Preparation method of Erbin gene knockout renal fibrosis animal model

Abstract

The invention discloses a preparation method of an Erbin gene knockout renal fibrosis animal model. The preparation method comprises the following steps: 1) obtaining an animal of which the Erbin gene is specifically knocked out from renal tubular epithelial cells; and 2) providing an effective dose of cis-platinum for the animal with the Erbin gene specifically knocked out of the renal tubular epithelial cells to obtain the renal fibrosis animal model. The cisplatin-induced renal tubular epithelial cell Erbin-KO specific knockout mouse renal fibrosis model constructed by the invention can be used for further researching the action and mechanism of the Erbin gene in chronic nephrosis, is further applied to the development of drugs for treating and / or preventing renal fibrosis, and has important application prospects and clinical significance.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of an Erbin gene knockout renal fibrosis animal model and belongs to the technical field of animal models. BACKGROUND

[0002] Chronic kidney disease (CKD) refers to abnormal structure or function of the kidney that lasts more than 3 months and affects health. Regardless of the cause, CKD is characterized by progressive and irreversible loss of nephron, microvascular injury, decreased regenerative capacity, and accompanied by pathological features such as inflammatory response, oxidative stress and metabolic disorder, eventually leading to renal failure and end-stage renal disease. The disease will affect 10% - 14% of the global population, and there is currently a lack of effective CKD treatment drugs in addition to dialysis and kidney transplantation, with more than one million people dying from CKD and its complications every year. With the arrival of population aging, the global incidence and mortality of CKD are still rising. By 2040, CKD will become the fifth leading cause of death worldwide and is considered one of the major global public health problems. Renal tubular epithelial cells, as the most important cell population in the renal cortex (about 90% of the total amount of renal cortical cells), are the main target cells of various acute and chronic kidney injuries and play an important role in the occurrence and development of chronic kidney disease. Renal fibrosis is a typical pathological feature and final manifestation of chronic kidney disease, and its morphological characteristics include glomerular sclerosis, tubular atrophy, interstitial chronic inflammation and fibrosis, and vascular rarefaction. This pathological process is caused by excessive deposition of extracellular matrix proteins (ECM) such as fibronectin and collagen due to the imbalance of the wound healing mechanism, which can cause destruction of the kidney tissue structure, renal dysfunction, and eventually lead to renal failure. At present, there is no effective treatment for renal fibrosis in clinical practice, therefore, the development of new therapeutic targets is particularly important for the treatment of CKD.

[0003] Erbin, also known as ErbB2 interacting protein (ErbB2ip), is a member of the LRR and PDZ domain-containing proteins (LAP) family, which is rich in leucine-rich repeats (LRR) and PDZ (Postsynaptic density-95 / Discslarge / Zonula occludens-1) domains. Erbin plays an important role in maintaining epithelial cell polarity and integrity, regulating cell growth and differentiation, and regulating intercellular signal transduction. Erbin is widely distributed in various tissues of humans and mice, including brain, liver, kidney, spleen, intestinal tract and skeletal muscle. According to the differences in cell type and microenvironment, Erbin can play an important role in inflammatory bowel disease, myocardial hypertrophy, colon cancer, breast cancer, liver cancer and lung cancer.

[0004] At present, the mechanism of Erbin gene in the regulation of kidney function of human or animal and the occurrence and development of chronic kidney disease is not clear. In order to further study the role of Erbin gene in the physiological function of kidney and the pathological process of chronic kidney disease, it is urgent to establish an animal model of kidney injury and fibrosis based on Erbin gene knockout. However, there is no public report on the establishment method and related application of the model in the prior art. SUMMARY

[0005] The purpose of the present application is to provide an animal model of kidney injury and fibrosis based on Erbin gene knockout.

[0006] The technical solution adopted by the present application comprises: A preparation method of an Erbin gene knockout kidney fibrosis animal model, comprising the following steps: 1) obtaining an animal with kidney tubular epithelial cell-specific Erbin gene knockout; 2) providing an effective dose of cisplatin to the animal with kidney tubular epithelial cell-specific Erbin gene knockout to obtain a kidney fibrosis animal model.

[0007] Preferably, the administration mode of cisplatin is intraperitoneal injection.

[0008] Preferably, the injection dose of cisplatin is 5-10 mg / kg body weight.

[0009] Preferably, the injection frequency of cisplatin is once a week, and the injection is continuous for three times.

[0010] Preferably, the animal is a mouse.

[0011] Preferably, the CDH16-Cre mice are crossed with Erbin-flox mice to obtain mice with genotype CDH16-Cre+ / -Erbinwt / flox, and then the CDH16-Cre+ / -Erbinwt / flox mice are self-crossed to obtain mice with genotype CDH16-Cre+ / -Erbinflox / flox by PCR identification, i.e. animals with kidney tubular epithelial cell-specific knockout of the Erbin gene.

[0012] The application also provides application of the renal fibrosis animal model obtained by the above method in screening drugs for preventing or treating renal fibrosis.

[0013] The application also provides application of Erbin gene knockout combined with cisplatin in preparing a renal fibrosis animal model.

[0014] Preferably, the Erbin gene knockout refers to knockout of the Erbin gene of kidney tubular epithelial cells of the animal.

[0015] Compared with the prior art, the application has the following advantages: In the cisplatin-induced renal fibrosis animal model, the Erbin gene knockout leads to obvious renal fibrosis, indicating that Erbin is a new target for renal fibrosis.

[0016] The cisplatin-induced renal tubular epithelial cell-specific Erbin gene knockout (Erbin-KO) mouse renal fibrosis model constructed by the application can be used for further research on the role and mechanism of the Erbin gene in chronic kidney disease, and is further applied to the development of drugs for treating and / or preventing renal fibrosis, and has important application prospects and clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : CDH16-cre; Erbin flox / flox Identification of mice.

[0018] Figure 2 : Changes in fibrosis markers in WT and Erbin-KO mice after injection of cisplatin.

[0019] Figure 3 : PAS staining of WT and Erbin-KO mice after injection of cisplatin.

[0020] Figure 4 : Collagen staining of WT and Erbin-KO mice after injection of cisplatin, wherein the left graph is a photograph of kidney staining, and the right graph is statistical data of fibrosis area. DETAILED DESCRIPTION

[0021] Specific implementation method: The application will be further described in connection with the following examples, which do not limit the scope of the application in any way.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the terms are used only in describing particular embodiments of the application and are not intended to limit the application. Also, unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Furthermore, the term "comprising" is used herein to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" when used herein in relation to a composition, means that the composition includes the recited elements, and also includes other elements that do not materially affect the basic and novel characteristics of the composition. "Consisting of" when used herein in relation to a composition, means the composition includes the recited elements exclusively.

[0023] The substances or instruments used in the following examples can be obtained from conventional commercial channels, if not specifically stated.

[0024] The WT mouse of the application refers to a wild-type (WT) mouse.

[0025] Example 1: Establishment of a kidney fibrosis model of a renal tubular epithelial cell-specific Erbin gene knockout mouse We obtained the CDH16-Cre + / - Erbin wt / flox mice by crossing CDH16-Cre mice (Nanjing Jisui Yaoke, model number: T004820) with Erbin-flox mice (Nanjing Jisui Yaoke, model number: T008906). Then, the CDH16-Cre + / - Erbin wt / flox mice were inbred, and the mouse tail DNA was extracted to determine the genotype by PCR. The genotype of the mice was CDH16-Cre + / - Erbin flox / flox mice (referred to as Erbin-KO) and the control mice (WT) with the genotype of CDH16-Cre + / - Erbin wt / wt were obtained. Figure 1

[0026] After the renal tubular epithelial cell-specific Erbin gene knockout mice described above reached the age of 10-12 weeks, Cisplatin solution prepared with normal saline was injected intraperitoneally, and the Cisplatin dose was 8 mg / kg of body weight.

[0027] Example 2: Comparison of the degree of kidney fibrosis 1. Method 1.1 Grouping and treatment ​Take 10-12 weeks old WT and Erbin-KO mice, 20 each, and randomly divide them into the following four groups, 10 each, and do the corresponding treatment: WT+sham group: wild type mice, injected with normal saline; Erbin-KO+sham group: Erbin-KO genotype mice, injected with normal saline; WT+Cisplatin group: wild type mice, injected with the Cisplatin solution described in Example 1.

[0028] Erbin-KO+Cisplatin group: Erbin-KO genotype mice, injected with the Cisplatin solution described in Example 1, representing the mouse kidney fibrosis model of the present application.

[0029] In the above treatment, the Cisplatin dose is 8 mg / kg body weight, and the mice are injected intraperitoneally once a week for 3 consecutive times. The mice are sacrificed on day 28, and the following tests are performed.

[0030] 1.2 Detection of renal tubular injury PAS staining (Periodic acid-Schiff staining) is used to observe the degree of renal tubular injury.

[0031] 1.3 Detection of kidney collagen content Masson staining is used to detect the collagen content of kidney tissue.

[0032] 2. Results 2.1 Changes in mRNA expression of kidney fibrosis markers The qRT-PCR results show that in the Cisplatin-induced kidney fibrosis model, the mRNA expression levels of collagen type 1 a1 (col1a1), collagen type 3 a1 (col3a1), transforming growth factor beta 1 (TGF-β1), tissue metalloproteinase inhibitor 1 (Timp1), and other fibrosis markers in the Erbin-KO group are more obviously elevated than in the WT group Figure 2 , indicating that the Erbin-KO group of mice exhibits a more severe fibrosis phenotype at the transcriptional level than the WT group of mice after Cisplatin treatment.

[0033] 2.2 Detection results of renal tubular injury PAS staining results showed that there was no obvious morphological difference between the kidney of Erbin-KO+sham group and WT+sham group mice. After cisplatin treatment, the kidney of WT+Cisplatin group mice showed significant tubular dilation, increased cast formation and epithelial cell shedding in the lumen and other pathological changes; while the above pathological damage of the kidney of Erbin-KO+Cisplatin group mice was more serious than that of WT+Cisplatin group, showing more extensive tubular structure destruction Figure 3 ). It is shown that compared with WT mice, Erbin-KO mice show more serious pathological damage of the kidney after cisplatin treatment.

[0034] 2.3 Kidney collagen content detection results Masson staining results showed that collagen deposition was observed in the kidney interstitium of WT+Cisplatin group mice, while the kidney interstitial collagen of Erbin-KO+Cisplatin group mice was significantly increased compared with that of WT+Cisplatin group, indicating that compared with WT mice, Erbin-KO mice showed more serious kidney fibrosis after cisplatin treatment Figure 4 )。

[0035] 3. Conclusion The above experimental results show that Erbin gene knockout can significantly aggravate cisplatin-induced kidney fibrosis in mice, confirming that Erbin is a key target gene for regulating kidney fibrosis and plays an important role in inhibiting the progression of kidney fibrosis. Based on this, the Erbin gene knockout mouse kidney fibrosis model provided by the present application has good application prospect and clinical value, and can be used for screening, evaluation and development of candidate drugs for treating and / or preventing kidney fibrosis, and provides important experimental tools and theoretical basis for related drug research and development.

[0036] The above examples are only for illustrating the technical concept and characteristics of the present application, and its purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing an animal model of kidney fibrosis with Erbin gene knockout, characterized in that, The steps include: 1) obtaining an animal with tubular epithelial cell-specific knockout of the Erbin gene; 2) providing an effective dose of cisplatin to the animal with tubular epithelial cell-specific knockout of the Erbin gene to obtain a renal fibrosis animal model.

2. The production method according to claim 1, characterized by, The cisplatin is administered by intraperitoneal injection.

3. The preparation method according to claim 2, characterized in that, The injection dose of cisplatin is 5-10 mg / kg body weight per time.

4. The production method according to claim 3, characterized by, The injection frequency of cisplatin is once a week, and the injection is continuous for three times.

5. The production method according to any one of claims 1 to 4, characterized by, The animal is a mouse.

6. The production method according to claim 5, wherein CDH16-Cre mice were crossed with Erbin-flox mice to obtain CDH16-Cre + / - Erbin wt / flox mice, and then CDH16-Cre + / - Erbin wt / flox mice were crossed with CDH16-Cre + / - Erbin flox / flox mice, i.e. animals with kidney tubular epithelial cell-specific knockout of the Erbin gene.

7. Use of the renal fibrosis animal model obtained by the preparation method of any one of claims 1-6 in screening drugs for preventing or treating renal fibrosis.

8. Use of Erbin gene knockout combined with cisplatin in the preparation of a renal fibrosis animal model.

9. Use according to claim 8, characterized in that, The Erbin gene knockout refers to the knockout of the Erbin gene of the tubular epithelial cells of the animal.

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