CN114982717A - Establishment, evaluation and application of animal model for establishing yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by coprophilous fungus transplantation method - Google Patents
Establishment, evaluation and application of animal model for establishing yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by coprophilous fungus transplantation method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2207/00—Modified animals
- A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention relates to a construction method of an animal model, in particular to establishment and evaluation of an animal model for establishing type 2 diabetes yin deficiency syndrome anthropogenic intestinal flora by using a fecal bacteria transplantation method. The invention obtains corresponding 2 type diabetes yin deficiency syndrome and/or non-yin deficiency syndrome animal models by preparing fecal bacteria liquid of 2 type diabetes yin deficiency syndrome and/or non-yin deficiency syndrome patients, and transplanting the prepared fecal bacteria liquid after the mice take quadruple antibiotic medicines by means of drenching and drinking water, and simultaneously adopts various methods to evaluate the animal models and confirms whether the model is established successfully. The invention provides an economical and practical animal model for yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes, which has the advantages of high stability, easy popularization, good repeatability and high success rate, provides a basis for the research of treating diabetes by traditional Chinese medicines, and provides a thought for realizing the standardization and normalization of the evaluation of the yin deficiency syndrome model.
Description
Technical Field
The invention relates to a construction method of an animal model, in particular to establishment, evaluation and application of an animal model for establishing type 2 diabetes yin deficiency syndrome and/or type 2 diabetes non-yin deficiency syndrome by using a fecal bacteria transplantation method.
Background
Diabetes has become one of the most serious public health problems worldwide in the 21 st century, most of which are type 2 diabetes. The clinical manifestations are "three more and one less", "three more" refers to polyuria, polydipsia and polyphagia, and "one less" refers to emaciation with symptoms of fatigue, polydipsia and the like. Type 2 diabetes belongs to the category of diabetes in traditional Chinese medicine, yin deficiency is the main pathological basis of the diabetes, yin deficiency syndrome is one of the basic symptoms of traditional Chinese medicine caused by deficiency of yin essence or body fluid of human body, and yin deficiency syndrome is widely distributed in clinical diseases of traditional Chinese medicine, wherein type 2 diabetes accounts for more than 90% of the total number of diabetes patients, most commonly diabetes.
The intestinal flora is a complex and large population, and in a healthy adult, the total weight of the intestinal flora can reach about 1 to 2 kg, the total number of cells contained in the intestinal flora is 1013 to 1014, which is about 10 times of the number of human cells, and the number of the coding genome of the intestinal microorganism is about 100 times of the coding genome of the human body, so the intestinal flora is also called as a second genome.
Fecal bacteria transplantation (FMT), which is defined as the effective means of reconstituting intestinal flora by transplanting functional flora in the feces of healthy people into the gastrointestinal tract of patients to reconstitute new intestinal flora and thus treat intestinal and parenteral diseases.
At present, a mouse model for a fecal transplantation test is complex to construct, poor in model effect and difficult to reflect the real situation; and reports related to animal models for establishing yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by a fecal transplantation method are not found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method, an evaluation method and application for establishing an animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes.
The invention is realized by the following technical scheme:
the invention provides a method for establishing an animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a fecal bacteria transplantation method, which specifically comprises the following steps:
(1) preparing a fecal strain liquid: preparing feces from a patient clinically diagnosed with yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes into fecal bacteria solution;
(2) the mice take quadruple antibiotic medicines in modes of drenching and drinking water, wherein the quadruple antibiotic is ampicillin, vancomycin, neomycin and metronidazole;
(3) model establishment: and (2) transplanting the fecal bacteria liquid prepared in the step (1) to the mice respectively to obtain a model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes.
Further, the method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using the fecal bacteria transplantation method specifically comprises the following steps of (1):
a, preparing a mixed solution of 0.9% of normal saline containing 10% of glycerol and 0.1% of L-cysteine as a feces protective solution, boiling the mixed solution in an autoclave at 115 ℃ for 20min, putting a feces sample into the feces protective solution, and uniformly mixing;
b, filtering the excrement protective solution of the uniformly mixed excrement obtained in the step A to obtain filtrate, performing centrifugal treatment, and discarding supernatant to obtain precipitate as collected thalli;
c, using a fecal protection solution to resuspend the precipitated thalli obtained in the step B;
and D, subpackaging the fecal strain liquid according to needs and preserving at-80 ℃.
Preferably, in the method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using the fecal strain transplantation method, the filtration is performed through a sterile 200-mesh nylon net in the step B; the centrifugation conditions were 4000rpm for 10 min.
Preferably, the step B, C, D of the method for establishing the animal model with yin-deficiency syndrome of type 2 diabetes and/or non-yin-deficiency syndrome of type 2 diabetes by using fecal transplantation method is performed in anaerobic environment, wherein the anaerobic environment requires O 2 Concentration of<2%。
Further, a method for establishing an animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a fecal bacteria transplantation method is characterized in that the step (2) specifically comprises the following steps:
step 1: selecting SPF male C57BL/6 mice with age of 6 weeks;
step 2: a mouse classification stage, namely classifying C57BL/6 mice into a sugar health bacterial liquid group (C group), a type 2 diabetes yin deficiency syndrome bacterial liquid group (TY group) and a type 2 diabetes non-yin deficiency syndrome bacterial liquid group (TNY group);
and step 3: and (3) a flora clearing stage, wherein the mice in the step 2 are administrated with the tetrad antibiotic daily in the 1 st week and are administrated with the tetrad antibiotic through drinking water in the 2 nd week.
And 4, step 4: in the molding stage, the mice are respectively implanted with lavage to transplant healthy volunteers, type 2 diabetes yin deficiency patients and non-yin deficiency patients with fecal bacteria liquid in step 3
Preferably, the method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using the coprophilous fungus transplantation method comprises the steps of drenching 5 mg/ampicillin, 5 mg/vancomycin, 5 mg/neomycin and 4 mg/metronidazole, and the amount of the tetraantibiotic given by drinking water is 1g/L ampicillin, 0.5g/L vancomycin, 1g/L neomycin and 1g/L metronidazole.
Preferably, the method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a fecal bacteria transplantation method is characterized in that the volume of the fecal bacteria for transplantation is 200 mu L/time, and the fecal bacteria is transplanted once a day and continuously for 7 weeks.
In addition, the invention provides an animal model evaluation method for establishing type 2 diabetes yin deficiency syndrome and/or type 2 diabetes non-yin deficiency syndrome by using a coprophilous fungi transplantation method, which comprises an open field experiment and an irritability score; mouse body temperature change, water intake, salivary flow rate, fecal moisture, skin moisture, body mass, 24h urine volume, urobilinogen and bilirubin and food intake, fasting plasma glucose, OGTT and ITT.
Preferably, the animal model evaluation method for establishing the yin-deficiency syndrome of type 2 diabetes and/or the non-yin-deficiency syndrome of type 2 diabetes by using the fecal strain transplantation method is characterized in that the open field test comprises the exploration behavior of a mouse, the residence time of the mouse in the middle lattice, the number of times of crossing the lattice, the movement distance and the movement speed of the open field.
In addition, the invention also provides application of a method for establishing a yin deficiency syndrome of type 2 diabetes and/or a non-yin deficiency syndrome animal model of type 2 diabetes by using a coprophilous bacterium transplantation method in diabetes treatment, and the diabetes animal model established by the method comprises an early diagnosis reagent and a method for researching and developing diabetes and a treatment medicine for researching and developing diabetes.
The invention has the following beneficial effects:
the research adopts a yin deficiency syndrome modeling method to construct a model, creatively combines yin deficiency syndrome typical expressions such as dysphoria with smothery sensation in five centers, hectic fever, dry mouth, dry skin, dry stool, emaciation and dark urine to formulate evaluation indexes, explores an animal model evaluation method of yin deficiency syndrome, and provides a thought for realizing standardization and normalization of yin deficiency syndrome model evaluation.
The invention provides an economical and practical animal model for yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes, which has the advantages of high stability, easy popularization, good repeatability and high success rate, and provides an experimental basis for the early clinical research of diabetes treatment by traditional Chinese medicines.
Drawings
FIG. 1 shows the activity of the mice in each group in the open field.
FIG. 2 groups of mice are stress-responsive scores.
FIG. 3 body temperature changes of mice in each group.
FIG. 4 variation in water intake for each group of mice.
Figure 5 saliva flow rate profile for each group of mice.
FIG. 6 fecal water content of groups of mice.
FIG. 7 moisture content of the skin of each group of mice.
FIG. 8 shows the variation of body mass of mice in each group.
FIG. 9 shows 24-hour urine volume of each group of mice.
FIG. 10 shows the food intake of mice in each group.
FIG. 11 FBG status of each group of mice.
FIG. 12 OGTT status of various groups of mice.
FIG. 13 ITT profile for each group of mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific embodiments. It should be understood that the embodiments described herein are merely illustrative of the present invention and that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.
Example 1.
Firstly, collecting and preparing fecal liquid.
1) Collecting fresh excrement
Preparing a feces protective solution: preparing 0.9% normal saline containing 10% glycerol and 0.1% L-cysteine mixed solution as feces protective solution, cooking and sterilizing at 115 deg.C in autoclave for 20min, placing feces sample in the feces protective solution, and mixing.
Fecal collection tube preparation is performed in an anaerobic workstation. Namely, 50 ml of sterile centrifuge tubes are taken and operated in an anaerobic workstation, 30ml of sterilized excrement protective solution is added into each tube, and a sealing film is used for sealing after a cover is screwed, so that the sterile excrement protective solution is used as a collecting tube.
20g of donor fresh excrement sample (the sampling part is the inner part of the excrement middle section) is collected and placed in the collecting tube, the tube cover is immediately screwed and sealed by a sealing film, and excrement is shaken after sealing. The excrement and the excrement protective liquid are fully and uniformly mixed and then placed on an ice bag as soon as possible, the ice bag is used for wrapping the periphery of the excrement and the excrement protective liquid, and in addition, two pipes of excrement for freezing storage are collected, wherein 2-3g of excrement is placed on ice. Transporting on ice, transporting to a laboratory of Jiangsu Jiejiaokang biotech company within 2h, immediately carrying out subsequent fecal strain liquid preparation work on the feces for preparing the fecal strain liquid, and storing the frozen feces in a refrigerator at the temperature of minus 80 ℃.
2) And a preparation process.
In an anaerobic workstation of a collection and extraction company, the collected human excrement sample is suspended in a prepared excrement bacterium protective solution, food residues are removed through a sterile 200-mesh nylon net, and the operation is repeated for 2 times. Screwing the tube cover of the centrifugal tube in the anaerobic workstation, sealing the tube cover by using a sealing film, taking out the anaerobic workstation for centrifugation, and centrifuging at 4000rpm for 10min to collect bacteria. And after the centrifugation is finished, transferring the biomass to an anaerobic workstation, removing supernatant, obtaining bottom sediment, namely the collected thalli, and using the excrement protective solution to resuspend the thalli. Initial volume of feces: resuspending and adding the feces protective solution to obtain a final volume ratio of 1:5, resuspending the thallus with the feces protective solution, subpackaging for transplanting, and immediately transferring to a refrigerator at-80 ℃ for storage after subpackaging the feces bacteria solution.
The anaerobic workstation used in the experimental process aims to provide an anaerobic environment for the experiment, and the anaerobic environment requires O 2 Concentration of<2%, anaerobic incubators may also be used to provide an anaerobic environment.
3) And fecal donor information.
Secondly, constructing an animal model.
SPF grade C57BL/6J male mice, 36, 6 weeks old, were purchased from Jiangsu Jiejiaokang Biotech, Inc.
SPF grade 6-week-old C57BL/6 mice were randomly divided into a healthy bacterial transplantation liquid group (referred to as group C), a type 2 diabetes yin deficiency syndrome patient bacterial transplantation liquid group (referred to as group TY), and a type 2 diabetes non-yin deficiency syndrome patient bacterial transplantation liquid group (referred to as group TNY), and each group had 12 mice. Each group of mice was given a quadruple antibiotic to sweep intestinal microbes for 2 weeks: antibiotics (5 mg/ampicillin, 5 mg/vancomycin, 5 mg/neomycin and 4 mg/metronidazole) are administered daily for 1 week; antibiotics (ampicillin 1g/L, vancomycin 0.5g/L, neomycin 1g/L, metronidazole 1 g/L) were given by drinking water on week 2. After 2 weeks of antibiotic cleaning, 200 mu L/piece of fecal bacterial liquid of healthy volunteers, type 2 diabetes patients with yin deficiency and non-yin deficiency patients are transplanted for 7 weeks continuously.
And thirdly, evaluating an animal model.
After the model construction is completed, the mice are observed for fasting blood glucose, water intake and body mass change every week. And (3) evaluating the glucose tolerance and the insulin response condition of the mice by adopting OGTT and ITT. Observing the dysphoria degree of the mice through an open field experiment and irritation-prone scores; the body temperature changes of the mice were dynamically observed, and the water intake, saliva flow rate, fecal water content, skin water content, body mass, 24h urine volume, urobilinogen and bilirubin and food intake changes of each group were compared to evaluate whether a yin deficiency phenotype was present.
And fourthly, evaluating results.
Measuring the result to
And s represents. Performing t test on data between two groups by using GraphPad 8.0 statistical software, performing one-factor variance analysis on data between multiple groups, and performing P<0.05 is statistically significant for differences, where P < 0.05, P < 0.01, P < 0.001, P < 0.0001; compared with the TNY group, the # P is less than 0.05, the # P is less than 0.01, the # P is less than 0.001 and the # P is less than 0.0001.
4.1 open field experiments.
And (4) exploring the autonomous behavior and behavior of the mouse and evaluating the dysphoria degree. Recording the exploration behavior of the mouse by using a video tracking system, observing the activity condition of the mouse within 5min, and analyzing the residence time, the crossing times, the movement distance and the movement speed of the mouse in the middle lattice.
Results are shown in figure 1, the percentage of TY group median residence time is increased compared to group C in open field experiments: (P< 0.05), the number of central cell punctures and the percentage of retention time decreased in the TNY group compared with those in the TY group (P＜0.05，P＜0.001）。
4.2 irritation score.
Judging and grading according to the reaction when the neck of the mouse is caught: level 1: when the neck is caught, screaming and frightened jump appear, and 1 point is recorded; and 2, stage: when the user wants to bite or sting the neck, 2 points are recorded: and 3, level: when the tail is lifted by fingers, screaming, frightened jump and even biting people appear or the tail and a mouse in the same cage are frequently dug, and 3 minutes are recorded; the case without the above is 0, and 0 is recorded. The degree of dysphoria is evaluated.
Results of irritation score as shown in fig. 2, the irritation score was significantly higher in TY group mice than in C and TNY groups (both of themP＜0.0001）。
4.3 body temperature detection (anal temperature, limb paw heart temperature, precordial region temperature).
An infrared thermometer is adopted to detect the anus temperature, the paw heart temperature of the four limbs and the precordial region temperature of the mouse at the 8 am, the 3 pm and the late 20 o' clock, and the dysphoria with smothery sensation degree of the five centers is evaluated.
As shown in fig. 3, the difference between TY group anal temperature, precordial temperature, and limb paw temperature increased at 15 o 'clock and anal temperature and limb paw temperature increased at 20 o' clock compared to group C and TNY, which were statistically significant.
4.4 drinking water amount.
The average daily water intake of mice was determined.
The results are shown in FIG. 4, where at week 1 of antibiotic washout, the TY and TNY groups had lower water intake than group C: (P＜0.01，PLess than 0.001), the difference among the groups at the 2 nd week of antibiotic drinking water is not obvious; during FMT stage, the TY and TNY groups at 1-2 weeks of FMT had decreased water intake compared with group C: (P< 0.0001), increase in TNY water intake at week 3-4 compared to group C: (P＜0.05，PLess than 0.01), the water drinking amount of TY group mice in the 5 th week is higher than that of C group and TNY group (both of them)P< 0.0001), the difference in water intake was not significant for each group by FMT week 6-7.
4.5 saliva flow rate test.
Cutting qualitative filter paper into equal-sized fan shapes with radius of about 2cm and weight difference below 10%, clamping with forceps, probing into mouse mouth, staying on tongue surface for 5s, taking out, and weighing with electronic balance to detect saliva flow rate. Saliva flow rate (mg/s) = (wet weight of filter paper-dry weight of filter paper)/5 s, and the degree of dry mouth is typically evaluated for the appearance of yin deficiency.
The results are shown in FIG. 5, where the saliva flow rate of TY mice is reduced compared to C and TNY groups (P＜0.01，P＜0.05）。
4.6 moisture content of feces.
Catching the mouse, massaging the anus part to promote defecation, taking 2-3 grains of excrement, immediately weighing, putting into a rapid moisture tester, selecting an automatic mode, heating at 105 ℃ until the machine stops, and measuring the dry weight. Stool water content = (wet-dry weight)/wet weight × 100%.
Results figure 6 shows that the moisture content of feces was reduced in TY mice compared to C and TNY mice (a)P＜0.01，P＜0.05）。
4.7. And (5) detecting the moisture content of the skin.
The moisture content of the skin on the back and the palms of the four limbs of the mouse was measured by using a portable elastic moisture oil light skin tester (HKJ-SK 03) to evaluate the degree of skin dryness.
As shown in FIG. 7, skin moisture content in limbs of TY group mice was decreased compared to those in C and TNY groups (both of themPLess than 0.01), the moisture content of the back skin was not significantly different.
4.8. And (5) detecting the physical quality.
The body mass of each mouse was determined and the body wasting degree was evaluated.
As shown in FIG. 8, the results of examining body mass showed that the body mass of the three groups of mice tended to increase with time, but the difference between the groups was not significant.
4.9.24 h urine volume.
And (3) placing the mouse in a metabolism cage, placing a measuring cylinder at a lower section interface to receive urine volume, taking out the mouse after 24h, and recording the urine volume of the mouse in each cage.
As shown in FIG. 9, the TY group mice had a decrease in 24h urine volume as compared with the C and TNY groups (both of themP＜0.05）。
4.10. Urine color (measurement of urobilinogen and bilirubin content).
Urine of a mouse is collected, and urine urobilinogen and urine bilirubin content are detected by using a URO urine analysis test strip (urine eight-linked 8N) and adopting urine dry chemical analysis. Fully immersing the test strip into urine, carrying out color comparison with a standard color code within a specified time on the color code, judging, reading and recording the result.
The results showed that the rate of weak positive for urobilinogen and the rate of positive for urobilirubin were increased in TY mice compared to C and TNY groups.
4.11. Food intake.
The food intake of each cage of mice was measured, and the results are shown in FIG. 10, in which there was no significant difference in the food intake of each group at week 1 of antibiotic washout, and the TY group at weeks 2 of antibiotic washout, 1 of FMT and 2 was lower than that of TNY group (T: (shown in FIGS.)P＜0.0001，P＜0.05，P< 0.01), increased food intake in the TNY group compared with the C group at week 3 of FMT (P< 0.05), increase in food intake of TY group at week 4 of FMT compared with C group (P< 0.05), the food intake of TY group and TNY group was increased in FMT week 5 compared to C group (P＜0.0001，P< 0.01), TNY diet was lower than that of group C at week 6 of FMT: (P< 0.01), TY group diet was higher than TNY group (P< 0.01), TNY diet lower than group C by FMT week 7: (P＜0.05）。
4.12. Fasting blood glucose.
The fasting blood glucose of the mice was measured weekly, and the results are shown in fig. 11, compared with the group C, the mice with the 3 rd to 7 th week of yin deficiency and the mice with the non-yin deficiency were observed phenotypically to have increased fasting blood glucose, and the mice with the yin deficiency group had an increased trend compared with the fasting blood glucose of the non-yin deficiency group, and were most significant at the 5 th week (b) (b)P＜0.05）。
4.13. OGTT。
After fasting for 12 h, the stomach was perfused with a 50% glucose solution at a body mass of 2 g/kg, blood was taken from the tail vein before (0 min) and 30, 60, 90, 120min after the administration of sugar, and the blood glucose level was measured, as shown in fig. 12, to verify whether the mice had impaired glucose tolerance.
4.14.ITT。
Fasting for 6 h, injecting normal short-acting insulin (0.75 u/kg body mass) into abdominal cavity, and measuring blood sugar level by taking blood from tail vein before (0 min) and 15, 30, 60, 90, 120min after insulin injection. As shown in fig. 13, compared to group C, blood glucose increased at 0, 15, 60, 90, 120min in the yin-deficiency group and increased at 0, 15, 30, 60, 90, 120min in the non-yin-deficiency group, and the differences were statistically significant; the area under the curve results show an increase in ITT AUC in the yin-deficiency and non-yin-deficiency groups compared to the healthy group (P＜0.0001）。
Claims (10)
1. A method for establishing an animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a fecal bacteria transplantation method is characterized by comprising the following steps:
(1) preparing a fecal strain liquid: preparing feces from clinically diagnosed yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes into fecal bacteria liquid;
(2) the mice take quadruple antibiotic medicines in modes of drenching and drinking water, wherein the quadruple antibiotic is ampicillin, vancomycin, neomycin and metronidazole;
(3) model establishment: and 2, transplanting the fecal bacteria liquid prepared in the step 1 to the mice respectively to obtain a model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes.
2. The method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a fecal bacteria transplantation method as claimed in claim 1, wherein the step (1) comprises the following steps:
a, preparing a mixed solution of 0.9% normal saline containing 10% glycerol and 0.1% L-cysteine as an excrement protective solution, boiling the mixed solution in an autoclave at 115 ℃ for 20min, putting an excrement sample into the mixed solution containing the excrement protective solution, and uniformly mixing;
b, filtering the excrement protective solution of the uniformly mixed excrement obtained in the step A to obtain filtrate, performing centrifugal treatment, and discarding supernatant to obtain precipitate as collected thalli;
c, using a fecal protection solution to resuspend the precipitated thalli obtained in the step B;
and D, subpackaging the fecal strain liquid according to needs and preserving at-80 ℃.
3. The method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using fecal bacteria transplantation as claimed in claim 2, wherein the centrifugation condition in step B is 4000rpm for 10 min.
4. The method of claim 2, wherein the yin deficiency syndrome of type 2 diabetes is established by fecal transplantationThe method for preparing non-yin deficiency animal model of type 2 diabetes is characterized in that step B, C, D is performed in anaerobic environment, wherein the anaerobic environment requires O 2 Concentration of<2%。
5. The method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using fecal bacteria transplantation as claimed in claims 1-4, wherein the step (2) comprises the following steps:
step 1: selecting SPF male C57BL/6 mice with age of 6 weeks;
step 2: a mouse classification stage, namely classifying C57BL/6 mice into a sugar health bacterial liquid group (group C), a type 2 diabetes yin-deficiency syndrome bacterial liquid group (group TY) and a type 2 diabetes non-yin-deficiency syndrome bacterial liquid group (group TNY);
and step 3: a flora clearing stage, wherein the mice in the step 2 take the tetrad antibiotic orally every day in the 1 st week and are given the tetrad antibiotic by drinking water in the 2 nd week;
and 4, step 4: and (3) in the molding stage, transplanting the mice in the step 3 into healthy volunteers, type 2 diabetes yin deficiency patients and non-yin deficiency patients respectively.
6. The method for establishing animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using fecal transplantation method as claimed in claim 5, wherein the amount of tetrad antibiotic orally administered in step (3) is 5 mg/piece ampicillin, 5 mg/piece vancomycin, 5 mg/piece neomycin and 4 mg/piece metronidazole, and the amount of tetrad antibiotic administered in drinking water is 1g/L ampicillin, 0.5g/L vancomycin, 1g/L neomycin and 1g/L metronidazole.
7. The method for establishing the animal model of yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using fecal bacteria transplantation as claimed in claim 5, wherein the amount of fecal bacteria transplanted in step (4) is 200 μ L/day, once a day for 7 weeks.
8. A method for establishing an animal model evaluation method for yin deficiency syndrome of type 2 diabetes and/or non-yin deficiency syndrome of type 2 diabetes by using a coprophilous fungus transplantation method is characterized by comprising the steps of performing an open field experiment, irritation-prone scoring, mouse body temperature change, water intake, saliva flow rate, feces water content, skin water content, body mass, 24-hour urine volume, urobilinogen, bilirubin, food intake, fasting blood glucose, OGTT and ITT for evaluation.
9. The method for evaluating the animal model of type 2 diabetes yin deficiency syndrome and/or type 2 diabetes non-yin deficiency syndrome by using fecal strain transplantation method as claimed in claim 8, wherein the open field test is the exploration behavior of the mouse, the residence time of the mouse in the middle lattice, the crossing times, the moving distance and the moving speed of the open field.
10. The use of any one of claims 1-9 in the development of diabetes mellitus, wherein the animal model of diabetes mellitus constructed by the method comprises reagents and methods for the early diagnosis of diabetes mellitus and drugs for the development of diabetes mellitus.
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