Method for constructing mouse hypothyroidism model

Abstract

The invention discloses a method for constructing a mouse hypothyroidism model, which comprises the following steps: injecting 131I into the abdominal cavity of a mouse after low-iodine diet for 2 weeks, and forming the model after one week, the mouse being a mouse with normal thyroidism. According to the application, the mouse with normal thyroid function is subjected to low-iodine diet for 2 weeks and then injected with a specific dose of 131I through the abdominal cavity, so that all thyroid tissues of the mouse can be removed after one week, and the application has the advantage of being capable of completely removing all thyroid tissues of the mouse with normal thyroid function.

Description

Technical Field

[0001] The present invention relates to the technical field of construction methods of animal models, and more specifically, to a method for constructing a hypothyroidism model in mice. Background Art

[0002] Thyroid cancer is the most common endocrine malignancy (accounting for 95%), and the incidence of thyroid cancer has increased extensively in the past 30 years. In the treatment of thyroid cancer, surgery is mainly used to remove cancerous foci, and radioactive iodine therapy can treat metastatic thyroid cancer foci that cannot be resected.

[0003] In the related art, radioactive iodine therapy is mainly applied to reduce the recurrence rate of patients with medium- and high-risk differentiated thyroid cancer. However, the uptake of radioactive iodine by normal thyroid follicular cells and thyroid cancer cells is different. Currently, there is no consensus on the dose and time of 131 I ablation therapy in mice.

[0004] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0005] The technical task of this application is to address the above deficiencies by providing a method for constructing a hypothyroidism model in mice. In this application, mice with normal thyroid function are given a low-iodine diet for 2 weeks and then intraperitoneally injected with a specific dose of 131 I. After one week, all thyroid tissues of the mice can be cleared, which has the advantage of completely clearing all thyroid tissues of mice with normal thyroid function.

[0006] To achieve the above object, this application provides the following technical solutions:

[0007] This application provides a method for constructing a hypothyroidism model in mice, including: after feeding the mice a low-iodine diet for 2 weeks, intraperitoneally injecting 131 I into the mice. After one week, the model can be established, where the mice are mice with normal thyroid function.

[0008] In some embodiments, the 131 activity of I is 10 uCi / g of mouse body weight.

[0009] In some embodiments, the mice are C57BL / 6 mice.

[0010] In some embodiments, the iodine content of the feed for the low-iodine diet is 0.01% or less.

[0011] Compared with the prior art, the advantages and positive effects of the present application are as follows: After 2 weeks of low-iodine diet for mice with normal thyroid function and then intraperitoneal injection of a specific dose of 131 I, all thyroid tissues of the mice can be removed after one week, solving the problem in the related art that the dosages and times for removing thyroid tissues are not unified only for diseased organisms, and having the advantages of being able to clarify the dosage and time for completely removing all thyroid tissues of mice with normal thyroid function. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0013] Figure 1 Show the 99m TcO 4 - Whole-body SPECT / CT imaging pictures (A is the control group, B is the low-iodine diet group, from left to right are transverse, coronal, and sagittal views);

[0014] Figure 2 Show the 99m TcO 4 - Whole-body SPECT / CT imaging pictures (A is the control group, B is the low-iodine diet group, from left to right are transverse, coronal, and sagittal views);

[0015] Figure 3 Show the 131 I whole-body SPECT / CT imaging pictures (A is the control group, B is the low-iodine diet group, from left to right are transverse, coronal, and sagittal views);

[0016] Figure 4 Show the bar chart of TSH levels in an embodiment of the present application;

[0017] Figure 5 Show the bar chart of urinary iodine levels in an embodiment of the present application;

[0018] Figure 6 Show the bar chart of radioactive counts in an embodiment of the present application;

[0019] Figure 7Show the HE staining pictures of thyroid tissue in an embodiment of the present application (A is the control group, B is the low-iodine diet group, the microscope scale bars are both 500um, and the magnification is 50X; a and b are the magnified images within the boxes in Figures A and B respectively, the microscope scale bars are both 100um, and the magnification is 200X). Detailed implementation manners

[0020] In order to more clearly understand the above objects, features and advantages of the present application, the following further describes the present application with reference to the drawings and embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0021] The following further describes the present application with reference to the drawings and specific embodiments.

[0022] 1. Experimental animals and grouping

[0023] (1) Experimental animals:

[0024] C57BL / 6 mice, at the SPF level, female, with a body weight of 17 - 18g. The mice are fed 5ml of Wahaha drinking pure water per day and 5g of mouse food (iodine content 310μg / kg) per day, that is, the total iodine intake of the mice per day is 1.55μg, and they are adapted for 1 week.

[0025] (2) Grouping:

[0026] The mice are randomly divided into a control group and a low-iodine diet group, with 5 mice in each group. The mice in the control group are given normal feed and fed Wahaha drinking pure water, and the iodine content of the normal feed is 310μg / kg. The mice in the low-iodine diet group are given continuous low-iodine diet for 2 weeks. The low-iodine diet feed is specially processed, and the iodine content is below 0.01%. After 2 weeks, the mice in the low-iodine diet group resume normal diet and drinking water.

[0027] 2. SPECT / CT imaging of mice

[0028] All mice are scanned and image collected through the InliView-3000B animal PET / SPECT / CT triple-mode machine provided by Beijing Yongxin Medical Equipment Co., Ltd. Before each mouse is scanned, it is first placed in a gas anesthesia machine for pre-anesthesia. After the mouse is anesthetized, it is moved to the examination bed and positioned, and gas anesthesia is performed throughout the examination process. The following parameters are used for acquisition: acquisition duration per frame: 10s, large field-of-view collimator, step angle 3.0, OSEM algorithm, Gaussian filtering, 120×120 image matrix, and 40 iterations.

[0029] The treatment methods for the mice are as follows:

[0030] Treatment 1: The mice in the low-iodine diet group were continuously fed a low-iodine diet for 2 weeks, and the mice in the control group were fed a normal diet for 2 weeks. After 2 weeks, both groups of mice were intraperitoneally injected with 99m TcO 4 - 600 μCi (22.2 MBq) (for imaging), and whole-body SPECT / CT imaging was performed 30 minutes later.

[0031] Treatment 2: The mice in the low-iodine diet group were continuously fed a low-iodine diet for 2 weeks, and the mice in the control group were fed a normal diet for 2 weeks. After 2 weeks, the mice in the low-iodine diet group were intraperitoneally injected with 131 I (10 μCi / g of mouse body weight) 0.1 ml, and the mice in the control group were intraperitoneally injected with the same volume of normal saline. One week later, they were intraperitoneally injected with 99m TcO 4 - 600 μCi (22.2 MBq) (for imaging), and whole-body SPECT / CT imaging was performed 30 minutes later.

[0032] Treatment 3: The mice in the low-iodine diet group were continuously fed a low-iodine diet for 2 weeks, and the mice in the control group were fed a normal diet for 2 weeks. After 2 weeks, the mice in the low-iodine diet group were intraperitoneally injected with 131 I (10 μCi / g of mouse body weight) 0.1 ml, and the mice in the control group were intraperitoneally injected with the same volume of normal saline. One week later, they were intraperitoneally injected with 50 μCi (1.85 MBq) of 131 I (for imaging), and whole-body SPECT / CT imaging was performed 2 hours later.

[0033] After SPECT / CT imaging of each mouse, the ROI of the thyroid tissue was outlined, the radioactive counts were read, and the back muscle tissue of the same area was taken as the background ROI.

[0034] Figures 1-3 The whole-body SPECT / CT imaging pictures of Treatments 1-3 in the embodiments of the present application are respectively shown. Among them, A is the control group, and B is the low-iodine diet group. In the pictures, from left to right are the transverse position, coronal position, and sagittal position. The white arrow in the sagittal picture indicates the position of the thyroid gland. As Figure 1 shown, whether it is a normal diet or a low-iodine diet, normal thyroid tissue imaging can be seen. As Figure 2 shown, normal thyroid tissue imaging can be seen in the mice of the control group, and no normal thyroid tissue imaging can be seen in the mice of the low-iodine diet group. As Figure 3 shown, normal thyroid tissue imaging can be seen in the mice of the control group, and no normal thyroid tissue imaging can be seen in the mice of the low-iodine diet group.

[0035] Table 1 shows the target-to-background ratio of the thyroid ROI of the mice in Treatments 1-3 in the embodiments of the present application.

[0036] Table 1. Mouse thyroid ROI target-to-background ratio

[0037]

[0038] As shown in Table 1, at 2 weeks of low-iodine diet, the target-to-background ratios of normal diet and low-iodine diet for 99m TcO 4 - were statistically analyzed and no significant difference was found. That is, whether it is a normal diet or a low-iodine diet, it does not affect 99m TcO 4 - uptake. At 1 week after ablation therapy, the 99m TcO 4 - target-to-background ratio and 131 I target-to-background ratio in the low-iodine diet group were both lower than those in the normal diet group. Statistical analysis found a significant difference. The 99m TcO 4 - target-to-background ratio and 131 I target-to-background ratio were both relatively low, basically approaching the background level, indicating successful ablation because the thyroid was successfully cleared, 99m TcO 4 - so the target-to-background ratio and 131 I target-to-background ratio were relatively low and close to the background level.

[0039] 3. Detection of mouse thyroid-stimulating hormone (TSH)

[0040] After continuously feeding the mice in the low-iodine diet group with low-iodine diet feed for 2 weeks and feeding the control group of mice with normal feed for 2 weeks, blood was taken from the medial canthus. And after ablation therapy for 1 week, blood was taken from the medial canthus of both groups of mice. The TSH level was measured by chemiluminescence immunoassay, and the detection range was 0.008 - 150.000 mU / L. Table 2 shows the TSH levels in the examples of this application. As shown in Table 2, at 1 week after ablation therapy, the TSH level in the low-iodine diet group was higher than that in the normal diet group, and there was a significant difference.

[0041] Table 2 Time-TSH levels (mU / L) of two groups of mice

[0042]

[0043]

[0044] Figure 4 shows the bar chart of TSH levels in the examples of this application. By Figure 4It can be seen that when the TSH levels of mice in the normal diet group and the low-iodine diet group were statistically analyzed at 2 weeks of low-iodine diet, there was no statistical difference, indicating that whether it was a normal diet or a low-iodine diet, iodine in food did not affect thyroid function. However, at 1 week after ablation therapy, there was a statistical difference in the TSH levels of the two groups of mice, and the TSH level of the low-iodine diet group of mice increased because 131 I has entered the thyroid tissue and caused a scavenging effect on the thyroid tissue, suggesting that a low-iodine diet is more conducive to clearing the thyroid tissue.

[0045] 4. Urinary iodine detection in mice

[0046] Each time urine was collected, mice in the same group were placed in an empty cage. After urination, urine was collected with a syringe, and urine was collected for about 2 - 3 days. Since the urine volume of mice is small and difficult to collect, in order to meet the measurement requirements, a method of multiple collections and pooling between groups was adopted. A urinary iodine quantitative detection kit produced by Wuhan Zhongsheng Biochemical Technology Co., Ltd. was used to detect urinary iodine and calculate the urinary iodine level.

[0047] Table 3 shows the urinary iodine levels at different times in the examples of the present application. Figure 5 shows the urinary iodine graph of mice at different times in the examples of the present application. From Table 3 and Figure 5 it can be seen that the urinary iodine level of the low-iodine diet group of mice was lower at 2 weeks of low-iodine diet, with a statistical difference from the normal diet group; after 1 week of resuming a normal diet, the urinary iodine level returned to a level similar to that of the normal diet. The iodine level in the normal body is reflected by the urinary iodine level. This result indicates that the urinary iodine level of the low-iodine diet is lower than that of the normal diet group of mice and is statistically significant, indicating that the low-iodine diet model was successfully established, and when the low-iodine diet group of mice resumed a normal diet for 1 week, the urinary iodine level in the body had already returned to the iodine level of the normal diet.

[0048] Table 3. Urinary iodine levels of two groups of mice at different times (μg / L)

[0049]

[0050]

[0051] 5. Thyroid radioactivity measurement in mice

[0052] 131 One week after ablation therapy with 131I 50 μCi (1.85 MBq). After 24 hours, approximately 0.5 ml of 10% chloral hydrate solution was intraperitoneally injected. After the mice died approximately 5 minutes later, the skin of the mice's necks was cut open along the midline of the neck with ophthalmic scissors. The submandibular glands were lifted and moved upward along the fascia to expose the thyroid cartilage and trachea. The muscles and fascia tissues on the ventral surface of the thyroid cartilage were carefully cut open to completely expose the thyroid cartilage. The cartilage and trachea were spread apart and cut at two places, one from the laryngeal direction and the other from the tracheal direction. The cut tissues were placed into a 1.5 ml centrifuge tube, and the centrifuge tube was quickly placed into a gamma counter to detect the radioactive count.

[0053] Table 4 shows the time-radioactivity of the thyroid tissues of two groups of mice in the embodiments of the present application. Figure 6 It shows the radioactive counts of the normal diet group and the low-iodine diet group of mice one week after the ablation treatment of the mice. As shown in Table 4 and Figure 6 shown, one week after the ablation treatment, the radioactive counts of the mice in the low-iodine diet group were very low, approaching the background level, indicating that the ablation of the mice was successful after one week. The iodine in the body (including 131 I) is mainly taken up by the thyroid tissue. If the thyroid tissue has been completely removed and there is no thyroid tissue taking up 131 I, the radioactive count of the thyroid is very low, approaching the background level of the surrounding environment.

[0054] Table 4. Time-radioactivity of the thyroid tissues of two groups of mice

[0055]

[0056] 6. HE staining of thyroid tissue

[0057] 6.1 Preparation of paraffin specimens

[0058] The mice were sacrificed one week after the ablation treatment with 131 I. The thyroid tissues were removed and fixed with formalin for at least 24 hours. Dehydration with gradient ethanol, clearing with xylene, embedding in paraffin, and cryosectioning (serial sections were cut from the laryngeal direction to the tracheal direction, with a distance of 200 μm between each section, a section thickness of 5 μm, mounted on glass slides, and baked in an oven at 60 °C for 1 hour for standby).

[0059] 6.2 HE staining

[0060] Put the sections into xylene I for 20 min and then into xylene II for 20 min for dewaxing in sequence; immerse them into ethanol with gradient concentrations for hydration in sequence: absolute ethanol I for 10 min, absolute ethanol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and rinse with distilled water. Then put the sections into hematoxylin for staining for 3 - 8 min and rinse with tap water; differentiate with 1% hydrochloric acid alcohol for 5 s and continue to rinse with tap water; blue with 0.6% ammonia water and rinse with running water; stain with eosin for 1 - 3 min. Dehydrate with gradient ethanol: 95% alcohol I for 5 min, 95% alcohol II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min for clearing. Then take out the sections from xylene, air-dry them, and seal the sections with neutral gum.

[0061] Figure 7 Showing the mouse in the embodiment of the present application 131 HE staining pictures of thyroid tissues of mice after 1 - week iodine-depleting therapy in the embodiment of the present application (A. Thyroid tissues of mice in the control group; B. Thyroid tissues of mice in the low-iodine diet group. The microscope scale bars are all 500 μm, and the magnification is 50X; a and b are the magnified images within the boxes in Figures A and B respectively. The microscope scale bars are all 100 μm, and the magnification is 200X). It can be Figure 7 seen that the thyroid follicular structure of mice in the control group is complete, with normal and relatively uniform size, rich colloid in the follicular cavity, and most of the epithelial cells being single-layered cuboid. No obvious thyroid follicular cells are seen in the low-iodine diet group.

[0062] ** in the above table and drawings indicates that there are significant differences in comparison between the low-iodine diet group and the normal diet group (control group), P < 0.01.

[0063] Through the above specific embodiments, those skilled in the art of the present application can easily implement the present application. However, it should be understood that the present application is not limited to the above specific embodiments. Based on the disclosed embodiments, those skilled in the art of the present application can arbitrarily combine different technical features to thus implement different technical solutions.

Claims

1. A method for constructing a mouse hypothyroidism model, characterized in that: include: Mice were given a low-iodine diet for 2 weeks and then injected intraperitoneally 131 I, the model can be formed after one week, wherein the mice have normal thyroid function.

2. The method for constructing a mouse hypothyroidism model according to claim 1, characterized in that: Said 131 The activity of I is 10uCi / g mouse body weight.

3. The method for constructing a mouse hypothyroidism model according to claim 1, characterized in that: The mice are C57BL / 6 mice.

4. The method for constructing a mouse hypothyroidism model according to claim 1, characterized in that: The iodine content of the feed in the low-iodine diet is below 0.01%.

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