Method for constructing podocyte injury animal model by utilizing miR-1

Abstract

The invention provides a method for constructing a podocyte injury animal model, and belongs to the technical field of experimental animal models. The method specifically comprises the following steps: injecting miR-1 agomir twice a week through caudal veins of a male C57BL / 6 mouse for 7 times in total to obtain a podocyte injury animal model; the miR-1 with high expression of skeletal muscle tissue is used for inducing podocyte damage, interference of damage of other organs and cells is reduced, and the miR-1 plays an important role in research on pathogenesis of chronic kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of experimental animal models, and in particular relates to a method for constructing a podocyte injury animal model by utilizing miR-1. Background Art

[0002] Podocytes are an important component of the filtration barrier, and podocyte damage is a key cause of proteinuric kidney disease.

[0003] The development of animal models has advanced our knowledge and understanding of glomerular diseases. Rodents have been widely used in experiments to better define the mechanisms of disease induction and progression and to identify potential targets and treatments. A variety of animals are used in the study of glomerular diseases, but rodent models are the first choice due to their low cost, low maintenance requirements and short gestation period. The animal models currently used to construct podocyte injury are mainly mice or rats injected with drugs that cause oxidative stress injury, such as doxorubicin and puromycin. They lack podocyte specificity and are prone to experimental animal death before the appearance of obvious podocyte phenotypes such as proteinuria. Therefore, it is a technical problem that needs to be solved in this field to provide an animal model of podocyte injury with good podocyte specificity, targeting the podocyte skeleton, and not easily causing the death of experimental animals. Summary of the invention

[0004] To solve the above problems, the present invention discloses a safe method for constructing a podocyte injury model targeting the podocyte skeleton and the application of miR-1. The method of the present invention has the advantages of low lethality, simplicity and good stability.

[0005] To achieve the above object, the technical solution of the present invention is as follows: The present invention provides application of miR-1 in constructing an animal model of podocyte injury.

[0006] Furthermore, the application comprises the following steps: slowly injecting miR-1 agomir through the tail vein of mice, injecting miR-1 agomir twice a week, for a total of 7 injections.

[0007] Furthermore, the dosage of miR-1 agomir injected each time is 5 nmol, dissolved in 200 μl of physiological saline for injection.

[0008] The present invention also provides the use of miR-1 in preparing a drug for inducing podocyte injury.

[0009] Furthermore, the drug induces podocyte injury by overexpressing miR-1.

[0010] Furthermore, the effective concentration of the miR-1 agomir is 25 μM.

[0011] The present invention also provides a method for constructing an animal model of podocyte injury using miR-1, comprising the following steps: fixing the mouse, disinfecting the skin at the tail injection site, slowly injecting miR-1 agomir through the tail vein, injecting miR-1 agomir twice a week, and injecting 7 times in total.

[0012] Furthermore, the mouse is a male C57BL / 6 mouse.

[0013] Furthermore, the dosage of miR-1 agomir injected each time is 5 nmol, dissolved in 200 μl of physiological saline for injection.

[0014] Furthermore, the method for determining podocyte injury is that the width of the podocyte foot process of the mice in the miR-1 agomir injection group is significantly different from that in the scramble injection group.

[0015] The present invention also provides the use of the podocyte injury animal model constructed by the above method in drugs for treating podocyte diseases.

[0016] The present invention also provides an animal model of podocyte injury, which is constructed by the above method. The mouse podocyte injury animal model is also used to study the pathogenesis of proteinuric nephropathy such as focal segmental glomerulosclerosis in humans. The animal model constructed by the present invention not only reflects the changes in podocyte injury from the urine albumin-creatinine ratio, a marker of podocyte injury, but also reflects the pathological changes of podocyte injury from microscopic levels such as tissue pathology.

[0017] The beneficial effects of the present invention are: Podocytes are important components of the glomerular filtration barrier. After podocyte damage, medium and large molecular blood components such as albumin leak from the filtration barrier. Therefore, microalbuminuria is an early manifestation of podocyte damage. The urine albumin-creatinine ratio corrected by creatinine is a reliable marker of podocyte damage. The present invention uses foot process width and podocyte markers as markers for judging podocyte damage, which is simple, easy, stable and reliable, and is also a commonly used method for evaluating modeling effects in the literature. The present invention uses safe miR-1 that acts on podocytes to induce podocyte damage, avoids drug-induced death of experimental animals, and reduces interference from confounding factors. The podocyte damage model constructed by the present invention provides a simple, safe and stable animal model for the study of the pathogenesis of podocyte diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Electron microscopic photographs of mouse kidneys after injection of miR-1 and random control sequences (Scramble); Figure 2 is the survival rate of mice after injection of miR-1 and random control sequence (Scramble); Figure 3 The immunohistochemical photos of CD2AP in mouse kidneys after injection of miR-1 and random control sequence (Scramble); Figure 4 The photo shows the results of immunoblotting of nephrin in the renal cortex of mice after injection of miR-1 and random control sequence (Scramble). DETAILED DESCRIPTION

[0019] The invention provides a method for constructing an animal model of podocyte injury, comprising the following steps: fixing mice, disinfecting the skin of the tail injection site, and slowly injecting miR-1 agomir into the tail vein, twice a week, for a total of 7 injections.

[0020] In the present invention, the animal is preferably a male animal, and the type of the animal is preferably SPF grade Balb / c or C57BL / 6 mouse. The experimental drug is miR-1 agomir, and there is no special restriction on the manufacturer. The drug is generally injected in the morning, and the mice are free to eat and drink before the operation. There is no special restriction on the method of fixing the mice and restricting the activities of the mice during the injection of the drug. Use 75% alcohol to apply the skin of the tail injection site for disinfection.

[0021] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example 1

[0022] Ten SPF-grade 7-week-old male C57BL / 6 mice were selected, weighing 20-25g, with good fur gloss, no hair loss, no trauma, disability, normal limb activity, and good spirits (animal production license). The mice were adaptively fed with a maintenance feed that meets national standards for one week. The animal feeding conditions were: 12 hours of lighting, 12 hours of darkness, and raised in an SPF barrier environment. The temperature was maintained at 21-24°C, and the humidity was 40-60%. The breeding environment was kept quiet to prevent noise. The animals were free to eat and drink. After one week of adaptive feeding, they were randomly divided into an experimental group (10) and a control group (10). The experiment was carried out after they adapted to the environment. After the mice were numbered, 5nmol of miR-1 / Scramble agomir was dissolved in 200μl sterile PBS and injected into the tail vein of the mice using an insulin syringe. After the injection, the injection point was pressed for one minute to stop bleeding. Each mouse in the experimental group was injected with 5nmol miR-1 agomir, and each mouse in the control group was injected with 5nmol Scramble agomir. The miR-1 agomir was injected twice a week for a total of 7 injections. There was no significant change in the appearance and mental state of the mice, and no mice died. The survival rate of mice after injection of miR-1 and random control sequence (Scramble) was as follows Figure 2As shown, there was no death in mice 9 months after the injection of miR-1; the results of this example show that the injection of miR-1 can induce podocyte injury in mice, which is a safe method for constructing an animal model of podocyte injury.

[0023] Electron microscopic images of mouse kidneys after injection of miR-1 and random control sequences (Scramble) Figure 1 As shown, podocyte foot processes fused after miR-1 injection; the width of podocyte foot processes of mice injected with miR-1 was wider than that after random control sequence (Scramble) injection, indicating damage to the podocyte skeleton.

[0024] CD2AP immunohistochemistry photos of mouse kidneys after injection of miR-1 and random control sequence (Scramble) Figure 3 As shown, CD2AP is a sensitive marker of podocyte injury. The CD2AP level in the kidneys of mice injected with miR-1 was lower than that after injection with the random control sequence (Scramble), indicating podocyte injury.

[0025] The results of immunoblotting of nephrin in the renal cortex of mice after injection of miR-1 and random control sequence (Scramble) are shown in the photo. Figure 4 As shown, nephrin is a podocyte marker. After miR-1 injection, the level of nephrin in the renal cortex of mice was reduced compared with that after random control sequence (Scramble) injection, indicating podocyte damage.

[0026] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. 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 all fall within the protection scope of the claims of the present invention.

Claims

1. Application of miR-1 in constructing animal model of podocyte injury.

2. The use of miR-1agomir according to claim 1 in constructing an animal model of podocyte injury, characterized in that: The application comprises the following steps: slowly injecting miR-1agomir through the tail vein of mice, injecting miR-1agomir twice a week, and injecting 7 times in total.

3. The use of miR-1 in constructing an animal model of podocyte injury according to claim 1, characterized in that: The dosage of miR-1 agomir injected each time was 5 nmol, which was dissolved in 200 μl of physiological saline for injection.

4. Application of miR-1 in the preparation of drugs that induce podocyte injury.

5. The use of miR-1 in the preparation of a drug for inducing podocyte injury according to claim 4, characterized in that: The drug induces podocyte injury by increasing miR-1.

6. The use of miR-1 in the preparation of a drug for inducing podocyte injury according to claim 4, characterized in that: The effective concentration of the miR-1 agomir is 25 μM.

7. A method for constructing an animal model of podocyte injury using miR-1, characterized in that: The method comprises the following steps: fixing the mouse, disinfecting the skin of the tail injection site, slowly injecting miR-1 agomir through the tail vein, injecting miR-1 agomir twice a week, and injecting 7 times in total.

8. The method for constructing an animal model of podocyte injury using miR-1 according to claim 7, characterized in that: The mice were male C57BL / 6 mice.

9. The method for constructing an animal model of podocyte injury using miR-1 according to claim 7, characterized in that: The dosage of miR-1 agomir injected each time was 5 nmol, which was dissolved in 200 μl of physiological saline for injection.

10. Use of the podocyte injury animal model constructed by the method according to any one of claims 7 to 9 in drugs for treating podocyte diseases.

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