System for controllably inducing pyroptosis of tumor cells and application thereof

Abstract

The invention discloses a system for controllably inducing pyroptosis of tumor cells and application of the system. The system comprises a red light activated Cre recombinase system and an expression vector of a GSDMDNT gene sequence of loxP flanking; the Cre recombinase system comprises: (1) an expression vector 1 containing a photosensitive protein PhyA gene sequence and a CreN59 sequence, and a connecting peptide located between the PhyA gene sequence and the CreN59 sequence; (2) an expression vector 2 containing a nucleoplasm shuttle protein FHY1 gene sequence and a CreC60 sequence, and a connecting peptide located between the FHY1 gene sequence and the CreC60 sequence; and (3) phycocyanobilin is contained. The system disclosed by the invention can be used for artificially and controllably regulating and controlling the expression of the cytotoxic gene GSDMDNT, so that directional and controllable pyroptosis induction is realized. The method can be applied to the fields of tumor therapy, gene therapy, cell biology and the like, and particularly has a wide application prospect in application scenarios requiring accurate control of gene expression.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a system for controllably inducing pyroptosis of tumor cells and its application. Background Art

[0002] Pyroptosis is a lytic and inflammatory form of cell death that can locally activate the immune system while the cell is dying. Increasing studies have shown that targeting the pyroptosis pathway is an important means for treating tumors. However, excessive and uncontrollable pyroptosis can lead to risks such as fever or tissue and organ damage. Therefore, reasonably regulating the occurrence of pyroptosis and artificially controllably inducing pyroptosis have important research and application values.

[0003] Currently, different strategies have been developed to induce pyroptosis. Commonly, the intrinsic pyroptosis signaling pathway Caspase3 / GSDME is activated by using chemotherapeutic drugs to induce pyroptosis of tumor cells. Selective pyroptosis of tumor cells can also be induced by using nanomaterials. For example, acid-sensitive nanophotosensitizers are designed to activate the phospholipase C / Caspase3 / GSDME signaling axis to achieve selective pyroptosis of tumor cells. Radiotherapy-controlled pyroptosis based on Caspase3 / GSDME can also be achieved by designing nano-sensitizers. All of the above technical means for inducing pyroptosis have the characteristic of relying on the gene expression of the cells themselves.

[0004] In addition to the pyroptosis induction pathway that relies on the expression of GSDME in tumor cells themselves, there are also reports of inducing pyroptosis based on bioorthogonal chemical systems. For example, by chemically modifying and coupling the NT and CT parts of GSDMA3, and by jointly using the decoupler Phe-BF3 to release GSDMA3-NT, pyroptosis can be induced without relying on endogenous gene expression in cells. Similarly, by exogenously delivering genes expressing the pyroptosis effector protein GSDM NT efficiently induces pyroptosis. For example, AAV is used to deliver GSDMD NT to actively induce pyroptosis of cells. It is worth noting that similar strategies based on exogenous delivery of therapeutic genes can overcome the dependence on the gene expression of tumor cells themselves.

[0005] Optogenetic regulation tools have both temporal and spatial controllability, and artificially designed and synthesized optogenetic tools usually possess bioorthogonality, making them ideal gene expression regulation tools. Currently, optogenetic regulation systems that respond to different wavelengths of light have been developed, such as the blue light-responsive PACre system, the red light-responsive REDMAP system, and the far-red light-responsive FISC system. By fusing the blue light-activated photosensitive protein CYR2 with inflammatory Caspase 1 and utilizing the photoactivated oligomerization property of CYR2, the photoactivation of Caspase1 activity can be achieved, thereby triggering Caspase1 / GSDMD-mediated pyroptosis. Based on this strategy, the spatiotemporal specific induction of pyroptosis can be realized. Additionally, to directly induce pyroptosis while avoiding crosstalk with other death pathways, the artificial directional and controllable induction of pyroptosis can also be achieved through the design of fusing the photosensitive protein LOV2 with GSDMD-NT.

[0006] However, the above existing technical solutions for inducing pyroptosis also have drawbacks in different aspects while achieving the induction of pyroptosis in cells. First, drugs or nano-material-based (such as chemotherapy) induction of pyroptosis in cells relies on the activation of the cell's own pyroptosis signaling axis, such as Caspase3 / GSDME, rather than directly activating and generating GSDME with pyroptosis-inducing activity. NT , Tumor cells can evade the induction of death signals by mutating Caspase3 and downregulating the expression of GSDME, etc., which leads to insufficient controllability and limited generality of the induction method characterized by relying on the cell's own gene expression. Methods based on exogenous delivery for treatment, such as delivering bioorthogonally chemically modified GSDMA3 or delivering GSDMD by AAV NT can directionally induce pyroptosis in cells, but have the characteristic of uncontrollable pyroptosis induction, which may lead to potential side effects such as excessive induction of pyroptosis causing fever or damaging normal tissues. Summary of the Invention

[0007] Aiming at the defects in the prior art, the present invention proposes a system for controllably inducing pyroptosis in tumor cells and its application. The present invention intends to develop a stringent red light-responsive Cre-On optogenetic switch RACS to regulate the expression of GSDMD NT so as to achieve the directional and controllable induction of pyroptosis.

[0008] The present invention provides a system for controllably inducing pyroptosis in tumor cells, including a Cre recombinase system and an expression vector of the GSDMD gene sequence flanked by loxP, and the GSDMD gene sequence is placed in the expression vector in a reverse manner; NT the GSDMD NT gene sequence;

[0009] The Cre recombinase system includes the following components:

[0010] (1) An expression vector 1 containing the gene sequence of photosensitive protein PhyA, the CreN59 sequence, and a linker peptide located between the PhyA gene sequence and the CreN59 gene sequence, wherein the PhyA gene sequence is as shown in SEQ ID NO.1 and the CreN59 gene sequence is as shown in SEQ ID NO.3;

[0011] (2) An expression vector 2 containing the gene sequence of nucleocytoplasmic shuttle protein FHY1, the CreC60 sequence, and a linker peptide located between the FHY1 gene sequence and the CreC60 gene sequence, wherein the FHY1 gene sequence is as shown in SEQ ID NO.2 and the CreC60 gene sequence is as shown in SEQ ID NO.4;

[0012] (3) Containing phycocyanobilin.

[0013] In some embodiments, the amino acid sequence of the linker peptide in (1) and (2) is as shown in SEQ ID NO.6.

[0014] In some embodiments, in the expression vector 1, the combination of the PhyA gene sequence, the CreN59 gene sequence, and the linker peptide is in turn: the PhyA gene sequence, the linker peptide, and the CreN59 gene sequence;

[0015] In the expression vector 2, the combination of the FHY1 gene sequence, the CreC60 gene sequence, and the linker peptide is in turn: the FHY1 gene sequence, the linker peptide, and the CreC60 gene sequence.

[0016] The present invention also provides the application of the described system in the preparation of products for mediating pyroptosis.

[0017] In some embodiments, the application is specifically: by irradiating with red light at a wavelength of 660 ± 20 nm, activating the Cre recombinase system to induce the expression of the GSDMD NT gene.

[0018] In some embodiments, the intensity of the red light irradiation is not less than 0.05 W / cm 2 , preferably 0.05 - 1 mW / cm 2 , and the duration is not less than 0.5 minutes.

[0019] In some embodiments, the products for mediating pyroptosis include but are not limited to anti-tumor drugs or cytotoxic reagents.

[0020] The present invention also provides the application of the described system in the preparation of drugs for tumor prevention or treatment.

[0021] In some embodiments, the tumor is bladder cancer.

[0022] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0023] 1. The system of the present invention can regulate the expression of GSDMD NT , thereby realizing the directed and controllable induction of pyroptosis. It can be applied to fields such as tumor treatment, gene therapy, and cell biology, and has broad application prospects especially in application scenarios that require precise control of gene expression.

[0024] 2. The system of the present invention regulates gene expression through light irradiation, has high spatial and temporal precision, and is sensitive in operation, and can induce pyroptosis of tumor cells at specific times and positions. Compared with the existing blue light-responsive PACre, red light-responsive RedCre, and far red light-responsive FISC Cre recombination systems, the system of the present invention has lower background expression activity, and the induction magnification is as high as 245.71 times.

[0025] 3. The present invention further reduces the background expression activity and improves the inducibility activity by screening and optimizing the connection modes of various genes and the Cre part in the system.

[0026] 4. The system of the present invention is completely based on exogenous gene expression without relying on the cell's own gene expression, thus making it widely applicable to different cell types.

[0027] 5. The good rigor of the system of the present invention can maximize the avoidance of background expression leakage in the previous regulation system and the resulting cell effects. It is especially suitable for cell death programming and precise cell tracing carried out by regulating the expression of cytotoxic genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is a schematic diagram of the principle design of the RACS for regulating the expression of target genes in Embodiment 1 of the present invention;

[0030] Figure 2 It is the screening test result of the component configuration method of RACS in Embodiment 2 of the present invention;

[0031] Figure 3 It is the screening result of the linker peptide in Embodiment 3 of the present invention;

[0032] Figure 4Results of in vitro regulation of Luc expression by 4RACS of the present invention;

[0033] Figure 5 Results of Western blot detection of Luc protein expression in Example 4 of the present invention;

[0034] Figure 6 Results of spatially specific induction of mCherry expression by 5RACS of the present invention;

[0035] Figure 7 Results of comparison of performance of different light-induced Crees in Example 6 of the present invention;

[0036] Figure 8 Comparison of background expression activities of RACS against core promoters in Example 6 of the present invention;

[0037] Figure 9 Uncontrolled pyroptosis of cells during the regulation of GSDMD expression by the Tet-On system in Example 7 of the present invention NT ;

[0038] Figure 10 Pyroptosis of cells caused by background expression of GSDMD under the control of the core promoter in Example 7 of the present invention NT ;

[0039] Figure 11 Controllable and directional induction of pyroptosis of 293T cells by RACS regulation of GSDMD expression in Example 8 of the present invention NT ;

[0040] Figure 12 Evaluation of the effects of different inhibitors on artificial cell pyroptosis in Example 8 of the present invention

[0041] Figure 13 Induction of pyroptosis of 5637 cells by RACS regulation of GSDMD expression in Example 8 of the present invention NT ;

[0042] Figure 14 Spatially specific regulation of GSDMD expression by RACS induces pyroptosis of 5637 cells in Example 8 of the present invention NT ; Detailed implementation manners

[0043] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0045] Example 1 Design of RACS

[0046] RACS consists of the photosensitive protein PhyA, the nucleocytoplasmic shuttle protein FHY1, the split Cre part (including the N-terminal part CreN59 and the C-terminal part CreC60 of Cre), and the pigment molecule phycocyanobilin (PCB) responsible for light energy capture and transfer. PhyA and CreN59 are connected by a linker peptide (sequence: LEASPSNPGASN (SEQ ID NO.6)) to form a fusion protein. Similarly, FHY1 and CreC60 are also connected by the same linker peptide to form a fusion protein. The corresponding coding DNA sequences of PhyA, FHY1, CreN59, CreC60, and the linker peptide are obtained by gene synthesis.

[0047] The recombinant ligation between the DNA sequences corresponding to PhyA, CreN59, and the linker peptide is completed by the universal multi-fragment one-step rapid cloning kit of YEASEN. FHY1, CreC60, and the linker peptide are also obtained by the same method. The obtained recombinant product is cloned into the multiple cloning site of the mammalian cell expression vector pcDNA3.1 to obtain the expression vector. The expression of the recombinant product is obtained by plasmid transfection of mammalian cells. Under red light irradiation at a wavelength of 660 ± 20 nm, PCB absorbs light energy and binds to PhyA, triggering a conformational change of PhyA, which enables PhyA to bind to FHY1. At the same time, it causes CreN59 and CreC60 to approach spatially to obtain Cre recombinase activity. Finally, it enters the nucleus under the leadership of FHY1 and recombinantly edits the sequence of the target gene GOI in the nucleus (characterized by the presence of loxP sites flanking). The principle of RACS regulating the expression of the target gene is as Figure 1 shown.

[0048] GOI includes the reporter gene Luciferase, the red fluorescent protein mCherry, and the pyroptosis-inducing gene GSDMD NT .

[0049] The nucleotide sequences of each gene are shown in Table 1:

[0050] Table 1

[0051] Name Sequence PhyA SEQ ID NO.1 FHY1 SEQ ID NO.2 CreN59 SEQ ID NO.3 CreC60 SEQ ID NO.4 <![CDATA[GSDMD NT > SEQ ID NO.5

[0052] In this example, the open reading frame (ORF) of GOI is placed in reverse to avoid the basal expression of the target gene in the non-induced state. Under the light-induced state, the direction of the target gene ORF is reset after recombination to obtain the expression of the target gene.

[0053] Example 2 Screening and Testing of the Combination Modes of RACS

[0054] To obtain a red light-activated Cre recombinase system that takes into account both excellent rigor and tunability. First, the combination modes of PhyA, FHY1, CreN, and CreC were tested and screened.

[0055] Among them, the nucleotide sequence of CreN104 is shown in SEQ ID NO.7, and the nucleotide sequence of CreC106 is shown in SEQ ID NO.8.

[0056] As Figure 2 shown, a total of 16 combination modes were screened and tested. In view of the fact that the first combination takes into account both low background expression activity and high inducibility activity, the first combination mode PhyA-CreN59, FHY1-CreC60 was selected as the most suitable combination.

[0057] During the screening implementation process, the firefly luciferase gene Firefly Luciferase (Luc) was used as the reporter gene. Three plasmids encoding the reporter gene Luc and the corresponding regulatory plasmids such as PhyA-CreN59 and FHY1-Cre60 were transiently transfected into 293T cells with a growth density of 70% by the Lip3000 method. The 293T cells were seeded in a 24-well plate. After 24 h of transfection, 10 μM PCB was added and incubated for 30 min, and the cells were irradiated with red light at 660 ± 20 nm with an intensity of 1 mW / cm 2 for 30 s to induce the expression of the Luc gene. The Luc activity was evaluated according to the instructions of the luciferase reporter gene monitoring kit of Yeasen Company.

[0058] Example 3 Optimization Design of the Linker Peptide in RACS

[0059] After confirming the combination configuration of PhyA CreN59 and FHY1 CreC60, to further improve the performance of RACS, the linker peptide between PhyA and CreN59 was optimized and designed, and at the same time, the linker peptide between FHY1 and CreC60 was optimized and designed. A total of 10 linker peptides were screened and tested. The implementation method of the screening test plan was the same as that in Example 2.

[0060] The amino acid sequences of each linker peptide are as follows:[[]]

[0061] L1: ASGSGGGGDV (SEQ ID NO.9);

[0062] L2: SDSAGSAGSAGSGSG (SEQ ID NO.10);

[0063] L3: GGGGSGGGGSGGGGR (SEQ ID NO.11);

[0064] L4: LEASPSNPGASN (SEQ ID NO.6);

[0065] L5: LEASPSNPGA (SEQ ID NO.12);

[0066] L6: LEASPSNPGASNGS (SEQ ID NO.13);

[0067] L7: LEASPSNPGASNGSGT (SEQ ID NO.14);

[0068] L8: APTLADLAVDLAALRPLEHPNPPLQRAAEALL (SEQ ID NO.15);

[0069] L9: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO.16);

[0070] L10: EAAAKEAAAKEAAAK (SEQ ID NO.17).

[0071] The results are as Figure 3 shown. Since the L4 linker peptide takes into account both low background expression activity and high induction fold, the L4 linker peptide was finally used for the subsequent implementation of the present invention.

[0072] Example 4 Characterization of the in vitro regulation of the firefly luciferase-encoding gene expression by RACS

[0073] To characterize the ability of RACS to regulate the expression of target genes in vitro, the firefly luciferase gene Firefly Luciferase (Luc) was used as a reporter gene. Three plasmids encoding Luc, PhyA-CreN59, and FHY1-CreC60 were transiently transfected into 293T cells with a growth density of 70% by the Lip3000 method. The 293T cells were seeded in 24-well plates. After 24 h of transfection, 10 μM PCB was added and incubated for 30 min, and the cells were irradiated with red light at 660 ± 20 nm with an intensity of 1 mW / cm 2 for 30 s to induce the expression of the Luc gene. The cells were collected at 1, 2, 4, 8, 16, 24, and 48 h after induction to evaluate the Luc activity, and the Luc activity was evaluated according to the instructions of the luciferase reporter gene monitoring kit from Yeasen.

[0074] The detection results are as Figure 4As shown, it indicates that RACS can effectively induce the expression of Luc. An obvious Luc activity signal can be detected 8 h after light induction, and the maximum Luc activity can be detected 48 h after induction. 293T cells were collected 48 h after light induction for Western blot analysis. The expression of Luc (the N-terminus of Luc is tagged with Flag) was detected with an anti-Flag antibody. Figure 5 The detection results show that RACS can effectively regulate the protein production of the reporter gene Luc. The above experimental results show that the RACS system has the sensitivity of second-level response and high controllability, with extremely low background expression signals and the characteristic of more than 100-fold improvement in expression level.

[0075] Example 5 Characterize the spatial-specific regulation of mCherry expression by RACS

[0076] To characterize the spatial-specific regulation characteristics of the RACS system, the plasmid encoding the red fluorescent protein mCherry was co-transfected into 293T cells with the plasmids encoding PhyACreN59 and FHY1CreC60 by Lip3000 method. 293T cells were cultured in a 6-cm culture dish, and the bottom of the culture dish was covered with a light-shielding film. A part of the light-shielding film was hollowed out in the shape of the letters "CS" to allow light to pass through. 24 h after transfection, the cells were irradiated from the bottom with a light intensity of 50 μW / cm 2 for 3 min, and after 48 h of light induction, pictures were taken in the mosaic shooting mode under a confocal microscope. The imaging results show that the light-transmitting area clearly presents the letter "CS" pattern by red fluorescent cells, while no mCherry signal is seen in the non-light-transmitting area, indicating that RACS shows excellent spatial specificity and controllability when inducing the expression of mCherry ( Figure 6 ).

[0077] Example 6 Comparison of RACS with other regulatory systems to characterize the controllability of RACS

[0078] To further highlight the controllability advantage of RACS, the RACS system was compared with other existing regulatory systems. These include existing light-responsive Cre such as PACre responsive to blue light, RedCre responsive to red light, and FISC Cre recombination system responsive to far-red light. In addition, RACS was compared with the regulatory system dependent on the core promoter subclass. In the experiment comparing the light-controlled Cre, the plasmids encoding the corresponding system components ( Figure 7 left) were co-transiently transfected into 293T cells cultured in a 24-well plate by Lip3000 method. The cell density before transfection was 70%. 24 h after transfection, light induction treatment was carried out.

[0079] Among them, the light condition for RACS is 1 mW / cm 2 for 30 s, and the light condition for PACre is 1 mW / cm 2, 30 s, RedCre illumination condition 5 mW / cm 2 , 16 h (during which the light is on for 15 min and off for 15 min, and this cycle repeats). FISC illumination condition 5 mW / cm 2 , 16 h (during which the light is on for 15 min and off for 15 min, and this cycle repeats). The illumination conditions corresponding to each of the above light control systems are to ensure the optimal performance of each system. 48 h after illumination, the expression activity of Luc was detected according to the luciferase reporter gene detection method of YEASEN Company, and the differences within and between groups were compared. Figure 7 The comparison results on the right show that the induction fold of RedCre is 3.88, that of FISC is 6.00, that of PACre is 14.95, while that of RACS is 245.71, and the background expression activity of RACS is the lowest. It shows that RACS exhibits the lowest background expression activity and the highest induction fold when regulating Luc expression, highlighting the unique controllability advantage of RACS. In addition, compared with the RedCre and FISC systems, RACS shows the advantages of flexible operating conditions and sensitive response.

[0080] In the experiment comparing RACS with the regulatory system dependent on the core promoter, in addition to the transfected group of RACS, the coding plasmid of Luc controlled by the core promoter was transfected into 293T cells in the same way, and no illumination was performed to compare the background expression activity. 48 h after transfection, the cells were collected and the expression activity of Luc was detected according to the above method, Figure 8 The results show that the background expression activity of Luc regulated by the RACS system is significantly lower than that of the core promoter control group.

[0081] Example 7 characterizes the pyroptosis caused by the background expression of GSDMD under the control of the core promoter NT

[0082] During the test of the classical induction system Tet-On regulating GSDMD NT to induce pyroptosis, it was found that the 293T cells transfected with the Tet-GSDMD NT plasmid underwent uncontrolled death. Due to the background expression activity of the core promoter, in further experiments, the plasmid containing only the core promoter controlling GSDMD NT expression was transfected into 293T cells, and the same phenomenon of uncontrolled cell death occurred, indicating that the background expression activity of the core promoter would lead to the occurrence of uncontrolled pyroptosis mediated by GSDMD NT

[0083] In the experiment evaluating the induction of pyroptosis by Tet-On regulating GSDMD NT the Tet-On-GSDMD with zsGreen label​​NT The expression plasmid was transfected into 293T cells in a 12-well plate by the Lip3000 method. After 24 h of transfection, in situ staining was performed according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, and confocal microscopy imaging analysis was carried out immediately after staining. The microscopy imaging results showed that pyroptosis occurred in 293T cells transfected with the Tet-On-GSDMD NT plasmid ( Figure 9 ).

[0084] In the experiment evaluating the induction of pyroptosis by the core promoter miniCMV regulating GSDMD NT The miniCMV-GSDMD NT expression plasmid with GFP label was transfected into 293T cells in a 12-well plate by the Lip3000 method. After 24 h of transfection, in situ staining was performed according to the instructions of the AnnexinV-FITC / PI apoptosis detection kit, and confocal microscopy imaging analysis was carried out immediately after staining. Figure 10 The microscopy imaging results showed that pyroptosis occurred in 293T cells transfected with the miniCMV-GSDMD NT plasmid.

[0085] Example 8 characterized that RACS regulated GSDMD NT expression mediated controllable and directional pyroptosis of cells

[0086] In view of the spatio-temporal specific regulation characteristics and strict expression regulation characteristics of RACS, GSDMD NT was used as the target gene for the directional and controllable induction test of pyroptosis. In the 293T cell test, the plasmids encoding RACS and DIO-GSDMD NT were transiently transfected into 293T cells by the Lip3000 method. After 24 h of transfection, light-induced GSDMD NT expression was performed. After 18 h of light induction, in situ staining was performed with the AnnexinV-FITC / PI apoptosis detection reagent and combined with confocal microscopy observation to analyze the occurrence of pyroptosis.

[0087] Figure 11 The staining results showed that the occurrence of pyroptosis in 293T cells could be effectively induced after light irradiation. In contrast, no pyroptotic cells were found in the non-light-irradiated treatment group. Under natural conditions, 293T cells do not undergo pyroptosis due to the lack of Gasdermin protein expression, while the introduction of RACS and GSDMD NT can promote the occurrence of pyroptosis. In addition, the addition of 10 μM of the Caspase inhibitor zVAD 30 min before the induction of pyroptosis did not prevent the occurrence of pyroptosis, while the addition of 20 μM of the GSDMD NT targeted inhibitor SCR-1481B1 significantly inhibited pyroptosis of cells (Figure 12 ), indicating that this process is independent of the activity of the cell's own Caspase and does not involve other forms of cell death. The above experimental results indicate that the regulation of GSDMD by RACS NT expression can directionally and controllably induce pyroptosis in 293T cells.

[0088] To further characterize the ability of RACS-based regulation of GSDMD NT expression to directionally and controllably induce pyroptosis in cells, tests were conducted on the artificial induction of pyroptosis in bladder cancer cells 5637. To achieve the intracellular delivery of RACS and GSDMD NT in 5637 cells, an adenovirus integrated with RACS and GSDMD NT was first constructed, and the adenovirus packaging followed the pAd-Easy system method. The adenovirus carrying the RACS and GSDMD NT expression modules was used to infect 5637 cells at a multiplicity of infection (MOI) of 100. After 8 h of virus infection, the medium was replaced with fresh complete medium, and cells were induced to undergo pyroptosis by light irradiation 36 h after virus infection. After 12 h of light irradiation, AnnexinV-FITC / PI apoptosis detection reagent was added to the cell culture medium, and immunofluorescence analysis was performed after in situ staining according to the aforementioned method. The experimental results showed that the regulation of GSDMD by RACS NT could efficiently induce pyroptosis in 5637 cells ( Figure 13 ).

[0089] To further characterize the property of RACS spatially specific regulation of GSDMD NT expression to induce pyroptosis in cells. 5637 cells were seeded in a 6-cm culture dish, and the bottom of the culture dish was covered with a light-shielding film. A rectangular frame was cut out in the center of the light-shielding film to allow light to pass through. After 24 h of seeding 5637 cells, the adenovirus carrying RACS and GSDMD NT was used to infect 5637 cells at an MOI of 100. And 36 h after infection, cells were induced to undergo pyroptosis under the condition of light irradiation at 50 μW / cm 2 , for 3 min. After 12 h of light induction, 2 drops of SytoxGreen dead cell dye were added to the cell culture medium (performed according to the product instructions of Thermo Fisher Scientific), and the cell death was observed under a confocal microscope after incubation at room temperature for 5 min. The results showed that cell death occurred specifically in the area where light passed through, further characterizing the spatial specificity of RACS-regulated GSDMD NT expression to induce pyroptosis ( Figure 14 ).

[0090] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

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[0092]

[0093]

Claims

1. A system for controllably inducing tumor cell pyroptosis, characterized in that: Includes red light-activated Cre recombinase system and loxP-flanked GSDMD NT The expression vector of the gene sequence, the GSDMD NT The gene sequence is placed in the expression vector in a reverse orientation; The Cre recombinase system includes the following components: (1) an expression vector 1 comprising a photosensitive protein PhyA gene sequence, a CreN59 gene sequence, and a connecting peptide between the PhyA gene sequence and the CreN59 sequence, wherein the PhyA gene sequence is shown in SEQ ID NO.1, and the CreN59 gene sequence is shown in SEQ ID NO.3; (2) an expression vector 2 comprising a nucleocytoplasmic shuttling protein FHY1 gene sequence, a CreC60 gene sequence, and a connecting peptide between the FHY1 gene sequence and the CreC60 gene sequence, wherein the FHY1 gene sequence is shown in SEQ ID NO.2, and the CreC60 gene sequence is shown in SEQ ID NO.4; (3) Contains phycocyanin.

2. The system according to claim 1, characterized in that The amino acid sequence of the connecting peptide described in (1) and (2) is shown in SEQ ID NO.

6.

3. The system according to claim 1, characterized in that In the expression vector 1, the combination of the PhyA gene sequence, the CreN59 gene sequence and the connecting peptide is: the PhyA gene sequence, the connecting peptide and the CreN59 gene sequence; In the expression vector 2, the combination of the FHY1 gene sequence, the CreC60 gene sequence and the connecting peptide is: FHY1 gene sequence, connecting peptide and CreC60 gene sequence.

4. Use of the system according to any one of claims 1 to 3 in the preparation of a product that mediates cell pyroptosis.

5. The use according to claim 4, characterized in that: The application is specifically: 660±20nm wavelength red light irradiation is used to activate the Cre recombinase system and induce GSDMD NT Gene expression.

6. The use according to claim 5, characterized in that: The intensity of the red light irradiation is not less than 0.05 mW / cm 2 , lasting not less than 0.5 minutes.

7. The use according to claim 4, characterized in that: The products that mediate cell pyroptosis include but are not limited to anti-tumor drugs or cytotoxic agents.

8. Use of the system according to any one of claims 1 to 3 in the preparation of drugs for tumor prevention or treatment.

9. The use according to claim 8, characterized in that: The tumor is bladder cancer.

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