Preparation of targeted Nptx2 siRNA and application of targeted Nptx2 siRNA in treatment of specific dermatitis

Abstract

The invention provides preparation of targeted Nptx2 siRNA and application of the targeted Nptx2 siRNA in treatment of specific dermatitis, and relates to the technical field of biomedicine, three groups of chemically modified nucleic acid siRNA molecules of targeted Nptx2 genes are provided, the three groups of chemically modified nucleic acid siRNA molecules can remarkably reduce Nptx2 gene expression in vitro, Nptx2Mus-380 and Nptx2Mus-707 can effectively reduce NPTX2 protein expression in vitro, and the protein level of the Nptx2Mus-707 group is the lowest; based on the data, the Nptx2Mus-707 has the most significant effect of inhibiting NPTX2 gene and protein expression, and specific verification experiments prove that the Nptx2Mus-707 molecule can effectively relieve itching and scratching caused by MC903-induced specific dermatitis, a foundation is laid for clinical treatment of chronic itching, and the Nptx2Mus-707 molecule has great application and popularization value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the preparation of Nptx2-targeted siRNA and its application in treating atopic dermatitis. Background Art

[0002] Itch is defined as an unpleasant sensation that evokes the desire to scratch. Like pain, acute itch is an important protective mechanism for detecting potentially harmful stimuli. However, chronic itch (i.e., itch cycles lasting more than 6 weeks in humans) no longer has a protective function, but instead causes pain and may impair quality of life, often to the same degree as chronic pain. Chronic itch is a debilitating symptom that accompanies various skin diseases, systemic diseases (such as chronic kidney disease or cholestatic liver disease), mental illness, and neuropathy. According to statistics, one-third of patients with skin diseases and nearly 15% of the general population experience chronic itch. Atopic dermatitis (AD) is a common, recurrent inflammatory skin disease in clinical practice. The etiology is very complex, involving multiple dimensions of pathogenic factors, including genetic factors, environmental factors, skin barrier disorders, skin flora imbalance, and Th2-biased inflammatory responses. The main clinical manifestations of AD are itching, rash, and dry skin. Severe and unbearable itching will cause scratching, which will aggravate the rash. Severe rash will also aggravate itching. It is particularly important to block this vicious cycle. In addition, long-term itching can significantly affect the patient's health, such as inattention, anxiety, behavioral disorders, autism, and social disorders. AD management will also increase social and family expenses. Epidemiological surveys have found that about 20% of children and about 1-3% of adults are affected by AD, and for an individual, the probability of developing AD in his or her lifetime is about 1 / 10. Survey data from the World Health Organization show that about 230 million people in the world suffer from AD, with a global incidence of 3.5%. At present, there is limited basic and clinical research on it, and there is a lack of antipruritic drugs with good therapeutic effects. Drugs used on the market include: antihistamines, hormone drugs, and immunomodulators. However, clinical antihistamines are not effective for all patients, and hormone drugs and immunomodulators are limited in their use due to their large side effects. Therefore, the development of new targets and antipruritic drugs is urgent.

[0003] Small interfering RNA (siRNA) is a short RNA molecule, usually a double-stranded RNA composed of 20 to 25 nucleotides. siRNA binds to the mRNA of the target gene through complementary binding, resulting in mRNA degradation or translation inhibition, thereby weakening or inactivating the function of the target gene. siRNA has been clinically used in a variety of diseases, such as tumors, arthritis and obesity. After entering the human body, siRNA interferes with the specific gene mRNA of the disease, making it impossible for the gene to express protein normally, thereby playing a role in treating the disease. Compared with commonly used therapeutic drugs, siRNA drugs have the advantages of low toxicity, fewer side effects, high efficiency and strong specificity, and are one of the most promising methods for treatment. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that the prior art lacks new targets for chronic pruritus and effective antipruritic drugs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The siRNA used to inhibit the expression of Nptx2, wherein the sequence of the siRNA is at least one of the following sequences:

[0007] Nptx2 Mus-380

[0008] Sense sequence 5′-GCUCCUUGCAAACCCUCAATT-3′

[0009] Antisense sequence 5′-UUGAGGGUUUGCAAGGAGCTT-3′

[0010] Nptx2 Mus-707

[0011] Sense sequence 5′-GCAAGAUCAAGAAGACAUUTT-3′

[0012] Antisense sequence 5′-AAUGUCUUCUUGAUCUUGCTT-3′

[0013] Nptx2 Mus-1209

[0014] Sense sequence 5′-GGUGGACAACAAUGUCGAUTT-3′

[0015] Antisense sequence 5′-AUCGACAUUGUUGUCCACCTT-3′

[0016] Preferably, the targets of the siRNAs are all designed in the conserved regions of the Nptx2 gene.

[0017] The present application also provides the use of siRNA that inhibits the expression of Nptx2 in the treatment of atopic dermatitis.

[0018] Preferably, the siRNA sequence for inhibiting the expression of Nptx2 comprises at least one of the above-mentioned siRNAs for inhibiting the expression of Nptx2.

[0019] Preferably, the drug is used to relieve the itching and scratching caused by MC903-induced atopic dermatitis.

[0020] The present application also provides a drug for treating atopic dermatitis, wherein the drug is used to inhibit the expression of Nptx2.

[0021] Preferably, the drug contains at least one of the above-mentioned siRNAs for inhibiting the expression of Nptx2.

[0022] The present application provides three groups of chemically modified nucleic acid siRNA molecules targeting the Nptx2 gene, all of which can significantly reduce the expression of the Nptx2 gene in vitro, among which Nptx2 Mus-380 and Nptx2 Mus-707 can effectively reduce the expression of NPTX2 protein in vitro, and the protein level of Nptx2 Mus-707 is the lowest; based on the above data, Nptx2 Mus-707 has the most significant effect in inhibiting the expression of NPTX2 gene and protein, and the present application proves through specific verification experiments that Nptx2 Mus-707 can effectively relieve the itching and scratching during MC903-induced atopic dermatitis, laying a foundation for the clinical treatment of chronic itch and having great application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 In one embodiment of the present invention, the Nptx2Mus-380 target sequence is located in the conserved region of the human and mouse Nptx2 gene sequence;

[0024] Figure 2 In one embodiment of the present invention, the Nptx2Mus-707 target sequence is located in the conserved region of the human and mouse Nptx2 gene sequences;

[0025] Figure 3 In one embodiment of the present invention, the Nptx2 Mus-1209 target sequence is located in the conserved region of the human and mouse Nptx2 gene sequence;

[0026] Figure 4This is a schematic diagram for verifying the increase in Nptx2 mRNA expression in the MC903-induced atopic dermatitis model TG in one embodiment of the present invention. There are 5 mice in each group. The real-time PCR results were tested using the Student's T-test. Compared with the solvent group at the corresponding time point, *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 5 The present invention is used to verify the increased expression of NPTX2 protein in TG of MC903-induced atopic dermatitis model mice in one embodiment, wherein the immunofluorescence single labeling result in Figure A shows that the NPTX2 fluorescence signal on the 10th day of MC903 is significantly enhanced compared with the Vehicle group; Figure B Western Blot shows that the NPTX2 gray value on the 10th day after MC903 is significantly enhanced compared with the Vehicle group, indicating that the number of NPTX2-expressing neurons and protein levels are increased 10 days after the model, scale bar = 20μm; Figure C NPTX-2 enzyme-linked immunosorbent assay shows that the NPTX2 protein content in the skin of patients with chronic pruritus is higher than that of normal people, revealing that the NPTX2 protein content in the skin of patients with chronic pruritus is significantly increased.

[0028] Figure 6 This is a comparison diagram of the silencing efficiency of Nptx2 mRNA in HEK293 cell line verified by in vitro experiments in one embodiment of the present invention, wherein the Real-time PCR results in Figure A show that the Nptx2 overexpression plasmid significantly increases the expression of the Nptx2 gene. The Real-time PCR results in Figure B show that the three groups of targeted Nptx2 siRNA can significantly inhibit the expression of the Nptx2 gene. The Western Blot results in Figure C show that Nptx2 Mus-380 and Nptx2 Mus-707 significantly inhibit the expression of NPTX2 protein; Figure D is a statistical graph of the grayscale value of Figure C. The statistical results were compared with the NC group using (One-way ANOVA), *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 7The figure is a result of relieving the scratching behavior of mouse specific dermatitis after siRNA interference in one embodiment of the present invention, wherein the behavioral results in Figure A show that Nptx2Mus-707 siRNA can relieve the itching caused by MC903, starting from 3 days after injection and lasting to the 4th day. Figure B Real-time PCR detects the knockdown efficiency of Nptx2 Mus-707 siRNA in vivo. Behavioral statistical analysis uses two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, compared with the NC group at the corresponding time point. The real-time PCR results are compared with the NC group at the corresponding time point using the t test (Student's T-test). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031] The present application provides a siRNA for inhibiting the expression of Nptx2, and the sequence of the siRNA is as follows:

[0032] Nptx2 Mus-380

[0033] Sense sequence 5′-GCUCCUUGCAAACCCUCAATT-3′

[0034] Antisense sequence 5′-UUGAGGGUUUGCAAGGAGCTT-3′

[0035] Nptx2 Mus-707

[0036] Sense sequence 5′-GCAAGAUCAAGAAGACAUUTT-3′

[0037] Antisense sequence 5′-AAUGUCUUCUUGAUCUUGCTT-3′

[0038] Nptx2 Mus-1209

[0039] Sense sequence 5′-GGUGGACAACAAUGUCGAUTT-3′

[0040] Antisense sequence 5′-AUCGACAUUGUUGUCCACCTT-3′

[0041] The targets of the three groups of siRNAs mentioned above are all designed in the conserved region of the Nptx2 gene.

[0042] Based on the three groups of siRNAs described above, the present application also provides the use of siRNAs that inhibit the expression of Nptx2 in the treatment of atopic dermatitis.

[0043] The drug is used to relieve the itching and scratching during MC903-induced atopic dermatitis.

[0044] The present application also provides a drug for treating atopic dermatitis, wherein the drug is used to inhibit the expression of Nptx2. In one embodiment, the drug comprises at least one of the following siRNAs: Nptx2 Mus-380

[0045] Sense sequence 5′-GCUCCUUGCAAACCCUCAATT-3′

[0046] Antisense sequence 5′-UUGAGGGUUUGCAAGGAGCTT-3′

[0047] Nptx2 Mus-707

[0048] Sense sequence 5′-GCAAGAUCAAGAAGACAUUTT-3′

[0049] Antisense sequence 5′-AAUGUCUUCUUGAUCUUGCTT-3′

[0050] Nptx2 Mus-1209

[0051] Sense sequence 5′-GGUGGACAACAAUGUCGAUTT-3′

[0052] Antisense sequence 5′-AUCGACAUUGUUGUCCACCTT-3′

[0053] The above contents are described below in conjunction with specific embodiments:

[0054] Example 1: Design and synthesis of siRNA

[0055] In the present application, RNAi Designer and other software are used to design the sequence of siRNA, and the parameters are set as follows: GC content is 35%-55%; the target region is the gene coding region and the evolutionary region.

[0056] The following three groups of double-stranded siRNA were designed and synthesized based on the Nptx2 mRNA sequence of mouse (NM_016789.3):

[0057]

[0058] In this application, Nptx2 siRNA is designed to target the Nptx2 gene of mice, and its target sequence is located in the coding region of the Nptx2 gene. Figure 1-Figure 3 shown.

[0059] Example 2: Changes in Nptx2 mRNA expression in TG of atopic dermatitis model mice

[0060] The specific steps include:

[0061] 2.1 Preparation of mice with atopic dermatitis model

[0062] Calcipotriol, MC903 for short, is a synthetic VitD 3 analogue with high affinity for vitamin D receptors. In this application, MC903 was used to induce a facial atopic dermatitis model, and the trigeminal ganglion (TG) of the model mouse was taken.

[0063] Wild-type mice (strain: C57BL / 6) were purchased from the Experimental Animal Center of Nantong University, aged 10 weeks, weighing 20±2g, and adapted for 3 days. The mice were anesthetized, and the cheeks were depilated with depilatory cream, and then adapted for 3 days. The mice were kept in anesthesia using a Reward mask anesthesia machine, and MC903 was applied to the face at a concentration of 200μM and a dose of 20μL, once a day, for 10 consecutive days. In the control group of mice, MC903 was changed to anhydrous ethanol (the solvent for dissolving MC903), and other operations were consistent with the model group.

[0064] 2.2 Extraction of animal tissue samples, RNA extraction, reverse transcription and real-time PCR process

[0065] 2.2.1 Extraction of tissue samples

[0066] (1) The mice were kept in an anesthesia state using a mask anesthesia machine, the chest cavity was cut open, and the heart was perfused with normal saline;

[0067] (2) After perfusion, cut open the skull, peel off the brain tissue, expose the trigeminal ganglion, separate the ganglion, and place the removed trigeminal ganglion in an RNAase Free EP tube containing 200 μL Trizol and place it on ice.

[0068] 2.2.2 RNA extraction

[0069] (1) Homogenize the sample once / 30s for 4 times until no tissue is visible, then add 800 μL Trizol and let stand for 5 min;

[0070] (2) Add 200 μL of chloroform to each tube, shake vigorously for 1 min, and let stand for 5 min;

[0071] (3) Centrifugation, conditions: 4°C, 12000 rpm, 15 min;

[0072] (4) Gently aspirate the supernatant into an RNAase Free EP tube, add an equal volume of isopropanol, mix gently until no filaments are visible to the naked eye, and let stand for 10 min;

[0073] (5) Centrifugation, conditions: 4°C, 12000 rpm, 15 min;

[0074] (6) Discard the supernatant, add 1 mL of anhydrous ethanol, gently mix, and centrifuge at 4°C, 12,000 rpm, for 15 min;

[0075] (7) Discard the supernatant, invert it onto filter paper, and place it at room temperature to dry until the precipitate becomes translucent;

[0076] (8) Add 20 μL RNase-free H2O to each tube, set the temperature to 60°C, and dissolve in a water bath for 10 min. After dissolution, place on ice.

[0077] (9) Measure RNA concentration using an OD meter.

[0078] 2.2.3 Reverse transcription of total RNA into cDNA

[0079] (1) Removal of DNA impurities in samples (10 μL system)

[0080]

[0081] (2) RNA reverse transcription into cDNA (20 μL system)

[0082]

[0083] After reverse transcription into cDNA, add deionized water to dilute 8 times and store at -20℃ for later use.

[0084] (3) Primer sequence

[0085] Primer design: This patent uses the mouse Nptx2 mRNA sequence in the NCBI database, designs primers on the NCBI website, and uses BLAST for specificity verification to predict the size of the amplified product. The primer specificity is specifically verified based on the melting curve and agarose gel results. The primer sequence is synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0086] The primer sequences required in the experiment are as follows

[0087]

[0088] 2.2.4 Real-time PCR experiment

[0089] (1) Prepare the real-time PCR reaction system (10 μL) according to the following table:

[0090]

[0091] (2) PCR reaction (Life Technology), the conditions are as follows:

[0092]

[0093] Real-time quantitative PCR results Figure 4 As shown, on day 10 of the model, Nptx2 mRNA expression in TG of the MC903-induced atopic dermatitis model was significantly increased.

[0094] Example 3: Detection of NPTX2 protein expression in TG of model mice

[0095] 3.1 Immunofluorescence sample preparation, staining, and filming

[0096] 3.1.1 Tissue section preparation

[0097] After the mice were anesthetized by isoflurane inhalation, they were perfused with saline and 4% paraformaldehyde and placed on ice for 2 hours for fixation. Then the TG was placed in a 5mL tube with 4% paraformaldehyde and placed at 4°C. After fixation, 20% sucrose was replaced. After the TG sank to the bottom of the tube, 30% sucrose was replaced. After the TG sank to the bottom again. The TG was taken out, the trimmed TG was placed in the model slot in order, and quickly frozen on the quick-freezing table of the slicer. After solidification, the embedded block was glued to the freezing table with OTC for frozen sectioning. The thickness of the TG tissue patch is 12μm, which is attached to the adhesive slide and dried. It can be directly placed in 0.01mol / L PBS for immunofluorescence staining, or frozen at -80°C for storage.

[0098] 3.1.2 Immunofluorescence staining

[0099] (1) Place the slices in a container containing 0.01 mol / L PBS and wash three times, 10 min each time;

[0100] (2) After washing, add immunofluorescence blocking solution and block at room temperature for 2 h;

[0101] (3) After blocking, add the primary antibody prepared with blocking solution and incubate at 4°C overnight;

[0102] (4) Take out the tube the next day, return it to room temperature, and wash it three times with 0.01 mol / L PBS, each time for 10 min;

[0103] (5) Dilute the secondary antibody in 0.01 mol / L PBS and incubate at room temperature in the dark for 2 h;

[0104] (6) Discard the secondary antibody and wash the slides three times with 0.01 mol / L PBS, each time for 10 min;

[0105] (7) Place the slide away from light at room temperature. After the slide is half dry, drop a sealing medium on the slide and cover it with a coverslip. After it is dry, use a fluorescence microscope to photograph it.

[0106] 3.2 Western Blot sample preparation, protein extraction, electrophoresis, membrane transfer, antibody incubation and chemiluminescence development

[0107] 3.2.1 Tissue sample preparation

[0108] Sampling was the same as in 2.2.1. Briefly, the mouse was anesthetized, the chest cavity was cut open, the heart was exposed, the heart was perfused, the skull was cut open, the brain tissue was separated, the TG was found, the TG was separated, and it was taken out and placed in an EP tube.

[0109] 3.2.2 Protein extraction

[0110] Prepare the lysis buffer according to the following table:

[0111]

[0112] (1) Add 100 μL of lysis buffer to each tube and homogenize until there is no tissue precipitation;

[0113] (2) Ice bath for 30 min, then centrifuge at 4°C, 14,000 rpm, for 20 min;

[0114] (3) Aspirate the supernatant into a new EP tube and measure the protein concentration;

[0115] (4) Add protein loading solution according to the reaction system and boil in a 100°C water bath for 10 min to denature the protein;

[0116]

[0117] 3.2.3 Electrophoresis

[0118] (1) Prepare SDS-PAGE gel of corresponding concentration according to the molecular weight of the target protein;

[0119] (2) Add the sample to the lane of SDS-PAGE gel and keep constant voltage at 110 V for 2 hours.

[0120] 3.2.4 Transfer

[0121] Transfer the proteins on the SDS-PAGE gel to a membrane (such as PVDF or nitrocellulose membrane) at a constant current of 250 mA for 90 min.

[0122] 3.2.5 Antibody incubation

[0123] (1) Wash the membrane once with TBST (1X) on a decolorizing shaker for 5 min;

[0124] (2) Block the membrane with a blocking solution prepared with 5% skim milk powder to prevent nonspecific binding at room temperature for 2 hours;

[0125] (3) Prepare specific primary antibody in blocking solution and incubate at 4°C overnight;

[0126] (4) The next day, remove the membrane, return it to room temperature, and wash it three times with TBST (1X) on a decolorizing shaker, each time for 10 min;

[0127] (5) Prepare secondary antibody in TBST (1X) and incubate at room temperature for 2 hours; then wash three times with TBST (1X), each time for 10 minutes.

[0128] 3.2.6 Chemiluminescence imaging

[0129] (1) Prepare the luminescent solution according to the reagent manufacturer's instructions;

[0130] (2) Add the luminescent solution to the washed membrane and incubate for 1 min;

[0131] (3) The membrane is placed on a high-sensitivity chemiluminescence system to develop the target protein band.

[0132] 3.2.7 Analysis of target protein bands using Image J software

[0133] (1) Import the target protein band into Image J software;

[0134] (2) Calculate the gray value of the band according to the Image J software operation guide;

[0135] (3) Draw a graph based on the grayscale value.

[0136] Immunofluorescence and Western Blot results Figure 5 As shown, the expression of NPTX2 protein and the number of positive neurons in TG of MC903 at 10 days were significantly increased compared with those in the control group.

[0137] 3.3 NPTX-2 ELISA sample preparation, protein extraction, and detection

[0138] 3.3.1 Sampling

[0139] Foreskins of patients with chronic itching and normal persons were obtained from the hospital, and samples of the same size were placed in EP tubes.

[0140] 3.3.2 Protein extraction and protein concentration determination are the same as 3.2.2.

[0141] 3.3.3 NPTX-2 ELISA kit for determination of NPTX2 content

[0142] (1) Dilution of standard: This kit provides one 1x original standard. Dilute in a small test tube according to the following chart;

[0143] 160ng / L Standard No. 5 150 μL of original standard is added to 150 μL of standard diluent 80ng / L Standard No. 4 150 μL of standard No. 5 was added to 150 μL of standard diluent 40ng / L Standard No. 3 150 μL of standard No. 4 was added to 150 μL of standard diluent 20ng / L Standard No. 2 150 μL of standard No. 3 was added to 150 μL of standard diluent 10ng / L Standard No. 1 150uL of standard No. 2 was added to 150μL of standard diluent

[0144] (2) Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50 μL of the standard sample to the enzyme-labeled plate, first add 40 μL of sample diluent to the test sample well, and then add 10 μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix;

[0145] (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min;

[0146] (4) Liquid preparation: dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use;

[0147] (5) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this process 5 times and pat dry.

[0148] (6) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well;

[0149] (7) Incubation: Same as 3;

[0150] (8) Washing: same operation as in step 5;

[0151] (9) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop the color at 37°C in the dark for 10 minutes;

[0152] (10) Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow);

[0153] (11) Determination: Use the blank well as the zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be performed within 15 minutes after adding the stop solution;

[0154] (12) Calculation: Draw a standard curve on coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Calculate the corresponding concentration from the standard curve based on the OD value of the sample; then multiply by the dilution factor to obtain the NPTX2 protein content in each well.

[0155] Example 4: Nptx2 siRNA knockdown efficiency verification

[0156] In this embodiment, Nptx2 overexpression plasmid was first transfected into HER293 cell line, and Real-time PCR was used to verify the overexpression of Nptx2 in HEK293 cells. Then, three groups of Nptx2 siRNA were transfected to verify the knockdown effect of siRNA, and the expression level of Nptx2 mRNA was detected by Real-time PCR. After transfection for 48 hours, Western Blot was used to verify the protein knockdown effect.

[0157] 4.1 HEK293 cell line culture method

[0158] 4.1.1 Recovery and passaging of HEK293 cell lines

[0159] (1) Take out the cryovial from the -80°C freezer and quickly place it in a 37°C water bath until it is completely thawed (the time should be controlled within 2 minutes). Transfer the cells to a 15 mL centrifuge tube containing pre-warmed complete medium and centrifuge for 5 minutes at 1000 rpm / min. Discard the supernatant.

[0160] (2) Resuspend the cell pellet with fresh culture medium and inoculate it into the culture dish. Replace the culture medium after 24 hours. Observe the cell growth status under an inverted microscope. If the cell density reaches 80%-90% and the cell status is good (tightly adhered to the wall, without vacuoles or particles), perform cell passaging;

[0161] (3) Pre-warm complete culture medium, PBS buffer and 0.25% trypsin before the experiment, discard the old culture medium in the culture dish, add an appropriate amount of PBS to gently wash the cell surface 1-2 times, and add trypsin;

[0162] (4) Place the culture dish in an incubator and incubate for 1 minute, then add an equal volume of complete culture to terminate digestion;

[0163] (5) Use a pipette to gently blow the cells off, transfer the cell suspension to a centrifuge tube, centrifuge for 5 minutes, discard the supernatant, resuspend the cells in fresh culture medium, and inoculate them into a new culture dish at a ratio of 1:3 to 1:6 for continued culture.

[0164] 4.2 HEK293 cells overexpressing Nptx2

[0165] (1) The second generation HEK293 cells were cultured at 1×10 5 Cells / well were inoculated into 12-well plates, and transfection was performed when the cell density reached about 50%;

[0166] (2) Use Lipofectamine 2000 transfection reagent. The transfection system for each well is configured as follows: add 1000 ng Nptx2 overexpression plasmid and 2 μL Lipofectamine 2000 to 50 μL Opti-MEM medium. Prepare a mixing tube according to the required amount, mix gently and let stand at room temperature for 15 min.

[0167] (3) Take out the 12-well plate from the cell culture incubator, aspirate the old culture medium, gently wash the cells 1-2 times with PBS, and add 1 mL of Opti-MEM to each well;

[0168] (4) Add the transfection mixture to the well, mix gently, and place in a cell culture incubator for culture;

[0169] (5) After 6 hours, replace with fresh culture medium and continue culturing, and observe the cell status regularly. 24 hours after transfection, extract total RNA from cells and reverse transcribe it into cDNA. qPCR was performed using Nptx2-specific primers to verify the overexpression of Nptx2 mRNA level.

[0170] 4.3 Validation of knockdown efficiency of Nptx2 siRNA

[0171] (1) HEK293 cells were cultured at 1×10 5 The density of cells / well was inoculated in a 12-well plate, and the co-transfection experiment was performed when the cell density reached about 50%. The transfection system for each well was configured as follows: 500ngNptx2 overexpression plasmid, 500ngNptx2 siRNA and 2μL Lipofectamine 2000 were added to 50μL Opti-MEM medium, and a mixing tube was configured. After gently mixing, it was placed at room temperature for 15min;

[0172] (2) Before co-transfection, aspirate the old culture medium, add pre-warmed PBS and gently wash the cells 1-2 times, and add 1 mL of Opti-MEM to each well;

[0173] (3) Add the co-transfection complex to the well, mix gently, and place in an incubator for culture;

[0174] (4) After 6 hours, replace with fresh culture medium and continue culturing, and observe the cell status regularly. 24 hours after transfection, extract total RNA from cells and perform qPCR to verify the knockdown efficiency of Nptx2 siRNA. 48 hours after transfection, extract total protein from cells, quantify it and perform SDS-PAGE electrophoresis. After transfer to the membrane, use NPTX2 antibody to detect the effect of Nptx2 siRNA on NPTX2 protein expression level.

[0175] Real-time PCR results are as follows Figure 6As shown, the NPTX2 overexpression plasmid successfully overexpressed NPTX2 in the HEK293 cell line, and the three groups of siRNA targeting Nptx2 could inhibit the expression of Nptx2.

[0176] The specific process of animal tissue sample extraction, RNA extraction, reverse transcription and Real-time PCR is as described in 2.2.

[0177] One siRNA with the best inhibitory effect (i.e., Nptx2 Mus-707 siRNA) was identified from three Nptx2 siRNAs. In order to improve the transfection efficiency of siRNA in mice, Nptx2 Mus-707 siRNA was delivered to Jima gene for methoxy and cholesterol modification.

[0178] Example 5: Effect of Nptx2 Mus-707 siRNA on the itch behavior of mice with atopic dermatitis

[0179] The specific experimental steps are as follows:

[0180] 5.1 Intra-trigeminal injection of Nptx2 Mus-707 siRNA in mice

[0181] Nptx2 Mus-707 siRNA was injected into the trigeminal nerve 5 days after the MC903 model was prepared (for the preparation of the MC903 model, see 2.1).

[0182] The siRNA was dissolved to a working concentration of 5 μg / 10 μL. On day 5 of MC903, 10 μL of Nptx2Mus-707 siRNA was drawn up using an insulin needle. The needle was placed at a 5-degree angle to the mouse face and entered the trigeminal nerve through the infraorbital foramen of the mouse.

[0183] The control group was NC siRNA, which was injected in the same way.

[0184] 5.2 Mouse behavioral testing

[0185] The mice were adapted three days in advance for behavioral testing, with the same experimental personnel and environment, and the double-blind method was used.

[0186] For the itch behavior test, mice were placed in a transparent nine-square grid on an iron frame, adapted for 30 minutes, and then recorded for 1 hour. The statistical method of scratching times: scratching the face with the hind paws, then licking, biting the paw fingers or putting it back on the bottom plate, completing this cycle of behavior is counted as one scratch; if the front paws wipe the face, it is not counted as itch behavior.

[0187] Experimental conclusion: In this application, Nptx2 Mus-707 siRNA was injected into the trigeminal nerve of MC903 for 5 days, and then the siRNA was tested to see whether it could alleviate the scratching caused by MC903. Figure 7 Nptx2 Mus-707 siRNA was able to alleviate the itch behavior induced by MC903, starting from 3 days after injection and lasting to the 4th day.

[0188] The experimental materials and sources of this application are as follows:

[0189]

[0190]

[0191] In summary, the present application provides three groups of chemically modified nucleic acid siRNA molecules targeting the Nptx2 gene, and all three groups can significantly reduce Nptx2 gene expression in vitro, among which Nptx2 Mus-380 and Nptx2 Mus-707 can effectively reduce NPTX2 protein expression in vitro, and Nptx2 Mus-707 has the lowest protein level; the present application proves through specific verification experiments that the expression of Nptx2 is significantly increased in transcriptome sequencing, and the in vivo experiment verifies that the expression of Nptx2 mRNA and protein in the model mouse TG is significantly increased; the present invention successfully designs and proves that three groups of siRNA can effectively inhibit the expression of Nptx2 gene, which can be used for drug development; Nptx2 siRNA in the trigeminal ganglion can relieve the itching and weight loss of MC903-induced atopic dermatitis, and at the same time downregulate the expression of Nptx2 mRNA. Therefore, downregulating or inhibiting the expression of NPTX2 gene and protein by applying small interfering RNA is an effective way to treat chronic itching.

Claims

1. siRNA for inhibiting Nptx2 expression, characterized in that: The sequence of the siRNA is at least one of the following sequences: Nptx2 Mus-380 Sense sequence 5′-GCUCCUUGCAAACCCUCAATT-3′ Antisense sequence 5′-UUGAGGGUUUGCAAGGAGCTT-3′ Nptx2 Mus-707 Sense sequence 5′-GCAAGAUCAAGAAGACAUUTT-3′ Antisense sequence 5′-AAUGUCUUCUUGAUCUUGCTT-3′ Nptx2 Mus-1209 Sense sequence 5′-GGUGGACAACAAUGUCGAUTT-3′ Antisense sequence 5′-AUCGACAUUGUUGUCCACCTT-3′.

2. The siRNA for inhibiting Nptx2 expression according to claim 1, characterized in that: The targets of the siRNAs are all designed in the conserved regions of the Nptx2 gene.

3. Application of siRNA inhibiting Nptx2 expression in the treatment of atopic dermatitis.

4. The use of the siRNA for inhibiting Nptx2 expression according to claim 3 in treating atopic dermatitis, characterized in that: The sequence of the siRNA for inhibiting Nptx2 expression comprises at least one of claim 1.

5. The use of siRNA for inhibiting Nptx2 expression in treating atopic dermatitis according to claim 4, characterized in that: The drug is used to relieve the itching and scratching during MC903-induced atopic dermatitis.

6. A drug for treating atopic dermatitis, characterized in that: The drug significantly inhibits the expression of Nptx2.

7. The drug for treating atopic dermatitis according to claim 6, characterized in that: The drug comprises at least one of the siRNAs for inhibiting the expression of Nptx2 according to claim 1.

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