Construction method of hypothyroidism model mouse and methylene hypothyroidism model mouse

Abstract

The invention relates to the technical field of construction of A subtraction and methylene subtraction models. The invention provides a construction method of a hypothyroidism model mouse and a hypothyroidism model mouse, which comprises the following steps of: (1) taking a mouse for a test, applying tamoxifen to the mouse for 4-6 days, stopping the drug for 12-16 days, and screening to obtain a hypothyroidism mouse model; and (2) taking a mouse for test, administering tamoxifen for 4-6 days, after stopping drug administration for 6-8 days, administering LT for 46-8 days, and screening to obtain the methylene hypothyroidism mouse model. Based on a Cre-loxP recombination principle, a TSHRflox / floxTG-CreERT2 mouse is utilized to induce thyroid TSHR knockout, namely a TSHRTko mouse model, and hypothyroidism and hypothyroidism mouse models can be constructed safely, quickly, efficiently and stably.

Description

Technical Field

[0001] The invention relates to the technical field of hypothyroidism and subacute hypothyroidism model construction, and in particular to a method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse. Background Art

[0002] The main existing techniques for hypothyroidism or subacute hypothyroidism models include: chemical induction, low iodine diet, radiotherapy 131 Ⅰ induction, surgical induction, etc.

[0003] (1) Chemical induction, i.e., antithyroid drugs, mainly including methimazole (MMI) and propylthiouracil (PTU), is a simple method with low cost. However, the toxicity of the drug itself will interfere with the body and the disease of concern. For example, MMI, as an antithyroid drug, has strong liver toxicity. The time period is long. The model is unstable and will quickly return to normal after drug withdrawal, which is not conducive to long-term research.

[0004] (2) Low-iodine diet. Iodine deficiency is the most common cause of hypothyroidism. This modeling method is simple, avoids complex surgery and invasive operations, and is highly similar to the actual clinical situation. It is mainly used to study iodine deficiency-related diseases. However, the availability is low, the preparation of commercial low-iodine food is complex and expensive, and the animal processing time is long, so the cost is high; it is difficult to accurately control its dosage and time during the experiment (poor controllability).

[0005] (3) Radioactivity 131 I induction, this modeling method is simple, has a high success rate, a short cycle, and is economical and feasible. However, this method has high requirements for modeling equipment and there is a risk of contamination by radioactive substances, so it is rarely used in general experiments.

[0006] (4) Surgical induction, including total thyroidectomy and subtotal thyroidectomy. Postoperative supplementation of levothyroxine can establish a subacute hypothyroidism model. This model is rapid and stable and is mainly used in rats. However, the operation is difficult and the surgery may damage nearby organs, including the thymus and large blood vessels. Animals die from complications such as excessive bleeding and postoperative infection. Therefore, the mortality rate is high and the postoperative survival rate is low, which greatly reduces the success rate of modeling. Summary of the invention

[0007] The purpose of the present invention is to provide a method for constructing a thyroid TSHR knockout mouse model and a method for constructing a hypothyroidism model and a subacute hypothyroidism model using the mouse, which can safely, quickly, efficiently and stably construct hypothyroidism and subacute hypothyroidism mouse models.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse, comprising the following steps:

[0010] (1) Get TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and the hypothyroidism mouse model was obtained after 12 to 16 days of drug withdrawal;

[0011] (2) Take TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and after stopping the drug for 6 to 8 days, LT4 was given for 6 to 8 days to screen out the subacute hypothyroidism mouse model.

[0012] Preferably, the TSHR in steps (1) and (2) flox / flox TG-CreERT2 mice were 7-9 week old male SPF grade mice.

[0013] Preferably, the dosage of tamoxifen in steps (1) and (2) is independently 30-50 mg / kg / d, and the tamoxifen is injected intraperitoneally using corn oil as a solvent.

[0014] Preferably, the screening method for the hypothyroidism mouse model in step (1) is: detecting the levels of TSH and FT4 in mouse serum, and if the TSH level is significantly higher than that of normal mice and the FT4 level is significantly lower than that of normal mice, the hypothyroidism model is successfully constructed.

[0015] Preferably, the dosage of LT4 in step (2) is independently 4-6 ug / kg / d, and the LT4 is injected intraperitoneally using corn oil solvent as a carrier.

[0016] Preferably, the screening method for the subacute hypothyroidism mouse model in step (2) is: detecting the levels of TSH and FT4 in mouse serum, the TSH level is significantly higher than that of normal mice, and the FT4 level is not significantly different from that of normal mice, and the subacute hypothyroidism model is successfully constructed.

[0017] The present invention provides a method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse, comprising the following steps: (1) obtaining TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and the hypothyroidism mouse model was obtained after 12 to 16 days of drug withdrawal; (2) TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and after 6 to 8 days of drug withdrawal, LT4 was given for 6 to 8 days to screen and obtain a subacute hypothyroidism mouse model. flox / flox TG-CreERT2 mice induce thyroid TSHR knockout, i.e. TSHR TkoMouse model, can safely, quickly, efficiently and stably construct hypothyroidism and subacute hypothyroidism mouse models.

[0018] The animal model of the present invention is based on the Cre-loxP recombination principle and utilizes TSHR flox / flox TG-CreERT2 mice induce thyroid TSHR knockout, which can safely, quickly, efficiently and stably construct hypothyroidism and subacute hypothyroidism mouse models, avoiding the shortcomings of other modeling methods, such as long time consumption, instability, low modeling success rate, etc. In particular, the construction of subacute hypothyroidism mouse models is an innovation in this field.

[0019] TSHR knockout mice flox / flox The modeling procedure of TG-CreERT2 mice is complicated, time-consuming, labor-intensive and costly; tamoxifen has toxic side effects, so the appropriate drug concentration and injection time are one of the keys to the success of this model.

[0020] In short, any modeling method has its advantages and disadvantages, but each modeling method also has its own pertinence, and it is necessary to choose an appropriate modeling method. This patent provides a new modeling method for hypothyroidism and subacute hypothyroidism mice, which provides a reference for research in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of the recombination reaction of the Cre-loxp system;

[0022] Figure 2 It is a tamoxifen-inducible Cre system;

[0023] Figure 3 TSHR of the present invention flox / flox TG-CreERT2 mouse construction process (customized by Saiye Biotechnology Co., Ltd.);

[0024] Figure 4 A brief flow chart of the modeling process;

[0025] Figure 5 TSHR flox / flox Construction process of TG-CreERT2 mice;

[0026] Figure 6 The expression of TSHR protein in mouse thyroid tissue was detected by WB (density quantification method); ****, P < 0.0001. DETAILED DESCRIPTION

[0027] The present invention provides a method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse, comprising the following steps:

[0028] (1) Get TSHR flox / floxTG-CreERT2 mice were given tamoxifen for 4 to 6 days, and the hypothyroidism mouse model was obtained after 12 to 16 days of drug withdrawal;

[0029] (2) Take TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and after stopping the drug for 6 to 8 days, LT4 was given for 6 to 8 days to screen out the subacute hypothyroidism mouse model.

[0030] In the present invention, the TSHR in steps (1) and (2) flox / flox TG-CreERT2 mice were 7-9 week old male SPF grade mice purchased from Saiye Biotechnology Co., Ltd.

[0031] In the present invention, the dosage of tamoxifen in steps (1) and (2) is preferably 30 to 50 mg / kg / d, more preferably 40 mg / kg / d. The tamoxifen is injected intraperitoneally with corn oil as the solvent, preferably as the carrier.

[0032] In the present invention, the screening method for the hypothyroidism mouse model in step (1) is preferably: detecting the levels of TSH and FT4 in mouse serum, the TSH level is significantly higher than that of normal mice, the FT4 level is significantly lower than that of normal mice, and the hypothyroidism model is successfully constructed, and the detection time is two weeks after tamoxifen is stopped (at this time, the thyroid TSHR is completely knocked out).

[0033] In the present invention, the significant determination method is: using one-way ANOVA analysis, and bilateral P<0.05 indicates statistical difference, that is, significant difference.

[0034] In the present invention, 2 weeks after tamoxifen withdrawal, the patient enters a hypothyroid state, and the patient remains in a stable hypothyroid state throughout the subsequent experimental period.

[0035] In the present invention, the dosage of LT4 in step (2) is preferably 4-6 ug / kg / d, more preferably 5 ug / kg / d. The LT4 is preferably injected intraperitoneally using corn oil as a solvent.

[0036] In the present invention, the screening method for the subacute hypothyroidism mouse model in step (2) is preferably: detecting the levels of TSH and FT4 in mouse serum, the TSH level is significantly higher than that of normal mice, and the FT4 level is not significantly different from that of normal mice, and the subacute hypothyroidism model is successfully constructed.

[0037] In the present invention, the purpose of injecting T4 is to achieve the sub-hypothyroidism state. The injection is chosen one week after tamoxifen is stopped (at this time, the thyroid TSHR has not yet been completely knocked out) to prevent it from progressing to the hypothyroidism state. The serum thyroid hormone level is detected one week after T4 injection (i.e., 2 weeks after tamoxifen is stopped) to verify whether it becomes a sub-hypothyroidism state after T4 injection. The time point for detection is only to detect whether the sub-hypothyroidism state is successful, and it is not the time to stop injecting T4.

[0038] In the present invention, T4 injection is started one week after tamoxifen is stopped. After one week, the patient enters a subacute hypothyroidism state. During the subsequent entire experimental period, T4 injection (5ug / kg / d) is required every day to maintain a stable subacute hypothyroidism state. Once the T4 injection is stopped, the patient will return to a hypothyroidism state.

[0039] In the present invention, mice are selected for gene knockout because the thyroid function and hypothalamus-pituitary-thyroid axis of mice are similar to human thyroid function, because the evolutionary process of mice is very similar to that of humans, both of which evolved from aquatic species to reptiles, amphibians, and mammals, and the homology between mouse genes and human genes is more than 90%, and the genes are easy to manipulate.

[0040] In the present invention, the Cre-loxP recombinase system, which was first discovered in bacteriophage P1, is currently the most widely used conditional gene editing tool. Bacteriophage P1 is a virus that infects Escherichia coli, and its genome contains a recombinase called Cre (Causes Recombination) and a specific DNA sequence loxP (Locus of crossing over (x) in P1). The Cre enzyme is a 38kDa protein composed of 343 amino acids. The loxP site is a 34-base-pair-long DNA sequence consisting of two 13-base-pair inverted repeat sequences and an 8-base-pair spacer sequence. The Cre recombinase can recognize and bind to the loxP site, inducing DNA recombination between the two sites. The effects of the Cre-loxP system are as follows: A. Deletion: When two loxP sites are on the same DNA chain and arranged in the same direction, the Cre recombinase will delete the DNA sequence between the two loxP sites and reconnect the remaining DNA fragments. This process is almost irreversible and is the most commonly used method; B. Inversion: When two loxP sites are on the same DNA chain but arranged in reverse, Cre recombinase will cause the DNA fragment between the two loxP sites to reverse 180 degrees. This process is reversible; C. Translocation: When two loxP sites are located on different DNA chains or chromosomes, Cre recombinase will cause the DNA fragments on the left and right sides of the two loxP sites to exchange and translocate. The translocation process is reversible, and this method is less used ( Figure 1 ).

[0041] Flox mice, or conditional knockout mice, are mice that contain paired loxP sites in the target gene. After mating with Cre tool mice, the target gene can be knocked out in specific tissues or cells.

[0042] CreER (Cre-Estrogen Receptor) is a fusion protein of Cre recombinase and estrogen receptor (ER). When no inducer is added, the expressed CreER binds to the HSP90 protein and is localized in the cytoplasm, unable to enter the nucleus to edit genomic DNA. Only after the addition of an inducer (here, an estrogen receptor antagonist Tamoxifen or 4-OHT (4-hydroxytamoxifen)), the inducer binds to the CreER protein, and CreER will dissociate from the anchor protein HSP90 through conformational changes, enter the nucleus, recognize the loxP site, and undergo recombination. In order to avoid the interference of endogenous estrogen, a point mutation (G521R) is made in the ligand binding region of the estrogen receptor to obtain CreERT, which can make CreER only respond to the induction of exogenous synthetic estrogens (such as Tamoxifen, 4-OHT [4-hydroxytamoxifen]). CreERT2 is a further improved version of CreERT, containing three point mutations C400V / M453A / L544A, which is more sensitive and faster in response to tamoxifen ( Figure 2 ).

[0043] In summary, TSHR flox / flox The mouse (referred to as FLOX mouse) refers to the insertion of loxP sites arranged in the same direction on both sides of the TSHR gene on both DNA strands; TG-CreERT2 mouse refers to the insertion of CreERT2 behind the thyroid-specific promoter; TSHR flox / flox Mating mice with TG-CreERT2 mice can achieve thyroid TSHR knockout, that is, TSHR Tko Mouse model (abbreviated as TKO mouse). TSHR flox / flox The construction process of TG-CreERT2 mice is shown in Figure 3 .

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Materials and drugs used in the examples of the present invention

[0046] 1. Animal source: TSHR in experimental group flox / flox TG-CreERT2 mice and control TSHR flox / flox Mice were purchased from Saiye Biotechnology Co., Ltd. and raised in the SPF-level Animal Experiment Center of Gansu University of Chinese Medicine.

[0047] 2. Drug sources: Tamoxifen, product number: S1238, purchased from Shanghai Lanmu Chemical Co., Ltd.; levothyroxine (LT4), product number: HY-18341, purchased from MedChemexpress (MCE) Biotechnology Company.

[0048] 3. Kit: Mouse thyrotropin (TSH) enzyme-linked immunosorbent assay kit, catalog number: E-EL-M1153, purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0049] Example 1

[0050] 1. Thyroid function test:

[0051] In the early morning, blood was collected from the rat tail vein and centrifuged at 4°C for 15 min (3000 rpm) to separate serum and clots. The upper serum was carefully aspirated into a sterile centrifuge tube with a pipette to avoid disturbing the red blood cells or clots in the lower layer. The serum in the sterile centrifuge tube was centrifuged again at 4°C for 15 min (3000 rpm), and the upper serum was carefully aspirated into a new sterile centrifuge tube with a pipette and stored at -80°C for later use.

[0052] (1) Detection of serum TSH concentration by enzyme-linked immunosorbent assay

[0053] a. Preparation: 20 minutes before the test, take the test kit and serum samples out of the refrigerator and place them at room temperature (25°C); dilute the concentrated washing solution with double distilled water (1:24); centrifuge the standard at 10,000×g for 1 minute, add 1mL of standard & sample diluent, cover the tube and let it stand for 10 minutes, then mix thoroughly until the standard solution is dissolved to prepare a standard working solution of 80ng / ml, and continue to prepare the following concentrations of standard solutions according to the instructions: 40, 20, 10, 5, 2.5, 1.25, 0ng / mL; centrifuge the biotinylated antibody concentrate at 800×g for 1 minute, and then dilute it 100 times with biotinylated antibody diluent to a working concentration of 1×; centrifuge the HRP enzyme-conjugated concentrate at 800×g for 1 minute, and then dilute it 100 times with diluent to a working concentration of 1×.

[0054] b. Operation steps: Set up standard wells, blank wells and sample wells respectively. Add 100μL of standard solution to the standard wells, 100μL of standard solution & sample diluent to the blank wells, and 100μL of sample diluent to the remaining wells; then, cover with a plate film and incubate at 37°C for 90 minutes; after incubation, shake off the liquid in the wells without washing, then add 100μL of biotinylated antibody working solution to each well, and continue to incubate at 37°C for 1 hour; after the incubation, shake off the liquid and pat dry on absorbent paper, fill each well with washing solution, soak for 1 minute, then shake off and pat dry, repeat this washing process 3 times; then add 100μL to each well Add HRP enzyme conjugate working solution, cover with plate film, incubate at 37°C for 30 minutes, and repeat washing 5 times; finally, add 90 μL of substrate solution (TMB) to each well, cover with plate film, and incubate at 37°C in the dark for 15 minutes; turn on the microplate reader 15 minutes in advance for preheating. When a clear gradient appears in the first 4 color-developing wells of the standard well, add 50 μL of stop solution to each well. After the reaction is completed, quickly use the microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0055] c. Calculation of results: Based on the known concentration of the standard and its corresponding OD value, draw a standard curve and further calculate the linear regression equation; substitute the OD value of the sample to be tested into the equation to deduce the concentration of the sample; finally, after the corresponding dilution multiple conversion, the exact concentration of TSH in the sample can be obtained.

[0056] (2) FT4 concentration was detected by fully automatic immunoassay analyzer, catalog number: i2000SR, purchased from Abbott Laboratories.

[0057] Example 2 Model building process:

[0058] (1)TSHR Tko Mouse model: 8-week-old male SPF TSHR flox / flox TG-CreERT2 mice were given tamoxifen (40 mg / kg / d) intraperitoneally for 5 consecutive days (corn oil solvent was used as carrier). After 2 weeks of drug withdrawal, thyroid TSHR was completely knocked out ( Figure 6 ).

[0059] (2) Hypothyroidism mouse model:

[0060] a. Construction process of hypothyroidism mouse model: 8-week-old male SPF grade TSHR flox / flox TG-CreERT2 mice were given tamoxifen (40 mg / kg / d) by intraperitoneal injection for 5 consecutive days (corn oil solvent was used as carrier). After 2 weeks of drug withdrawal, blood samples were collected by tail cutting to detect thyroid function (serum TSH and FT4 levels).

[0061] b. Signs of successful construction of the hypothyroidism mouse model: The serum TSH and FT4 levels were detected. The TSH level was significantly higher than that of the control group mice, and the FT4 level was significantly lower than that of the control group mice, indicating that the hypothyroidism model was successfully constructed (Table 1).

[0062] (3) Subacute hypothyroidism mouse model:

[0063] a. Construction process of subacute hypothyroidism mouse model: 8-week-old male SPF grade TSHR flox / flox TG-CreERT2 mice were given 40 mg / kg / d of tamoxifen by intraperitoneal injection for 5 consecutive days (corn oil solvent was used as carrier), and LT was given 45 ug / kg / d by intraperitoneal injection for 7 consecutive days (corn oil solvent was used as carrier) after drug withdrawal for 1 week. The tail was cut off and blood was collected to detect thyroid function (serum TSH and FT4 levels).

[0064] b. Signs of successful construction of subacute hypothyroidism mouse model: Serum TSH and FT4 levels were detected. The TSH level was significantly higher than that of the control group mice, while the FT4 level was not significantly different from that of the control group mice, indicating that the subacute hypothyroidism model was successfully constructed (Table 2).

[0065] Table 1 Statistical analysis of serum TSH and FT4 levels in each group of mice

[0066]

[0067] Note: Compared with the baseline, # P<0.05, *P<0.01; compared with the control group at the same time point, @ P<0.05, & P<0.01; TSH, thyroid stimulating hormone; FT4, free thyroxin.

[0068] a The experimental group stopped taking Tamoxifen for 1 week

[0069] b The experimental group stopped taking Tamoxifen for 2 weeks

[0070] Table 2 Statistical analysis of serum TSH and FT4 levels in each group of mice

[0071]

[0072]

[0073] Note: Compared with the baseline, # P<0.05, *P<0.01; compared with the control group at the same time point,@ P<0.05, & P<0.01; TSH, thyroid stimulating hormone; FT4, free thyroxin.

[0074] a Experimental group T4 injection for one week

[0075] As can be seen from the above examples, the present invention provides a method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse, comprising the following steps: (1) obtaining TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and the hypothyroidism mouse model was obtained after 12 to 16 days of drug withdrawal; (2) TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and after 6 to 8 days of drug withdrawal, LT4 was given for 6 to 8 days to screen and obtain a subacute hypothyroidism mouse model. flox / flox TG-CreERT2 mice induce thyroid TSHR knockout, i.e. TSHR Tko Mouse model, can safely, quickly, efficiently and stably construct hypothyroidism and subacute hypothyroidism mouse models.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a hypothyroidism model mouse and a subacute hypothyroidism model mouse, characterized in that: The steps include: (1) Get TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and the hypothyroidism mouse model was obtained after 12 to 16 days of drug withdrawal; (2) Take TSHR flox / flox TG-CreERT2 mice were given tamoxifen for 4 to 6 days, and after stopping the drug for 6 to 8 days, LT4 was given for 6 to 8 days to screen out the subacute hypothyroidism mouse model.

2. The construction method according to claim 1, characterized in that: TSHR described in steps (1) and (2) flox / flox TG-CreERT2 mice were 7-9 week old male SPF grade mice.

3. The construction method according to claim 1, characterized in that: The dosage of tamoxifen in steps (1) and (2) is independently 30-50 mg / kg / d, and the tamoxifen is injected intraperitoneally using corn oil as a solvent.

4. The construction method according to claim 1, characterized in that: The screening method for the hypothyroidism mouse model in step (1) is: detecting the levels of TSH and FT4 in mouse serum, the TSH level is significantly higher than that of normal mice, and the FT4 level is significantly lower than that of normal mice, and the hypothyroidism model is successfully constructed.

5. The construction method according to claim 1, characterized in that: The dosage of LT4 in step (2) is independently 4-6ug / kg / d, and the LT4 is injected intraperitoneally using corn oil solvent as a carrier.

6. The construction method according to claim 1, characterized in that: The screening method for the subacute hypothyroidism mouse model in step (2) is: detecting the levels of TSH and FT4 in mouse serum. The TSH level is significantly higher than that of normal mice, and the FT4 level is not significantly different from that of normal mice. The subacute hypothyroidism model is successfully constructed.

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