Application of PNO1 in construction of ulcerative colitis animal model

Abstract

The invention discloses an application of PNO1 in construction of an ulcerative colitis animal model. PNO1 is applied to at least one of the following items: (1) preparing a product for diagnosing ulcerative colitis; (2) screening candidate drugs for preventing and / or treating ulcerative colitis as targets; and (3) constructing the ulcerative colitis animal model. The invention firstly finds that the PNO1 gene is positively correlated with the ulcerative colitis and can be used as a marker for diagnosing the ulcerative colitis, so that the ulcerative colitis is more accurately and quickly diagnosed, and a new therapeutic target and a new therapeutic approach are provided for treating colon cancer. The invention further constructs an ulcerative colitis animal model which can be better used for pathogenic mechanism and treatment research of ulcerative colitis and screening of colitis candidate drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the use of PNO1 in constructing an ulcerative colitis animal model. Background Art

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic idiopathic intestinal inflammation involving dynamic changes in multiple cell types, including epithelial cells, mesenchymal cells, and immune cells. Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease characterized by continuous and diffuse inflammatory changes in the colorectal mucosa. The pathological changes are mainly colonic ulcers and erosions, which are diffuse, superficial, and continuous, with a long course and repeated attacks.

[0003] The construction of animal models provides a good means for the study of disease mechanisms and the screening of therapeutic drugs. At present, the methods for establishing animal models of inflammatory bowel disease are mainly divided into: spontaneous models, induced models and gene-modified models, among which the induced models include chemical drug induction, immunological method induction, cell transplantation induction, etc. Spontaneous models have the problem of being difficult to use in large numbers as animal experimental models due to the inconsistency of onset time and severity. Chemical induction models, often induced by trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS), are the most commonly used modeling methods because of their low cost, controllability and strong repeatability. However, they are challenging to determine the appropriate dose of chemical substances. Too low a dose may not produce the desired pathological changes, while too high a dose may cause excessive pathological damage or animal death. In addition, animals with different genetic backgrounds may respond differently to the same chemical substance, which may affect the universality and repeatability of the model. Immune-induced animal models use allogeneic or xenogeneic tissues to sensitize experimental animals. They have the problems of complex preparation methods, long experimental cycles, and limited ability to distinguish between the immune responses of mice and humans. Cell transplantation induction models are difficult to standardize, lack stable reproducibility, and are difficult to effectively study pathogenic mechanisms. Genetically modified models refer to the use of gene editing technology to insert exogenous genes into the animal genome or mutate, knock out, overexpress, etc. the animal's own genes to give the animal UC characteristics. The onset and severity of the disease depend largely on environmental factors, resulting in high variability in research.

[0004] At present, the UC model is achieved by allowing mice to freely drink dextran sulfate sodium (DSS). DSS is a sulfated polysaccharide synthesized from sucrose and has an anticoagulant effect similar to heparin. DSS is given to rats by free drinking. According to the medication time and medication cycle, two types of colitis models, acute and chronic, can be made. Acute colitis models usually use higher concentrations of DSS and a shorter administration time, such as 3% to 5% DSS free drinking for one week; while chronic colitis models use low concentrations of DSS and a longer administration time, such as 1% to 3% DSS continuous drinking for several weeks. Wang Qian et al. studied and compared the results of rat models of acute colitis induced by different concentrations of DSS. That is, rats were allowed to freely drink different concentrations of DSS solutions for 7 days to construct a rat UC model. The effect of DSS solution concentration on the severity of the disease was explored and it was found that 30g / L DSS could construct an acute UC rat model (Wang Qian, Bao Yongxin. Construction and comparison of rat models of acute ulcerative colitis induced by two mass concentrations of DSS [J]. Journal of Guangzhou University of Chinese Medicine, 2019, 36(04): 574-577). However, the DSS-induced colitis model used free drinking water to administer the drug, which resulted in large differences within the group. Some individuals drank less water, resulting in less obvious colitis symptoms.

[0005] Currently, there are few reports on the construction of colitis animal models using gene modification. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of PNO1 in constructing an ulcerative colitis animal model, thereby solving the problems in the prior art.

[0007] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0008] The first aspect of the present invention protects the use of PNO1 in at least one of the following: (1) preparing a product for diagnosing ulcerative colitis; (2) as a target for screening candidate drugs for preventing and / or treating ulcerative colitis; (3) constructing an ulcerative colitis animal model.

[0009] The second aspect of the present invention protects a method for constructing an ulcerative colitis animal model, comprising the following steps: inserting an expression frame containing the PNO1 gene into the genome of the animal to obtain the PNO1 gene knock-in animal.

[0010] The third aspect of the present invention protects the use of the ulcerative colitis animal model obtained by the construction method described above in at least one of the following: A1) screening drugs for preventing and / or treating ulcerative colitis; A2) studying the pathology and etiology of ulcerative colitis.

[0011] The fourth aspect of the present invention protects a method for screening candidate drugs for preventing and / or treating ulcerative colitis, comprising the following steps: administering the drug to the ulcerative colitis animal model obtained by the construction method as described above, and screening the candidate drug by comparing the changes in the ulcerative colitis animal model before and after administration of the drug.

[0012] The technical solution of the present invention has the following beneficial effects:

[0013] 1) The present invention found that the PNO1 gene is positively correlated with ulcerative colitis at the molecular level. The mRNA, gene and protein expression levels of PNO1 are significantly increased in ulcerative colitis tissues. Therefore, PNO1 can be used as a biomarker for ulcerative colitis. Whether a patient has ulcerative colitis can be determined by detecting PNO1.

[0014] 2) The present invention knocks in the PNO1 gene in animals and uses DSS induction to construct an ulcerative colitis animal model. This combination of gene modification and chemical induction is consistent with the etiology that acute colitis is induced by multiple factors, and can better simulate the characteristics of human ulcerative colitis. The ulcerative colitis animal model of the present invention basically meets the evaluation of the three dimensions of disease activity index score, histological change score and colon length. Whether from the perspective of pathophysiological theory or the perspective of simulating typical clinical symptoms and treatment prediction, it is an ideal animal model, providing a theoretical basis for the development of new treatment plans for ulcerative colitis.

[0015] 3) The animal model constructed by the present invention has stable effects and small individual differences; the gene and chemical inductions work together to construct a more accurate disease model. The DAI index, histological change score, and colon length severity of the ulcerative colitis in this model are higher than those of animals induced by DSS alone and animals knocked into the PNO1 gene. In addition, the expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) are also higher than those of animals induced by DSS alone and animals knocked into the PNO1 gene. This shows that the ulcerative colitis animal model constructed by this application can be better used for the pathogenic mechanism and treatment research of ulcerative colitis, as well as the screening of candidate drugs for ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is a statistical graph of the expression level of PNO1 mRNA in UC tissues and normal tissues in Example 1 of the present invention.

[0017] Figure 2 Shown is a statistical chart of the expression level of PNO1 protein in UC tissues and normal tissues in Example 1 of the present invention.

[0018] Figure 3 Shown is a graph showing the body weight results of each group of mice in Example 2 of the present invention.

[0019] Figure 4 Shown is the DAI index result graph of each group of mice in Example 2 of the present invention.

[0020] Figure 5 Shown is the colon length result graph of each group of mice in Example 2 of the present invention.

[0021] Figure 6 Shown is the spleen weight result of each group of mice in Example 2 of the present invention.

[0022] Figure 7 Shown is a graph showing the degree of colon pathological damage in each group of mice in Example 2 of the present invention.

[0023] Figure 8 The graph shows the expression results of PNO1 protein in the colon tissue of each group of mice in Example 2 of the present invention.

[0024] Fig. 9 Shown is the expression result of inflammatory factors in the colon tissue of each group of mice in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The intestine is the largest immune organ in the body. It can effectively respond to pathogens that enter the intestine. It is the body's first line of defense against foreign invaders and is also the place where the body regulates systemic immune function. The intestine is an organ that communicates with the outside world. Different pathogens and microorganisms invade the intestine every day. When the body is stressed, traumatized, or the body's resistance is reduced, these foreign molecules enter the intestine, destroy the intestinal mucosal barrier, shorten or disappear the intestinal villi, and destroy intestinal epithelial cells, leading to the accumulation of inflammatory cells and the release of a large number of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α. These inflammatory factors can cause chemotaxis of macrophages and further synthesize cyclooxygenase, leading to intestinal inflammation in animals, and then abdominal pain, diarrhea, and blood in the stool.

[0026] Currently, there are many animal models that are widely used to study the etiology and pathogenesis of ulcerative colitis and test the efficacy of newly developed drugs. In particular, the DSS-induced colitis model is the most widely used. It is constructed by giving animals free drinking of DSS aqueous solutions of different concentrations. The symptoms of the model are very similar to those of human UC, mainly manifested as diarrhea, mucous stools, fecal occult blood, macroscopic blood in stools, weight loss, decreased activity, and poor coat color. Because DSS-induced ulcerative colitis animal models mostly use free drinking, the amount of drinking for each animal cannot be accurately controlled, and individual differences may occur, which will have a certain impact on the results.

[0027] The present invention found that PNO1 was significantly increased in UC tissues at the mRNA, gene and protein expression levels. Then, PNO1 knock-in animals were constructed by gene editing and DSS induction was used to construct an ulcerative colitis animal model. The model constructed by the present invention has the advantages of being suitable for the disease, good repeatability and strong stability. Therefore, the model of the present invention can be used for the subsequent pathological pathology and etiology of UC, as well as the diagnosis of UC disease and the screening of UC treatment drugs. On this basis, the present invention was completed.

[0028] The first aspect of the present invention protects the use of PNO1 in at least one of the following:

[0029] (1) Preparation of products for diagnosing ulcerative colitis;

[0030] (2) as a target for screening candidate drugs for the prevention and / or treatment of ulcerative colitis;

[0031] (3) Construct an animal model of ulcerative colitis.

[0032] The present invention firstly discovered that the expression level of PON1 mRNA in UC tissue was significantly higher than that in normal colon tissue. Then, through the GEO database, it was found that the expression level of PNO1 gene in UC tissue was significantly higher than that in normal colon tissue. Further detection of the expression level of PNO1 protein in UC tissue was significantly higher than that in normal colitis tissue. Therefore, PNO1 can be used as a diagnostic indicator for ulcerative colitis.

[0033] In certain embodiments, screening for drugs to prevent and / or treat colitis refers to using PNO1 as the target of the drug, screening or designing drugs that can antagonize the biological functions mediated by the target PNO1, and thus screening drugs for the prevention or treatment of ulcerative colitis. Drug screening is usually achieved through drug screening models, which include in vivo and in vitro models. In vivo models usually use animal models, such as mice, rats, pigs, monkeys, etc.; in vitro models usually refer to studies conducted in vitro, such as in vitro cell experiments, tissue slice experiments, etc. In vitro cell experiments include human cells, animal cells or bacteria, etc.

[0034] In certain embodiments, the PNO1 gene is numbered NM_025443.2 in the NCBI database and ENSMUSG0000002016 in the Ensembl database, and is located on mouse chromosome 11. The CDS sequence comprises the sequence shown in SEQ ID NO.1.

[0035] The second aspect of the present invention protects a method for constructing an ulcerative colitis animal model, comprising the following steps: using DSS to induce PNO1 gene knock-in animals to obtain the ulcerative colitis animal model.

[0036] The chemical induction regulation system of gene expression has the advantages of high gene expression level and artificial control. The biggest advantage of the chemical induction gene expression system is that the precise control of gene timing and localization expression is achieved through the administration of chemical induction substances. The present application successfully constructed an ulcerative colitis animal model by combining gene modification and chemical induction, which has typical symptoms of ulcerative colitis (UC), such as weight loss, occult blood in stool, shortened colon length, colon tissue pathological changes, etc. Compared with the ulcerative colitis animal model constructed by DSS induction alone or the PNO1 gene knock-in animal alone, the model constructed by the present invention using the combination of PNO1 knock-in and DSS induction has a higher DAI index, histological change score, severity of colon length, and early appearance of typical symptoms of UC; in addition, the expression levels of inflammatory factors TNF-α, IL-6 and IL-1β are also higher than those of the DSS-induced animal model and the PNO1 gene knock-in animal alone, so it can be better used for the pathogenesis and treatment of ulcerative colitis.

[0037] In certain embodiments, the animal is selected from one or more of mice, rats, hamsters, zebrafish, pigs, fruit flies and rabbits. Preferably, it is a mouse. Specifically, it is a C57BL / 6 mouse. The mouse is an 8-week-old mouse with a body weight of about 26 g.

[0038] In certain embodiments, the method for obtaining the PNO1 gene knock-in animal is as follows: inserting an expression frame containing the PNO1 gene into the genome of the animal to obtain the PNO1 gene knock-in animal.

[0039] In certain embodiments, the nucleotide sequence of the PNO1 gene comprises the sequence shown in SEQ ID NO. 1. The PNO1 gene is derived from mice.

[0040] In certain specific embodiments, the expression cassette containing the PNO1 gene is inserted by using the CRISPR / Cas9 gene editing system or the Cre / loxp gene editing system. Preferably, the expression cassette containing the PNO1 gene is inserted by using the Cre / loxp gene editing system.

[0041] In certain specific embodiments, the site where the expression cassette containing the PNO1 gene is inserted is selected from the Rosa26, Hprt and hipp11 sites, preferably the Rosa26 site.

[0042] In certain more specific embodiments, the expression cassette containing the PNO1 gene comprises: promoter-Loxp-3*SV40-Loxp-PNO1-polyA. The Loxp comprises the sequence shown in SEQ ID NO.2. The 3*SV40 (same as 3*ployA) comprises the sequence shown in SEQ ID NO.3. The promoter is a CAG promoter. The CAG promoter comprises the sequence shown in SEQ ID NO.4. The polyA comprises the sequence shown in SEQ ID NO.5.

[0043] In some more specific embodiments, the expression cassette containing the PNO1 gene further comprises a DTA element, and the DTA is located upstream of the promoter. The DTA comprises the sequence shown in SEQ ID NO.6.

[0044] In some more specific embodiments, the expression cassette containing the PNO1 gene further comprises a Neo element, and the Neo is located downstream of poly A. The Neo comprises the sequence shown in SEQ ID NO.7.

[0045] In certain more specific embodiments, the expression frame containing the PNO1 gene is site-specifically inserted into a safe site of an animal via a targeting vector, wherein the targeting vector comprises the sequences shown in SEQ ID NO.8 and SEQ ID NO.9.

[0046] In one embodiment, the method for obtaining a PNO1 gene knock-in animal by inserting an expression cassette containing the PNO1 gene at a specific site based on the Cre / Loxp system is as follows:

[0047] 1) constructing a targeting vector containing the expression frame of the PNO1 gene;

[0048] 2) Introducing the targeting vector into embryonic stem cells (ES) to obtain positive clones, and transplanting the positive clones into pseudo-pregnant female animals to obtain gene knock-in animals, i.e., F0 generation positive animals;

[0049] 3) Crossbreeding the F0 generation positive animals with wild-type animals to obtain F1 generation positive animals;

[0050] 4) self-pollinating the F1 generation positive animals to obtain F2 generation positive animals;

[0051] 5) Crossbreeding the F2 generation positive animals with systemic Cre tool animals to obtain F3 generation positive animals;

[0052] 6) The positive F3 animals were self-fertilized to obtain F4 animals, i.e., PNO1 gene knock-in animals.

[0053] Further, in 1), the expression cassette containing PNO1 is inserted into the plasmid VB009-fv through the homologous recombination arm to obtain a targeting vector. The targeting vector comprises the following expression cassette: DTA-5'arm-CAG Promoter-Loxp-3*SV40-Loxp-Pno1CDS-polyA-SDA-Neo-SDA-3'arm. Specifically, the sequence of the targeting vector is shown in SEQ ID NO.8 and SEQ ID NO.9.

[0054] Further, in 2), the targeting vector is introduced into ES cells by various methods such as electroporation, calcium phosphate / DNA precipitation and direct injection, and the method is introduced here. Preferably, the targeting vector is introduced into ES cells by electroporation. The standard electroporation procedure of cyagen is followed, and the parameters of electroporation are set to 240V, 500μF.

[0055] Furthermore, in 2), the means of transplanting into pseudo-pregnant female animals include but are not limited to various transformation or transfection methods, such as calcium phosphate, liposome-mediated chemical transfection, gene gun, electroporation, microinjection and other physical methods. Microinjection is preferred.

[0056] In certain embodiments, the weight average molecular weight of the DSS is 36,000 to 50,000 Da.

[0057] In certain embodiments, the DSS is administered via a DSS aqueous solution, the concentration of which is 1-5wt%, 1-3wt%, 2.5-4wt%, 3-5wt%, 1wt%, 2wt%, 3wt%, 4wt%, or 5wt%. The concentration of the DSS aqueous solution can be adjusted as needed. Generally, when constructing a chronic colitis model, the DSS aqueous solution is 1-3wt%; and when constructing an acute colitis model, the concentration of the DSS aqueous solution is 3-5wt%.

[0058] In certain specific embodiments, based on the animal body weight, the dosage of the DSS aqueous solution is 150-250 mL / kg body weight, or 150-195 mL / kg body weight, or 180-220 mL / kg body weight, or 205-250 mL / kg body weight, or 150 mL / kg body weight, 165 mL / kg body weight, 180 mL / kg body weight, 205 mL / kg body weight, 221 mL / kg body weight, 240 mL / kg body weight, or 250 mL / kg body weight.

[0059] In certain specific embodiments, the DSS induction comprises the following steps:

[0060] 1) First administration: Continue to administer DSS for 5 to 7 days;

[0061] 2) Second administration: After the first administration, the animals were fed in the usual way for 7 to 14 days, and then DSS was continuously administered for another 5 to 7 days;

[0062] 3) Third administration: After the second administration, the animals were fed in the usual way for 7 to 14 days, and then DSS was continuously administered for another 5 to 7 days;

[0063] After the third administration, the ulcerative colitis animal model was obtained.

[0064] The success of ulcerative colitis modeling is evaluated and scored from three aspects, namely, the Disease Activity Index score (DAI score), the histological change score, and the colon length. The DAI score is the sum of three indicators: body weight, stool viscosity, and fecal occult blood; the standard method for histological analysis is HE staining; and the colon length is shortened. The severity of the DAI index, histological change score, and colon length of the animal enteritis model of the present application is higher than that of the DSS-induced animal model and the PNO1 gene knock-in animal alone.

[0065] The third aspect of the present invention protects the use of the ulcerative colitis animal model obtained by the construction method as described above in at least one of the following: A1) screening drugs for preventing and / or treating ulcerative colitis; A2) studying the pathology and etiology of ulcerative colitis.

[0066] The fourth aspect of the present invention protects a method for screening candidate drugs for preventing and / or treating ulcerative colitis, comprising the following steps: administering the drug to an ulcerative colitis animal model obtained by the construction method described above, and screening out the candidate drug by comparing the changes in the ulcerative colitis animal model before and after administration of the drug.

[0067] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0068] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0069] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, as those skilled in the art grasp the prior art and record the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0070] In the following examples of this application, the information of animals, experimental drugs and main reagents is as follows:

[0071] Experimental drugs and main reagents

[0072] DSS (batch number 160110), DSS molecular weight range is 36000~50000Da, MP Company, USA; anhydrous ethanol (batch number 64-17-5), Xilong Science Co., Ltd.; xylene (batch number 1330-20-7), Shanghai Wokai Biotechnology Co., Ltd.; eosin staining solution (batch number G1100), hematoxylin staining solution (batch number G1080), Beijing Solebow Company; paraformaldehyde (batch number LA0427), Fuzhou Feijing Biotechnology Co., Ltd.; PNO1 antibody (batch number 21059-1-AP), Wuhan Sanying Biotechnology Co., Ltd.; IL-6 antibody (batch number ab208113), Abcam Company, USA; IL-1β antibody (batch number 12242), CST Company, USA; TNF-α antibody (batch number 41504), Signalway Biotechnology Co., Ltd., USA Biotechnology Company; Immunohistochemistry kit (Batch No. KIT-9720), Sodium citrate antigen retrieval solution (Batch No. MVS-0066), DAB staining kit (Batch No. DAB-0031), Fuzhou Maixin Biotechnology Development Co., Ltd.

[0073] Main experimental instruments

[0074] Pipette (Raining, USA); electronic balance (Ohaus Instruments, Shanghai); automatic biological tissue dehydrator (Hubei Xiaogan Yaguang Medical Electronics Technology Co., Ltd.); paraffin embedding machine (Hubei Xiaogan Yaguang Medical Electronics Technology Co., Ltd.); pathological slicer (Leica, Germany), ultrapure water system (Merck Millipore, USA); inverted microscope system (Leica, Germany).

[0075] Data Statistical Analysis

[0076] All experimental results were statistically analyzed using SPSS26.0 software for significant differences. The experimental results were expressed as ±s. If the results were in accordance with the normal distribution, the independent sample T test (two-group comparison) or one-way variance (ANOVA) (three or more groups) was used for inter-group comparison; if the results were not in accordance with the normal distribution, Tamhane's T2 (two-group comparison) or rank sum test (three or more groups) was used for analysis. P<0.05 was considered to be statistically significant.

[0077] Example 1

[0078] In this Example 1, the expression difference of PNO1 in colitis tissue and normal tissue was studied from the mRNA level, gene level and protein level.

[0079] 1) Q-PCR detection of PNO1 expression at the mRNA level in UC tissues and normal intestinal tissues in cDNA chips

[0080] UC-related cDNA chips included 40 UC tissues and 7 normal intestinal tissues. The experiment was performed according to the instructions provided by the company. The PCR reaction system was prepared according to the table below and placed in a 7500Fast Real time PCR instrument for mRNA expression detection. The PCR reaction system and reaction time are shown in the table below.

[0081] Table 1 PCR reaction system preparation

[0082]

[0083] Table 2 PCR reaction conditions

[0084]

[0085] Expression level calculation: After amplification, the fluorescence signal was collected at each cycle extension and the data were analyzed and sorted.

[0086] mRNA expression calculation formula:

[0087] △Ct of mRNA = Ct(sample)-CT(internal reference);

[0088] △△Ct=△Ct-△Ct(mean);

[0089] The relative expression of mRNA was 2 -△△ct

[0090] Table 3 PNO1 and GAPDH primer sequences

[0091]

[0092] The expression of PNO1 mRNA in UC tissues and normal intestinal tissues was detected by Q-PCR on cDNA chip. Figure 1 As shown in D.

[0093] from Figure 1 As shown in D, the expression of PNO1 mRNA in 40 UC tissues was significantly higher than that in 7 normal intestinal tissues (*P<0.05, compared with normal intestinal tissues).

[0094] 2) GEO2R analysis of PNO1 gene expression in UC tissues and normal intestinal tissues

[0095] In the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), the GEO Profiles search was used to search for datasets GSE87473, GSE75214, and GSE87466 related to the clinical sample data of "Ulcerative colitis". The differentially expressed genes were analyzed by GEO2R, ​​and the PNO1 gene was selected to analyze the differences in PNO1 between UC tissues and normal intestinal tissues. The results showed that Figure 1 A, B and C.

[0096] from Figure 1 As shown in Figures A, B and C, compared with normal intestinal tissue, the expression of PNO1 in UC tissue was significantly higher than that in normal tissue in multiple data sets (*P<0.05).

[0097] 3) Immunohistochemical detection of PNO1 protein expression levels in UC tissues and normal tissues

[0098] Ulcerative colitis samples (including 11 UC tissues and 7 normal tissues) were collected in the Second People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine from 2020 to 2021, and a portion of the fresh tissue was fixed with 4% paraformaldehyde.

[0099] (1) Remove wax and enter water:

[0100] The tissue chip was dewaxed with xylene for 15 min × 2 times, 100% ethanol for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, tap water for 5 min, and PBS for 5 min × 3 times.

[0101] (2) Repair, blocking, antibody incubation, and color development

[0102] Pour the antigen repair solution into a beaker and heat it to boiling in a microwave oven. Place the paraffin sections in the beaker and simmer for 10 minutes, then take out the beaker and cool it to room temperature. Use PBS to wash off the antigen repair solution (5 minutes × 3 times), add 100 μL of endogenous peroxide blocker, incubate in a wet box at room temperature for 10 minutes, wash off the endogenous enzyme blocker with PBS (5 minutes × 3 times), dry the liquid, add non-specific staining blocker, and block in a wet box at room temperature for 1 hour; after drying the liquid, add JPH1 antibody (1:50 dilution), incubate at 4°C overnight, and wash with PBS (5 minutes × 3 times). Add the appropriate secondary antibody and incubate at room temperature for 1 hour. After the incubation, wash with PBS (5 minutes × 3 times). Add an appropriate amount of horseradish enzyme avidin working solution and incubate at room temperature for 1 hour, then wash with PBS (5 minutes × 3 times). Then use DAB for color development, hematoxylin counterstaining and seal the slide. Finally, the microscope was used to observe and capture images. Six fields of view were randomly selected at 400 times the field of view. The number of positive expression cells and the total number of cells were counted and the percentage of positive cells was calculated. The positive expression percentage was <5% for 0 points, 5% to 25% for 1 point, 26% to 50% for 2 points, 50% to 75% for 3 points, and >75% for 4 points. The evaluation criteria for cell staining intensity were determined by the depth of staining in positive expression cells: basically no staining (negative) was 0 points, weak staining (light yellow) but significantly stronger than the negative control was 1 point, medium staining intensity (yellow) was 2 points, and strong staining (brown yellow) was 3 points.

[0103] PNO1 expression (IHC score) = positive percentage × staining depth of positive expression cells;

[0104] Results Figure 2 shown.

[0105] from Figure 2 It can be seen that compared with normal intestinal tissue, the expression of PNO1 in the colon tissue of UC patients was significantly upregulated (*P<0.05).

[0106] Example 2

[0107] Furthermore, in Example 2, PNO1 was knocked into mice and DSS was used to induce colitis to construct an animal model.

[0108] These include:

[0109] 2.1. Obtaining PNO1 gene knock-in animals

[0110] The linearized targeting vector was electroporated. The construction of the targeting vector refers to CN2024104977067. The targeting vector was introduced into mouse ES cells, and the screened positive cell clones that underwent homologous recombination were injected into C57BL / 6 donor mouse blastocysts, and the blastocysts were transplanted into the uterus of CD-1 pseudo-pregnant mice to produce F0 generation positive chimeric mice.

[0111] The F0 positive mice were bred with tissue-specific Cre mice to obtain the F1 PNO1 flox / wt Mouse.

[0112] F1 generation PNO1 flox / wt The mice were self-bred together to obtain the F2 generation PNO1 flox / flox Homozygous mice.

[0113] F2 generation PNO1 flox / flox Homozygous mice were mated with EIIa-Cre (systemic Cre) positive mice to obtain F3 generation PNO1 heterozygous Cre positive mice.

[0114] The F3 generation mice were self-bred in the same cage, and the tails of the mice were identified after weaning and separation, and finally the F4 generation PNO1 knock-in (PNO1-KI) mice, also known as PNO1 gene knock-in mice, were obtained for subsequent experiments.

[0115] 2.2 Experimental Animal Grouping and Animal Model Construction

[0116] Eight-week-old experimental mice weighing approximately 26 g were randomly divided into four groups, with eight mice in each group:

[0117] Group 1 (WT): C57BL / 6 mice;

[0118] Group 2 (WT+DSS): C57BL / 6 mice induced with DSS;

[0119] Group 3 (PNO1-KI, or KI): PNO1-KI mice constructed in step 2.1;

[0120] Group 4 (PNO1-KI+DSS, or KI+DSS): PNO1-KI mice constructed in step 2.1 and induced with DSS.

[0121] The specific steps of DSS induction are as follows:

[0122] The mice were given 2% DSS drinking water for 7 consecutive days, and then switched to normal drinking water for 14 consecutive days (the first cycle); repeated administration of 2% DSS drinking water for 7 days, and normal drinking water for 14 days (the second cycle); repeated administration of 2% DSS drinking water for 7 days (the third cycle), and the entire laboratory process totaled 49 days. The mice in the same group were housed in the same cage, and each cage was equipped with a drinking bottle. The drinking water volume of each group was 200 mL of 2% DSS aqueous solution; fresh 2% DSS aqueous solution was replaced every 2 days.

[0123] The weight, blood in stool and diarrhea of ​​mice were recorded every two days, and the disease activity index was calculated. After the experiment, blood was collected by anesthesia, and the corresponding organs of each group were taken for detection.

[0124] After the experiment, the colon tissues of mice in each group were separated, observed and photographed, and the length and weight of the colon were measured. The colon tissues were packaged and stored in a -80°C refrigerator or fixed with 4% paraformaldehyde for subsequent detection of relevant experimental indicators.

[0125] 2.2. Investigation of colitis indicators

[0126] 2.2.1. Investigation of mouse body weight and disease index

[0127] The vital activities and physical signs of mice in each group were observed every two days, and the weight, diarrhea, and blood in the stool of the mice were recorded. The disease activity index (DAI) of mice in each group was scored according to the weight, diarrhea, and blood in the stool, and the DAI score was calculated according to the calculation method in Table 4. Disease activity index score = (weight loss rate score + stool characteristics score + blood in the stool score) / 3.

[0128] Table 4 DAI calculation method

[0129]

[0130] Note: Normal stool refers to formed stool; loose stool refers to mushy, semi-formed stool that does not stick to the anus; loose stool refers to watery stool that can stick to the anus.

[0131] The results of the body weight of mice in each group are as follows Figure 3 shown.

[0132] from Figure 3It can be seen that the body weight of mice in the PNO1-KI group and the WT group showed a gradual upward trend, which was consistent with the growth of mice, and the weight increase trend of PNO1-KI mice was significantly higher than that of WT mice; the WT+DSS group and the PNO1-KI+DSS group showed obvious weight loss during and after the 2% DSS administration, and there were periodic fluctuations with the cyclic changes of 2% DSS administration, which was consistent with the symptoms of enteritis; in addition, after the experiment, compared with the WT+DSS group, the weight loss trend of mice in the PNO1-KI+DSS group was more significantly reduced, and the weight loss rate decreased from an average of 101.00% to 96.59%, a decrease of 4.4%.

[0133] The results of DAI index of each group of mice are as follows Figure 4 shown.

[0134] from Figure 4 It can be seen that in the first cycle (i.e., continuous administration of 2% DSS aqueous solution + 14 days of water):

[0135] On the 3rd day, mice in the PNO1-KI+DSS group began to gradually show symptoms such as decreased activity, mental depression, occult blood in stool or visible blood in stool. On the 5th day, 5 / 8 mice showed varying degrees of visible blood in stool, which was most serious on the 7th day, with 8 / 8 mice showing blood in stool, which lasted until the 13th day, when the blood disappeared. On the 4th day, the WT+DSS group began to show obvious UC symptoms, including occult blood in stool and diarrhea. On the 6th day, 2 / 8 mice showed visible blood in stool, which was most serious on the 7th day, with 4 / 8 mice showing blood in stool, which lasted until the 10th day, when the blood disappeared.

[0136] In the second cycle (i.e., continuous administration of 2% DSS aqueous solution + 14 days of water):

[0137] In the PNO1-KI+DSS group, 5 / 8 mice had bloody stools to varying degrees starting on day 24, which was the most severe on day 29, with 8 / 8 mice having bloody stools, which lasted until day 34, when the bloody stools disappeared. In the WT+DSS group, obvious UC symptoms began to appear on day 25, with 2 / 8 mice having bloody stools, which was the most severe on day 29, with 4 / 8 mice having bloody stools, which lasted until day 32, when the bloody stools disappeared.

[0138] In the third cycle (i.e., continuous administration of 2% DSS aqueous solution + 14 days of water):

[0139] In the PNO1-KI+DSS group, 3 / 8 mice had visible bloody stools on day 44, and the bloody stools worsened until the end of the experiment. In the WT+DSS group, obvious UC symptoms began to appear on day 44, with 2 / 8 mice having visible bloody stools, and the bloody stools worsened until the end of the experiment.

[0140] At the end of the experiment, compared with the WT+DSS group, the DAI value of the PNO1-KI+DSS group changed more severely and significantly, with the average value increasing from 5.29 to 8.25, an increase of 56.0% (*P<0.05).

[0141] In summary, in DSS-induced PNO1 knock-in mice, ulcerative colitis develops earlier and the DAI value is higher.

[0142] 2.2.2. Investigation of mouse colon length

[0143] Actual photos of the colon tissues of mice in each group and statistical graphs of colon lengths. The results are shown in Figure 5 In the colon real-life photos, the left column shows the colon after the experiment, and the right column shows the colon before the experiment.

[0144] from Figure 5 It can be seen that the colon appearance of mice in the WT group and the PNO1-KI group was similar, and the colon lengths of the two groups were similar; compared with the WT group, the colon lengths of mice in the WT+DSS group and the PNO1-KI+DSS group were significantly shortened; compared with the WT+DSS group, the colon length of mice in the PNO1-KI+DSS group was shortened more significantly, from an average value of 6.45 cm to 5.84 cm, a shortening of nearly 9.5% (*P<0.05).

[0145] In addition, pathological results showed that the colon edema and congestion in the WT+DSS group were milder, with less stasis and ulceration, and less colon morphological changes; while the colon damage in the PNO1-KI+DSS group was more severe, with extensive colon erosion and ulceration, obvious mucosal edema, and petechiae scattered on the colon mucosa. There was no damage to the colon morphology in the WT and PNO1-KI groups.

[0146] 2.2.3. Investigation of mouse spleen weight

[0147] After the experiment, the spleen of each group of mice was removed to observe the gross appearance and weight. The actual photos of the spleen and the statistical results of spleen weight are shown in Figure 6 shown.

[0148] from Figure 6 It can be seen that the spleen weights of mice in the WT group and the PNO1-KI group were similar; compared with the WT group, the spleen weights of mice in the WT+DSS group and the PNO1-KI+DSS group were increased; compared with the WT+DSS group, the spleen weight of mice in the PNO1-KI+DSS group was increased, from an average value of 0.13 to 0.15, an increase of 20%; compared with the WT group, the spleen weight of mice in the PNO1-KI+DSS group was significantly increased (*P<0.05).

[0149] 2.2.4. Investigation of the degree of pathological damage in mouse colon

[0150] HE staining was used to detect the pathological changes in the colon tissue of each group of mice. The specific operation of HE staining was as follows:

[0151] After the tissue was fixed in 4% paraformaldehyde for 48 hours, it was replaced with 70% alcohol for 24 hours. After the tissue was trimmed the next day, the tissue was dehydrated and waxed in the corresponding reagents: 70% ethanol, 30 minutes; 80% ethanol, 30 minutes; 90% ethanol, 30 minutes; 95% ethanol, 30 minutes; anhydrous ethanol I, 10 minutes; anhydrous ethanol II, 10 minutes; anhydrous ethanol III, 30 minutes; xylene I, 5 minutes; xylene II, 10 minutes; paraffin I, 10 minutes; paraffin II, 10 minutes; paraffin III, 25 minutes. After paraffin embedding, adjust the thickness to 4 μm for sectioning, and pick up after the tissue is flattened in 37°C water, and fix it on the cover glass for use. Before the experiment, bake the slices at 60°C for 4 hours, and wait until the temperature returns to room temperature, and then dewax the slices in the corresponding reagents. Stain with hematoxylin solution for 1 minute and rinse with tap water. Separate the colors with 1% hydrochloric acid ethanol solution for a few seconds and rinse with tap water. The samples were stained with eosin solution for 5 seconds, washed with tap water, and finally dehydrated and sealed.

[0152] The results are as follows Figure 7 shown.

[0153] from Figure 7 It can be seen that the colon tissues of the WT group and the PNO1-KI group were intact in structure, the epithelial cells were neatly arranged in a cylindrical shape, the cell nucleus was located in the lower part of the cell, multiple goblet cells were scattered, and no inflammatory cells were observed; while in the colon tissues of the WT+DSS group and the PNO1-KI+DSS group, varying degrees of inflammatory cell infiltration were observed, the epithelial cells in the colon tissue were disordered and deformed, the crypts disappeared, the goblet cells decreased, and varying degrees of tissue dysplasia were observed, the cell morphology was diverse, and the arrangement was disordered; the cell deformation in the PNO1-KI+DSS group was more obvious, and the pathological damage was more serious.

[0154] The degree of colonic inflammation in the WT+DSS group was mild, with the depth of lesions mostly reaching the muscular layer, the destruction area of ​​the basal crypts was small, the range of lesions was also small, and the degree of colonic injury was mild; while the colonic tissue inflammation in the PNO1-KI+DSS group was severe, with the depth of lesions reaching the serosal layer, the destruction of the crypts was enlarged, and the epithelial tissue was destroyed. There was no significant change in colonic pathology between the WT group and the PNO1-KI group.

[0155] 2.2.5. Investigation of PNO1 protein expression in mouse colon tissue

[0156] Immunohistochemistry was used to detect the expression of PNO1 protein in the colon tissue of mice. The specific immunohistochemistry method is as follows:

[0157] After the wax block was sliced, dewaxed and dehydrated, it was placed in 100℃ sodium citrate for antigen retrieval reaction. After the temperature returned to room temperature, it was washed with PBS for 5min. 0.2% Triton X-100 was added for 10min and 0.3% H2O2 for 10min respectively to permeabilize the membrane and remove endogenous peroxidase. After washing with PBS, the blocking solution was added for blocking at room temperature for 1h. After removing the blocking solution, an appropriate amount of primary antibody was added and incubated at 4℃ overnight. After the incubation, it was washed with PBS for 10min, and the biotin-labeled anti-rabbit / mouse secondary antibody was added and incubated at room temperature for 1h. After washing with PBS, an appropriate amount of horseradish enzyme-labeled streptavidin working solution (SA / HRP) was added and incubated at room temperature for 30min, and then DAB color development solution (20× reagent A, 20× reagent B, 20× reagent C were configured in proportion to 1× developer) was prepared for color development, and hematoxylin was counterstained and dehydrated and sealed. Leica upright microscope was used for observation and photography, and 5 fields of view were randomly selected for photography and analysis. The protein expression score (IHC score) of PNO1 was determined by the product of the positive rate (0%-25%, level 1; 26-50%, level 2; 51-75%, level 3; 76%-100%, level 4) and the expression intensity (negative, 0; weak positive, 1; moderately positive, 2; strongly positive, 3). The primary antibody used for the detection of PNO1 was PNO1 antibody.

[0158] The results are as follows Figure 8 shown.

[0159] from Figure 8 It can be seen that the expression of PNO1 protein in the colon tissue of the WT group was low, and the expression of PNO1 in the colon tissue of the PNO1-KI group was significantly higher than that of the WT group. After the PNO1-KI+DSS group was successfully established, the expression of PNO1 protein in the colon tissue increased significantly, and the PNO1-KI+DSS group was higher than the PNO1-KI group and the WT+DSS group, indicating that DSS can induce a large amount of PNO1 expression.

[0160] 2.2.6. Investigation of the expression of inflammatory factors in mouse colon tissue

[0161] UC is a type of ulcerative colitis, and its pathogenic mechanism is characterized by an abnormal immune response to intestinal contents. IL-6 is a multifunctional cytokine that promotes inflammatory responses. TNF-α and IL-1β can promote the proliferation of intestinal stem cells and promote the proliferation of intestinal epithelial cells. In a variety of inflammatory responses, elevated levels of TNF-α, IL-6, and IL-1β can be detected.

[0162] Immunohistochemistry was used to detect the expression of inflammatory factors TNF-α, IL-6 and IL-1β proteins in the colon tissue of mice. The immunohistochemistry method was the same as step 2.5. The primary antibodies used to detect the inflammatory factors TNF-α, IL-6 and IL-1β proteins were TNF-α antibody, IL-6 antibody and IL-1β antibody, respectively.

[0163] The results are as follows Fig. 9 As shown in the figure, the bar graph of inflammatory factor expression on the right shows TNF-α, IL-6 and IL-1β from top to bottom.

[0164] from Fig. 9 It can be seen that the expressions of IL-6, IL-1β and TNF-α in the colon tissues of the WT group and the PNO1-KI group were at low levels; while the expressions of IL-6, IL-1β and TNF-α in the colon tissues of the WT+DSS group and the PNO1-KI+DSS group were at high levels, and the expression of inflammatory factors in the PNO1-KI+DSS group was significantly higher than that in the WT+DSS group.

[0165] In addition, the present application uses 8 WT and 8 PNO1-KI mice, and the same method as step 2.2 in Example 2 is used to construct ulcerative colitis mouse models. After the first cycle, the proportion of different DAI score segments on the day of the end of the statistics are calculated. The results are shown in Table 5.

[0166] Table 5

[0167]

[0168] As shown in Table 5, 100% of the mice in the PNO1-KI+DSS group showed typical symptoms of ulcerative colitis, and 75% of the mice had DAI scores ranging from 1.0 to 4.9, indicating a high degree of severity; while 87.5% of the mice in the WT+DSS group showed typical symptoms of ulcerative colitis, and only 25% of the mice had DAI scores ranging from 1.0 to 4.9, indicating a low degree of severity. This indicates that the model of the present application has small intra-group differences, good repeatability, and strong stability.

[0169] In summary, the animal colitis model constructed by knocking in PNO1 in animals in this application has a higher DAI index, histological change score, and severity of colon length than the colitis model constructed by DSS induction alone; in addition, the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α are also higher than those of the animal model induced by DSS alone. Therefore, the animal colitis model of the present invention has the advantages of strong controllability, good repeatability, and strong stability, and can be better used for the pathogenic mechanism and treatment research of ulcerative colitis, as well as the screening of candidate drugs for enteritis.

[0170] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of PNO1 in at least one of the following: (1) preparing a product for diagnosing ulcerative colitis; (2) as a target for screening candidate drugs for preventing and / or treating ulcerative colitis; (3) constructing an animal model of ulcerative colitis.

2. A method for constructing an ulcerative colitis animal model, characterized in that: The steps include: The ulcerative colitis animal model was obtained by using DSS to induce PNO1 gene knock-in animals.

3. The construction method according to claim 2, characterized in that: The method for obtaining the PNO1 gene knock-in animal is as follows: inserting an expression frame containing the PNO1 gene into the genome of the animal to obtain the PNO1 gene knock-in animal.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of the PNO1 gene comprises the sequence shown in SEQ ID NO.1; And / or, inserting the expression frame containing the PNO1 gene through the CRISPR / Cas9 gene editing system or the Cre / loxp gene editing system.

5. The construction method according to claim 4, characterized in that: The expression cassette containing the PNO1 gene comprises: promoter-Loxp-3*SV40-Loxp-PNO1-polyA; And / or, the site where the expression frame containing the PNO1 gene is inserted is selected from one of the Rosa26, Hprt and hipp11 sites.

6. The construction method according to claim 2, characterized in that: The DSS is administered via a DSS aqueous solution, and the concentration of the DSS aqueous solution is 1 to 5 wt %.

7. The construction method according to claim 2, characterized in that: The DSS induction comprises the following steps: 1) First administration: Continue to administer DSS for 5 to 7 days; 2) Second administration: After the first administration, the animals were fed in the usual way for 7 to 14 days, and then DSS was continuously administered for another 5 to 7 days; 3) Third administration: After the second administration, the animals were fed in the usual way for 7 to 14 days, and then DSS was continuously administered for another 5 to 7 days; After the third administration, the ulcerative colitis animal model was obtained.

8. The construction method according to claim 1, characterized in that: The animal is selected from one or more of mice, rats, hamsters, zebrafish, pigs, fruit flies and rabbits; preferably, the animal is a mouse.

9. Use of the ulcerative colitis animal model obtained by the construction method according to any one of claims 2 to 8 in at least one of the following: A1) Screening for drugs to prevent and / or treat ulcerative colitis; A2) Studying the pathogenesis and etiology of ulcerative colitis.

10. A method for screening candidate drugs for preventing or treating ulcerative colitis, characterized in that: The method comprises the following steps: administering the candidate drug to the ulcerative colitis animal model obtained by the construction method according to any one of claims 2 to 8.

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