Dual genetically engineered mesenchymal stem cells and uses thereof

Abstract

The application discloses double-gene engineered mesenchymal stem cells and application thereof, relates to the technical field of biological medicine. The double-gene engineered mesenchymal stem cells provided by the application co-express CXCR4 and TNFSF14 proteins, exhibit enhanced tumor tropism and immune-mediated anti-tumor activity. Specifically, based on the principle that CXCR4 can enhance the active homing ability of mesenchymal stem cells to gastric cancer lesions expressing its ligand SDF-1 (SDF-1 / CXCL12), and TNFSF14 (LIGHT) can interact with HVEM and LTbetaR receptors to promote the activation of T cells and NK cells, the mesenchymal stem cells co-expressing CXCR4 and LIGHT overcome the defects of insufficient targeting of natural MSCs and weak immune activation ability, and have excellent anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to dual-gene engineered mesenchymal stem cells and their applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) are considered ideal carriers for targeted cancer therapy due to their excellent tumor homing properties, low immunogenicity, and ease of in vitro expansion and genetic modification. MSCs derived from tissues such as bone marrow, adipose tissue, and umbilical cord possess the ability to migrate into the tumor microenvironment (TME) and deliver therapeutic agents. Human umbilical cord-derived MSCs (HUC-MSCs) are particularly attractive due to their non-invasive origin, higher proliferative capacity, and potent immunomodulatory functions. Some preclinical studies have demonstrated the efficacy of MSCs in delivering pro-apoptotic genes, cytokines, and oncolytic viruses directly to the tumor site. However, their clinical translation remains limited by insufficient tumor targeting, the risk of promoting tumor growth under certain conditions, and insufficient anti-tumor immune stimulation. These limitations underscore the need for genetic enhancement of MSCs to fully realize their potential in cancer treatment.

[0003] A key approach to improvement is to enhance the tumor homing ability of MSCs through overexpression of specific chemokine receptors. Among these, CXC chemokine receptor type 4 (CXCR4) has been extensively studied for its role in the SDF-1 / CXCR4 axis, which plays a crucial role in mediating cell migration and retention within the tumor microenvironment. Many solid tumors, including gastric cancer, exhibit elevated levels of stromal cell-derived factor-1 (SDF-1), which attracts CXCR4-expressing cells to the tumor site. Overexpression of CXCR4 in engineered HUC-MSCs has shown promise in improving their homing efficiency and persistence at the tumor site, thereby increasing the therapeutic load delivered to malignant tissues.

[0004] In addition to CXCR4's enhanced localization ability, TNFSF14, also known as LIGHT (homogeneous with lymphotoxin and exhibiting inducible expression), functions as a potent immunomodulatory molecule. By competing with HSV glycoprotein D for the receptor HVEM expressed on T lymphocytes, LIGHT effectively triggers anti-tumor immune responses. A member of the TNF superfamily, LIGHT binds to two key receptors, HVEM and LTβR, thereby stimulating T cell activation, enhancing dendritic cell function, and promoting the disruption of the tumor vascular system. It has also been shown to remodel the immunosuppressive tumor microenvironment and increase the infiltration of cytotoxic T lymphocytes. When expressed by MSCs, LIGHT has the potential to transform these otherwise immunosuppressive cells into immune activators within the tumor niche. Therefore, LIGHT represents a highly attractive therapeutic gene for use in engineered MSC-based cancer therapies.

[0005] Although existing studies have explored the roles of CXCR4 or LIGHT in cancer treatment separately, research on integrating both into MSCs is still lacking. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a mesenchymal stem cell co-expressing CXCR4 and TNFSF14 and its application in the preparation of a drug for treating gastric cancer.

[0007] To address the above problems, the present invention proposes the following technical solution:

[0008] This invention provides dual-gene engineered mesenchymal stem cells that co-express CXCR4 and TNFSF14 proteins.

[0009] Furthermore, the mesenchymal stem cells include mesenchymal stem cells derived from human umbilical cord.

[0010] Furthermore, the CXCR4 and TNFSF14 protein genes are integrated into the genome of the mesenchymal stem cells via lentiviral vector transduction.

[0011] Furthermore, the lentiviral vector contains the EF1α promoter.

[0012] Furthermore, the dual-gene engineered mesenchymal stem cells exhibit migration inhibition and apoptosis induction effects on gastric cancer cells.

[0013] The present invention also provides a pharmaceutical composition comprising the aforementioned dual-gene-engineered mesenchymal stem cells and a pharmaceutically acceptable carrier.

[0014] The present invention also provides a method for preparing the aforementioned dual-gene engineered mesenchymal stem cells, comprising the following steps:

[0015] Provide mesenchymal stem cells;

[0016] The mesenchymal stem cells were transduced using a lentiviral vector carrying the CXCR4 and TNFSF14 protein genes.

[0017] Engineered mesenchymal stem cells stably co-expressing CXCR4 and TNFSF14 proteins were obtained through screening.

[0018] Furthermore, the multiplicity of infection of the lentiviral vector is 5-20.

[0019] The present invention also provides the use of the aforementioned dual-gene engineered mesenchymal stem cells in the preparation of drugs for inhibiting gastric cancer cell migration and / or inducing gastric cancer cell apoptosis.

[0020] The present invention also provides the use of the aforementioned dual-gene engineered mesenchymal stem cells in the preparation of drugs for the treatment of gastric cancer.

[0021] Compared with the prior art, the technical effects achieved by the present invention include:

[0022] The present invention provides dual-gene engineered mesenchymal stem cells, which co-express CXCR4 and TNFSF14 proteins, exhibiting enhanced tumor tropism and immune-mediated antitumor activity. Specifically, based on the principles that CXCR4 can enhance the active homing ability of mesenchymal stem cells to gastric cancer lesions expressing its ligand SDF-1 (SDF-1 / CXCL12), and that TNFSF14 (LIGHT) can interact with HVEM and LTβR receptors to promote the activation of T cells and NK cells, mesenchymal stem cells co-expressing CXCR4 and LIGHT overcome the defects of insufficient targeting and weak immune activation capacity of natural MSCs, and have excellent antitumor effects.

[0023] The dual-gene engineered mesenchymal stem cells provided by this invention exhibit migration inhibition and apoptosis induction effects on gastric cancer cells. This offers a novel cellular immunotherapy strategy for gastric cancer with active targeting and strong immune activation capabilities, thus broadening the treatment options for gastric cancer. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This invention provides a schematic diagram of the construction of lentiviral vectors in dual-gene engineered mesenchymal stem cells and a schematic diagram of the in vivo experimental timeline. Specifically, a is a map of plasmid pEF1α-CXCR4-TNFSF14, showing the insertion of CXCR4 and TNFSF14 genes and regulatory elements into the mesenchymal stem cell genome; b is a schematic diagram of mesenchymal stem cells carrying CXCR4 and TNFSF14 for tumor-targeted immunotherapy; c is the experimental timeline of animal experiments, indicating mouse environmental adaptation, tumor cell injection, stem cell therapy, and sample collection on day 34.

[0026] Figure 2 The graph shows the results of flow cytometry detection of CXCR4 and LIGHT protein expression rates in the dual-gene engineered mesenchymal stem cells provided in this embodiment of the invention under different viral transduction doses.

[0027] Figure 3The results of cell killing assays of dual-gene engineered mesenchymal stem cells at different effector-target ratios;

[0028] Figure 4 Images of dual-gene engineered mesenchymal stem cells at 12 and 24 hours, provided in an embodiment of the present invention;

[0029] Figure 5 The results are as follows: a) is a flow cytometry image of Annexin V / PI cell apoptosis assay; b) is the quantitative data of apoptosis percentage in the control group, MSC group and Tg-MSC group.

[0030] Figure 6 This is a graph evaluating the in vivo antitumor efficacy and safety; where a is a tumor photograph; b is a tumor growth curve; c is a weight change curve; and d is a Ki-67 immunohistochemical staining image of tumor tissue; scale bar: 200 μM.

[0031] Figure 7 H&E staining images of the heart, kidneys, liver, lungs and spleen, scale bar: 200 μM. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Similar component reference numerals in the drawings represent similar components. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0034] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0035] It should also be understood that experimental methods without specific conditions are generally performed according to standard conditions in the field or the manufacturer's recommendations. Reagents or instruments used without a specified manufacturer are all commercially available, standard products. All experiments and procedures involved comply with all necessary biosafety and ethical standards.

[0036] Example 1: Packaging of Lentiviral Vectors

[0037] See Figure 1 Using a commercially available lentiviral backbone plasmid (pRRLSIN.cPPT.PGK-GFP.WPRE), the coding sequences (CDS) of the human CXCR4 gene (NM_003467, CDS 96-1154) and the TNFSF14 (LIGHT) gene (NM_001376887, CDS 92-814) were inserted into the multiple cloning site using molecular cloning technology to construct the co-expression vector pEF1α-CXCR4-TNFSF14 (see schematic diagram). Figure 1 (a) This construct is designed for gene expression under the control of the EF1α promoter and contains appropriate regulatory elements to enhance transcription.

[0038] Human embryonic kidney 293T cells (purchased from ATCC) were seeded at a density of 1.25E+5 / mL in T175 culture flasks and cultured under standard conditions (37℃, 5% CO2) in Dulbecco modified Eagle medium (DMEM; 22400-089, Gibco, USA) supplemented with 10% fetal bovine serum (FBS; 10091-148, Gibco, USA). For lentiviral packaging, polyethyleneimine hydrochloride (PEI; 24765-1, Polysciences, USA) was used as the transfection reagent. The core plasmid (pEF1α-CXCR4-TNFSF14) was co-transfected into 293T cells with the packaging plasmids pMDLg / pRRE, pRSV-Rev, and the envelope plasmid pMD2.G (all purchased from Addgene, USA).

[0039] Lentiviral supernatant was collected at 24, 48, and 72 hours post-transfection. The collected virus-containing medium was filtered through a 0.45 μm filter to remove cell debris, then centrifuged at 8000 × g for 15 minutes using PEG6000 (528877-1KG, Millipore, USA) and concentrated 100-fold. The resulting concentrated lentiviral particles were stored at -80°C for later use.

[0040] Example 2: Preparation and Identification of Dual-Gene Engineered Human Umbilical Cord Mesenchymal Stem Cells (Tg-MSCs)

[0041] Human umbilical cord-derived mesenchymal stem cells (HUC-MSCs, purchased from Beike Biotechnology, Shenzhen, China) were cultured in low-glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). Cells were maintained in a humidified atmosphere at 37°C with 5% CO2 and underwent routine culture and passage. When cells reached approximately 70-80% confluence, concentrated lentiviral particles prepared in Example 1 were added at a multiplicity of infection (MOI) of 10. Twenty-four hours after transduction, the medium was replaced with fresh complete medium, and cells were cultured further to maintain appropriate density. The medium was changed every 2 to 3 days to support optimal cell growth and transgene expression.

[0042] Cells were collected 72 hours after transduction using trypsin digestion. Flow cytometry was performed using a BD LSRFortessa™ flow cytometer to detect the expression of CXCR4 and LIGHT proteins, and the data were analyzed using FlowJo software (v10). Transduced HUC-MSCs (hereinafter referred to as Tg-MSCs) were stained with fluorescent dyes conjugated to antibodies targeting mesenchymal stem cell surface markers, including CD184 (CXCR4), CD258 (LIGHT), CD44, CD105, CD29, and CD90, as well as negative markers CD38, CD117, CD45, and CD34. Allotype controls were included to gating and verify specificity, and marker expression and transduction efficiency were quantified based on fluorescence intensity spectra.

[0043] See results Figure 2 Compared with untransduced HUC-MSCs (hereinafter referred to as MSCs), the proportion of positive expression of CXCR4 and LIGHT in Tg-MSCs transduced with 20 μL lentivirus exceeded 80%, and the expression level increased in a dose-dependent manner (5, 10, 20 μL). These results confirm the stable expression of CXCR4 and LIGHT in Tg-MSCs after lentivirus transduction.

[0044] Example 3: Evaluation of the in vitro killing effect of Tg-MSCs on gastric cancer cells

[0045] HGC-27 cells expressing luciferase (5000 cells / well) were seeded in 96-well plates and incubated for 24 hours. MSCs (positive control) and Tg-MSCs (experimental group) were added at effector-to-target ratios (E:T) of 1:1, 5:1, and 10:1, respectively, with MSC-free medium serving as a negative control.

[0046] After co-culturing for 48 hours, 100 µL of D-luciferin (150 µg / mL; Abcam, USA) was added to each well. After incubation in the dark for 10 minutes, the chemiluminescence value was detected using a multi-mode microplate reader. Background signal < 1% of the signal in the target cell wells only. Cell viability (%) was calculated using the formula: (Chemical luminescence value of experimental group / Clinical luminescence value of negative control group) × 100%, and the percentage of cytotoxic lysis was calculated as 100 (%) - Cell viability (%).

[0047] The results are as follows Figure 3 As shown, at effector-to-target ratios of 1:1, 5:1, and 10:1, Tg-MSCs induced significantly stronger tumor cell killing effects compared to MSCs and the control group (p < 0.001 at all ratios), exhibiting dose-dependent cytotoxicity. At an effector-to-target ratio of 10:1, Tg-MSCs induced a decrease in HGC-27 cell viability of over 70%, and its cytotoxicity was significantly stronger than that of the unmodified MSCs group under the same conditions (p < 0.001), indicating that Tg-MSCs possess potent in vitro anti-gastric cancer cell activity.

[0048] Example 4: Effects of Tg-MSCs on gastric cancer cell migration and apoptosis

[0049] Scratch assay: To assess the effects of MSCs and Tg-MSCs on cancer cell migration, HGC-27 cells were seeded in 12-well plates and cultured until approximately 90% confluence was achieved, forming a homogeneous and dense monolayer. A straight line was drawn in the center of each well using a 200 μL pipette tip, and cells were gently washed with PBS to remove detached cells. Transwell chambers containing positive controls (MSCs), experimental groups (Tg-MSCs), or negative controls (no MSCs) were placed above the scratched cancer cell layer. Cells were co-cultured for 48 hours, and images were captured at 0, 12, 24, and 36 hours to monitor wound closure. The scratch area was measured and analyzed using OLYMPUS cellSens Dimension 2.1 software (OLYMPUS, Japan).

[0050] Apoptosis analysis: To assess the apoptosis-inducing effect on cancer cells, HGC-27 cells were seeded in 12-well plates and incubated for 24 hours. Transwell chambers containing positive control (MSCs), experimental (Tg-MSCs), or negative control (no MSCs) were then placed above the cancer cell layer. After another 24 hours of co-culture, HGC-27 cells were collected and stained using an Annexin V / PI apoptosis detection kit (e.g., BD Pharmingen™), and apoptosis was detected by flow cytometry.

[0051] The results are as follows Figure 4As shown, Tg-MSCs significantly inhibited HGC-27 migration compared to MSCs and the negative control group. At 12 hours, wound closure was reduced by approximately 65% ​​in the Tg-MSC treatment group, while MSC-treated cells showed only a reduction of approximately 25% (p < 0.001). At 24 hours, wound closure in the Tg-MSC group remained incomplete, while the negative control and MSC groups showed near-complete closure. Figure 5 As shown, flow cytometry analysis revealed that the total apoptosis rate (early + late apoptosis) in the Tg-MSC co-culture group was significantly higher than that in the unmodified MSC group and the control group (approximately 45% vs. 18% vs. 10%, p<0.001), demonstrating that it can effectively induce apoptosis in gastric cancer cells.

[0052] Example 5: In vivo efficacy evaluation of Tg-MSCs in a nude mouse model of gastric cancer

[0053] Animal model establishment: BALB / c nude mice (6-8 weeks old) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Shanghai, China) and raised under specific pathogen-free (SPF) conditions. To establish a xenotransplantation model, HGC-27 gastric cancer cells in the logarithmic growth phase were introduced at a rate of 1×10⁻⁶. 8 Cells were suspended in sterile saline at a density of 50 μL / mL, and 50 μL of the cell suspension was subcutaneously injected into the right back of each mouse. Tumor development was assessed seven days after inoculation.

[0054] Grouping and Administration: Mice were anesthetized after intraperitoneal injection of D-fluorescein (150 mg / kg; Abcam, USA) and imaged using an IVIS system. The tumors were allowed to grow to an average size of approximately 50 mm. 3 (Calculation formula: (length × width)) 2 When ) / 2), begin treatment assessment ( Figure 1 c). In this phase, mice were randomly divided into three groups: negative control group (no MSCs, tail vein injection of equal volume of PBS), positive control group (tail vein injection of 5×10^5 cells / time, unmodified MSCs), and experimental group (tail vein injection of 5×10^5 cells / time, Tg*-MSCs).

[0055] Indicator monitoring: Tumor size, weight, and bioluminescence intensity were measured every four days, and imaging data were analyzed using Living Image software (Caliper Life Sciences, USA).

[0056] The results are as follows Figure 6As shown in Figures ab, in BALB / c nude mice carrying HGC-27 xenografts, tumor volume growth in the Tg-MSCs treatment group was significantly inhibited compared with the MSC-treated group and the negative control group (p < 0.001). At the experimental endpoint, the mean tumor volume in the Tg-MSCs group was reduced by more than 60% compared with the negative control group (p < 0.001). There were no significant differences in body weight changes among the negative control group, the MSC group, and the Tg-MSCs treatment group. Figure 6 (c) indicates that Tg-MSCs did not cause systemic toxicity. Importantly, treatment with Tg-MSCs alone showed significant tumor growth inhibition and a significant reduction in tumor volume, highlighting the superior efficacy of dual-gene-engineered MSCs.

[0057] Histological analysis:

[0058] When the tumor volume exceeds 2000 mm 3 Or, when other humane endpoints are reached, the mice are euthanized in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC).

[0059] Sample Collection: Mice were sacrificed on day 34 after the last administration, and tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were dissected. Tumor tissue and organs were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μM thick sections. Hematoxylin-eosin (H&E) staining and immunohistochemical staining for Ki-67 antigen (using anti-Ki-67 antibody, Abcam) were performed to assess tumor morphology, necrosis, and cell proliferation. Ki-67 positive cells showed brown nuclear staining, and the proliferation index was calculated as the percentage of Ki-67 positive cells in five randomly selected high-power fields for each sample.

[0060] Tumor tissue: H&E staining showed that control tumors exhibited strong and widespread nuclear positivity, consistent with high proliferative activity, with a proliferation index of approximately 75%. MSC-treated tumors showed less and more patchy Ki-67 expression, with a proliferation index reduced to approximately 55%. Tg-MSC tumors showed significantly reduced Ki-67 staining, large areas lacking nuclear staining, and a proliferation index reduced to below 20% (p < 0.001). Figure 6 (d in the text)

[0061] Histological examination results of major organs (heart, liver, kidneys, lungs, and spleen) as follows Figure 7As shown, no significant pathological damage or structural abnormalities were observed in the major organs of mice in each treatment group. Myocardial fibers remained intact, liver lobule structure was well preserved, glomeruli and renal tubules appeared normal, and no structural changes were observed in the alveoli and splenic follicles. These findings indicate that both MSC and Tg-MSC administration are well tolerable in vivo and did not induce detectable off-target tissue damage. This demonstrates the good biosafety of Tg-MSCs in vivo.

[0062] In summary, the above results indicate that Tg-MSCs exert superior anti-tumor cell proliferation and cytotoxic effects compared with MSCs or the negative control group.

[0063] The above embodiments, using gastric cancer as an example, verify the antitumor activity of the dual-gene engineered MSCs co-expressing CXCR4 and LIGHT provided by the present invention in vitro and in vivo. The dual-gene engineered HUC-MSCs of the present invention can also be used to prepare antitumor drugs for lung cancer, liver cancer, kidney cancer, etc.

[0064] Example 7: Preparation of the pharmaceutical composition

[0065] The Tg-MSCs prepared in Example 2 were washed twice with sterile PBS, resuspended in cell cryopreservation medium containing 5% human serum albumin and 10% DMSO, and the cell density was adjusted to 1×10^7 cells / mL. The cells were then aliquoted into sterile cryovials to obtain the pharmaceutical composition described in this invention. This pharmaceutical composition can be stored long-term in liquid nitrogen and can be used for injection therapy after thawing.

[0066] The above examples demonstrate that the dual-gene engineered HUC-MSCs co-expressing CXCR4 and LIGHT provided by this invention significantly enhance antitumor activity against gastric cancer both in vitro and in vivo. Compared with unmodified HUC-MSCs, Tg-MSCs exhibit superior tumor homing ability, induce strong apoptosis, inhibit migration, and suppress tumor proliferation, while also demonstrating good safety. These examples show that genetic modification of MSCs with chemokine receptor (CXCR4) and co-stimulatory ligand (LIGHT) can simultaneously improve tumor targeting and immune-mediated tumor cell killing.

[0067] Typically, HUC-MSCs exhibit inherent tumor tropism but limited therapeutic efficacy, primarily due to insufficient tumor infiltration and weak immune activation. This invention, through the dual expression of CXCR4 and LIGHT in MSCs, provides both effective tumor localization and robust immune activation, demonstrating superior anti-tumor results.

[0068] Meanwhile, in vitro studies showed that overexpression of CXCR4 and LIGHT exhibited a synergistic effect: Tg-MSCs inhibited gastric cancer cell migration, promoted apoptosis, and demonstrated stronger cytotoxicity than unmodified MSCs. In vivo, xenograft models further validated these effects, with Tg-MSCs significantly inhibiting tumor growth and reducing the Ki-67 proliferation index. Histopathological evidence of increased necrosis confirmed the functional validation data, highlighting the ability of Tg-MSCs to remodel tumor tissue structure. This invention, by combining the transport receptor (CXCR4) with the immune ligand (LIGHT), leverages the principles that CXCR4 enhances the active homing ability of mesenchymal stem cells to gastric cancer lesions expressing its ligand SDF-1 (SDF-1 / CXCL12), and that TNFSF14 (LIGHT) can interact with HVEM and LTβR receptors to promote T cell and NK cell activation. Mesenchymal stem cells co-expressing CXCR4 and LIGHT overcome the shortcomings of insufficient targeting and weak immune activation of natural MSCs, while simultaneously addressing delivery challenges and the immunosuppressive tumor microenvironment. This dual-gene approach may serve as a superior cell therapy platform for the treatment of solid tumors, addressing the challenges of homing and immune evasion in solid tumor therapy.

[0069] In a gastric cancer model, dual-gene engineered Tg-MSCs expressing CXCR4 and LIGHT exhibited enhanced tumor tropism, stronger immune-mediated killing, and superior tumor suppression compared to unmodified MSCs. By simultaneously improving tumor targeting and overcoming immune evasion, this strategy provides a promising and innovative cell therapy for solid tumors.

[0070] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0071] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A dual-gene engineered mesenchymal stem cell, characterized in that, The mesenchymal stem cells co-express human CXCR4 and TNFSF14 proteins; the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells; The human CXCR4 and TNFSF14 protein genes are integrated into the genome of the mesenchymal stem cells via lentiviral vector transduction; the lentiviral vector contains the EF1α promoter.

2. The dual-gene engineered mesenchymal stem cells as described in claim 1, characterized in that, The dual-gene engineered mesenchymal stem cells exhibit migration inhibition and apoptosis induction effects on gastric cancer cells.

3. A pharmaceutical composition, characterized in that, It includes the dual-gene engineered mesenchymal stem cells as described in claim 1 and a pharmaceutically acceptable vector.

4. A method for preparing dual-gene engineered mesenchymal stem cells as described in claim 1, characterized in that, Includes the following steps: Provide mesenchymal stem cells; The mesenchymal stem cells were transduced using a lentiviral vector carrying the CXCR4 and TNFSF14 protein genes. Engineered mesenchymal stem cells stably co-expressing CXCR4 and TNFSF14 proteins were obtained through screening.

5. The preparation method according to claim 4, characterized in that, The lentiviral vector has an infection multiplicity of 5-20.

6. The use of the dual-gene engineered mesenchymal stem cells as described in claim 1 in the preparation of a drug for treating gastric cancer.

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