Method for constructing an animal model of kidney stones based on UOX gene knockout mice

Abstract

The present invention relates to the technical field of animal models and their application, and discloses a method for constructing a kidney stone animal model based on UOX gene knockout mice, comprising: ‑ / ‑ The acquisition of mice; identification of hyperuricemia mouse model; construction of hyperuricemia kidney stone mouse model and its identification scheme. The specific steps of the method are to purchase UOX ‑ / ‑ Male C57BL / 6 mice were used, and the hyperuricemia mouse animal model was identified by serum uric acid detection combined with gene knockout reporter. The hyperuricemia kidney stone model was constructed by giving mice NH4CI free drinking water and benzbromarone gavage for 3 weeks. The success of the hyperuricemia kidney stone mouse model was comprehensively determined by color Doppler ultrasound of the mouse urinary system, urine crystal detection and observation of kidney tissue pathological sections. This model can better simulate the production and metabolic pathways of uric acid in the human body, and provide a good and stable model for studying the mechanism of action of a certain drug or a certain target on hyperuricemia kidney stones.

Description

Technical Field

[0001] The invention relates to the technical field of animal models and their application, and more specifically, to a method for constructing a kidney stone animal model based on UOX gene knockout mice. Background Art

[0002] Hyperuricemia (HUA) is mainly caused by increased blood uric acid due to reduced uric acid excretion or purine metabolism disorder. The incidence of HUA is increasing year by year, and 26-40% of patients with kidney stones have hyperuricemia. HUA is a high-risk factor for kidney stones. With the changes in human diet and living habits, the incidence of kidney stones will further increase, which will seriously endanger human health. Therefore, actively and effectively controlling blood uric acid levels can reverse the causes of kidney stone formation and reduce the recurrence rate, which is of positive significance.

[0003] In evolution, the gene encoding urate oxidase (UOX) in humans has been silenced and inactivated (therefore the risk of hyperuricemia is greatly increased), while the gene encoding urate oxidase in rodents is normally expressed, and UOX can break down uric acid into allantoin and excrete it from the body. This feature makes it difficult to replicate human hyperuricemia in rodents. Therefore, genetic engineering technology is used to make animals produce uricase gene deletion, obtain UOX-deficient mice, and create a hyperuricemia model. This model can better simulate the production and metabolic pathways of uric acid in humans, and provide a good and stable model for studying the mechanism of action of a certain drug or a certain target on hyperuricemia or hyperuricemia kidney stone model. Summary of the invention

[0004] In order to overcome the above-mentioned defects existing in the prior art, the present invention provides a method for constructing an animal model of kidney stones based on UOX gene knockout mice, which can better simulate the production and metabolic pathways of uric acid in humans, and provide a good and stable model for studying the mechanism of action of a certain drug or a certain target on hyperuricemia or hyperuricemia kidney stone model.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions: A method for constructing a kidney stone animal model based on UOX gene knockout mice comprises the following steps:

[0006] S1. Selection of mice: Select healthy, mentally healthy UOX mice without trauma or disability. - / - Male C57BL / 6 mice were used as experimental animals and were adaptively raised;

[0007] S2. Identification of mice: After one week of feeding, 0.2 ml of blood samples were collected from mice, and serum uric acid was tested;

[0008] S3. Feeding of mice: Two weeks of recovery feeding were conducted, during which mice were given standard feed, appropriate amount of melon seeds and clean drinking water. Drinking water, bedding and feed were changed every day, and the life and health of the mice were observed;

[0009] S4. Mouse modeling: The mice were given standard feed, free drinking water with a well-proportioned NH4CI concentration (0.25%) and a dose of 5 mg kg -1 The mice were treated with benzbromarone by gavage for 3 weeks, and the drinking water, bedding and feed were changed every day and the life and health conditions of the mice were observed.

[0010] S5. Model identification, including:

[0011] 1) After the mice have completed the three-week treatment, the urinary system of the mice is examined abdominally using an ultrasound imager to observe the kidneys of the mice. If there are stones or urine salt deposits, the model is successfully identified;

[0012] 2) If no stones or urine salt deposits are found in the mouse kidneys in step (1), urine of the mouse is collected for urine crystal detection, and after the mouse urine is centrifuged in a centrifuge, the number and morphology of the crystals are observed under a microscope, and finally the crystals are observed under a polarizing microscope;

[0013] 3) The mice are then dissected and the kidney tissue is observed with the naked eye or HE staining is performed on the kidney tissue sections. If crystals or stones are formed, the model is successfully identified.

[0014] Furthermore, in step S2, blood samples of mice are collected by sampling blood from the mouse orbital venous plexus.

[0015] Furthermore, in step S2, a uric acid content detection kit is used to detect serum uric acid in mice.

[0016] Application of the kidney stone animal model based on UOX gene knockout mice in anti-hyperuricemia treatment.

[0017] Application of the kidney stone animal model based on UOX gene knockout mice in the treatment of kidney stones.

[0018] In summary, the present invention has the following beneficial effects:

[0019] (1) Currently, no animal model of hyperuricemia kidney stones has been established.

[0020] (2) Most of the existing animal models of kidney stones are about the construction method of calcium oxalate kidney stone model, while this patent constructs an animal model of hyperuricemia kidney stones. In addition, the stone model constructed by this method is not a single model, but can also construct a calcium oxalate stone model, a pure uric acid stone model or a mixed stone model. This is consistent with the results of postoperative stone composition analysis of patients with hyperuricemia kidney stones in the clinic.

[0021] (3) The present application is constructed by using UOX gene knockout mice, and there is no need to use other drugs or things that induce hyperuricemia in mice, which greatly reduces the burden on mice and the mortality rate of mice in constructing the model; at the same time, gene knockout mice can enable mice to maintain high levels of hyperuricemia, while models constructed using drugs or things cannot construct a stable hyperuricemia model due to factors such as metabolism.

[0022] (4) Gene knockout mice can more closely simulate the uric acid metabolic pathway in the human body, providing a more stable model for the study of clinical drug treatment and pathological mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is an abdominal ultrasound image of a mouse in an embodiment of the present invention;

[0024] Figure 2 is a graph of urine crystal detection of mice in an embodiment of the present invention;

[0025] Figure 3 It is a HE staining picture of mouse kidney tissue in the embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following is combined with Figure 1-3 The present invention is described in further detail.

[0027] Example 1: A method for constructing a kidney stone animal model based on UOX gene knockout mice, comprising the following steps:

[0028] S1. Selection of mice: Select healthy, mentally healthy UOX mice without trauma or disability. - / - Male C57BL / 6 mice were used as experimental animals and were adaptively raised;

[0029] S2. Identification of mice: After one week of feeding, 0.2 ml of blood samples were collected from mice, and serum uric acid was tested;

[0030] S3. Feeding of mice: Two weeks of recovery feeding were conducted, during which mice were given standard feed, appropriate amount of melon seeds and clean drinking water. Drinking water, bedding and feed were changed every day, and the life and health of the mice were observed;

[0031] S4. Mouse modeling: The mice were given standard feed, free drinking water with a well-proportioned NH4CI concentration (0.25%) and a dose of 5 mg kg -1 The mice were treated with benzbromarone by gavage for 3 weeks, and the drinking water, bedding and feed were changed every day and the life and health conditions of the mice were observed.

[0032] S5. Model identification, including:

[0033] 1) After the mice have completed the three-week treatment, the mouse's urinary system is examined abdominally using an ultrasound imager to observe the mouse's kidneys. If there are stones or urine salt deposits, the model is successfully identified;

[0034] 2) If no stones or urine salt deposits are found in the mouse kidneys in step (1), urine of the mouse is collected for urine crystal detection, and after the mouse urine is centrifuged in a centrifuge, the number and morphology of the crystals are observed under a microscope, and finally the crystals are observed under a polarizing microscope;

[0035] 3) The mice are then dissected and the kidney tissue is observed with the naked eye or HE staining is performed on the kidney tissue sections. If crystals or stones are formed, the model is successfully identified.

[0036] It is worth mentioning that in step S2, blood samples of mice are collected by sampling blood from the mouse orbital venous plexus, and the serum uric acid of mice is detected using a uric acid content detection kit.

[0037] Example 2: Experiment on the construction of hyperuricemia kidney stone model mice by combining ammonium chloride and benzbromarone

[0038] (1) Selection of experimental model mice

[0039] Ten 4-week-old healthy UOX mice with good mental condition and no trauma or disability were selected. - / - Male C57BL / 6 mice were used as experimental animals and were adaptively raised for one week;

[0040] (2) Identification of experimental model mice

[0041] After one week of feeding, 0.2 ml of blood samples were collected from the mouse orbital venous plexus, and the serum uric acid of the mice was tested using a uric acid content detection kit;

[0042] (3) Feeding of experimental model mice

[0043] The mice were fed with standard feed, appropriate amount of melon seeds and clean drinking water for two weeks of recovery feeding. The drinking water, bedding and feed were changed every day and the health of the mice was observed.

[0044] (4) Establishment of experimental model mice

[0045] The mice were given standard feed, free drinking water with a well-proportioned NH4CI concentration (0.25%) and a dose of 5 mg kg -1 The mice were treated with benzbromarone by gavage for 3 weeks, and the drinking water, bedding and feed were changed every day and the life and health conditions of the mice were observed.

[0046] Experimental results:

[0047] (1) Serum uric acid levels in mice

[0048] Use a high-speed centrifuge at 4 ℃ , 3000rpm for 10 minutes, and the supernatant was used for uric acid detection; the uric acid concentration of each mouse was detected using an automatic microplate reader, and the results are shown in the table. The reference literature suggests that the serum uric acid concentration of ordinary C57BL / 6J male mice is 133.10±26.88umol / L, and the serum uric acid concentration of animals with successful modeling is 505.13±179.16umol / L. The serum uric acid concentration of ten mice in this experiment was 912.66±106.07umol / L, which was significantly higher than the concentration shown in the literature. Combined with the gene detection report, the results showed that the mice in this experiment were indeed hyperuricemia animal models.

[0049]

[0050] (2) Health status of mice after modeling

[0051] After three weeks of modeling intervention, all 10 mice survived healthily, responded quickly, were in good spirits, had normal diet and normal activities.

[0052] Example 3: Identification of Hyperuricemia Kidney Stone Model Mice

[0053] (1) Urinary system ultrasound of experimental model mice

[0054] The mice were anesthetized using a multifunctional ventilator for model mice, and all the hair on the abdomen of the model mice was removed. The urinary system of the model mice was detected using a mouse ultrasound imager.

[0055] (2) HE staining of kidney tissue of experimental model mice

[0056] The mice were dissected and the kidneys were observed with the naked eye to see if there were any crystals or stones formed. The kidney tissues of the mice were stained with HE to see if there were any crystals or stones formed in the kidney tissue sections.

[0057] (3) Detection of urinary crystals in experimental model mice

[0058] The urine of mice was collected for urine crystal detection, and after the urine of mice was centrifuged in a centrifuge, the number and morphology of the crystals were observed under a microscope, and finally the crystals were observed under a polarizing microscope.

[0059] Experimental results:

[0060] (1) Urinary system ultrasound results

[0061] like Figure 1 As shown, the kidneys of the model mice were normal in size, the parenchymal echo was homogeneous, and multiple strong echo light masses were clearly observed in the middle of the mouse renal calyx.

[0062] (2) HE staining of kidney tissue

[0063] The mice were dissected and the kidney tissues were stained with HE. The results were as follows: Figure 3 As shown, after the modeling period was over, the model mice were dissected to obtain the kidney tissues of the mice.

[0064] The mouse kidney tissue was dissected at the coronal level. After the dissection, the crystals or stones in the mouse renal pelvis and calyces were observed with the naked eye. A large number of fine granular substances in yellow-brown color were found in the mouse renal pelvis and calyces. The observation results are as follows: Figure 3 As shown in A.

[0065] The mouse kidney tissue was placed in an EP tube containing 4% paraformaldehyde for fixation. A large number of stone-like particles were visible in the liquid and kidney tissue. The results were as follows: Figure 3 As shown in B.

[0066] The fixed kidney tissue was stained with HE. The pathological tissue sections after HE staining showed a large number of crystals or stone precipitates deposited in the glomeruli and renal tubular lumens. The observation results were as follows: Figure 3 As shown in C.

[0067] (3) Urine crystal detection

[0068] Collect the urine of mice for urine crystal detection and observe the crystals under a polarizing microscope. Figure 2 As shown, urine samples of mice were collected using 24-hour metabolic cages after modeling, and urine crystals were observed under a microscope. Figure 2 A, a large number of irregular transparent crystals of different sizes can be observed under the 400x microscope; the above urine crystals were observed separately using a polarized light microscope, and the results were as follows Figure 2 As shown in B, a single rectangular transparent crystal can be seen under the field of view of an ordinary optical microscope (400x). After replacing the polarized light objective lens, it can be seen that the crystal is colorful, as shown in Figure 2 As shown in C.

[0069] (4) Success rate of mouse modeling

[0070] According to the above criteria, all 10 mice were successfully modeled. See the table below

[0071]

[0072] In summary, the method for constructing an animal model of kidney stones based on UOX gene knockout mice provided in the present application can construct a stable kidney stone model.

[0073] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for constructing a kidney stone animal model based on UOX gene knockout mice, characterized by: The following steps are involved: S1. Selection of mice: Select healthy, mentally healthy UOX mice without trauma or disability. - / - Male C57BL / 6 mice were used as experimental animals and were adaptively raised; S2. Identification of mice: After one week of feeding, 0.2 ml of blood samples were collected from mice, and serum uric acid was tested; S3. Feeding of mice: Two weeks of recovery feeding were conducted, during which mice were given standard feed, appropriate amount of melon seeds and clean drinking water. Drinking water, bedding and feed were changed every day, and the life and health of the mice were observed; S4. Mouse modeling: The mice were given standard feed, free drinking water with a well-proportioned concentration of 0.25% NH4CI, and a dose of 5 mg kg -1 The mice were treated with benzbromarone by gavage for 3 weeks, and the drinking water, bedding and feed were changed every day and the life and health of the mice were observed; S5. Model identification, including: 1) After the mice have completed three weeks of drug treatment, use a mouse ultrasound imager to perform an abdominal examination of the mouse's urinary system and observe the mouse's kidneys. If there are stones or urine salt deposits, the model is successfully identified; 2) If no stones or urine salt deposits are found in the mouse kidneys in step (1), collect the mouse urine for urine crystal detection, centrifuge the mouse urine, observe the number and morphology of crystals under a microscope, and finally observe the crystals under a polarizing microscope; 3) The mice are then dissected and the kidney tissue is observed with the naked eye or HE staining is performed on the kidney tissue sections. If crystals or stones are formed, the model is successfully identified.

2. The method for constructing a kidney stone animal model based on UOX gene knockout mice according to claim 1, characterized in that: In step S2, blood samples of mice are collected by sampling blood from the orbital venous plexus of mice.

3. The method for constructing a kidney stone animal model based on UOX gene knockout mice according to claim 1, characterized in that: In step S2, the serum uric acid of the mice is detected using a uric acid content detection kit.

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