SiRNA targeting serping1 gene and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a siRNA targeting SERPING1 gene and application thereof. The application provides a siRNA targeting SERPING1 gene, the siRNA can efficiently knock down the expression of SERPING1 (knock-down efficiency > 80%), and firstly discloses the negative regulation function of SERPING1 in airway epithelial cell inflammation and the action mechanism thereof relying on secretory factors. The siRNA can be used as an experimental tool for researching the function of SERPING1 gene, and can also be used for constructing an inflammatory cell model, thereby providing an effective tool for researching the function of SERPING1 gene, constructing an inflammatory cell model and screening anti-inflammatory drugs, and having important scientific research value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a siRNA targeting the SERPING1 gene and its application. Background Technology

[0002] The SERPING1 gene (serpin family G member 1) encodes a C1 esterase inhibitor (C1-INH), a serine protease inhibitor that plays a crucial negative regulatory role in inflammatory responses, complement system activation, the kallikrein-kinin system, and the coagulation cascade. The main physiological functions of C1-INH include inhibiting C1r and C1s proteases in the classical complement pathway, MASP-1 and MASP-2 in the lectin pathway, and regulating FXIIa and kallikrein in the coagulation contact pathway, thereby inhibiting excessive bradykinin production. Defects in the SERPING1 gene have been clearly demonstrated to be closely associated with hereditary angioedema (HAE), with approximately 85% of HAE patients exhibiting SERPING1 gene mutations, leading to decreased C1-INH protein levels or abnormal function. Recent large-scale population cohort studies have also found a significant association between SERPING1 insufficiency and a markedly increased risk of thrombotic diseases such as venous thrombosis, non-cardiac stroke, and peripheral artery disease.

[0003] RNA interference (RNAi) technology is an important tool for studying gene function and developing therapeutics. By designing and synthesizing small interfering RNAs (siRNAs) that specifically target target genes, the expression of target genes can be silenced at the posttranscriptional level, and it has been widely used in gene function research and drug development. Currently, there are reports on RNA interference targeting the SERPING1 gene. For example, some studies have investigated the effects of injecting Serping1 siRNA into MPTP-induced Parkinson's disease mouse models on dopaminergic neurons; others have used siRNA to knock down GRP75, thereby improving cellular calcium overload and mitochondrial damage caused by SERPING1 mutations.

[0004] However, current research on SERPING1 siRNA mainly focuses on two directions: one is to explore the mechanisms of SERPING1 gene mutation-related diseases (such as HAE), and the other is to conduct preliminary explorations using SERPING1 as a target in other disease models. There are currently no reports on the functional validation of SERPING1-targeting siRNAs in airway epithelial cell inflammation models, nor is the impact of SERPING1 knockdown on cellular secretome function clarified. In particular, whether SERPING1 participates in regulating inflammatory factor expression in CSE (cigarette smoke extract)-induced bronchial epithelial cell inflammation models, and whether its regulatory mechanism is related to secretory factors in cell supernatant, remains unreported. Furthermore, current technology does not provide experimentally validated siRNA sequences that can effectively knock down SERPING1 expression in human bronchial epithelial cells and possess clearly defined functional activity. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention develops an experimentally validated siRNA sequence targeting the SERPING1 gene. This siRNA can efficiently knock down SERPING1 expression (knockdown efficiency >80%) and, for the first time, reveals the negative regulatory function of SERPING1 in airway epithelial cell inflammation, as well as its mechanism of action relying on secreted factors. This siRNA can serve as an experimental tool for studying SERPING1 gene function and can also be used to construct inflammatory cell models, laying an important foundation for building an anti-inflammatory drug screening platform based on the SERPING1 target, and possessing significant scientific research value and broad application prospects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides the application of siRNA targeting the human SERPING1 gene in the preparation of a reagent for knocking down the expression of the SERPING1 gene in human bronchial epithelial cells, wherein the sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, the antisense strand nucleotide sequence is shown in SEQ ID NO.2, and the siRNA has a dTdT overhang at its 3' end.

[0007] This invention provides a siRNA sequence capable of efficiently and specifically knocking down the expression level of the SERPING1 gene mRNA in human bronchial epithelial cells. This invention involves the rational design and screening of multiple candidate siRNAs, followed by verification of their knockdown efficiency using qPCR technology, ultimately yielding a siRNA that significantly reduces SERPING1 expression.

[0008] Preferably, the human bronchial epithelial cells are Beas-2b human bronchial epithelial cells.

[0009] Preferably, siRNA is introduced into human bronchial epithelial cells using liposome-mediated transfection, with a final siRNA transfection concentration of 10-50 nM.

[0010] A second aspect of this invention provides the application of siRNA targeting the human SERPING1 gene in the preparation of a tool for studying the function of the SERPING1 gene. The siRNA has the sense strand nucleotide sequence shown in SEQ ID NO.1 and the antisense strand nucleotide sequence shown in SEQ ID NO.2. The siRNA has a dTdT overhang at its 3' end. The tool for studying the function of the SERPING1 gene is a cell model constructed based on human bronchial epithelial cells for in vitro studies of the loss of SERPING1 gene function. The tool is used to verify the negative regulatory function of SERPING1 in airway epithelial inflammation and / or to verify that SERPING1 exerts an anti-inflammatory effect through secretory factors.

[0011] This invention clarifies the functional role of SERPING1 in airway epithelial inflammation. Existing technologies have not reported the related functions of SERPING1 in airway epithelial cell inflammatory responses. This invention uses screened and effective siRNA to knock down SERPING1 expression in Beas-2b cells, demonstrating that this treatment significantly upregulates CSE-induced inflammatory cytokine expression. This result is the first to confirm, based on an airway epithelial cell model, that SERPING1 is a negative regulator of inflammatory responses, providing crucial experimental evidence for elucidating the biological function of SERPING1 in airway inflammation.

[0012] A third aspect of the present invention also provides the application of siRNA targeting the human SERPING1 gene in constructing a CSE-induced airway epithelial inflammation cell model, wherein the sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, the antisense strand nucleotide sequence is shown in SEQ ID NO.2, and the siRNA has a dTdT overhang at its 3' end.

[0013] This invention also reveals the mechanism of action of SERPING1 in regulating inflammation. In a cell supernatant transfer experiment, supernatant from airway basal stem cells with high background SERPING1 expression and supernatant from stem cells with SERPING1 knocked down by siRNA were used to incubate CSE-induced Beas-2b cells. The results showed that the supernatant with high background SERPING1 expression significantly inhibited the expression of CSE-induced inflammatory factors, while the supernatant treated with siRNA lost this inhibitory effect. This result is the first functional demonstration that SERPING1 exerts its anti-inflammatory effect by regulating the cell secretome, and does not depend on the cell contact pathway.

[0014] Preferably, the CSE-induced airway epithelial inflammatory cell model is used for anti-inflammatory drug screening and / or for efficacy evaluation of SERPING1-targeted drugs.

[0015] Preferably, the final volume concentration induced by CSE is 1%-10%.

[0016] A fourth aspect of this invention provides the application of siRNA targeting the human SERPING1 gene in the preparation of a SERPING1 function detection kit, characterized in that the sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, the antisense strand nucleotide sequence is shown in SEQ ID NO.2, and the siRNA has a dTdT overhang at its 3' end; the kit is used to detect the functional status of SERPING1 and / or evaluate the effect of the test compound on SERPING1 gene expression.

[0017] The fifth aspect of the invention also provides the use of a conditioned medium containing human airway basal stem cells highly expressing SERPING1 in the preparation of a formulation for inhibiting smoking-related airway epithelial inflammation.

[0018] Preferably, the method for preparing the conditioned medium for human airway basal stem cells with high SERPING1 expression includes: screening airway basal stem cells with high background SERPING1 expression, culturing for more than 24 hours, collecting the culture supernatant, filtering to remove cell debris, and centrifuging and concentrating the medium 9-11 times using a 10 kDa ultrafiltration tube (Merck Millipore, UFC901096) to obtain the conditioned medium.

[0019] Compared with the prior art, the beneficial effects of the present invention are: (1) A highly efficient and specific SERPING1-targeting siRNA tool is provided. The siRNA (siRNA-2) has a knockdown efficiency of over 80% in Beas-2b cells and no obvious cytotoxicity. It can be used to construct SERPING1-deficient cell models and CSE-induced inflammatory cell models, providing an effective tool for studying the function of SERPING1 in multiple biological pathways and screening anti-inflammatory drugs.

[0020] (2) The negative regulatory function of SERPING1 in airway epithelial inflammation was revealed for the first time. After knocking down SERPING1 with the siRNA, the expression of CSE-induced IL-6, IL-8 and TNF-α was significantly increased, proving that SERPING1 is an important negative regulator of airway inflammation.

[0021] (3) The mechanism by which SERPING1 exerts its anti-inflammatory effect through secreted factors was elucidated for the first time. Cell supernatant transfer experiments demonstrated that the stem cell supernatant lost its anti-inflammatory function after SERPING1 knockdown, providing a new direction for the development of anti-inflammatory drugs based on SERPING1 secreted factors.

[0022] (4) The siRNA sequence designed in this invention is well-defined, simple to prepare, and has sufficient functional verification. It also has good reproducibility and promising prospects for widespread application. Attached Figure Description

[0023] Figure 1 Fluorescence imaging results 24 hours after siRNA transfection; Figure 2 The knockout efficiency of three SERPING1 siRNAs was detected by qPCR. Figure 3 To detect the effect of si-SERPING1-1 on the beas-2b cell model by qPCR; Figure 4 To investigate the effects of CM-BC and si-SERPING1-1-CM-BC conditioned medium on the expression of inflammatory factor mRNA in CSE-induced Beas-2b inflammation model cells, qPCR was used. Detailed Implementation

[0024] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0026] In current technologies, the biological function of SERPING1 in airway epithelial cell inflammatory responses remains unclear, and effective research tools are lacking for studying its loss-of-function. Furthermore, no siRNA sequences targeting SERPING1 have been reported, and the knockdown efficiency and specificity of existing gene silencing tools in airway epithelial cells need improvement. In addition, whether SERPING1 regulates inflammation through secreted factors, and its feasibility as an anti-inflammatory target, lacks direct functional evidence.

[0027] To address the above problems, the present invention proposes the following solutions: In a first aspect, the present invention provides an siRNA targeting the SERPING1 gene. The siRNA is designed as follows: based on the human SERPING1 gene mRNA sequence (GenBank accession number: NM_000062), a target sequence is selected from its CDS region. Design principles include: length 19-21 nt; GC content 35%-55%; avoidance of consecutive single-base repetitions (≤4 times); and exclusion of homology with other genes through BLAST alignment. The 3' end of the siRNA can be dTdT or UU overhangs (see Example 1).

[0028] Based on the above principles, this invention designed and synthesized three candidate siRNAs: the sense strand of siRNA-1 is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2; the sense strand of siRNA-2 is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; the sense strand of siRNA-3 is shown in SEQ ID NO.5, and the antisense strand is shown in SEQ ID NO.6. Cell transfection and qPCR verification showed that siRNA-2 could efficiently and specifically knock down the expression of SERPING1 mRNA in Beas-2b cells, with a knockdown efficiency of over 80% (see Example 3 and...). Figure 2 The optimal sequence is described above. The siRNA can undergo chemical modifications such as 2'-O-methyl, 2'-fluoro, thiophosphate bonds, cholesterol, or GalNAc.

[0029] Secondly, this invention also provides the application of the above-mentioned siRNA in knocking down SERPING1 gene expression. The siRNA is transfected into mammalian cells (human bronchial epithelial cells Beas-2b) via liposome-mediated transfection (Lipofectamine 3000), degrading SERPING1 mRNA through RNA interference. The final concentration of siRNA is 10-50 nM, preferably 25-30 nM. Specific procedures: siRNA and transfection reagent are diluted separately with Opti-MEM and mixed, incubated at room temperature for 15 minutes to form a complex; then, after 6 hours of cell transfection, the medium is replaced with complete medium, and the cells are cultured for another 24 hours post-transfection (see Example 2 and...). Figure 1 ).

[0030] Thirdly, this invention also provides the application of the above-mentioned siRNA in promoting CSE-induced expression of inflammatory factors. The specific method is as follows: (1) Beas-2b cells are cultured to 60%-80% confluence; (2) siRNA is mixed with Lipofectamine 3000 and incubated for 15 minutes to form a complex; (3) the complex is added to the cells for transfection, and the medium is changed after 6 hours; (4) fresh medium containing CSE (final concentration 1%-10%, preferably 5%) is added and incubated for another 24 hours; (5) cells are collected, and qPCR is used to detect the mRNA expression levels of inflammatory factors (IL-6, IL-8, TNF-α). CSE stimulation after transfection can significantly upregulate the expression of inflammatory factors (see Examples 4, 5, and 6). Figure 3 , 4 ).

[0031] Fourthly, the present invention also provides the application of the above-mentioned siRNA in the preparation of tools for studying the function of the SERPING1 gene, and in the construction of CSE-induced inflammatory cell models, which can be used for screening anti-inflammatory drugs or evaluating the efficacy of SERPING1-targeting drugs.

[0032] Fifthly, the present invention also provides a pharmaceutical composition comprising the above-mentioned siRNA, wherein the active ingredient is preferably siRNA-1, and further comprises a pharmaceutically acceptable carrier and / or excipients, and the final concentration of siRNA is 10-100 nM, preferably 30 nM. Dosage forms include aerosols, powders, sprays, solutions, liposomes, or nanoformulations; the carrier includes liposomes, cationic polymers, GalNAc conjugates, or exosomes.

[0033] In a sixth aspect, the present invention provides the application of the above-mentioned siRNA in the preparation of a detection kit, the kit comprising the siRNA for detecting the functional state of SERPING1 or evaluating the effect of a test compound on SERPING1 expression.

[0034] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1: Design and synthesis of siRNA targeting the SERPING1 gene Based on the mRNA sequence of the human SERPING1 gene (GenBank accession number: NM_000062.3, transcript variant 1), siRNAs were designed using the target sequences in its CDS region [target 1 (SEQ ID NO.9): CCAAGATGCTATTCGTTGA, target 2 (SEQ ID NO.10): GTTTCCAGCTTGCCGACAA, target 3 (SEQ ID NO.11): TCTGACTTGGAGAGTCATT]. Design principles: length 21 nt; GC content 40%-55%; consecutive single base repeats ≤ 4 times; BLAST alignment to exclude homology with other genes; dTdT overhang added to the 3' end. Three candidate siRNAs were designed and synthesized according to these principles, named si-SERPING1-1, si-SERPING1-2, and si-SERPING1-3, respectively. The negative control siNC was a sequence with randomly arranged bases verified as having no homology by BLAST alignment. The sequence information is shown in Table 1. All siRNAs were synthesized by commercial companies, purified by HPLC, and had a purity of >95%.

[0036] Table 1: Candidate siRNA sequence information targeting the SERPING1 gene Example 2: Cell Culture and siRNA Transfection Human bronchial epithelial Beas-2b cells (ATCC) were seeded in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37°C and 5% CO2. 24 hours before transfection, cells were seeded into 6-well plates (2 × 10⁶ cells per well). 5 To ensure confluence of 60%-70% on the day of transfection, the siRNA was then transfected according to the instructions of Lipofectamine 3000 (Sangon Biotech, catalog number E607402-0100): the siRNA and Lipofectamine 3000 were diluted separately with Opti-MEM and mixed, and incubated at room temperature for 15 minutes to form a complex; then the complex was added dropwise to each well (final siRNA concentration 30 nM). After 6 hours of transfection, the complete medium was replaced and the culture was continued until 24 hours after transfection.

[0037] After obtaining the siRNA and transfection reagents, to investigate the transfection effect, siRNA-FAM was simultaneously transfected. Fluorescence imaging was performed 24 hours later, and the results are as follows: Figure 1 As shown, cells transfected with siRNA-FAM exhibited significant fluorescence intensity, while siRNA-NC showed relatively little fluorescence. This indicates that siRNA can be successfully transfected into cells under these conditions.

[0038] Example 3: qPCR detection of siRNA knockdown efficiency of SERPING1 mRNA Cells were collected 24 hours after transfection, and total RNA was extracted using the TRIzol method. The concentration and purity were determined by NanoDrop. 1 μg of total RNA was reverse transcribed into cDNA (HiScript III RT SuperMix). Using GAPDH as an internal control, qPCR was performed using ChamQ Universal SYBR qPCR Master Mix. The reaction mixture consisted of 20 μL: 2 μL cDNA, 0.4 μL each of forward and reverse primers (final concentration 200 nM), 10 μL 2×SYBR premix, and ddH2O to make up the volume. The amplification program was: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 10 seconds; 60℃ annealing and extension for 30 seconds, for a total of 40 cycles; melting curve analysis was performed at 65-95℃. Each sample was tested in triplicate. -ΔΔCt The relative expression level was calculated using the method shown in Table 2.

[0039] result( Figure 2 The results showed that, compared with siNC, the expression of SERPING1 mRNA in Beas-2b cells transfected with si-SERPING1-1 was significantly reduced (P<0.001), with a knockdown efficiency of over 80%; the knockdown effects of si-SERPING1-2 and si-SERPING1-3 were not significant (P>0.05). Therefore, si-SERPING1-1 (hereinafter referred to as "the siRNA of this invention") was used in subsequent examples.

[0040] Table 2: qPCR primer sequences Example 4: Effects of the siRNA of the present invention on the expression of inflammatory factors in CSE-induced Beas-2b cells 1. Preparation of CSE: Following the method described in the literature (Mio T, Romberger DJ, Thompson AB, et al. Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells[J]. American Journal of Respiratory and Critical Care Medicine, 1997, 155(5): 1770-1776.), cigarette smoke was introduced through a rubber tube into a flask containing 20 mL of serum-free DMEM / F12. A vacuum pump was used to continuously evacuate the flask until the smoke was completely dissolved. After filtration and sterilization using a 0.22 μm filter, CSE was obtained. OD 320 =1.0 is defined as 100% concentration of CSE stock solution, stored at -80℃ for later use.

[0041] 2. The experiment was divided into four groups: blank control group, CSE treatment group, siNC+CSE group, and siSERPING1+CSE group. siRNA transfection was performed as in Example 2; 24 hours after transfection, the culture medium was discarded, and fresh complete culture medium containing 5% (v, v) CSE was added, and incubation continued for another 24 hours. Cells were collected to extract RNA, and qPCR was used to detect the expression levels of IL-6, IL-8, and TNF-α mRNA (primer sequences are shown in Table 3), with GAPDH as an internal control.

[0042] result( Figure 3 The results showed that, compared with the blank control group, the expression of IL-6, IL-8, and TNF-α was significantly upregulated in the CSE-only treatment group (P<0.01), indicating that CSE successfully induced an inflammatory response. Compared with the siNC+CSE group, the expression of IL-6, IL-8, and TNF-α was further significantly increased in the si-SERPING1+CSE group (P<0.01 or P<0.001), demonstrating that si-SERPING1-1 promotes the expression of inflammatory factors in beas-2b cells induced by CSE. This demonstrates that SERPING1 plays a negative regulatory role in inflammation in airway epithelial cells.

[0043] Table 3: Primer sequences for qPCR of inflammatory factors Example 5: Verification using the siRNA of this invention to show that SERPING1 regulates inflammation through secretory factors To investigate the mechanism by which SERPING1 regulates inflammation, a cell supernatant transfer experiment was conducted to verify whether it exerts its effects through secretory factors.

[0044] (1) Five airway basal stem cells from different human sources were revived [Reference for cell acquisition method: Du S, JinJ, Tang C, et al. Airway Basal Stem Cells Inflammatory Alterations in COVID-19 and Mitigation by Mesenchymal Stem Cells[J]. Cell Proliferation, 2025, 58(6): e13812.], and the mRNA expression level of SERPING1 in each cell was detected by qPCR to screen cell lines with high background expression of SERPING1; (2) Preparation of supernatant: Human airway basal stem cells with high background expression of SERPING1 were seeded in 6-well plates and divided into a control group and a SERPING1 knockdown siRNA group. The transfection method was the same as in Example 2. The control group was transfected with negative control siRNA, and the siRNA group was transfected with SERPING1-targeting siRNA. After 24 h of transfection, the culture medium was replaced with fresh complete medium and cultured for another 24 h. Then, the culture medium was replaced with DMEM and cultured for more than 24 h. The cell culture supernatants of the two groups were collected and filtered through a 0.22 μm filter to remove cell debris. The supernatants were then concentrated 10 times by centrifugation using a 10 kDa ultrafiltration tube (Merck Millipore, UFC901096) and stored at -80℃ for later use. The supernatant of the control group was labeled CM-BC, and the supernatant of the SERPING1 knockdown group was labeled si-SERPING1-1-CM-BC. The cells of the two groups were collected simultaneously, and the SERPING1 mRNA level was detected by qPCR to confirm the gene knockdown efficiency.

[0045] (3) Conditioned supernatant incubation treatment: Beas-2b was seeded in 6-well plates. When the cell confluence reached 70%, 5% CSE was added, followed by the addition of CM-BC and si-SERPING1-1-CM-BC conditional supernatants (the conditional supernatant was mixed with fresh complete culture medium at a 1:1 volume ratio), and incubation was continued for 24 h. Cells from each group were collected, and the mRNA expression levels of IL-6, IL-8, and TNF-α were detected by qPCR.

[0046] (4) Experimental results (combined with) Figure 4 ): 1) Among the five airway basal stem cell lines, two showed high basal SERPING1 mRNA expression. The cell lines with high basal SERPING1 expression were defined as those where, under basal culture conditions without gene intervention or drug stimulation, the relative expression level of SERPING1 mRNA, as detected by qPCR, was significantly higher than that of the other cell lines. Figure 4 Cell lines numbered 4 and 5 showed significantly higher SERPING1 mRNA expression levels than cell lines numbered 1, 2, and 3 (at least 10-fold higher relative expression), and were identified as cell lines with high background SERPING1 expression, which were used for subsequent conditional supernatant preparation.

[0047] 2) CSE treatment significantly upregulated the mRNA levels of inflammatory factors IL-6, IL-8, and TNF-α; in the CSE inflammation model: Cell supernatant with high expression of SERPING1 (CM-BC) can significantly inhibit the increase of inflammatory factors induced by CSE; After treatment with the cell supernatant of SERPING1 knockdown (si-SERPING1-1-CM-BC), the mRNA expression levels of IL-6, IL-8, and TNF-α were significantly higher than those of the control supernatant treatment group (#marked, P<0.01), and the anti-inflammatory effect disappeared.

[0048] The above results demonstrate that SERPING1 exerts its anti-inflammatory effect through secretory factors.

[0049] As demonstrated by the above embodiments, the SERPING1-targeting siRNA provided by this invention can efficiently knock down SERPING1 expression (knockdown efficiency >80%), and for the first time proves the negative regulatory function of SERPING1 in airway epithelial cell inflammation and its mechanism of action through secretory factors. This invention provides an effective tool for studying SERPING1 gene function, constructing inflammatory cell models, and screening anti-inflammatory drugs, and has significant scientific research value and broad application prospects.

[0050] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of a siRNA targeting the human SERPING1 gene in the preparation of a reagent for knocking down the expression of the SERPING1 gene in human bronchial epithelial cells, characterized in that, The sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence is shown in SEQ ID NO.

2. The siRNA has a dTdT overhang at its 3' end.

2. The application according to claim 1, characterized in that, The human bronchial epithelial cells mentioned are Beas-2b human bronchial epithelial cells.

3. The application according to claim 1, characterized in that, siRNA was introduced into human bronchial epithelial cells using liposome-mediated transfection, with a final siRNA transfection concentration of 10-50 nM.

4. The application of a siRNA targeting the human SERPING1 gene in the preparation of tools for SERPING1 gene function research, characterized in that, The sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence is shown in SEQ ID NO.

2. The siRNA has a dTdT overhang at its 3' end. The SERPING1 gene function research tool is a cell model constructed based on human bronchial epithelial cells for in vitro research on the loss of SERPING1 gene function. The research tool is used to verify the negative regulatory function of SERPING1 in airway epithelial inflammatory response and / or to verify that SERPING1 exerts anti-inflammatory effects through secretory factors.

5. The application of a siRNA targeting the human SERPING1 gene in constructing a CSE-induced airway epithelial inflammation cell model, characterized in that, The sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence is shown in SEQ ID NO.

2. The siRNA has a dTdT overhang at its 3' end.

6. The application according to claim 5, characterized in that, The CSE-induced airway epithelial inflammatory cell model is used for anti-inflammatory drug screening and / or for efficacy evaluation of SERPING1-targeted drugs.

7. The application according to claim 5, characterized in that, The final volumetric concentration induced by CSE is 1%-10%.

8. The application of a siRNA targeting the human SERPING1 gene in the preparation of a SERPING1 function detection kit, characterized in that, The sense strand nucleotide sequence of the siRNA is shown in SEQ ID NO.1, and the antisense strand nucleotide sequence is shown in SEQ ID NO.

2. The siRNA has a dTdT overhang at its 3' end. The kit is used to detect the functional status of SERPING1 and / or evaluate the effect of the test compound on SERPING1 gene expression.

9. Application of conditioned medium containing human airway basal stem cells with high expression of SERPING1 in the preparation of formulations for inhibiting smoking-related airway epithelial inflammation.

10. The application according to claim 9, characterized in that, The method for preparing the conditioned medium for human airway basal stem cells with high SERPING1 expression includes: screening airway basal stem cells with high background SERPING1 expression, culturing for more than 24 hours, collecting the culture supernatant, filtering to remove cell debris, and concentrating 9-11 times to obtain the conditioned medium.

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