A cannabinoid receptor defective in vitro cell model and its construction method and application

Abstract

The present invention belongs to the technical field of cannabinoid receptor defective in vitro cell models, and specifically relates to a cannabinoid receptor defective in vitro cell model and a construction method and application thereof. The construction method comprises the following steps: (1) constructing a siRNA-CB1 vector for knocking out the CB1 gene of HaCaT cells; (2) constructing a siRNA-CB2 vector for knocking out the CB2 gene of HaCaT cells; (3) culturing HaCaT cells; (4) transfecting the HaCaT cells in step (3) with the vectors in step (1) and step (2); and (5) culturing the transfected HaCaT cells. The cannabinoid receptor defective in vitro cell model of the present invention utilizes HaCaT cells and transforms cannabinoid receptors to construct an in vitro cell model, which can facilitate the study of the mechanism of nodular prurigo to develop corresponding treatment plans.

Description

Technical Field

[0001] The present invention belongs to the technical field of cannabinoid receptor defective in vitro cell models, and in particular relates to a cannabinoid receptor defective in vitro cell model and a construction method and application thereof. Background Art

[0002] Prurigo nodularis (PN) is a chronic inflammatory skin disease with severe itching. Its main lesions are several to dozens of hyperkeratotic papules or nodules with severe itching, and occasionally rupture or ulcers may occur. Patients feel persistent and unbearable itching, mainly distributed on the sides of the limbs, head and neck, back, etc. The endocannabinoid system (eCB system, ECS) is involved in the regulation of skin cell proliferation, survival and differentiation, and is a key determinant in maintaining skin homeostasis. Cannabinoids belong to a family of lipophilic compounds and contain psychoactive ingredients. Cannabinoids exert a variety of biological activities by activating cannabinoid receptors, triggering a variety of physiological and pathological regulatory processes, such as pain, itching, inflammation, feeding and bone homeostasis.

[0003] Cannabinoid receptors are a type of G protein-coupled receptors containing seven transmembrane domains. In the skin, they are involved in skin balance, barrier formation and regeneration, mediate the skin's perception of heat, cold and pressure, and participate in inflammatory responses. However, the specific mechanism of PN is still unclear, and there are many difficulties in collecting samples to study the disease under experimental conditions. First, patients may have atopic dermatitis or systemic diseases such as chronic kidney disease, diabetes and thyroid diseases. Some even have mental illnesses. The patients are not very cooperative and are not suitable to be directly used as research subjects. At the same time, directly using patients as research subjects also faces many ethical issues. Secondly, there is currently no mature disease model for studying nodular prurigo in the world, including mice, rhesus monkeys and in vitro cell models. Summary of the invention

[0004] In order to solve the problems of difficulty in sampling PN patients and lack of in vitro research models in the prior art, the present invention provides a cannabinoid receptor-deficient in vitro cell model and its construction method and application. By using human immortalized epidermal cells (HaCaT cells), an in vitro cell model is constructed by modifying the cannabinoid receptors, which can facilitate the study of the mechanism of nodular prurigo and develop corresponding treatment plans.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] The present invention aims to provide a method for constructing a cannabinoid receptor-deficient in vitro cell model, comprising the following steps:

[0007] (1) Construction of siRNA-CB1 vector for CB1 gene knockout in HaCaT cells;

[0008] (2) Construction of siRNA-CB2 vector for CB2 gene knockout in HaCaT cells;

[0009] (3) Cultivating HaCaT cells;

[0010] (4) transfecting the HaCaT cells in step (3) with the vectors in step (1) and step (2);

[0011] (5) Cultivate the transfected HaCaT cells.

[0012] Furthermore, in the siRNA-CB1 sequence, the target sequence is SEQ ID NO: 1, and the RNA oligonucleotide sequence is SEQ ID NO: 2; or the target sequence is SEQ ID NO: 3, and the RNA oligonucleotide sequence is SEQ ID NO: 4; or the target sequence is SEQ ID NO: 5, and the RNA oligonucleotide sequence is SEQ ID NO: 6; or the target sequence is SEQ ID NO: 7, and the RNA oligonucleotide sequence is SEQ ID NO: 8; or the target sequence is SEQ ID NO: 9, and the RNA oligonucleotide sequence is SEQ ID NO: 10.

[0013] Furthermore, in the siRNA-CB2 sequence, the target sequence is SEQ ID NO: 11, and the RNA oligonucleotide sequence is SEQ ID NO: 12; or the target sequence is SEQ ID NO: 13, and the RNA oligonucleotide sequence is SEQ ID NO: 14; or the target sequence is SEQ ID NO: 15, and the RNA oligonucleotide sequence is SEQ ID NO: 16; or the target sequence is SEQ ID NO: 17, and the RNA oligonucleotide sequence is SEQ ID NO: 18; or the target sequence is SEQ ID NO: 19, and the RNA oligonucleotide sequence is SEQ ID NO: 20.

[0014] Furthermore, the expression plasmids in steps (1) and (2) are both pSilencer 1.0-U6.

[0015] Furthermore, the transfection method in step (4) comprises the following steps:

[0016] 1) Cell plating: HaCaT cells are plated and cultured in culture medium;

[0017] 2) Cell transfection: prepare siRNA duplex-Lipofectamine RNAiMAX complex, add RNAi duplex-Lipofectamine RNAiMAX complex to each well containing cells and mix well;

[0018] 3) Medium change: Change the medium after transfecting cells.

[0019] Furthermore, the preparation method of the siRNA duplex-Lipofectamine RNAiMAX complex is as follows: dilute the siRNA duplex with serum-free Opti-MEM I reduced serum medium, mix well and let stand; mix Lipofectamine TM RNAiMAX was then diluted in Opti-MEM I reduced serum medium and allowed to stand; the diluted RNAi duplex was mixed with the diluted Lipofectamine TM RNAi MAX was mixed, mixed and incubated to obtain a siRNA duplex-Lipofectamine RNAiMAX complex.

[0020] Further, in step (5), the cells are incubated at 37° C. in a CO 2 incubator.

[0021] A cannabinoid receptor-deficient in vitro cell model is constructed by any of the construction methods described above.

[0022] A method for constructing a cannabinoid receptor-deficient in vitro cell model or an application of a cannabinoid receptor-deficient in vitro cell model in the pathological study of prurigo nodularis.

[0023] The invention provides a method for constructing an in vitro cell model siRNA CB1 / 2 of cannabinoid receptor deficiency, comprising a method for constructing and screening a siRNA-CB1 vector and a method for constructing and screening a siRNA-CB2 vector, and then using the two vectors to knock out CB1 and CB2 genes in HaCaT cells; verifying and evaluating the effect of constructing the siRNA CB1 / 2 model by verifying key keratin in a signaling pathway of prurigo nodularis disease; and evaluating the consistency of the in vitro cell model by comparing the expression of key proteins in siRNA CB1 / 2 of prurigo nodularis patients and an in vitro cell model; constructing an in vitro cell model using the method can simulate the pathogenesis of prurigo nodularis in vitro, and is used for research on the treatment of prurigo nodularis disease.

[0024] Compared with the prior art, the beneficial technical effects of the present invention are:

[0025] 1) The present invention constructs a cannabinoid receptor-deficient in vitro cell model siRNA CB1 / 2 by modifying the CB1 and CB2 genes, which is used to simulate the pathogenesis of nodular prurigo and facilitate the study of the nodular prurigo signaling pathway. Using the model in this invention patent to screen out key target molecules can provide a new idea for the treatment of cell hyperproliferation, abnormal differentiation and inflammatory skin diseases.

[0026] 2) The present invention is the first to construct a cannabinoid receptor-deficient in vitro cell model siRNACB1 / 2, which can be conveniently used in the study of the pathogenesis of prurigo nodularis and solve the problem of difficulty in sample collection during the research process.

[0027] 3) The present invention is easy to implement, has low cost and better consistency of clinical indicators compared with mouse and monkey models, and can provide a new research idea for the treatment of nodular prurigo.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the construction process of a cannabinoid receptor-deficient in vitro cell model of the present invention.

[0030] Figure 2 This is a graph showing the expression changes of CB1 and CB2 in the in vitro cell model siRNACB1 / 2 strain of the present invention.

[0031] Figure 3 This is a graph showing the expression changes of keratin K6 and K17 in the in vitro cell model siRNACB1 / 2 strain of the present invention.

[0032] Figure 4 This is a graph showing the expression changes of STAT3 / 6 in the in vitro cell model siRNACB1 / 2 strain of the present invention.

[0033] Figure 5 This is a graph showing the consistency evaluation results between the in vitro cell model siRNACB1 / 2 of the present invention and clinical samples. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0035] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0036] 1. In vitro cell model design

[0037] In order to study the pathogenesis of nodular prurigo, the inventor has read a lot of literature, and the inventor's laboratory has also conducted a lot of preliminary exploratory research. The inventor found that in the lesion area of ​​PN, compared with healthy skin, the expression of cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) was downregulated, especially CB1 expression was significantly downregulated, the expression of proliferative keratin (K5 / K14) and activated keratin (K6A, K6B, K16, K17) was significantly upregulated, and the expression of differentiated K1 / K10 was also upregulated. Therefore, it is speculated that the induction of keratin expression by CB1 and CB2 receptors in keratinocytes through the activation of STAT3 may be one of the pathogenesis of nodular prurigo. Therefore, verifying the expression pattern of keratin in the epidermis of nodular prurigo can help us understand the pathological characteristics of epidermal hyperplasia and hyperkeratosis, and explore the signal pathways of keratin expression regulation involving cannabinoid receptors. Combined with the previous research work, in order to more conveniently continue to study the pathogenesis of PN, the inventor designed an in vitro cell model (such as Figure 1 ). The specific steps are as follows:

[0038] (1) constructing multiple siRNA-CB1 vectors for knocking out the CB1 gene in HaCaT cells, screening multiple vectors, and selecting the vector with the best knockout effect;

[0039] (2) construct multiple siRNA-CB2 vectors for knocking out the CB2 gene in HaCaT cells, screen the multiple vectors, and select the vector with the best knockout effect;

[0040] (3) Cultivate HaCaT cells and optimize them to the optimal state;

[0041] (4) transfecting the HaCaT cells in step (3) with the vector with the best effect screened in step (1) and step (2);

[0042] (5) Cultivating the transfected HaCaT cells, and screening the successfully transfected HaCaT cell lines by PCR and other technical means, and naming them as siRNACB1 / 2;

[0043] (6) Treating transfected HaCaT cells with ACE / ACEA and detecting the expression of key proteins;

[0044] (7) Conduct clinical consistency evaluation on the constructed in vitro cell model siRNACB1 / 2.

[0045] 2. Construction and screening of siRNA-CB1 vector

[0046] 1) siRNA-CB1 target sequence design

[0047] In order to design siRNA specifically for silencing the CB1 gene in HaCaT cells, the inventors analyzed the human CB1 gene, downloaded transcript information through NIBI data, and designed a series of target sequences. We found the sequence of CB1 mRNA on pubmed, downloaded the sequence of RNA transcript in FASTA format, and the accession number containing the transcript is as follows: NM_001370546.1, NM_001424094.1, NM_001370547.1, NM_001424096.1, NM_001424097.1, NM_001424095.1, NM_001424095.1, NM_016083.6, NM_001160226.3, NM_001365870.2, NM_001365869.2, NM_001365870.3, NM_001365869.3, NM_001365871.4, NM_001365869.5, NM_001365869.6, NM_001365869.7, NM_001365869.8, NM_001365869.9, NM_001365869.1, NM_001365869.1, NM_001365869. 001160258.3, NM_001424098.1, XM_047418171.1, NM_001365872.2, NM_001365874.3, XM_047418172.1, NM_033181.4, XM_054354204.1,

[0048] First, select the target fragment position of the target gene CB1 and display the representative siRNA sequence; secondly, exclude the fragments containing SNP sites and non-open reading frames from the candidate siRNA; next, remove some unfavorable siRNA sequences, such as degenerate bases, toxic domains, repeated sequences, high GC / low GC sequences; then score from the hot chemistry, high-order structure, etc., and list the sequences from high to low; then perform Blast analysis on the siRNA fragments to remove non-specific binding sequences; finally, select and design 5 siRNAs. The specific rules for the sequence are as follows:

[0049] Starting from the transcription AUG start codon of CB1, search for downstream AA sequences and record the 19 nucleotides adjacent to each AA 3' end as candidate siRNA target sites. The GC content of each target sequence is required to be around 30%-50%, and the siRNA sequence length is between 19 and 29nt. Compare the candidate target sequences with the NCBI database (www.ncbi.nl m.nih.gov / BLAST / ) to exclude those sequences that are homologous to other coding sequences / ESTs. This design scheme ultimately tested 5 candidate target sequences, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The inventors finally found an optimal siRNA sequence through a series of verification experiments, numbered siRNA-CB1-04, for subsequent research.

[0050] The sequence of siRNA-CB1 is as follows:

[0051]

[0052] 2) Preparation of siRNA-CB1 expression vector

[0053] Studies have shown that siRNA obtained in vitro is easily degraded when introduced into cells, so the inventors designed an expression vector for siRNA-CB1 to ensure that siRNA-CB1 is continuously expressed in HaCaT cells and the CB1 gene is silenced for a long time.

[0054] In order to achieve stable expression of siRNA-CB1 in cells, the inventors used the commercial pSilencer 1.0-U6 expression plasmid provided by Thermo Fisher Scientific, and cloned the DNA double-stranded template sequence corresponding to siRNA into the promoter of RNA polymerase III of the vector, so that the desired siRNA molecules can be expressed in vivo. The inventors then amplified the expression vector through Escherichia coli, and prepared a large number of expression vector plasmids through cloning and screening for use.

[0055] 3. Construction and screening of siRNA-CB2 vector

[0056] 1) siRNA-CB2 target sequence design

[0057] In order to design siRNA specifically for silencing the CB2 gene in HaCaT cells, the inventors analyzed the human CB2 gene, downloaded transcript information through NIBI data, and designed a series of target sequences. We found the sequence of CB2 mRNA on pubmed, downloaded the sequence of RNA transcript in FASTA format, and the accession number of the transcript is as follows:

[0058] NM_001841.3, XM_011540629.4, XM_017000261.3, XM_047444833.1,

[0059] A series of target sequences were designed, and the specific rules are as follows:

[0060] Starting from the transcription AUG start codon of CB2, search for downstream AA sequences and record the 19 nucleotides adjacent to each AA 3' end as candidate siRNA target sites. The GC content of each target sequence is required to be around 30%-50%. Compare the candidate target sequences with the NCBI database (www.ncbi.nlm.nih.gov / BLAST / ) to exclude those sequences that are homologous to other coding sequences / ESTs. This design scheme ultimately tested 5 candidate target sequences, and through a series of verification tests, an optimal siRNA sequence was finally found, named siRNA-CB2-03, for subsequent research. The siRNA-CB2 sequence is as follows:

[0061]

[0062]

[0063] 2) Preparation of siRNA-CB2 expression vector

[0064] Studies have shown that siRNA obtained in vitro is easily degraded when introduced into cells, so the inventors designed an expression vector for siRNA-CB2 to ensure that siRNA-CB2 is continuously expressed in HaCaT cells and the CB2 gene is silenced for a long time.

[0065] In order to achieve stable expression of siRNA-CB2 in cells, the inventors used the pSilencer 1.0-U6 expression plasmid provided by Thermo Fisher Scientific, cloned the DNA double-stranded template sequence corresponding to siRNA into the promoter of RNA polymerase III of the vector, and then expressed the desired siRNA molecules in vivo. The inventors then amplified the expression vector through Escherichia coli, and prepared a large number of expression vector plasmids through cloning and screening.

[0066] 4. Knockout of CB1 and CB2 genes in HaCaT cells

[0067] The present invention uses the siRNA-CB1 and siRNA-CB2 expression vectors screened in the above steps to co-infect HaCaT cells, and then further screen. Generally speaking, the cell fusion rate reaches 30-50% after about 24 hours of passage, and the transfection efficiency is good. For adherent cells, they should be repeatedly broken up to prepare single cells for planting (but they should not be over-digested mechanically or enzymatically, and a good balance should be found). For suspended cells, they should also have a certain number and cell state during transfection. Taking a 24-well plate as an example, the number of cells during transfection should be 4-8×10 5 / well (cells with strong stemness should be about half as few). In order to improve the efficiency, the present invention uses the commercial Lipofectamine produced by Thermo Fisher Scientific TM Transfect cells with transfection reagent. The method is as follows:

[0068] 1) Cell plating

[0069] Usually, cells are plated one day before transfection. Cells are plated after counting according to the cell growth rate, with 5000 cells per well of a 24-well plate. Cells are cultured in 500ul of complete culture medium without DMED to ensure that the cell density reaches about 80% to 90% when transfected the next day.

[0070] 2) Cell transfection

[0071] For each well to be transfected, prepare the siRNA duplex-Lipofectamine RNAiMAX complex and set up a negative control for the experiment. Fluorescently labeled non-target siRNA has been synthesized siRNA. The specific steps are as follows:

[0072] Dilute 6 pmol of siRNA duplex with 50 μL of serum-free Opti-MEM I reduced serum medium, mix gently, and let stand for 5 minutes. TM RNAiMAX, then dilute 1 μL in 50 μL Opti-MEM I reduced serum medium, avoid rough blowing when mixing (because it may cause liposome ineffectiveness), and let it stand for 3 to 5 minutes. TM RNAi MAX mix, mix gently and incubate at room temperature for 10-20 minutes.

[0073] Add RNAi duplex-Lipofectamine RNAiMAX complex to each well containing cells. This makes a final volume of 600 μL and a final RNA concentration of 10 nM. Gently rock the plate back and forth to mix.

[0074] 3) Change the fluid

[0075] Transfection aids (such as Lipofectamine RNAi MAX) have certain cytotoxicity, so it is necessary to change the medium in time 4 to 8 hours after plasmid or siRNA transfection of cells (replace serum-free medium with complete medium).

[0076] 4) Detection

[0077] Incubate the cells at 37°C in a CO2 incubator. 48-72 hours after transfection, the silencing / overexpression effect can be detected by QPCR or Western blot. Take the cells out of the incubator, which are siRNA CB1 / 2, and then perform subsequent related tests or store them at -80°C for future use.

[0078] 5. Verification of key proteins in siRNA CB1 / 2 in in vitro cell models

[0079] 1) Verification of CB1 and CB2 expression levels

[0080] First, we used fluorescence PCR to verify the changes in CB1 and CB2 expression levels in knockout cell lines. By comparing the changes in RNA levels in normal cell lines (control), siRNA CB1 / 2 knockout lines (siRNA CB1 / 2) and empty vector cell lines (0-vector), we found that the RNA levels of CB1 and CB2 genes in the siRNA knockout lines were significantly downregulated (see Figure 2 ), indicating that the in vitro model was successfully constructed.

[0081] 2) Key keratin expression

[0082] Existing studies have shown that the expression of activated keratin K6 / K16 and K17 in normal human epidermis is extremely low, and their expression is upregulated in patients with psoriasis, PN, etc. In normal skin lesions, AEA and ACEA act on keratinocytes and can induce a decrease in K6 expression. However, when the CB1 ligand is abnormal, the action of ACEA leads to an upregulation of K6 expression. K17 expression is similar to K6. Under the action of AEA and ACEA, CB1 / CB2 dysfunction will also be upregulated. Therefore, we used K6 and K17 as markers to verify the effect of the model. The study found that after AEA and ACEA were added to the constructed cell model siRNA CB1 / 2, K6 and K17 were significantly upregulated (see Figure 3 ).

[0083] 3) Changes in STAT3 / 6 phosphorylation

[0084] Previous studies have shown that the transcription factor STAT3 / 6 is activated in the nucleus in PN lesions, and STAT3 / 6 phosphorylation is upregulated in PN lesions. We detected the changes of two indicators in siRNA CB1 / 2 by western blot, and the expression of these two factors increased (see Appendix Figure 4 ).

[0085] Therefore, the in vitro cell model siRNA CB1 / 2 was successfully constructed, and the key protein verification was consistent with the results of clinical patient studies.

[0086] 6. Evaluation of the consistency of in vitro cell model simulating prurigo nodularis

[0087] In order to verify the consistency of the evaluation results of siRNA CB1 / 2 in clinical samples and cell models, we used the Department of Dermatology of Peking University Shenzhen Hospital as the clinical research center and recruited a total of 38 patients with confirmed nodular prurigo, mainly from the Greater South China region. The case diagnosis criteria all met the clinical diagnostic criteria for nodular prurigo. The skin lesions and skin tissues around the lesions were collected from the patients, and peripheral blood was collected. At the same time, personal information (age, gender, course of disease, occupation, treatment within the past month), blood routine, total IgE, thyroid function and other data were recorded, and the patient's skin disease quality of life index was collected. In addition, 12 normal human skin samples were collected as the experimental control group. The specimens were collected from the skin excision samples of the Department of Stomatology of Peking University Shenzhen Hospital. The collection of skin tissue and blood involved was in accordance with the patient's informed consent system and was reviewed and approved by the Ethics Committee of Peking University Shenzhen Hospital.

[0088] This protocol selected four indicators, namely K6, K17, STAT3 and STAT6, and used them to evaluate the consistency between siRNA CB1 / 2 strains and clinical samples by detecting changes in RNA level expression. The consistency between siRNA CB1 / 2 strains and PN patients was evaluated by fitting curves, and the results showed that the consistency reached 90.4% (see Figure 5 ). Therefore, the siRNA CB1 / 2 strain model constructed by the present invention is highly consistent with clinical PN patient samples and can be used for PN patient mechanism research.

[0089] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0090] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A method for constructing an in vitro cell model of cannabinoid receptor deficiency, characterized in that: The following steps are involved: (1) Construction of siRNA-CB1 vector for CB1 gene knockout in HaCaT cells; (2) Construction of siRNA-CB2 vector for CB2 gene knockout in HaCaT cells; (3) Cultivating HaCaT cells; (4) transfecting the HaCaT cells in step (3) with the vectors in step (1) and step (2); (5) Cultivate the transfected HaCaT cells.

2. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 1, characterized in that: In the siRNA-CB1 sequence, the target sequence is SEQ ID NO: 1, and the RNA oligonucleotide sequence is SEQ ID NO: 2; or the target sequence is SEQ ID NO: 3, and the RNA oligonucleotide sequence is SEQ ID NO: 4; or the target sequence is SEQ ID NO: 5, and the RNA oligonucleotide sequence is SEQ ID NO: 6; or the target sequence is SEQ ID NO: 7, and the RNA oligonucleotide sequence is SEQ ID NO: 8; or the target sequence is SEQ ID NO: 9, and the RNA oligonucleotide sequence is SEQ ID NO:

10.

3. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 1, characterized in that: In the siRNA-CB2 sequence, the target sequence is SEQ ID NO: 11 and the RNA oligonucleotide sequence is SEQ ID NO: 12; or the target sequence is SEQ ID NO: 13 and the RNA oligonucleotide sequence is SEQ ID NO: 14; or the target sequence is SEQ ID NO: 15 and the RNA oligonucleotide sequence is SEQ ID NO: 16; or the target sequence is SEQ ID NO: 17 and the RNA oligonucleotide sequence is SEQ ID NO: 18; or the target sequence is SEQ ID NO: 19 and the RNA oligonucleotide sequence is SEQ ID NO:

20.

4. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 1, characterized in that: The expression plasmids in steps (1) and (2) are both pSilencer 1.0-U6.

5. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 1, characterized in that: Step (4) The transfection method comprises the following steps: 1) Cell plating: HaCaT cells are plated and cultured in culture medium; 2) Cell transfection: prepare siRNA duplex-Lipofectamine RNAiMAX complex, add RNAi duplex-Lipofectamine RNAiMAX complex to each well containing cells and mix well; 3) Medium change: Change the medium after transfecting cells.

6. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 5, characterized in that: The preparation method of siRNA duplex-Lipofectamine RNAiMAX complex is as follows: dilute siRNA duplex with serum-free Opti-ME MI reduced serum medium, mix well and let stand; mix Lipofectamine TM RNAiMAX was then diluted in Opti-MEM I reduced serum medium and allowed to stand; the diluted RNAi duplex was mixed with diluted Lipofectamine TM RNAiMAX was mixed, mixed and incubated to obtain a siRNA duplex-Lipofectamine RNAiMAX complex.

7. The method for constructing a cannabinoid receptor-deficient in vitro cell model according to claim 1, characterized in that: Step (5), incubate the cells at 37°C in a CO2 incubator.

8. An in vitro cell model of cannabinoid receptor deficiency, characterized in that: It is obtained by the construction method described in any one of claims 1 to 7.

9. A method for constructing a cannabinoid receptor-deficient in vitro cell model according to any one of claims 1 to 7 or use of a cannabinoid receptor-deficient in vitro cell model according to claim 8 in pathological research of prurigo nodularis.

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