Construction method and application of sterile Chinese hamster model
Through cesarean section and sterile ICR mouse milk replacement technology, the problem of lack of sterile Chinese gopher population was solved, and the sterile Chinese gopher population was successfully established for disease research, especially type 2 diabetes research.
Patent Information
- Application Number
- CN202510390685.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-05-13
AI Technical Summary
The current lack of sterile Chinese gopher populations has limited research on the relationship between intestinal flora and specific diseases, especially in the study of type 2 diabetes.
Chinese gopher milk mice to be purified were obtained through cesarean section, and sterile ICR mice were used for milk replacement to establish a sterile Chinese gopher population.
There were 9 Chinese gophers successfully, 5 males and 4 females, with a survival rate of 56%. The sterile status was confirmed through GB/T 14926.41-2001 detection, and the sterile Chinese gophers population was successfully established in China for the first time.
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Figure CN119969343A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of animal experiment model construction, and in particular relates to a construction method and application of a sterile Chinese hamster model. Background Art
[0002] Type 2 diabetes mellitus (T2DM) accounts for more than 90% of diabetes. Its occurrence is related to multiple factors such as genetics and environment. It has insulin resistance and β-cell damage as its pathological basis, and is mainly manifested as hyperglycemia. The specific pathogenesis is still unclear. Animal models are indispensable tools for studying the pathogenesis and pharmacodynamics of diabetes. There are many ways to construct T2DM animal models, among which the pathogenesis and metabolic characteristics of spontaneous T2DM animal models are similar to those of humans. As early as 1959, Chinese hamsters were first reported as a potential spontaneous hereditary diabetes model. Inbreeding in this group can spontaneously produce diabetes. Wang Lu et al. initially established a T2DM animal model group through selective inbreeding and observed that the fasting blood glucose of the animals increased significantly at 12 months of age and began to spontaneously develop T2DM-related symptoms (WANGL, WANG-C, ZHANG·R, et al. Phenotypic characterization of anovel type 2.diabetes animal model. in a SHANXI·MU·colony of Chinese hamsters [J]. Endocrine, 2019, 65(1): 61-72.). In recent years, some studies have shown that intestinal flora can affect sugar and lipid metabolism by regulating energy intake, and intestinal flora disorders can promote sugar and lipid metabolism disorders, which may be related to the occurrence and development of diabetes. Germ-free animals have the clearest microbial background and can effectively control environmental factors such as flora, genes and diet. They are one of the most effective animal models for studying the effects of gut microbiota (GM) on human health and disease. However, there are no reports on germ-free Chinese hamster populations, and there is a lack of Chinese hamster models for studying the relationship between gut flora and specific diseases. Therefore, the development of more diverse T2DM animal models will not only help to study the pathogenesis of T2DM, but also provide new model materials for its treatment-related research. Summary of the invention
[0003] In view of this, the purpose of the present invention is to provide a method for constructing a sterile Chinese hamster model and its application, using cesarean section to obtain suckling mice to be purified, using sterile-grade ICR mice as milk substitutes to biologically purify Chinese hamsters, and establishing a sterile Chinese hamster population.
[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0005] The present invention provides a method for constructing a sterile Chinese hamster model, comprising the following steps:
[0006] 1) The SPF-grade Chinese hamsters were mated together in cages of male and female, and the uterus was removed when the pregnant Chinese hamsters were about to give birth. The hamsters were placed in a purification isolator, and the uterus was removed to obtain the Chinese hamster sucklings to be purified;
[0007] 2) Mating sterile-grade ICR mice of both sexes in the same cage to obtain ICR female mice after delivery;
[0008] 3) Use ICR mother mice after delivery to provide milk for the Chinese hamster pups to be purified, and obtain the sterile Chinese hamster model after successful weaning.
[0009] Preferably, the aseptic-grade ICR mice in step 2) are housed 1 to 2 days earlier than the SPF-grade Chinese hamsters.
[0010] Preferably, in step 1), the number of SPF-grade Chinese hamsters in a cage is 24 to 36, with a male-female ratio of 1:1.
[0011] Preferably, the hysterectomy in step 1) is performed during the gestation period of SPF-grade Chinese hamsters for 19 to 21 days.
[0012] Preferably, the number of the sterile-grade ICR mice is 16 to 24, with a male-female ratio of 1:1.
[0013] Preferably, the time of the substitute milk in step 3) is 1 day after the ICR mother mouse gives birth.
[0014] The present invention also provides application of the sterile Chinese hamster model constructed by the construction method in disease research.
[0015] Preferably, the disease comprises type 2 diabetes.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The invention increases the number of animals, accurately calculates the gestation period of ICR mice and Chinese hamsters, optimizes the cage time, shortens the time difference of delivery, selects the surrogate mother mouse with the closest delivery time for surrogate milk, successfully purifies the surrogate milk by the second cesarean section, successfully weans 9 Chinese hamsters, including 5 males and 4 females, with a weaning survival rate of 56%, and all are qualified in the sterile Chinese hamster state detection according to GB / T 14926.41-2001. The invention utilizes the cesarean section purification technology, uses the sterile ICR mice for surrogate milk, successfully weans Chinese hamsters, and successfully establishes the sterile Chinese hamster population in China for the first time. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 It is a technical roadmap for cesarean section purification;
[0019] Figure 2 are germ-free Chinese hamsters of different ages;
[0020] Figure 3 It is the cecum of the sterile Chinese hamster;
[0021] Figure 4 It is the lactation situation of germ-free Chinese hamsters;
[0022] Figure 5 This is the Chinese hamster sterility test report. DETAILED DESCRIPTION
[0023] The present invention provides a method for constructing a sterile Chinese hamster model, comprising the following steps:
[0024] 1) The SPF-grade Chinese hamsters were mated together in cages of male and female, and the uterus was removed when the pregnant Chinese hamsters were about to give birth. The hamsters were placed in a purification isolator, and the uterus was removed to obtain the Chinese hamster sucklings to be purified;
[0025] 2) Mating sterile-grade ICR mice of both sexes in the same cage to obtain ICR female mice after delivery;
[0026] 3) Use ICR mother mice after delivery to provide milk for the Chinese hamster pups to be purified, and obtain the sterile Chinese hamster model after successful weaning.
[0027] In the present invention, male and female SPF Chinese hamsters are caged together for mating, and a hysterectomy is performed when the pregnant Chinese hamster is about to give birth, and the hamster is transferred into a purification isolator, and the uterus is peeled off to obtain the Chinese hamster sucklings to be purified. The number of SPF Chinese hamsters in the cage is preferably 24 to 36, more preferably 26 to 34, and the male-female ratio is preferably 1:1; the time of the hysterectomy is preferably 19 to 21 days during the gestation period of the SPF Chinese hamster pregnant mouse; the environmental temperature of the hysterectomy is preferably 36 to 38°C, more preferably 37°C.
[0028] In the present invention, sterile-grade ICR mice are mated together to obtain ICR female mice after delivery. The number of the sterile-grade ICR mice is preferably 16 to 24, more preferably 18 to 22, and the male-female ratio is preferably 1:1; the sterile-grade ICR mice are caged 1 to 2 days earlier than the SPF-grade Chinese hamsters.
[0029] In the present invention, the ICR mother mice after delivery are used to replace the milk of the Chinese hamster milk mice to be purified, and the sterile Chinese hamster model is obtained after successful weaning. The time of the replacement milk is preferably 1 day after the ICR mother mice give birth.
[0030] The present invention also provides the application of the sterile Chinese hamster model constructed by the construction method in disease research. In the present invention, the disease includes type 2 diabetes.
[0031] 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.
[0032] Example 1
[0033] 1. Experimental Animals
[0034] 50 SPF Chinese hamsters aged 8-10 weeks, 25 males and 25 females. Purchased from Beijing Huafukang Biotechnology Co., Ltd. [SCXK (Beijing) 2024-0003]; 40 sterile ICR mice, 20 males and 20 females, bred by our laboratory, and kept in a special sterile isolator of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences [SYXK (Beijing) 2023-0037]. During the feeding period, the animals freely consumed customized sterile hamster feed sterilized by 50kGy 60Coγ-ray irradiation, provided by Keao Xieli (Tianjin) Feed Co., Ltd. [SCXK (Tianjin) 2020-0004]. Free drinking water, drinking water was sterilized at 132℃ for 1h. Feeding environment: temperature 21-25℃, humidity 40%-70%, light-dark ratio of 12h:12h. This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animals, Chinese Academy of Medical Sciences, with approval number ZH23004.
[0035] 2. Main reagents and instruments
[0036] Peracetic acid disinfectant (Beijing Xidebao Disinfection Products Co., Ltd., HY-XDB-500), surgical isolator (Beijing Huanyu Zhongke Purification Engineering Co., Ltd., 1160cm×650cm×600cm), high-pressure steam sterilizer (TOMY, SX-700), clean bench (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., ZHJH-C1118C).
[0037] 3. Experimental methods
[0038] Biological purification is the only economical and effective method to establish a high-grade animal population. The technology currently used in China is cesarean section purification, which is to obtain suckling mice through hysterectomy and then use the same or different species of animals as milk substitutes. Figure 1 .
[0039] First cesarean section cleansing:
[0040] (1) Animal preparation
[0041] For surrogate mothers, sterile ICR mice with good maternal instincts, healthy and non-eating offspring were selected; for donor mothers, SPF-grade Chinese hamsters with 2 to 3 litters were selected, healthy and disease-free. The gestation period of sterile ICR mice is 19 to 21 days, and the gestation period of SPF-grade Chinese hamsters is 19 to 21 days. The time of ICR mice and Chinese hamsters being put into the cage was accurately calculated according to their gestation period. 20 Chinese hamsters and 20 ICR mice were selected, and the ratio of male to female was 1:1. The date of the plugging was recorded, and the male mice were kept separately after the plugging was seen. The time of putting surrogate mothers into the cage was 2 days earlier than that of the donor mothers to ensure that the surrogate mothers gave birth in advance and were well prepared for surrogate milk.
[0042] (2) Preoperative preparation
[0043] Sterilize the cesarean section purification isolation bag with 2% peracetic acid solution and keep it ventilated for 48 hours before use. The ambient temperature for cesarean section should reach 36-38℃. Connect the U-shaped aqueduct to the transfer port of the isolation bag, prepare 2% peracetic acid solution with 37℃ distilled water, and pour it into the U-shaped aqueduct. Make sure to prepare a set of surgical instruments for each pregnant mouse, and sterilize them at 121℃ for 60 minutes before use.
[0044] (3) Cesarean section
[0045] When the gestation period of Chinese hamsters is 19-21 days, close observation begins. When the pregnant mice show loss of appetite, restlessness, sitting behavior, palpation of their pubic symphysis to an open state, and flushing, swelling, and a small amount of secretions on the vulva, the pregnant mice are in the state of labor and can be performed by cesarean section and hysterectomy. Pregnant mice killed by cervical dislocation are placed in a 2% peracetic acid solution and soaked for 3 seconds. They are placed on a dissecting board and their limbs and head are fixed. The abdomen of the pregnant mice is disinfected with iodine cotton balls against the hair, and the abdomen is cut open along the midline of the abdomen to fully expose the uterus. The cervix and bilateral uterine horns are clamped with hemostats, and the uterus is cut off on the outside of the cervix to separate the uterus. The uterus is passed into a purification isolation bag through a U-shaped aqueduct filled with 37℃ 2% peracetic acid solution. After using sterile water to rinse the residual peracetic acid on the surface of the uterus, it is placed on gauze, the uterus and amniotic membrane are torn open to remove the suckling mouse, and the amniotic membrane and amniotic fluid around the nose and mouth of the suckling mouse are quickly wiped off. Healthy mice begin to breathe with their mouths open. For mice that are not breathing, gently and rhythmically press their chests to stimulate their breathing. Or use tweezers to gently pinch the base of their tails to make them make sounds and induce breathing. The color of mice that begin to breathe will gradually change from purple to bright red.
[0046] Pay attention to the details during the operation: use sufficient force when killing pregnant mice by cervical dislocation to avoid injuring the fetus; when selecting donor mice, be careful not to carry pathogens that can be vertically transmitted through the placental barrier; during the experimental operation, strictly perform aseptic operations.
[0047] (4) Milk substitute
[0048] After the cesarean section, select the large suckling mice without external injuries during the operation, mix them with the feces, urine, bedding, etc. of the suckling mother mouse, and put them in the original box of the suckling mother mouse. The ICR mother mouse will suckle the suckling mice 1 day after delivery, and keep 1 to 2 pups. Observe the crescent-shaped white milk on the abdomen of the suckling mice delivered by cesarean section, indicating that the suckling mother mouse accepts the suckling milk. After the Chinese hamster suckling mice open their eyes, observe the suckling milk at any time, and separate them from the ICR suckling mother mouse in advance and feed them with soft food artificially until weaning.
[0049] Experimental results: as shown in Table 1.
[0050] Table 1. Milk replacement status of ICR mothers after the first cesarean section
[0051]
[0052] The first cesarean section yielded 4 suckling mice, which were mixed and then fed with 5 sterile ICR mother mice. All the Chinese hamster suckling mice died on the second day, and the purification failed. The reasons for the failure were: ① ICR mice and Chinese hamsters are two species. ICR mice belong to the order Rodentia, the family Muridae, and the genus Mouse. Chinese hamsters belong to the order Rodentia, the family Cricetidae, and the genus Hamster. There are individual differences between Chinese hamsters and ICR suckling mice. ② All 5 suckling mother mice gave birth at the 19th day of pregnancy. Due to the lack of experience in breeding Chinese hamsters, only 10 pairs were caged together. Chinese hamsters are ferocious and difficult to raise. After the cages were combined, most male mice were bitten to death or injured. One mother mouse successfully conceived and gave birth at the 21st day of pregnancy. This resulted in a 4-day age difference between ICR suckling mice and Chinese hamster suckling mice, missing the best suckling time, causing the ICR suckling mother mice to refuse suckling, and the purification failed.
[0053] Second cesarean section purification:
[0054] (1) Animal preparation
[0055] For surrogate mothers, sterile ICR mice with good maternal instincts, healthy and non-eating offspring were selected; for donor mothers, SPF-grade Chinese hamsters with 2 to 3 litters were selected, healthy and disease-free. The gestation period of sterile ICR mice is 19 to 21 days, and the gestation period of SPF-grade Chinese hamsters is 19 to 21 days. The time for ICR mice and Chinese hamsters to be caged together was accurately calculated based on their gestation period. 30 Chinese hamsters and 20 ICR mice were selected, and the ratio of male to female was 1:1 for caged together. The date of the plugging was recorded, and the surrogate mice were raised separately from the male mice after the plugging was seen. The time for the surrogate mothers to be caged together should be 1 day earlier than the donor mothers to ensure that the surrogate mothers gave birth in advance and were well prepared for surrogate milk.
[0056] Steps (2), (3) and (4) are the same as for the first cesarean section cleansing.
[0057] Experimental results: as shown in Table 2.
[0058] Table 2 Milk replacement status of ICR mothers after the second cesarean section
[0059]
[0060] The second cesarean section operation yielded 18 suckling mice, 2 of which died after cesarean section. The remaining 16 suckling mice were mixed with milk and then fed by 7 sterile ICR mother mice. The cesarean section was successful in purifying the milk substitute, and 9 Chinese hamsters were successfully weaned, including 4 females and 5 males (such as Figure 2 shown).
[0061] (5) SPF-grade Chinese hamsters and successfully purified sterile Chinese hamsters were dissected and the size of the cecum was observed.
[0062] Experimental results: Figure 3 As shown in Figure 2, the cecum of the sterile Chinese hamster was huge during dissection.
[0063] (6) Production and breeding testing of sterile Chinese hamsters
[0064] When the sterile Chinese hamsters were weaned, they were separated into cages at a ratio of 1:1 between male and female. At 12 to 14 weeks of age, 2 of the 4 female hamsters successfully became pregnant, with female 1 giving birth to 4 pups and female 2 giving birth to 6 pups, resulting in 9 Chinese hamsters successfully weaned. Figure 4 ).
[0065] Table 3 Reproduction of germ-free Chinese hamsters
[0066]
[0067] (7) Maintenance and detection of sterility in Chinese hamsters
[0068] The successfully weaned sterile Chinese hamsters are transferred to isolation bags for breeding. Every month, feces samples, feed, bedding and drinking water of the Chinese hamsters are collected and sent to the China Food and Drug Inspection Institute for sterility testing, and a test report is issued.
[0069] Experimental results: Figure 5 As shown. A total of 9 Chinese hamsters were successfully weaned, 4 females and 5 males. Fecal samples of Chinese hamsters were collected and tested for sterility according to GB / T 14926.41-2001, and the test results were qualified.
[0070] As can be seen from the above embodiments and experimental examples, the present invention increases the number of animals, accurately calculates the gestation period of ICR mice and Chinese hamsters, optimizes the cage time, shortens the time difference of delivery, selects the surrogate mother mouse with the closest delivery time for surrogate milk, and successfully purifies the surrogate milk by cesarean section for the second time, successfully weaning 9 Chinese hamsters, 5 males and 4 females, and the weaning survival rate is 56%. According to GB / T14926.41-2001, the state detection of sterile Chinese hamsters is qualified. The present invention utilizes cesarean section purification technology, uses sterile ICR mice for surrogate milk, successfully weaning Chinese hamsters, and successfully establishes a sterile Chinese hamster population for the first time in China.
[0071] 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 sterile Chinese hamster model, characterized in that: The following steps are involved: 1) The SPF-grade Chinese hamsters were mated together in cages of male and female, and the uterus was removed when the pregnant Chinese hamsters were about to give birth. The hamsters were placed in a purification isolator, and the uterus was removed to obtain the Chinese hamster sucklings to be purified; 2) Mating sterile-grade ICR mice of both sexes in the same cage to obtain ICR female mice after delivery; 3) Use ICR mother mice after delivery to provide milk for the Chinese hamster pups to be purified, and obtain the sterile Chinese hamster model after successful weaning.
2. The construction method according to claim 1, characterized in that: Step 2) The aseptic-grade ICR mice are housed 1 to 2 days earlier than the SPF-grade Chinese hamsters.
3. The construction method according to claim 1, characterized in that: Step 1) The number of SPF Chinese hamsters in a cage is 24 to 36, with a male-female ratio of 1:
1.
4. The construction method according to claim 1, characterized in that: The hysterectomy in step 1) is performed at a time of 19 to 21 days during the gestation period of the SPF Chinese hamsters.
5. The construction method according to claim 1, characterized in that: Step 2) The number of the sterile-grade ICR mice is 16 to 24, with a male-female ratio of 1:
1.
6. The construction method according to claim 1, characterized in that: Step 3) The time for the substitute milk is 1 day after the ICR mother mouse gives birth.
7. Application of the germ-free Chinese hamster model constructed by the construction method according to any one of claims 1 to 6 in disease research.
8. The use according to claim 7, characterized in that: Such diseases include type 2 diabetes.