Engineering fibroblast as well as preparation method and application thereof

Abstract

The invention discloses an engineering fibroblast as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the engineering fibroblast provided by the invention, a proinflammatory factor IL-1 is neutralized by secreting IL-1RA, an inflammatory loop mediated by the proinflammatory factor IL-1 is broken, and a tumor immune microenvironment is remodeled, so that inhibition on immune effector cells (such as T cells and NK cells) is indirectly and effectively relieved, and favorable conditions are created for other immunotherapy methods or body autoimmune clearance; the problem that the immunosuppressive tumor microenvironment cannot be regulated and controlled well is solved. Meanwhile, the defects that the half-life period of the recombinant IL-1RA protein is short, frequent administration is needed, the local drug concentration of the focus is difficult to maintain and the like are overcome, stable and high-concentration delivery of the therapeutic protein is realized, and the pharmacokinetic bottleneck of the traditional protein drug is overcome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to engineered fibroblasts, their preparation methods, and applications. Background Technology

[0002] The IL-1 family plays a crucial role in promoting tumor growth within the tumor microenvironment (TME). IL-1 can be secreted by tumor cells, stromal cells, and immune cells. It promotes the expression of inflammation-related genes by activating signaling pathways such as NF-κB, stimulating the proliferation of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the TME. These cells can suppress anti-tumor immune responses. Furthermore, it can promote T cell exhaustion by upregulating PD-L1 expression and, synergistically with vascular endothelial growth factor (VEGF), promote tumor angiogenesis, thus favoring tumor growth and metastasis.

[0003] Interleukin-1 receptor antagonists (IL-1RAs) are an important member of the interleukin-1 (IL-1) family, composed of... IL- 1RN Encoded in the gene, IL-1RA plays a crucial role in regulating inflammatory responses and immune responses. IL-1RA binds to the IL-1 receptor IL-1R1 without activating the receptor, thereby preventing pro-inflammatory cytokines such as IL-1α and IL-1β from binding to their receptors and inhibiting the activation of downstream inflammatory signaling pathways, thus exerting an anti-inflammatory effect. IL-1RA also has potential applications in gene therapy. In some animal models and clinical trials, introducing the IL-1RA gene into joints via gene transfer technology has effectively inhibited the development of osteoarthritis (OA), reducing cartilage damage, osteophyte formation, and inflammatory responses. This gene therapy strategy utilizes the anti-inflammatory properties of IL-1RA, providing new ideas and methods for treating chronic inflammatory diseases such as osteoarthritis. Recombinant IL-1RA has been approved for RA treatment and has shown potential therapeutic value in other inflammatory diseases and cardiovascular diseases.

[0004] The currently marketed drug is a recombinant protein drug produced using recombinant DNA technology, which is structurally similar to the endogenous interleukin-1 receptor antagonist (IL-1RA). The production of this protein is based on recombinant DNA technology. First, the gene encoding human IL-1RA is inserted into a plasmid vector to construct a recombinant expression vector, which is then transformed into E. coli host bacteria. Through large-scale fermentation culture, the engineered bacteria can efficiently express the target protein. Since the protein often exists in the form of inactive inclusion bodies in the bacteria, it must undergo cell disruption, denaturant dissolution, and a delicate refolding process to correctly fold into a biologically active three-dimensional structure. Then, it is highly purified by multi-step chromatographic techniques such as ion exchange, hydrophobic interaction, and molecular sieving to remove impurities such as host proteins, nucleic acids, and endotoxins. Finally, the high-purity protein is placed in a buffer containing stabilizers to prepare a sterile preparation. Although this technology can achieve large-scale production of proteins, its refolding step is inefficient and easily affects the yield. Furthermore, the final product has inherent defects due to the lack of post-translational modifications (such as glycosylation), short half-life, and the risk of immunogenicity. Furthermore, this recombinant protein exhibits significant pharmacokinetic defects: due to its small molecular weight and lack of structures that prolong half-life, such as the Fc fragment, it is cleared from the human body extremely rapidly, with a terminal half-life of only about 4 to 6 hours. This necessitates a high-frequency dosing regimen of once-daily subcutaneous injection, undoubtedly imposing a heavy medication burden on patients with chronic diseases requiring long-term treatment and affecting treatment adherence. Additionally, systemic administration may result in insufficient local drug concentration at the lesion site, and there is a potential immunogenic risk of inducing drug-resistant antibodies.

[0005] Human dermal fibroblasts, harvested and cryopreserved after large-scale in vitro culture, are subsequently used either as cell suspensions for local injection or implanted into lesion areas in combination with simple scaffold materials. They are applied to lumbar disc degeneration, repair of chronic, difficult-to-heal wounds, and skin regeneration. They function by naturally secreting extracellular matrix and various bioactive factors (such as growth factors and cytokines) to replenish damaged tissue, promote repair, or inhibit inflammation. However, unmodified fibroblasts have limited secretory function, secreting very little IL-1RA, making it difficult to enhance and precisely regulate the pathological processes of complex diseases. Furthermore, their role in the tumor microenvironment has not been effectively studied and applied. Many existing immunotherapies (such as checkpoint inhibitors) act directly on immune cells, but their effectiveness is limited in the strongly immunosuppressive microenvironment dominated by factors such as IL-1. Summary of the Invention

[0006] 1. The problem to be solved Firstly, this invention addresses the shortcomings of existing recombinant IL-1RA proteins, such as short half-life, frequent administration, and difficulty in maintaining local drug concentration at the lesion site. By modifying human fibroblasts into engineered fibroblasts that can sustainably and autonomously secrete IL-1RA, this invention achieves stable and high-concentration delivery of therapeutic proteins, overcoming the pharmacokinetic bottleneck of traditional protein drugs.

[0007] Secondly, the engineered fibroblasts provided by this invention neutralize the pro-inflammatory factor IL-1 by secreting IL-1RA, thereby breaking the inflammatory circuit mediated by IL-1, reshaping the tumor immune microenvironment, and thus indirectly and effectively relieving the inhibition of immune effector cells (such as T cells and NK cells), creating favorable conditions for other immunotherapies or the body's own immune clearance, and solving the problem of ineffective regulation of the immunosuppressive tumor microenvironment.

[0008] 2. Technical Solution In a first aspect, the present invention provides an engineered fibroblast that overexpresses an interleukin-1 receptor antagonist (IL-1RA).

[0009] Furthermore, the aforementioned fibroblasts contain exogenous... IL-1RN Gene, IL-1RN The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] Furthermore, the aforementioned fibroblasts include any one or more of the following: tissue-isolated fibroblasts, stem cell-differentiated fibroblasts, and fibroblasts transdifferentiated from other cells.

[0011] Furthermore, the fibroblasts isolated from the aforementioned tissues include fibroblasts isolated from skin tissue.

[0012] Furthermore, the aforementioned fibroblasts are dermal fibroblasts, such as those from the foreskin. Human fibroblasts derived from the foreskin can regulate immune responses by secreting TGFβ, inhibiting PBMC proliferation, and suppressing macrophage polarization towards a pro-inflammatory phenotype, thereby suppressing the inflammatory response and exerting an inhibitory effect on the local immune microenvironment. By introducing the gene for secretory IL-1RA into modified human fibroblasts using lentiviruses, IL-1RA-secreting human fibroblasts can be obtained, enhancing the immunosuppressive effect on the local tumor microenvironment and enabling the immune system to capture immune-escaped tumor cells.

[0013] Furthermore, the aforementioned stem cell-differentiated fibroblasts include fibroblasts differentiated from induced pluripotent stem cells (iPSCs).

[0014] Secondly, the present invention provides a method for preparing the above-mentioned engineered fibroblasts, the method comprising: Will IL-1RN Genes are introduced into fibroblasts to obtain cells containing exogenous genes. IL-1RN Gene-engineered fibroblasts.

[0015] Furthermore, the aforementioned importation includes lentiviral infection.

[0016] Furthermore, the aforementioned lentiviral infections include: suspension infections.

[0017] Furthermore, the above-mentioned suspension infection includes: preparing fibroblasts into a suspension, adding viral infection solution, centrifuging at low speed, adding fibroblast culture medium containing histone deacetylase inhibitor and ROCK signaling pathway inhibitor, and co-culturing to obtain engineered fibroblasts.

[0018] Furthermore, the aforementioned histone deacetylase inhibitor is Entinostat.

[0019] Furthermore, the aforementioned ROCK signaling pathway inhibitor is Y-27632.

[0020] Thirdly, the present invention also provides a biological agent, which is the supernatant of the above-mentioned engineered fibroblasts. As a further explanation of the present invention, the supernatant contains IL-1RA.

[0021] Fourthly, the present invention also provides the use of the engineered fibroblasts of the first aspect or the biological agents of the third aspect in the preparation of drugs for treating tumors.

[0022] Furthermore, the dosage form of the aforementioned tumor treatment drugs is an injection.

[0023] Fifthly, the present invention also provides a pharmaceutical composition comprising the engineered fibroblasts of the first aspect or the biological agent of the third aspect, and pharmaceutical excipients.

[0024] Furthermore, the dosage form of the above-mentioned pharmaceutical composition is an injection.

[0025] 3. Technical Effects Compared with the prior art, the advantages of this invention are as follows: (1) The engineered fibroblasts, their preparation method, and their applications provided by this invention, wherein the engineered fibroblasts are IL-1RA overexpressing, when used to prepare anti-tumor drugs, especially when prepared as injectable preparations for local injection, can neutralize the key pro-inflammatory and immunosuppressive factor IL-1 in the tumor microenvironment by secreting IL-1RA, thereby breaking the IL-1-mediated inflammatory circuit, effectively relieving the inhibition of immune effector cells such as T cells and NK cells, reshaping the tumor immune microenvironment (TME), and enhancing the immunotherapy effect. At the same time, compared with unmodified human fibroblasts, they have increased immunomodulatory function, breaking through the limitations of their natural secretion spectrum, and achieving enhanced therapeutic function.

[0026] (2) The engineered fibroblasts and their preparation method and application provided by the present invention can continuously and autonomously secrete IL-1RA in vivo, realizing continuous and efficient local drug delivery of IL-1RA, and solving the pharmacokinetic bottlenecks of recombinant protein therapy such as short half-life, frequent administration, and insufficient target concentration at systemic administration sites.

[0027] (3) The engineered fibroblasts, their preparation method, and their applications provided by this invention use human fibroblasts (especially those derived from the foreskin) as the chassis cells for gene modification, which have the advantages of being easy to obtain, having strong amplification capabilities, and possessing natural immune regulatory characteristics. At the same time, the use of lentiviral infection, especially the innovative "suspension infection" process, significantly improves the modification efficiency and efficiently integrates the IL-1RA gene into the genome of human fibroblasts. Attached Figure Description

[0028] Figure 1 This is a map of lentiviral expression vectors.

[0029] Figure 2 This is the result of an ELISA test for IL-1RA secretion.

[0030] Figure 3 This is the result of a Western blot (WB) test for IL-1RA secretion.

[0031] Figure 4 This is the result of flow cytometry analysis of IL-1RA secretion.

[0032] Figure 5 This is the result of the transwell test.

[0033] Figure 6 This is the result of the transwell test count.

[0034] Figure 7 This is a picture of a subcutaneous tumor.

[0035] Figure 8 This is a graph showing the growth of tumor volume.

[0036] Figure 9 This is a tumor weight graph.

[0037] Figure 10 It is a trend chart of animal weight. Detailed Implementation

[0038] The present application will be further described below with reference to specific embodiments.

[0039] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0040] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0042] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0043] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0044] As used herein, an antagonist is a class of drugs that can prevent or weaken the binding of other drugs or endogenous substances (such as hormones, neurotransmitters, etc.) to receptors. The mechanism of action of antagonists is primarily based on receptor competition. Receptors are specialized proteins on the cell surface that can bind to ligands (such as hormones, neurotransmitters, drugs, etc.) and elicit a series of physiological responses. Antagonists inhibit the pharmacological effects of agonists by occupying receptor sites and preventing the binding of agonists to receptors. In this invention, IL-1RA binds to IL-1R, preventing the binding of IL-1 to IL-1R.

[0045] In this invention, the source of fibroblasts is not limited. As an example, this invention provides an isolation of dermal fibroblasts. Foreskin tissue was collected from boys under 15 years of age and transferred to a biosafety cabinet for tissue digestion and separation. The specific process was as follows: (a) The tissue was washed three times with PBS buffer, disinfected with 10 mL of povidone-iodine for 3 min, then disinfected with 10 mL of 75% ethanol for 3 min, and washed three times with PBS; (b) The tissue was minced to about 1 mm, and 6 mL of type I collagenase (brand: Gibco, catalog number: 17100017) with a final concentration of 0.2 U / mL was prepared. The tissue was transferred to the type I collagenase solution and placed in a shaker at 70 r / min for 2 h at 37°C; (c) The digested tissue was transferred to a biosafety cabinet, and 12 mL of PBS buffer was added to terminate the digestion; the tissue was filtered using a 70 μm cell filter, the filtrate was collected, and centrifuged twice at 300 g for 5 min; (d) The centrifuged precipitate was collected and counted, and DMEM + 10% FBS complete medium (DMEM complete medium) was used to digest the precipitate at a rate of (1-2) × 10⁻⁶. 4 pcs / cm 2 Primary fibroblasts were cultured at high density.

[0046] Example 1 This embodiment provides engineered fibroblasts and their preparation method.

[0047] In this embodiment, the engineered fibroblasts are dermal fibroblasts that overexpress interleukin-1 receptor antagonist (IL-1RA).

[0048] In this embodiment, the engineered fibroblasts are infected using lentivirus infection, which transmits the IL-1RA-encoding protein. IL-1RN Genes were introduced into fibroblasts derived from foreskin to obtain engineered fibroblasts.

[0049] Specifically, the steps include the following: (1) Construction of lentiviral expression vector like Figure 1 As shown, IL-1RN The gene was inserted into a lentiviral expression vector, and the virus was prepared to construct a lentivirus expressing IL-1RA. This process was outsourced to a supplier (Yunzhou Biotechnology Co., Ltd., brand: VectorBuilder). IL-1RN The nucleotide sequence of the gene encodes the secretory peptide +IL-1RA: 5'--3' (SEQ ID NO.1).

[0050] (2) lentiviral expression vector introduced into fibroblasts 1) Take frozen human fibroblasts (from foreskin tissue, passage P3), thaw them rapidly in a 37°C water bath, and then feed them at a rate of 15,000 cells / cm³. 2 Cells were seeded at a density in culture dishes and cultured at 37°C and 5% CO2 for approximately 4 days. The complete culture medium for human fibroblasts was high-glucose DMEM supplemented with 10% fetal bovine serum (FBS). The high-glucose DMEM medium was Gibco. TMBASICDMEM (High Glucose, Pyruvate), purchased from Thermo Fisher Scientific, catalog number C11995500BT; fetal bovine serum was Gibco. TM Fetal bovine serum, purchased from Thermo Fisher Scientific, catalog number 10091148. Viral infection can be performed once cells have grown to a suitable density.

[0051] 2) Quickly remove the lentivirus (provided by Yunzhou Biotechnology Co., Ltd.) from the -80°C freezer and immediately place it on ice to thaw. Dilute the lentivirus stock solution to MOI=5 with complete culture medium (high glucose DMEM + 10% fetal bovine serum) and add polyethylene infection reagent (Sigma, TR-1003-G) to a final concentration of 5-8 μg / mL.

[0052] 3) The human fibroblasts cultured in step 1) were treated with trypsin-EDTA (Gibco) TM ,25200072) digestion, PBS buffer (Gibco) TM After terminating digestion (10010023), centrifuge at 300×g for 5 minutes; remove the supernatant and resuspend the cells in complete culture medium, preparing a cell suspension at 300,000 cells / mL; mix the cell suspension with the virus infection solution from step 2) and centrifuge at low speed (300×g, centrifuge for 10 minutes) to promote virus contact with cells and infect cells; after centrifugation, let stand for 20 minutes, then remove the supernatant containing the virus, add complete human fibroblast culture medium containing a combination of histone deacetylase inhibitor 5 μM Entinostat (Selleck, S1053) and ROCK signaling pathway inhibitor 1 μM Y-27632 dihydrochloride (MedChemExpress, HY-10583), co-culture for 24 hours, remove the supernatant, replace with complete human fibroblast culture medium and continue culturing until the cell confluence reaches 80%, then proceed to the next step of cell passage or sample collection and detection.

[0053] The control group was infected with the control virus using the same method. The control virus did not contain the target gene shown in SEQ ID NO.1.

[0054] For the adherent lentiviral infection group, using the same lentivirus, the human fibroblasts cultured in step 1) were digested with trypsin-EDTA (Gibco™, 25200072), digestion was terminated with 0.01M PBS (Gibco™, 10010023), and centrifuged at 300×g for 5 minutes. After removing the supernatant, the cells were resuspended in complete culture medium and seeded at 300,000 cells / well in 6-well cell culture plates. After overnight culture to allow cell adhesion, the viral infection solution from step 2) was added, and the cells were co-cultured for 12 hours. After removing the supernatant, the culture medium was replaced with complete human fibroblast culture medium, and the cells were cultured until the confluence reached 80%. At this point, the cells could be passaged or sampled for testing.

[0055] (3) Detection of IL-1RA expression in engineered fibroblasts The expression of IL-1RA was compared with that of the control group and the traditional adherent lentiviral infection method. The expression of IL-1RA in cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), the expression of IL-1RA in cell lysate was detected by Western blotting, and the expression of IL-1RA on cell surface was detected by flow cytometry.

[0056] (a) ELISA detection During ELISA testing, the cell samples to be tested were seeded into cell culture plates at the same density, and high-glucose DMEM medium (without serum) was added. After culturing for 24 h, the cell culture supernatant was aspirated, and the cells were digested with trypsin-EDTA and counted. The cell number was recorded, and the amount of IL-1RA that 1 million cells could secrete per 24 hours (pg / million cells / 24h) was calculated based on the ELISA test results.

[0057] ELISA assays were performed using the Human IL-1RA ELISA kit (brand: Absin, catalog number: abs551328). Following the kit instructions, 100 μL of sample or standards of different concentrations were added to each well, and 100 μL of universal diluent was added to each blank well. The plate was sealed and incubated at 37°C for 60 minutes. Next, the plate was removed, the liquid was discarded, and without washing, 100 μL of biotinylated antibody working solution was added directly to each well. The plate was sealed and incubated at 37°C for 60 minutes. Afterward, the liquid was discarded, and 300 μL of 1× washing buffer was added to each well. The plate was allowed to stand for 1 minute, the washing buffer was discarded, and the plate was patted dry on absorbent paper. This washing process was repeated 3 times. Then, 100 μL of enzyme conjugate working solution was added to each well, the plate was sealed, and incubated at 37°C for 30 minutes. The liquid was discarded again, and the plate was washed 5 times according to the washing method in step 4. Next, add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes. Finally, remove the microplate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm. Calculate the secretion of IL-1RA based on the standard curve.

[0058] The results are as follows Figure 2 As shown, con-lenti is the control group, lenti (adherence) is the adherent lentivirus group, and lenti (suspension) is the suspension infection group of this invention. It can be seen that the control group has almost no IL-1RA protein, while the IL-1RA protein secretion in the suspension infection group is significantly higher than that in the traditional adherent lentivirus group.

[0059] (b) WB detection Cell samples were collected and lysed with RIPA lysis buffer (Beyotime Biotechnology Co., Ltd., P0013B) to extract total protein. The protein concentration in the lysis buffer was determined using a protein quantification kit (BCA method) (Beyotime, P0012). Protein standards were prepared according to the kit instructions; 1.2 mL of standard preparation solution was added to 30 mg of protein standard (BSA) to prepare a 25 mg / mL protein standard solution, which was then diluted to 0.5 mg / mL for the experiment. BCA working solution was then prepared by mixing BCA reagents A and B in a 50:1 ratio and allowing it to stabilize at room temperature for 24 hours. To determine protein concentration, the standards and samples were added to a 96-well plate, followed by the BCA working solution. The plate was incubated at 37°C for 20-30 minutes, and the absorbance at wavelength A562 was measured using a microplate reader. The sample protein concentration was calculated based on the standard curve, following the specific procedures outlined in the kit instructions.

[0060] After determining the protein concentration, the concentrations of different samples were adjusted to be the same. Then, the lysis buffer samples were mixed with 5× loading buffer (Beyotime, P0015) and boiled for 10 minutes for denaturation. SDS-PAGE electrophoresis was performed using a precast gel (Beyotime, P0052A) with electrophoresis buffer P0014D. The electrophoresis program was: stacking gel 80 V, separating gel 120 V. Protein samples of different sizes were separated by SDS-PAGE gel electrophoresis using Bio-Rad Mini-PROTEAN gels. ® Electrophoresis can be performed on other equivalent products.

[0061] After electrophoresis, the protein was transferred from the gel to a PVDF membrane using a wet transfer method at a constant current of 300 mA for 60 minutes (ice bath) in transfer buffer (Beyotime, P0021A). After transfer, non-specific binding sites on the membrane were blocked with blocking buffer (TBST solution containing 5% skim milk powder (Beyotime, ST673)) at room temperature for 1 hour. Then, the membrane was incubated overnight at 4°C with the corresponding anti-IL-1RA primary antibody (Abcam, ab303490). After incubation, the membrane was washed with wash buffer (TBST) (Beyotime, ST673) to remove unbound primary antibody. Then, the membrane was incubated with the corresponding secondary antibody-HRP-labeled protein (Proteintech, SA00001-2) to detect IL-1RA protein bound by the primary antibody. After washing again, the membrane was treated with chemiluminescent substrate (ECL) (Beyotime, P0018S) to induce luminescence. Finally, the signal was captured using an imaging system (BioRad's ChemiDoc™ or other equivalent products), and the expression level of IL-1RA protein was quantitatively analyzed using image analysis software.

[0062] The results are as follows Figure 3 As shown, the IL-1RA content in the suspended infection group of the present invention was significantly higher than that in the traditional adherent lentivirus infection group.

[0063] (c) Flow cytometry detection First, take the cell suspension, wash the cells twice with PBS, centrifuge at 200×g for 5 minutes, and discard the supernatant. Resuspend the cells in 1 mL of PBS. Then, perform antibody incubation by adding the fluorescently labeled antibody (anti-human IL-1RA, Abcam, ab317288) to the cell suspension in the centrifuge tube, mixing well, and incubating at room temperature in the dark for 30 minutes. Add 1 mL of buffer per tube, centrifuge at 200×g for 5 minutes, discard the supernatant, resuspend the cell pellet in 200 μL of buffer, incubate at 4℃ in the dark, and analyze within 30 minutes. Unstained cell controls and cells stained with the target antibody are analyzed separately. Parameters are set using the control parameters, and sample data are collected using these parameters, collecting 10,000 effective cells.

[0064] The results are as follows Figure 4 As shown, the proportion of IL-1RA positive cells in the suspension infection group of this invention was the highest at 99.4%, the positive rate in the traditional adherent infection group was 66.0%, and the expression level in the control group was extremely low.

[0065] Example 2 This embodiment provides a study on the effect of engineered fibroblasts on tumor cell migration.

[0066] Lung cancer cell line A549 (Chinese Academy of Sciences Type Culture Collection Committee, catalog number: THu 150) and prostate cancer cell line 22RV1 (American Center for Type Culture Collection (ATCC), CRL-2505) were cultured separately in RPMI 1640 (Gibco, 11875093) + 10% FBS (volume ratio). When the cell confluence reached 90%, the cells were digested with trypsin and resuspended to prepare single-cell suspensions, which were then counted. A549 and 22RV1 cells were seeded at 100,000 cells per well in the upper chamber of a Transwell (6-well plate) with serum-free medium (RPMI 1640 (Gibco, 11875093)). 200,000 M2 macrophages were seeded in the lower chamber. M2 macrophages can promote local inflammatory responses and tumor cell migration. Lower chamber culture medium: The blank group (NC) used complete culture medium (RPMI 1640 containing 10% fetal bovine serum), the control group (IL-1RA) used complete culture medium supplemented with 50 ng / mL recombinant human IL-1RA protein (Proteintech, Ag1277), and the experimental group (Lenti-IL-1RA) used the supernatant of genetically engineered fibroblasts (cells prepared by introducing the lentiviral expression vector into fibroblasts in step (2)) after 24 hours of culture (the supernatant collected in the ELISA detection step, after detecting the IL-1RA content in the supernatant with an ELISA kit, and then diluted with complete culture medium to the same concentration as the control group). The cells were incubated statically in a 37℃, 5% CO2 incubator for 72 hours. Then remove the upper chamber, carefully wipe away the cells in the upper layer of the upper chamber with a cotton swab, gently rinse with PBS, fix the upper chamber in 4% paraformaldehyde (Servicebio, G1101) for 30 minutes, discard the fixative, rinse twice with PBS, add an appropriate amount of crystal violet staining solution (Sigma-Aldrich, C0775) and stain for 20-30 minutes. Finally, slowly wash away excess staining solution with PBS and take pictures under a microscope.

[0067] The results are as follows Figure 5 and Figure 6As shown, the engineered cell line used in this invention can significantly inhibit cell migration and is significantly superior to the recombinant IL-1RA proteome.

[0068] Example 3 This embodiment provides a study on the role of engineered fibroblasts in tumor suppression in vivo.

[0069] Lung cancer cell line A549 was cultured in culture medium. When the cell confluence reached 90%, the cells were digested with trypsin and resuspended to prepare single-cell suspensions, which were then counted. The cell density was adjusted to 5 million cells / mL with injectable saline, with 500,000 cells per 100 μL suspension. The cell suspension was aspirated using a 1 mL sterile syringe.

[0070] Anesthetizing immunodeficient mice (BALB / c nude) (Beijing Vital River Laboratory Animal Technology Co., Ltd.): Using the inhaled anesthetic isoflurane, mice were placed in an induction box, and a gas mixture containing 3%-4% (v / v) isoflurane was introduced. After approximately 1-2 minutes, once the mice were unconscious and their breathing was stable, they were removed. The skin on one side of the mouse's back was disinfected with an alcohol swab at a predetermined location. The skin was pinched open with one hand, and the needle was inserted subcutaneously parallel to the skin, slowly injecting 100 μL of cell suspension. A distinct subcutaneous blister was observed. The needle was slowly withdrawn, and the injection site was gently pressed for a moment to prevent leakage of the suspension. The inoculated mice were placed on a 37°C warm pad or clean bedding and closely observed until they were fully awake (usually 2-5 minutes), then returned to their cages for normal rearing. On the second day after inoculation, at the same location where tumor cells were injected subcutaneously, the test group received a subcutaneous injection of 100,000 engineered fibroblasts constructed in this invention per 100 μL, the control group received a subcutaneous injection of 100 μL of physiological saline, and the IL-1RA control group (IL-1RA group) received a subcutaneous injection of 700 pg / 100 μL of recombinant human IL-1RA protein (Proteintech, Ag1277).

[0071] Starting from day 7 post-inoculation, the length (a) and width (b) of the tumor were measured every 5 days using calipers, and the tumor volume was calculated until an animal reached the experimental endpoint (≤1000 mm). 3 Ultimately, the animals were sacrificed using CO2 inhalation, and the tumors were surgically removed for weighing and photographing.

[0072] Figure 7 The image shows a subcutaneous tumor. As can be seen from the image, the tumor volume in the experimental group (test substance group) injected with engineered fibroblasts was significantly smaller than that in the control group (saline group) and the group injected with only recombinant IL-1RA protein (IL-IRA group).

[0073] Figure 8The tumor volume growth graph shows that the tumor volume growth rate in the engineered fibroblast group was significantly lower than that in the control group and the IL-IRA group.

[0074] Figure 9 The tumor weight graph shows that the tumor weight in the engineered fibroblast group was significantly lower than that in the control group and the IL-IRA group.

[0075] Figure 10 The figure shows the trend of animal body weight. The body weight of mice in each group remained stable during the experiment, with no significant differences.

[0076] Animal experiments have demonstrated that engineered fibroblasts significantly inhibit tumor growth in vivo. Compared with the control group and the group injected with recombinant IL-1RA protein only, engineered fibroblasts significantly reduced tumor volume and weight, and this inhibitory effect was persistent and stable. These results confirm the effectiveness and safety of engineered fibroblasts as a novel tumor treatment strategy.

Claims

1. An engineered fibroblast, characterized in that, The fibroblast overexpresses IL-1RA.

2. The fibroblast cells according to claim 1, characterized in that, The fibroblast contains an exogenous IL-1RN gene, the nucleotide sequence of which is shown as SEQ ID NO.

1. IL-1RN gene, the nucleotide sequence of which is shown as SEQ ID NO.

1.

3. The fibroblast cells according to claim 1 or 2, characterized in that, The fibroblast comprises any one or more of a tissue-isolated fibroblast, a stem cell-differentiated fibroblast, and a cell-transdifferentiated fibroblast.

4. The method for preparing fibroblasts according to any one of claims 1 to 3, characterized in that, The method comprises: IL-1RN Gene introduction into fibroblasts to obtain engineered fibroblasts containing exogenous IL-1RN The nucleotide sequence of the gene is shown as SEQ ID NO.

1. IL-1RN The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

5. The preparation method according to claim 4, characterized in that, The introducing comprises a lentivirus infection.

6. The production method according to claim 5, characterized by, The lentivirus infection comprises a suspension infection, which comprises preparing the fibroblast into a suspension, adding a virus infection solution, adding a fibroblast culture medium containing a histone deacetylase inhibitor and a ROCK signaling pathway inhibitor after low-speed centrifugation, and obtaining the engineered fibroblast after co-culturing.

7. A biological agent, characterized in that, The biological agent is a supernatant of the engineered fibroblast according to any one of claims 1-3.

8. Use of the fibroblast according to any one of claims 1-3 or the biological agent according to claim 7 in the preparation of a tumor treatment drug.

9. Use according to claim 7, characterized in that, The tumor treatment drug is in the form of an injection.

10. A pharmaceutical composition, characterized by, The tumor treatment drug comprises the fibroblast according to any one of claims 1-3 or the biological agent according to claim 7, and a pharmaceutically acceptable excipient.

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