CN116649284A

CN116649284A - Construction method and application of spontaneous skin type lupus erythematosus animal model

Info

Publication number: CN116649284A
Application number: CN202310521593.5A
Authority: CN
Inventors: 陆前进; 田婧汝; 史丽晴; 张丁垚
Original assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Current assignee: Institute of Dermatology and Skin Disease Hospital of CAMS
Priority date: 2023-05-10
Filing date: 2023-05-10
Publication date: 2023-08-29
Anticipated expiration: 2043-05-10
Also published as: CN116649284B

Abstract

The invention discloses a construction method of a spontaneous cutaneous lupus erythematosus animal model, which leads a mouse to spontaneously generate cutaneous lupus erythematosus phenotypes through knocking out PPAR gamma (Peroxisome proliferator-activated receptor gamma) in keratinocytes in a small range, wherein the spontaneous cutaneous lupus erythematosus phenotypes comprise local skin inflammatory phenotypes and dehairing, and the histological examination has dermal immune cell infiltration and basal lamina zone IgG deposition; and meanwhile, systemic lupus erythematosus like changes such as proteinuria, glomerular IgG deposition, serum autoantibodies and the like do not exist. The animal model simulates the clinical symptoms of the cutaneous lupus erythematosus and provides an important tool for the related research of the cutaneous lupus erythematosus.

Description

Construction method and application of spontaneous skin type lupus erythematosus animal model

Technical Field

The invention belongs to the technical field of disease animal model construction methods, and particularly relates to construction of an animal model of cutaneous lupus erythematosus.

Background

Cutaneous lupus erythematosus (Cutaneous lupus erythematosus, CLE) is a group of autoimmune skin diseases with various clinical manifestations, which can be divided into multiple subtypes according to different disease progression, and the manifestations, course and prognosis of skin lesions of different subtypes are different, including acute, subacute, chronic and intermittent CLE. The chronic CLE can be further classified into a disc LE, a wart LE, a deep LE, a chilblain LE, a blastko linear LE, and the like. Common histological features of CLE include interfacial dermatitis and deposition of autoantibodies at the dermis-epidermis interface. The incidence of CLE is about 4.2/10 ten thousand, slightly higher than SLE (3/10 ten thousand). Female incidence rate is higher than male (5.8/10 ten thousand ratio 2.4/10 ten thousand), and incidence age is between 30 and 69 years. The prevalence of CLE is 70.4/10 ten thousand, and female prevalence is also higher than male (85.1/10 ten thousand: 56.9/10 ten thousand), and the prevalence peak age is 50-59 years. Between 5% and 25% of CLE patients can develop systemic lupus erythematosus (Systemic lupus erythematosus, SLE) during the course of the disease, severely threatening the patient's health.

The pathogenesis of CLE involves a variety of factors including genetic, epigenetic and environmental factors, among others, various genetic and environmental causes promoting infiltration of T cells, B cells, neutrophils, antigen presenting cells and NK cells into diseased skin. The current view suggests that the pathogenic pathways of CLE, in addition to dendritic cell activation, T cell imbalance, cytokine imbalance, B lymphocyte deficiency and autoantibody production, also uv irradiation stimulates keratinocytes to produce innate immunity-related cytokines and trigger cell death, thereby activating nucleic acid signaling pathways. First line therapeutic agents for CLE are mainly antimalarial drugs and glucocorticoids, whereas immunosuppressants, thalidomide and avermectin are used for the treatment of refractory CLE. At present, CLE still lacks an effective treatment mode, so that genetic, environmental and immunoregulatory factors driving CLE can be better understood, and important basis is provided for the treatment of CLE. However, little is known about the pathogenesis of CLE or the mechanisms that lead CLE patients to progress to SLE, in part because of the lack of suitable animal models. Therefore, the model has similar genetic background to CLE patients, can simulate clinical symptoms and has spontaneous animal models with cost advantages, and has important significance for researching the pathogenesis of CLE and a safe and effective novel therapy.

To date, most LE models have focused on SLE, including NZB/W F1 mice, MRL/lpr mice, BXSB/Yaa mice, and newly developed PD-1H KO mice. These typical lupus mouse models rarely exhibit significant skin lesions and are very heterogeneous in skin lesions, with large differences in pathological processes from patient to patient, and lack many of the key features of human CLE. Furthermore, these models require 6 months or more for the appearance of the skin loss phenotype, which takes a long time. The newly developed CLE mouse model has an IL-21 induced humanized CLE mouse model which requires injection of abnormal immune cells derived from active SLE patients and induction of development of lupus-like skin manifestations using UVB irradiation, which has disadvantages in that the operation is complicated, and the mouse model has an acute injury phenotype caused by UVB, which is not in agreement with the clinical manifestations of CLE patients.

PPARgamma (Peroxisome proliferator-activated receptor gamma ) is a key molecule of the PPAR pathway, can regulate adipocyte differentiation and function, regulate maturation and function of immune cells, affect cell proliferation of tissues and organs, and are also involved in tumor generation. There is no report of constructing an animal model of self-hairing CLE using keratinocyte-conditioned pparγ knockout mice.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention provides a novel spontaneous CLE animal model for researching and developing pathogenesis, pathological characteristics and treatment methods of the CLE.

In order to achieve the technical aim, the invention discloses a method for constructing a spontaneous skin type lupus erythematosus animal model, which comprises the following steps:

(1) Constructing a keratinocyte-conditioned PPARgamma knockout mouse which is a C57BL/6 mouse of Krt 5-Creet2+/-PPARgamma flox/flox-/-;

(2) Knocking out pparγ genes in keratinocytes to mediate disease phenotype occurrence using a gene knockout activator in a small range; the small range knockout is that the skin on the ventral side or the dorsal side of the single ear of a diagonal forming cell conditional PPARgamma knockout mouse or the body is less than or equal to 4cm ² Is applied to the hairless or shaved skin.

Wherein, the gene knockout activator is a compound which can be combined with an estrogen receptor mutant ERT after metabolism to lead the CreERT2 to exert the activity of Cre recombinase for gene knockout.

In one embodiment, the gene knockout activator is 4-hydroxy tamoxifen.

Wherein, the use mode of the 4-hydroxy tamoxifen is any one of external application, skin application, subcutaneous injection with or without needle or intradermal injection.

Specifically, when 4-hydroxy tamoxifen is used as a gene knockout activator, 4cm or less of hairless or shaved PPARgamma knockout mice conditional on keratinocytes ² The area of skin is smeared with 4-hydroxy tamoxifen solution for 1-7 days once a day, and the skin on the abdomen side and the back side of a single ear can be selected for smearing for the convenience of operation.

Wherein the 4-hydroxy tamoxifen solution is prepared according to the proportion of 50mg of 4-hydroxy tamoxifen, 1ml of DMSO and 3-49ml of corn oil, preferably the 4-hydroxy tamoxifen solution is prepared according to the proportion of 50mg of 4-hydroxy tamoxifen, 1ml of DMSO and 9ml of corn oil; the dose of the 4-hydroxy tamoxifen solution applied externally to the mice is 10-80 mu l each time.

Wherein the mice develop disease phenotype 7-12 days after the initial external application of 4-hydroxy tamoxifen.

Specifically, the keratinocyte conditional pparγ knockout mice were constructed by the following method:

and mating PPARγflox/flox and Krt5-CreERT2 mice to obtain a hybrid generation, reserving heterozygote mice with the genotype of Krt5-CreERT2+/-PPARγflox/flox +/-and mutually mating and breeding the heterozygote mice to obtain mice with the genotype of Krt5-CreERT2+/-PPARγflox/flox-/-.

The invention further provides application of the spontaneous skin type lupus erythematosus animal model constructed by the construction method in researching CLE disease mechanism.

The invention also provides application of the spontaneous skin type lupus erythematosus animal model constructed by the construction method in screening medicaments for targeted treatment of lupus skin lesions.

The beneficial effects are that: the invention constructs a spontaneous CLE animal model by knocking out PPARgamma in mouse keratinocytes, which does not need artificial induction and can conditionally appear CLE-like phenotype, including local skin inflammatory phenotype, dehairing, and dermal immune cell infiltration in histological examination, and the basal lamina tape IgG deposition; meanwhile, systemic lupus erythematosus like changes such as proteinuria, glomerular IgG deposition and serum autoantibodies do not exist, the clinical symptoms of the CLE are simulated, and an important tool is provided for the related research of the CLE. Compared with other CLE models, the model has the advantages of rapid onset, high efficiency and stability due to genetic background instead of drug induction. All mice with a model built will have a phenotype of a certain degree 1-2 weeks after induction of PPARgamma knockout of keratinocytes, the success rate of modeling is 100%, the heterogeneity in groups is small, and the repeatability is strong. In addition, krt5-CreERT2+/-PPARγflox/flox-/-mice can be obtained continuously through breeding, and are economical and convenient.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 is a graph showing genotyping of keratinocyte PPARgamma conditional knockout mice;

FIG. 2 shows the visual appearance of depilatory and inflammatory lesions after PPARgamma knockdown in a local mouse keratinocyte, showing hyperkeratosis, follicular angle plugs, thickening of the stratum spinosum, liquefied degeneration of basal cells, and immune cell infiltration of the dermis layer by histological examination of the skin;

FIG. 3 shows basal lamina tape IgG deposition as indicated by frozen sections of skin tissue after PPARgamma knockout of local mouse keratinocytes;

FIG. 4 shows that there is no significant IgG deposition in the glomeruli of mice following a partial keratinocyte PPARgamma knockout;

FIG. 5 shows that mice do not exhibit urinary proteins following a partial keratinocyte PPARgamma knockout;

FIG. 6 shows that there is no significant increase in the level of anti-nuclear antibodies and anti-dsDNA antibodies in the peripheral blood of mice after PPARgamma knockdown of local keratinocytes.

Detailed Description

The present invention will be further described with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. Various substitutions and alterations are made according to the general technical knowledge and the conventional methods in the field without departing from the technical idea of the present invention, and all such substitutions and alterations are included in the protection scope of the present invention.

Example 1 4-hydroxy tamoxifen monaural application activated topical keratinocyte pparγ knockout induced CLE model.

1. Experimental materials

1. Experimental drugs and reagents: DMSO (Sigma-Aldrich), 4-hydroxy tamoxifen (Sigma-Aldrich), corn oil (Beyotime), proteinuria test paper (Ulipite), urine protein quantification kit (Nanjing Jiancheng Bioengineering Institute), anti-nuclear and anti-double stranded DNA ELISA kit (Cusabio), 4% paraformaldehyde (Biosharp), OCT embedding medium (Sakura), goat anti-mouse IgG-FITC (abcam).

1.2 laboratory animals

The mice used in this experiment were Krt5-CreERT2+/-PPARγflox/flox-/-mice, about 5-12 weeks of week old, and 16-30g body weight, available from Shanghai Nannon model biotechnology Co., ltd. Krt5-CreERT2 mice are able to express the CreERT2 fusion protein targeted in keratinocytes by means of the endogenous promoter/enhancer element of the Krt5 locus. The CreERT2 fusion protein consists of a ligand binding region mutant (ERT) of the estrogen receptor (estrogen receptor, ER) and a Cre recombinase protein. In the absence of tamoxifen induction, the Creet 2 protein is in an inactive state within the cytoplasm; after tamoxifen induction, the metabolite 4-hydroxy tamoxifen of tamoxifen is combined with ERT to make CreERT2 enter nucleus to generate the recombinant enzyme activity of Cre. PPARγflox/flox mice, i.e., PPARγ conditional knockout mice, have a loxP site inserted at both ends of the specific exon of the PPARγ gene, which can be cleaved by the active Cre recombinase. The use of Krt5-CreERT2 mice mated with mice containing loxP flanking sequences resulted in offspring mice of genotype Krt5-CreERT2+/-PPARγflox/flox-/-where the PPARγ gene in Krt5 expressing cells could be knocked out by tamoxifen-induced Cre-mediated gene recombination. The constructed Krt5-CreERT2+/-PPARγflox/flox-/-mice can precisely directionally knock out PPARγgene in keratinocytes. Compared with a common conditional knockout mouse, the gene knockout time can be accurately regulated, the phenotype of the mouse before and after gene knockout can be compared on the same individual, the disease occurrence process can be simulated to the greatest extent, and the model mouse is an ideal CLE disease model mouse.

Feeding conditions: the room temperature is 18-20 ℃, the humidity is 50-60%, the brightness is alternate (12 hours), the luminosity is moderate, and the ventilation is clean. All experiments were approved and conducted as directed by the ethical committee of the dermatology hospital of the national academy of medical science (the dermatology institute of the national academy of medical science).

2. Experimental method

2.1 local keratinocyte knockout of PPARgamma in mice

PPARγflox/flox-/-Krt 5-Creet2+/-mice were grouped as required: control and model groups. 50mg of 4-hydroxy tamoxifen was mixed in 1ml of DMSO, 9ml of corn oil was added, the suspension was thoroughly mixed using a vortexing device, and sonicated in an ultrasonic bath at 37℃for 20 minutes to prepare a solution of 4-hydroxy tamoxifen (5 mg/ml) ready for use. Corn oil containing 10% DMSO was applied to one ear of the control group; model set single ear paint (about 3cm area) ² ) 50 μl of a solution of 5mg/ml 4-hydroxy tamoxifen. Two groups of mice were continuously applied with the corresponding solution on a single ear for 6 days.

2.2PCR identification of keratinocyte PPARgamma conditional knockout mouse genotype

Mouse tail genomic DNA was extracted according to the instructions using a rapid mouse tail genotype identification kit (beyotide). Fresh mouse tail was taken: scissors and forceps were rinsed with 70% ethanol prior to the experiment. Cutting tail tip of a mouse with the length of 0.2 cm to 1cm to prepare template DNA, and preparing a PCR reaction system:

TABLE 1 PCR reaction System

Reagent(s)	Final concentration	Volume of
			Double distilled water or Milli-Q water	-	7.4μl
Stencil (digestion products)	2-20ng/μl	1μl
			Primer mix (10 uM each)	0.8uM	1.6μl
Easy-Load ^TM PCR Master Mix(Green,2X)	1X	10μl
			Total volume of	-	20μl

TABLE 2PCR reaction parameters

After the completion of the PCR reaction, agarose gel electrophoresis was performed. The genotype of the mice was detected. The primer sequences were as follows:

PPARγflox/flox primer 1:5'-CTTCCCCTTCCCCAAAATGAGTC-3';

PPARγflox/flox primer 2:5'-TCTGTGGCTGGACTACAGGA-3';

krt5-e (2A-CreERT 2) primer 1:5'-GTGGCTTACATTCTGCAACATTTT-3';

krt5-e (2A-CreERT 2) primer 2:5'-GGCCCACGCTTCACCAG-3';

krt5-e (2A-CreERT 2) primer 3:5'-GGATCCGCCGCATAACCAGT-3'.

Wherein for pparγflox: the size of the variant (Mutant) is 572bp, the size of the Heterozygote (heteozygate) is 572bp, and the Wild type (Wild type) is 445bp;

for Krt5-Cre: the variant (Mutant) was 610bp in size, the Heterozygote (Heterozygate) was 610bp in size, and the Wild-type (Wild type) was 453bp.

2.3 mouse skin lesions, urine proteins and autoantibody monitoring

Mice were observed for skin lesions weekly and photographed with a camera for survival. The mice were periodically cleaned of midrange urine and urine protein was detected using a test paper for detection of ulide proteinuria. The urinary protein content of mice was quantified using CBB. The content of peripheral blood serum antinuclear antibodies and anti-double-stranded DNA antibodies was detected using ELISA kit (Cusabio).

2.4 histopathology

On day 17, mice were sacrificed after anesthesia for cervical dislocation. Binaural skin of the same portion of the three groups of mice was cut with a sharp scalpel and ophthalmology, approximately 0.5 cm x 0.5 cm, spread on tinfoil, and snap frozen with liquid nitrogen or fixed to 4% paraformaldehyde. And (5) after fixation, dehydrating, waxing and embedding. And (3) slicing the tissue block, spreading, baking, dewaxing, hematoxylin and eosin staining, sealing, taking a view picture by using a microscope, and analyzing inflammatory cell infiltration in the skin damage of the mice.

2.5 immunofluorescent staining

After the skin tissue or kidney subjected to liquid nitrogen quick freezing is embedded by OCT, the skin tissue or kidney is placed on a frozen section mould, the frozen section mould is used for slicing, OCT is washed off, a goat anti-mouse IgG-FITC (1:200) is added after the immune fluorescence sealing liquid (Beyotime) is sealed for 1 hour at 37 ℃ for incubation at 4 ℃ overnight, the cell nucleus is stained with DAPI working liquid at room temperature for 10 minutes after PBS is cleaned, the sealing sheet is cleaned by PBS, and the skin and glomerular IgG deposition is observed by a fluorescence microscope imaging.

3. Experimental results

FIGS. 1-6 are graphs showing the phenotypic outcome of spontaneous CLE-like disease induced in mice 17 days after PPARgamma knockdown using localized keratinocytes with single ear smeared with 4-hydroxy tamoxifen as gene knockout activator.

FIG. 1 is a graph showing genotyping of keratinocyte PPARgamma conditional knockout mice. The genotype of the target mouse is Krt5-CreERT2+/-PPARγflox/flox-/-, such as 07.

Fig. 2 is a photograph of a local keratinocyte pparγ knockout day 17 mouse. The mice in the model group can see dehairing and inflammatory skin lesions, and the mice in the control group have no skin lesions. The skin histological examination of the mice in the model group showed the existence of hyperkeratosis, hair follicle horn plug, thickening of the stratum spinosum, liquefied degeneration of basal cells, and infiltration of dermal layer immune cells. The skin of the same part of the mice in the control group has no obvious inflammatory change. Indicating that mice with reduced levels of PPARgamma protein in a small range of keratinocytes spontaneously develop CLE-like inflammatory lesions.

FIG. 3 shows the deposition of basal lamina IgG in mice on day 17 of PPARgamma knockout of local mouse keratinocytes. Frozen sections of skin tissue from mice in the model group indicated the presence of basal lamina tape IgG deposition. Control mice had no basal lamina with IgG deposition on the skin at the same site. Indicating that mice with reduced levels of PPARgamma protein in a small range of keratinocytes spontaneously develop skin basement membrane band IgG deposition.

FIG. 4 shows the deposition of mouse glomerular IgG on day 17 of local keratinocyte PPARgamma knockout. The glomeruli of the mice in the model group had no significant IgG deposition compared to the control group. The above results indicate that a decrease in pparγ in a small range of keratinocytes does not lead to glomerular IgG deposition in mice.

Fig. 5 is the urinary protein content of the mice on day 17 of local keratinocyte pparγ knockout. The urine protein of the model group mice has no obvious change compared with the urine protein of the control group mice. The above results indicate that a decrease in pparγ in a small range of keratinocytes does not interfere with mouse kidney function and cause proteinuria.

FIG. 6 shows the levels of anti-nuclear antibodies and anti-dsDNA antibodies in peripheral blood of mice from day 17 of local keratinocyte PPARgamma knockout. The content of the anti-nuclear antibody and the anti-double-stranded DNA antibody of the peripheral blood of the model group mice is not obviously different from that of the control group mice. The above results indicate that pparγ reduction in a small range of keratinocytes does not result in autoantibody in mice.

We used 7 keratinocyte pparγ conditional knockout mice in total for molding, 7 mice were successful in molding, and the molding success rate was 100%. The above results indicate that the animal model phenotype is similar to CLE clinical symptoms.

In summary, we develop a CLE mouse model which can be induced rapidly and efficiently by knocking out the epidermal pparγ, the model is similar to the genetic background of a patient, the self-initiated disease does not need artificial interference, the phenotype stability heterogeneity is small, so that we can explore the pathophysiology mechanism of CLE and effectively perform drug screening, and a construction method of a keratinocyte pparγ knocking-out induced self-initiated CLE animal model is established, so as to solve the difficulties of large clinical difference, high price and instability of the current CLE model.

The invention provides a thought and a method for preparing a self-hairing CLE animal model by knocking out keratinocyte PPARgamma in a small range, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. The method for constructing the spontaneous skin type lupus erythematosus animal model is characterized by comprising the following steps of:

(2) Small-scale knockout of PPARgamma genes in keratinocytes using gene knockout activatorsTo mediate disease phenotype occurrence; the small range knockout is that the skin on the ventral side or the dorsal side of the single ear of a diagonal forming cell conditional PPARgamma knockout mouse or the body is less than or equal to 4cm ² Is applied to the hairless or shaved skin.

2. The method of claim 1, wherein the knock-out activator is a compound that is metabolized to bind to the estrogen receptor mutant ERT to allow CreERT2 to exert Cre recombinase activity for gene knockout.

3. The method of claim 2, wherein the gene knockout activator is 4-hydroxy tamoxifen.

4. A method of construction according to claim 3, wherein 4-hydroxy tamoxifen is applied by any one of topical application, dermal application, subcutaneous injection with or without needle, or intradermal injection.

5. The method according to claim 3, wherein the skin on the ventral and dorsal sides, or on the body of a keratinocyte conditional PPARgamma knockout mouse is 4cm ² Continuously applying 4-hydroxy tamoxifen solution to the hairless or shaved skin for 1-7 days, once a day.

6. The method according to claim 5, wherein the 4-hydroxy tamoxifen solution is prepared in a ratio of 50mg of 4-hydroxy tamoxifen, 1ml of DMSO, 3 to 49ml of corn oil; the dose of the 4-hydroxy tamoxifen solution applied externally to the mice is 10-80 mu l each time.

7. The method of claim 1, wherein the mice develop disease phenotype 7-12 days after the initial topical application of 4-hydroxy tamoxifen.

8. The method of claim 1, wherein the keratinocyte conditional pparγ knockout mouse is constructed by:

and mating PPARγflox/flox and Krt5-CreERT2 mice to obtain a hybrid generation, reserving heterozygote mice with the genotype of PPARγflox/flox +/-Krt 5-CreERT2+/-, and mating and breeding the heterozygote mice with each other to obtain the mice with the genotype of PPARγflox/flox-/-Krt5-CreERT2 +/-.

9. Use of the spontaneous cutaneous lupus erythematosus animal model constructed by the construction method of any one of claims 1-8 in researching CLE disease mechanism.

10. Use of the spontaneous cutaneous lupus erythematosus animal model constructed by the construction method of any one of claims 1-8 in screening drugs for targeted treatment of lupus skin lesions.