-bearing mouse model may comprise the following steps:
step 1: four antibiotics, vancomycin (about 100mg/kg mouse weight), neomycin sulfate (about 200mg/kg mouse weight), metronidazole (about 200mg/kg mouse weight) and ampicillin (about 200mg/kg mouse weight), were prepared as solutions using sterile physiological saline, and then gavage was performed 1 time a day for 3 consecutive days according to the mouse weight to obtain a sterile animal model.
Step 2: human fresh faeces were taken, weighed, mixed in an equal volume (w/v) of PBS containing 20% glycerol and stored in a refrigerator at-80 ℃. Before the coprophilous fungi is transplanted, human fecal samples are unfrozen and suspended in PBS with the same volume, after uniform mixing, the mixture is filtered by a filter screen with 100 meshes, filtrate is collected by a centrifugal tube, the volume of the filtrate is recorded, the filtrate is centrifuged at 7000r/min for 5min, supernatant is poured off, normal saline is added to the original volume and mixed uniformly, the step is repeated for 3 times, the lower-layer precipitate is dissolved by the normal saline again, and the mixture is vortex-mixed uniformly to obtain coprophilous fungi liquid which is used for coprophilous fungi transplantation.
And step 3: about 200. Mu.l of fecal bacteria solution was transplanted to the sterilized animals by intragastric administration 1 time per day for 3 consecutive days.
Approximately 100. Mu.l of fecal inoculum was applied to the fur of each mouse. 5 mice were housed per cage.
And 4, step 4: on day 3 after fecal strain transplantation, mice were inoculated subcutaneously with 5.0X 10 5 And MC38 cells. After 5 days (the average tumor volume reaches 100-150 mm) 3 ) The PD-1 inhibitor or isotype control IgG antibody was injected intraperitoneally every 3 days for 3 times.
And 5: the fecal inoculum transplant animals were fed about 10mg/kg pectin 2 times a day for 4 consecutive days by gavage.
Step 6: evaluation of transplantation efficiency: fecal fungi were transplanted for 3 days (1/day) using human feces. After 10 days, the intestinal flora of the mice is sequenced by adopting a high-throughput sequencing method, and bioinformatics analysis is carried out to detect the structure and diversity of the fecal flora and evaluate the transplantation efficiency.
The invention has the advantages of the following aspects;
1. compared with the prior art, the method has the advantages that no complex and expensive equipment is needed when the mouse model is constructed, the method is friendly to experimental animals, the construction method is high in efficiency, only 9 days are needed from modeling to model building, the method is simple and easy to implement, the success rate is high, and the repeatability is good; the SPF-level mouse is used for replacing a sterile mouse, so that the cost is reduced, and the constructed experimental mouse model is relatively stable, has a good effect, can truly and reliably reflect the environment in the intestinal tract of a human body, and has a good practical application value.
2. The modeling method has no side effect and lower cost, and the established humanized flora tumor-bearing mouse model is an indispensable animal model for intestinal flora and human related diseases and has wider application.
In a preferred embodiment, the kit may further comprise at least one of the following items in addition to the broad spectrum antibiotic composition, the human intestinal flora, the dietary fiber pectin, the gavage instrument, the cell injector, respectively: a carrying case, the space of which is divided into defined spaces for accommodating one or more containers, such as vials, tubes, and the like, each containing a separate component for use in the method of the invention; instructions, which may be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, for example paper with a download window for the operation demonstration video APP, such as a two-dimensional code, or in the form of multimedia, such as a usb-disc, a web-disc, etc.
The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.
Examples
The addition amounts, contents and concentrations of various substances are referred to herein, wherein the percentages refer to weight percent (wt%) or volume/weight percent (w/v%) unless otherwise specified.
In the examples herein, if no specific description is made about the operating temperature, the temperature is generally referred to as room temperature (10-40 ℃).
Materials and instruments:
SPF grade 6-8 week old female C57BL/6 mice, weight 18.0 + -2.9 g (Shanghaisi Leike laboratory animal technology Co., ltd.) 5 groups, total 20, centrifuge (Eppendorf 5810R), SW-CJ-2F clean bench (Suzhou clean-up facility Co., ltd.), RPMI 1640/DMEM medium (Solarbio Co., ltd.), EP tube (Axygen), anticoagulated blood collection tube, pipette gun, sterile gun tip, physiological saline, slide caliper, sterile 100mm petri dish, iodophor, 75% alcohol.
Sequencing of microbial genome was performed by Shanghai microbial technology Co., ltd.
Example 1: establishing humanized flora tumor-bearing mouse model
1. Construction of sterile animal models
Preparing four antibiotics including vancomycin (100 mg/kg of mouse weight), neomycin sulfate (200 mg/kg of mouse weight), metronidazole (200 mg/kg of mouse weight) and ampicillin (200 mg/kg of mouse weight) into a solution by using sterile physiological saline, and performing intragastric administration for 1 time per day for 3 consecutive days according to the weight of the mouse; obtaining the sterile animal model.
2. Patient specimen collection
Tumor patients were enrolled as standard: the new patient with solid tumor has no infectious disease, clear pathological diagnosis, proper tumor size, complete clinical information and signed informed consent.
Establishing a group patient database comprising: name, sex, age, contact details, pre-operative data (including blood routine, biochemistry, gastroscopy, imaging examination, etc.), post-operative data (blood routine, biochemistry, pathology diagnosis report, TNM staging, etc.), treatment regimen, treatment effect, survival, etc.
Fresh stool specimens from 4 newly diagnosed patients with malignant solid tumors were collected from the secondary Zhongshan Hospital at the university of Fudan, weighed, mixed in an equal volume (w/v) of PBS containing 20% glycerol, and stored in a freezer at-80 ℃. Before the coprophilous fungi is transplanted, human fecal samples are unfrozen and suspended in PBS with the same volume, after uniform mixing, the human fecal samples are filtered by a filter screen with 100 meshes, filtrate is collected by a centrifugal tube, the volume of the filtrate is recorded, 7000r/min is centrifuged for 5min, supernatant is poured off, normal saline is added to the original volume, the mixture is uniformly mixed, the step is repeated for 3 times, the lower-layer precipitate is dissolved by the normal saline again, and the mixture is uniformly vortex-mixed to obtain coprophilous fungi liquid which is used for coprophilous fungi transplantation.
Inclusion criteria for patient specimens:
1) No combined infectious diseases, no serious dysfunction of heart, liver and hematopoietic system, no diarrhea or constipation, serious malnutrition and the like in 2 months before the composition is added;
2) At least 3 months prior to stool sample collection, without any antibiotics, probiotics, or antimicrobial agents;
3) No other history of malignancy or receiving anti-tumor therapy;
4) No history of receiving fecal bacteria transplantation;
5) No diet or strict vegetarian diet;
6) There is no history of serious cardiovascular and cerebrovascular diseases.
3. Feces collection and storage
1) Directly discharging the excrement into a clean container, avoiding the pollution of urine, a toilet wall and the like to an excrement sample as much as possible, and taking a sample at the same time or uniformly mixing all the samples and then sampling;
2) Digging a fecal sample by using a sterile spoon, putting a proper amount of fecal sample into a sterile storage tube, subpackaging the sample at 200 mg/tube, and quickly putting the sample into liquid nitrogen for more than 15min for quenching treatment;
3) The collected sample is immediately put into a refrigerator at minus 80 ℃ for storage for later use.
4) If the sample needs to be detected repeatedly, the sample can be uniformly mixed by using a sterile rod and then is subpackaged by a plurality of pipes so as to avoid repeated freeze thawing, and the sample is immediately stored in a refrigerator at the temperature of 80 ℃ below zero for later use after being collected.
4. Fecal treatment and intragastric transplantation
Before the coprophilous fungi is transplanted, human fecal samples are unfrozen and suspended in PBS with the same volume, after uniform mixing, the mixture is filtered by a filter screen with 100 meshes, filtrate is collected by a centrifugal tube, the volume of the filtrate is recorded, the filtrate is centrifuged at 7000r/min for 5min, supernatant is poured off, normal saline is added to the original volume and mixed uniformly, the step is repeated for 3 times, the lower-layer precipitate is dissolved by the normal saline again, and the mixture is vortex-mixed uniformly to obtain coprophilous fungi liquid which is used for coprophilous fungi transplantation.
Equal amounts of the fecal suspension were mixed for each patient, and 300. Mu.l of the mixed fecal suspension was injected into the stomach of the mouse through a gavage needle 1 time a day for 3 consecutive days.
In addition, 100. Mu.l of fecal bacteria solution was applied to the fur of each mouse. 5 mice were housed per cage.
5. Intragastric treatment of dietary fiber pectin
In the mouse model after the fecal bacteria transplantation, 10mg/kg pectin was injected into the stomach of the mouse 2 times a day for 4 consecutive days.
6. Establishment of tumor model
1) Culturing enough MC38 cells to ensure good cell state, no pollution and no impurity. When the cell fusion degree reaches 70-80%, digesting with 0.25% pancreatin, collecting in 15ml centrifuge tube, centrifuging at 1000r/min for 5min, re-suspending the single cell suspension with PBS, counting with cell counter, and formulating with PBS to concentration of 5.0 × 10 6 Each/ml.
2) Placing the mice on a dry and clean laboratory bench, under aseptic conditions, extracting 200 μ l of tumor cell suspension with a l ml syringe, injecting subcutaneously into right axilla of the mice, wherein the number of inoculated cells in each mouse is 5.0 × 10 5 And (4) respectively.
3) The cell tumorigenesis was observed, and after 1 week, the tumor volume and body weight of the mice were measured and recorded.
4) When the tumor volume of the mouse reaches 100-150mm 3 When the administration is started. The tumor volume calculation formula of the mice is as follows: v =0.5 × long diameter × short diameter.
5) On day 30, collecting samples of feces, blood and the like of each group of mice, dislocating cervical vertebrae, killing the mice, fixing the mice on an animal operating table, sterilizing the whole body with alcohol, taking heart, liver, spleen, lung, kidney, colon and tumor tissues, and repeatedly washing the tissues with precooled PBS. And weighing and measuring the tumor tissue, and photographing and recording. Dividing the taken tissue into two parts, and putting one part into formalin solution; one part is put into liquid nitrogen for quick freezing, and then the frozen part is put into a refrigerator at the temperature of 80 ℃ below zero for storage.
The experimental results of this example are as follows:
1. results of sterile mouse modeling
In the construction and verification of a humanized flora tumor-bearing mouse, after an SPF (specific pathogen free) level mouse is subjected to gastric lavage treatment by using the broad-spectrum antibiotic composition for 1 week, the mouse is killed, an intestinal tract is taken out, the cecum is cut off, observation and photographing are carried out, the cecum of the antibiotic treatment group mouse is subjected to compensatory enlargement, the diameter of the intestinal tract is increased, the cecum content is greatly reserved, and the characteristic accords with the cecum appearance of a sterile mouse (A in figure 1). 16srDNA gene sequencing revealed that the sparse curve of intestinal flora was much lower in mice treated with the antibiotic combination than in the control group (B in FIG. 1), indicating that the intestinal flora was overall reduced. Meanwhile, the broad-spectrum antibiotic composition greatly reduces the diversity of intestinal flora of mice, including the Chao index, shannon index and ACE index (C-E in fig. 1), and seriously destroys the normal structure of the intestinal flora. Species composition analysis based on phylum (phylum) levels suggested that bacteroidetes and firmicutes were the predominant species in the gut flora in the control group, while the original commensal flora was effectively eliminated after feeding the broad-spectrum antibiotic composition (a in fig. 2). Species composition analysis based on genus (genus) level also suggested that bacteroides, akkermansia, lactobacillus, bifidobacterium etc. were all cleared compared to before feeding the antibiotic composition (B in fig. 2). These results suggest that the bacterial flora in the intestinal tract of the mice has been substantially cleared and that a sterile mouse model was successfully obtained.
2. Evaluation of transplantation efficiency
We used the feces of the patients for 3 days (1/day) of fecal transplantation. After 10 days, intestinal flora of the mice is sequenced by a high-throughput sequencing method and subjected to bioinformatics analysis to detect the structure and diversity of fecal flora, a flora evolutionary tree is constructed based on the dissimilarity coefficient of Bray-Curtis distance, and the result shows that the flora of each human donor and the flora of the recipient mice have obvious aggregative property, which indicates that the human donor and the recipient mice have similar community structures (A in figure 3). Further, source Tracker analysis showed that over 70% of the microorganisms in the gut of recipient mice were derived from human donors. These results suggest that intestinal microbes of human donors essentially successfully colonize the intestines of recipient mice (fig. 3B).
Example 2: application of humanized flora tumor-bearing mouse model in treatment of immune checkpoint inhibitor
The application of the humanized flora tumor-bearing mouse model in the treatment of the immune checkpoint inhibitor is explored, and a foundation is laid for formulating an individualized accurate medication scheme.
Recent studies have shown that the efficacy and complications of tumor immunotherapy are related to the composition of the patient's intestinal flora, and are characteristic of the composition of the patient's intestinal flora that is susceptible to treatment or susceptible to adverse reactions. These features may serve as biomarkers to predict the prognosis of immunotherapy. However, the structure and composition of the intestinal flora are influenced by various factors, including host genetic factors, geographical conditions, dietary habits, health conditions and drug use conditions, and thus the difference between individual intestinal flora is as high as 80% -90%. The complexity of the research on the intestinal flora is increased, and the development opportunity is brought to the application of the intestinal flora in precise medical treatment and personalized medical treatment.
In view of the influence and high heterogeneity of intestinal flora on tumor immunotherapy, the invention discloses a humanized flora tumor-bearing mouse model with human physiological characteristics obtained by using feces of patients before treatment as donors, which can be used for testing various immunotherapy drugs and combined drugs and effectively screening malignant tumor drugs.
With the experimental mouse model of fecal bacteria transplantation in example 1 as the subject, 1 fresh fecal specimen of a newly diagnosed patient with colon cancer was collected. And simultaneously collecting 1 excrement sample of healthy people with similar body types and weights and no basic diseases, using the excrement sample as a donor of human excrement, and constructing a humanized flora tumor-bearing mouse model by adopting a colon cancer cell line MC 38. The immune checkpoint inhibitor is a PD-1 inhibitor (InVivoMAb anti-mouse PD-1, bio X cell Co. Ltd. USA, # BE 0146).
The experimental results of this example are as follows:
1. test results of therapeutic effects of humanized colony tumor-bearing mouse model on PD-1 inhibitor (InVivoMAb anti-mouse PD-1)
All mice were well tolerated during the construction of the model. After the model is successfully constructed, the spirit is good, the hair is smooth, and the diet and the defecation are normal. The therapeutic effect of PD-1 inhibitors was different in each group of mice. Referring to fig. 4, panel a shows that the tumor volume of the original mice (Control) without the construction procedure was the largest, the tumor volume of the humanized intestinal flora tumor-bearing mouse model (COL) was the next, the tumor volume of the tumor-bearing mice (HV) receiving healthy human fecal bacteria was significantly lower, the next time the mice were fed with the broad-spectrum antibiotic composition (ABX cocktail). In fig. C, it is shown that the tumor weight of the original mouse (Control), the humanized intestinal flora tumor-bearing mouse model (COL), and the mouse fed with the broad-spectrum antibiotic composition (ABX cocktail) without the construction procedure is large, and the tumor weight of the tumor-bearing mouse (HV) receiving healthy human fecal bacteria is significantly reduced. The result shows that the PD-1 inhibitor has poor inhibition effect on tumor growth by using a mouse model (COL) constructed by colon cancer patient flora; in contrast, the therapeutic effect of the PD-1 inhibitor was superior in the mouse model (HV) constructed using the healthy volunteer population (A, C in FIG. 4). There was also a significant statistical difference between the survival times of the two groups (B in fig. 4). These results suggest that the colon cancer patient may fail in subsequent PD-1 inhibitor treatment, while the intestinal flora of healthy volunteers may rescue their resistance to PD-1 inhibitor treatment.
2. Evaluation and analysis of tumor immune microenvironment of humanized flora tumor-bearing mouse model
There is increasing evidence that the gut flora is able to modulate the host's immune function and response to tumor therapy. To explore the molecular and cellular mechanisms of the intestinal flora in the treatment of PD-1 inhibitors, we performed immunohistochemical analysis of tumor tissues in each group of mice. We observed a mouse model (COL) constructed using the flora of colon cancer patients, with T cell infiltration in tumor tissuesSignificantly reduced, including CD4 + T cells and CD8 + T cells, both of which play a key role in the body's anti-tumor immune response (figure 5). In addition, intestinal flora (HV) of healthy persons may increase the tumor immune microenvironment CD4 + And CD8 + Infiltration of T cells forms a "hot" tumor microenvironment. Referring to fig. 4B, the IHC score of CD4 and the IHC score of CD8 in tumor tissues of the original mouse (Control), the humanized intestinal flora tumor-bearing mouse model (COL), the mice fed with the broad-spectrum antibiotic composition (ABX cocktail), which did not perform the construction procedure, were relatively low, and the tumor-bearing mice (HV) receiving healthy human fecal bacteria were significantly improved. This result shows that the humanized flora tumor-bearing mouse model (COL) can effectively reflect the tumor immune microenvironment status of the patient.
The embodiments described above are presented to facilitate a person of ordinary skill in the art to understand and use the invention. It will be readily apparent to those skilled in the art that modifications may be made to the embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (10)
1. A kit for constructing a humanized flora mouse model, comprising: broad spectrum antibiotic composition, human intestinal flora, dietary fiber, and gastric lavage device.
2. The kit of claim 1, wherein the human intestinal flora is derived from stool from a cancer patient.
3. The kit of claim 2, further comprising: mouse cancer cells for constructing a cancer mouse model.
4. The kit of claim 3, wherein the mouse cancer cell is a mouse tumor cell, such as a mouse colon cancer cell, used to construct a mouse model of tumor, such as a mouse model of colon cancer.
5. The kit of claim 1, further comprising: a tool for isolating intestinal bacteria from stool, the tool comprising: a sampling spoon, a filter, a centrifuge tube, and a buffer agent/buffer solution suitable for storing viable bacteria.
6. The kit of claim 1, wherein the broad spectrum antibiotic composition comprises: vancomycin, neomycin sulfate, metronidazole and ampicillin.
7. The kit of claim 1, wherein the dietary fiber is pectin.
8. A method of constructing a humanized flora mouse model using the kit of claim 1, comprising the steps of:
1) Isolating human intestinal flora: separating fecal bacteria from human fresh or frozen fecal samples;
2) The broad-spectrum antibiotic composition is used for gastric perfusion of a mouse, so that endogenous flora of the mouse is eliminated, the physiological state of the mouse is not obviously influenced, and a sterile animal model is obtained;
3) Perfusing human intestinal flora into a gastric mouse;
4) Smearing fecal bacteria liquid on the skin and hair of the mouse;
5) Feeding pectin to the fecal strain transplanted animal through intragastric administration;
6) Evaluation of transplantation efficiency: and sequencing the intestinal flora of the mouse by adopting a high-throughput sequencing method, carrying out bioinformatics analysis, detecting the structure and diversity of the fecal flora, and evaluating the transplanting efficiency.
9. The method as recited in claim 8, further comprising the step of:
7) Constructing a tumor model: and culturing sufficient tumor cells in vitro, and performing subcutaneous injection on the mouse to cause the tumor formation in the mouse, thereby obtaining the humanized flora tumor-bearing mouse model.
10. Use of a mouse model of a tumor as defined in claim 4 in screening for evaluation of immune checkpoint inhibitors.
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