Anti-tumor stem cell preparations and their preparation methods

Abstract

This invention discloses an anti-tumor stem cell preparation and its preparation method, belonging to the field of biomedical technology. The stem cell genome contains an introduced target gene, a hypoxia-responsive element, and a regulatory element. The target gene is linked downstream of the regulatory element and expressed under its regulation. The regulatory element is located downstream of the hypoxia-responsive element. The preparation process of the anti-tumor stem cell preparation includes the following steps: S1, plasmid construction; S2, lentiviral packaging; S3, stem cell culture; S4, cell transfection; S5, cell screening. This invention uses the NTRK1 promoter and VEGFA-1 enhancer to synergistically regulate the highly efficient and specific expression of the NADH oxidase gene in the adrenocortical tumor environment, catalyzing the generation of a large amount of reactive oxygen species to induce cancer cell apoptosis. This invention solves the problem in existing technologies where stem cell-targeted gene therapy relies solely on the homing effect of stem cells, resulting in a lack of specific targeting ability to the lesion site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to anti-tumor stem cell preparations and their preparation methods. Background Technology

[0002] Adrenocortical tumor (ACC) is a rare malignant tumor. Due to its high malignancy and rapid progression, many patients present with tumor infiltration of surrounding tissues or distant metastasis, making surgery impossible. Clinically, ACC presents as either hormone-secreting functional tumors or non-functional tumors that do not secrete hormones. Adult ACC is mostly non-functional, while pediatric ACC is predominantly functional. Cortisol secretion is mainly associated with tumor recognition, advanced disease stages, and poor prognosis. Non-functional ACC mainly manifests as compression or hemorrhage after tumor rupture.

[0003] Currently, complete surgical removal of the lesion is the only curative treatment for adrenocortical tumors (ACC), making early diagnosis and radical surgery crucial. For patients who cannot undergo surgery, adjuvant therapies such as mitotane, cytotoxic drugs, and targeted therapies can be considered, but their efficacy is currently unsatisfactory. Furthermore, ACC has a very high prognosis, with a 5-year overall survival rate of only 35%-60%.

[0004] Targeted gene therapy can target specific sites on tumors, enhance the local therapeutic effect, and reduce systemic adverse reactions. Stem cells are widely available and possess strong self-renewal capabilities, making them suitable as carrier cells for gene-targeted cancer therapy. However, current methods for delivering drugs using stem cells largely rely on the homing effect of stem cells, lacking specific targeting capabilities to lesion sites. Therefore, there is an urgent need to develop a method for precisely targeted drug delivery to treat adrenocortical tumors. Summary of the Invention

[0005] Therefore, the present invention provides an anti-tumor stem cell preparation and its preparation method to solve the problem that the existing technology lacks specific targeting ability to lesion sites because stem cell targeted gene therapy relies solely on the homing effect of stem cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the invention, an anti-tumor stem cell preparation is provided, the preparation comprising genetically modified stem cells, wherein the stem cells are dispersed in a medically acceptable carrier to obtain the anti-tumor stem cell preparation.

[0007] Furthermore, the concentration of the genetically modified stem cells is 1×10⁻⁶. 5 -2×10 5 per mL.

[0008] Furthermore, the genome of the genetically modified stem cells includes an introduced target gene, a hypoxia response element, and a regulatory element. The target gene is located downstream of the regulatory element, and its expression is regulated by the regulatory element. The regulatory element is located downstream of the hypoxia response element, and the three are operatively connected.

[0009] Furthermore, the target gene is the NADH oxidase encoding gene, the hypoxia response element is the VEGFA-1 enhancer, and the regulatory element is the NTRK1 promoter.

[0010] Furthermore, the sequences of the NADH oxidase encoding gene, the NTRK1 promoter, and the VEGFA-1 enhancer are SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively.

[0011] Furthermore, the genetically modified stem cells are umbilical cord mesenchymal stem cells.

[0012] Furthermore, the tumor is an adrenocortical tumor.

[0013] Furthermore, the medically acceptable carrier is phosphate buffer or LB liquid culture medium.

[0014] According to a second aspect of the present invention, a method for preparing an anti-tumor stem cell preparation is provided, comprising the following steps: S1, Plasmid Construction: Obtain the required gene sequences from the database and construct plasmids; S2, Lentiviral Packaging: Lentiviral packaging was performed on the plasmid constructed in step S1 using a liposome transfection kit. S3, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1% penicillin-streptomycin and cultured and passaged in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics. S4. Cell transfection: After incubating 4th generation umbilical cord mesenchymal stem cells in 12-well culture plates for 12 hours, lentiviral particles were mixed with umbilical cord mesenchymal stem cells, polybrene was added, and transfection was carried out for 3-4 days to obtain VEGFA-NTRK1-NOX-MSCs. S5. Cell screening: The transfected VEGFA-NTRK1-NOX-MSCs were screened with puromycin to obtain cell clones that stably expressed the target gene. After expansion culture, the anti-tumor stem cell preparation was obtained. Flow cytometry was used to detect the cells to confirm their stem cell characteristics.

[0015] According to a third aspect of the present invention, the use of an anti-tumor stem cell preparation in the preparation of a drug for treating adrenocortical tumors is provided.

[0016] The present invention has the following advantages: 1. This invention uses the NTRK1 promoter as a regulatory element and introduces the VEGFA-1 enhancer as a hypoxia response element to regulate the expression of the NADH oxidase gene. NTRK1 is highly expressed only in adrenal cancer environments and normal blood environments; cancer cells typically exhibit a hypoxic microenvironment. Therefore, the hypoxia-activated-tissue-specific dual-regulatory expression system constructed in this invention can specifically regulate the expression of the NADH oxidase gene in the adrenal cancer environment and effectively avoid non-specific expression of the target gene.

[0017] 2. Cancer cells have extremely fragile redox homeostasis. NADH oxidase can catalyze the production of a large amount of reactive oxygen species, which can disrupt the redox balance of cancer cells and induce apoptosis.

[0018] 3. Mesenchymal stem cells have the ability to migrate directionally to tumor tissues, actively penetrate and enter the tumor microenvironment, and have self-renewal capabilities. After modification with the NTRK1 promoter and VEGFA-1 enhancer, they can work synergistically with the homing effect of stem cells to efficiently target cancer lesions. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0021] Figure 1The image shows the recombinant lentiviral expression plasmid pLVX-VEGFA-NTRK1-NOX provided in the embodiments of the present invention. Detailed Implementation

[0022] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] According to a first aspect of the present invention, an anti-tumor stem cell preparation is provided, the preparation comprising genetically modified stem cells, wherein the stem cells are dispersed in a medically acceptable carrier to obtain the anti-tumor stem cell preparation.

[0024] The concentration of genetically modified stem cells was 1×10⁻⁶. 5 -2×10 5 per mL.

[0025] The genome of the genetically modified stem cells contains the introduced target gene, hypoxia response element, and regulatory element. The target gene is located downstream of the regulatory element, and its expression is regulated by the regulatory element. The regulatory element is located downstream of the hypoxia response element, and the three are operatively connected.

[0026] The target gene is the NADH oxidase encoding gene, the hypoxia response element is the VEGFA-1 enhancer, and the regulatory element is the NTRK1 promoter.

[0027] The sequences of the NADH oxidase encoding gene, the NTRK1 promoter, and the VEGFA-1 enhancer are SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively.

[0028] Among them, the genetically modified stem cells are umbilical cord mesenchymal stem cells.

[0029] The tumor was an adrenocortical tumor.

[0030] Among them, the medically acceptable carriers are phosphate buffer or LB liquid culture medium.

[0031] According to a second aspect of the present invention, a method for preparing an anti-tumor stem cell preparation is provided, comprising the following steps: S1, Plasmid Construction: Obtain the required gene sequences from the database and construct plasmids; S2, Lentiviral Packaging: Lentiviral packaging was performed on the plasmid constructed in step S1 using a liposome transfection kit. S3, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1% penicillin-streptomycin and cultured and passaged in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics. S4. Cell transfection: After incubating 4th generation umbilical cord mesenchymal stem cells in 12-well culture plates for 12 hours, lentiviral particles were mixed with umbilical cord mesenchymal stem cells, polybrene was added, and transfection was carried out for 3-4 days to obtain VEGFA-NTRK1-NOX-MSCs. S5. Cell screening: Transfected VEGFA-NTRK1-NOX-MSCs were screened with puromycin to obtain cell clones that stably expressed the target gene. After expansion culture, anti-tumor stem cell preparations were obtained. Flow cytometry was used to detect the cells to confirm their stem cell characteristics.

[0032] The usage process of this invention embodiment is as follows: Example S1, Plasmid Construction: a. Obtain the coding region of the NADH oxidase gene, the VEGFA-1 enhancer, and the NTRK1 promoter sequence from Genebank and synthesize the three gene sequences respectively (Shanghai Jikai Gene Medical Technology Co., Ltd.). b. The pLVX-EF1α-IRES-puro plasmid vector was digested with Sph I and BAMH I restriction enzymes, and the linearized vector fragment was recovered. c. Using In-Fusion cloning technology, the sequences encoding the VEGFA-1 enhancer, NTRK1 promoter, and NADH oxidase gene were cloned into a linearized vector fragment in the order of enhancer-promoter-target gene, yielding the recombinant lentiviral expression plasmid pLVX-VEGFA-NTRK1-NOX, which was then identified by sequencing. The map of the recombinant lentiviral expression plasmid pLVX-VEGFA-NTRK1-NOX is shown below. Figure 1 ; S2, Lentiviral Packaging: 293T cells in good growth condition were used at a dose of 5 × 10 3 cells / cm 2The cells were seeded at a density of [missing information - likely a specific density] in culture dishes. When cell confluence reached 75%, the cells were transfected into 5 × 10⁶ cells using a liposome transfection kit according to the manufacturer's instructions. 7.5 μg of the recombinant lentiviral expression plasmid pLVX-VEGFA-NTRK1-NOX constructed in step S1, 5.63 μg of the packaging plasmid psPAX2, and 1.88 μg of the envelope plasmid pMD2.G were co-transfected into each culture dish. 6 293T cells were transfected; 48h and 72h after transfection, the cell supernatant was collected and filtered through a 0.45μm filter membrane to obtain lentiviral particles containing the target gene, which were stored at -80℃ for later use. S3, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells (purchased from Shanghai Yuchun Biotechnology Co., Ltd.) were used at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1%, and each flask was incubated in 5 mL of serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics at 37°C for 24 h at 5% CO2. b. After culturing for 24 hours, remove cells that are not attached to the bottom of the culture dish by changing the medium. Change the medium every 3 days. When the cell density reaches 60%, passage the cells at a ratio of 1:3. S4. Cell transfection: a. Disperse umbilical cord mesenchymal stem cells cultured to passage 4 in DEME / F12 medium at a concentration of 1×10⁻⁶. 5 Cells / mL, and 1×10⁻⁶ cells / mL per well 5 The cells were seeded at a density of 100 cells per well into 12-well culture plates, and 1 mL of DMEM / F12 medium containing 10% FBS was added to each well and incubated for 12 h. b. Add 20 μL of lentivirus particles to each well into the umbilical cord mesenchymal stem cell culture medium, then add 1 μg of polybrene. After transfection for 24 h, replace with fresh DMEM / F12 medium and continue transfection for 48 h to obtain VEGFA-NTRK1-NOX-MSCs. S5. Cell screening: Add 2 μg puromycin to 1 mL of transfected VEGFA-NTRK1-NOX-MSCs in each well for selection. Replace the medium with fresh puromycin every 3 days. After 12 days of selection, cell clones stably expressing the target gene are obtained. After expansion culture, resuspend the stem cells in phosphate buffer (pH 7.2-7.4) to a concentration of 1 × 10⁻⁶. 5 The anti-tumor stem cell preparation A1 was obtained by measuring 1 cell / mL. Flow cytometry was used to detect the positive expression rates of CD73, CD90 and CD105 on the cell surface (>95%) and CD34, CD45 and CD14 (<2%) to confirm its stem cell characteristics.

[0033] Comparative Example 1 This comparative example is based on the previous example, except that the recombinant plasmid does not contain the VEGFA-1 enhancer sequence, while the other specific processing parameters are the same as in the previous example, resulting in the anti-tumor stem cell preparation B1.

[0034] Comparative Example 2 S1, Plasmid Construction: a. Log in to Genebank to obtain the NTRK1 promoter sequence of Homo sapiens and synthesize the gene sequence; b. The pLVX-EF1α-IRES-puro plasmid vector was digested with EcoRI and BAMH I restriction enzymes, and the linearized vector fragment was recovered. c. The NADH oxidase gene sequence was cloned into a linearized vector fragment using In-Fusion cloning technology to obtain the recombinant lentiviral expression plasmid pLVX-EF1α-NOX, which was then identified by sequencing. S2, Lentiviral Packaging: 293T cells in good growth condition were used at a dose of 5 × 10 3 cells / cm 2 The cells were seeded at a density of [missing information - likely a specific density] in culture dishes. When the cell confluence reached 75%, the recombinant lentiviral expression plasmid pLVX-EF1α-NOX constructed in step S1 was co-transfected with 5.63 μg of packaging plasmid psPAX2 and 1.88 μg of envelope plasmid pMD2.G into 5 × 10⁶ cells / mL culture dishes using the liposome transfection kit according to the manufacturer's instructions. 6 293T cells were transfected; 48h and 72h after transfection, the cell supernatant was collected and filtered through a 0.45μm filter membrane to obtain lentiviral particles containing the target gene, which were stored at -80℃ for later use. S3, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1%, and each flask was incubated in 5 mL of serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics at 37°C for 24 h at 5% CO2. b. After culturing for 24 hours, remove cells that are not attached to the bottom of the culture dish by changing the medium. Change the medium every 3 days. When the cell density reaches 60%, passage the cells at a ratio of 1:3. S4. Cell transfection: a. Disperse umbilical cord mesenchymal stem cells cultured to passage 4 in DEME / F12 medium at a concentration of 1×10⁻⁶. 5 Cells / mL, and 1×10⁻⁶ cells / mL per well 5The cells were seeded at a density of 100 cells per well into 12-well culture plates, and 1 mL of DMEM / F12 medium containing 10% FBS was added to each well and incubated for 12 h. b. Add 20 μL of lentivirus particles to each well into the umbilical cord mesenchymal stem cell culture medium, then add 1 μg of polybrene. After transfection for 24 h, replace with fresh DMEM / F12 medium and continue transfection for 48 h to obtain EF1α-NOX-MSCs. S5. Cell screening: 2 μg of puromycin was added to 1 mL of transfected EF1α-NOX-MSCs in each well for selection. The puromycin-containing medium was replaced every 3 days. After 12 days of selection, cell clones stably expressing the target gene were obtained. After expansion culture, the stem cells were resuspended in phosphate buffer (pH 7.2-7.4) to a concentration of 1 × 10⁻⁶. 5 Cells / mL; flow cytometry was used to detect the positive expression rates of CD73, CD90 and CD105 on the cell surface >95% and the positive expression rates of CD34, CD45 and CD14 <2% to confirm their stem cell characteristics.

[0035] This comparative example is based on the previous example, except that the NTRK1 promoter gene and VEGFA-1 enhancer gene were replaced with the EF-1α promoter gene in the recombinant plasmid. That is, the recombinant plasmid was reconstructed using the promoter of the original plasmid vector pLVX-EF1α-IRES-puro to transfect stem cells and prepare anti-tumor stem cell preparations. The other specific processing parameters are the same as in the previous example, and anti-tumor stem cell preparation B2 is obtained. The sequence of the EF-1α promoter gene is shown in SEQ ID No: 4.

[0036] Comparative Example 3 S1, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1%, and each flask was incubated in 5 mL of serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics at 37°C for 24 h at 5% CO2. b. After culturing for 24 hours, remove cells that are not attached to the bottom of the culture dish by changing the medium. Change the medium every 3 days. When the cell density reaches 60%, passage the cells at a ratio of 1:3. S2, Stem Cell Drug Delivery: a. Disperse umbilical cord mesenchymal stem cells cultured to passage 4 in DEME / F12 medium at a concentration of 1×10⁻⁶. 5 Cells / mL, and 1×10⁻⁶ cells / mL per well 5The cells were seeded at a density of 100 cells per well into 12-well culture plates. 1 mL of DMEM / F12 medium containing 10% FBS was added to each well and the plates were incubated for 12 h. After incubation, the medium was discarded and the plates were washed twice with phosphate buffer at pH 7.2-7.4. b. Add 1 mL of mitotane (Maclean, CAS: 53-19-0) at a concentration of 100 μg / mL to each well and incubate at 37°C for 6 h in 5% CO2. c. Discard the drug-containing culture medium, wash three times with PBS to remove free drug, add 0.25% trypsin to digest and collect cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant; d. Resuspend cells in phosphate buffer (pH 7.2–7.4) to a final volume of 1 × 10⁻⁶. 5 cells / mL; S3, Cell Screening: Flow cytometry was used to detect the positive expression rates of CD73, CD90, and CD105 on the cell surface (>95%) and the positive expression rates of CD34, CD45, and CD14 (<2%) to confirm their stem cell characteristics.

[0037] This comparative example is based on the previous example, except that only umbilical cord mesenchymal stem cells loaded with mitotane were used to prepare the anti-tumor stem cell preparation, resulting in anti-tumor stem cell preparation B3.

[0038] Test Example 1 The stem cells of the anti-tumor stem cell preparations prepared in the examples and comparative examples were characterized by flow cytometry. The results are shown in Table 1.

[0039] Table 1. Stem cell characterization results of anti-tumor stem cell preparations

[0040] Experimental results show that the positive expression rates of CD73, CD90 and CD105 of the anti-tumor stem cell preparations prepared in the embodiments of the present invention and Comparative Examples 1-3 are all higher than 96%, and the negative rates of CD34, CD45 and CD14 are all lower than 1.5%, indicating that the umbilical cord mesenchymal stem cell characteristics of the stem cell preparations prepared in the embodiments of the present invention and Comparative Examples 1-3 are well maintained.

[0041] Test Example 2 The cell viability in the anti-tumor stem cell preparations prepared in the embodiments and comparative examples of the present invention was determined by flow cytometry, specifically including the following steps: The stem cell preparation was washed with phosphate buffer (pH 7.2-7.4) and resuspended to a final concentration of 1×10⁻⁶. 4A stem cell suspension was prepared by measuring cells per mL. 1 μL of the stem cell suspension was added to 5 μL of 1 μg / mL 7-AAD staining solution and incubated at room temperature in the dark for 15 min. The stem cell viability was determined using flow cytometry with an excitation wavelength of 488 nm and detection channel FL3. To more accurately verify the efficacy of the anti-tumor stem cell preparation prepared in this invention, untransfected stem cells were used as the control group, and stem cells transfected with the blank pLVX-EF1α-IRES-puro plasmid were used as the empty plasmid group. The cell viability of each group of stem cell preparations is shown in Table 2.

[0042] Table 2 Cell viability of stem cell preparations

[0043] As can be seen, groups A1 and B1 exhibited the highest cell viability, approaching that of the untransfected plasmid control group and the empty plasmid group, indicating that the pLVX-EF1α-IRES-puro plasmid vector, the complete stem cell preparation of this invention, and the stem cell preparation without the VEGFA-1 enhancer gene do not affect stem cell activity. Compared with the control group, the cell viability of group B2 decreased by 24.1%, which may be due to the non-specific expression of NADH oxidase driven by the EF-1α promoter, which generates a large amount of reactive oxygen species (ROS) even in the normal environment of non-adrenocortical tumors, causing damage to stem cells. Compared with the control group, the cell viability of group B3 decreased by 32.8%, significantly lower than that of groups A1, B1, and B2, which were transfected with plasmids, indicating that mitotane has significant cytotoxicity to umbilical cord mesenchymal stem cells.

[0044] Test Example 3 To verify the tumor cell proliferation inhibition effect of the anti-tumor stem cell preparation prepared in this invention, the anti-tumor stem cell preparations prepared in the examples and comparative examples were co-cultured with mouse Y-1 cells (mouse adrenocortical tumor cells, purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) in a non-contact manner, and the cell proliferation inhibition rate was detected. The specific steps included: S1 and Y-1 cell seeding: Y-1 cells in the logarithmic growth phase were resuspended in DMEM / F12 medium containing 10% FBS to a concentration of 5 × 10⁻⁶ cells / mL. 4 Cells / mL, add 500 μL of cell suspension to the lower chamber of Transwell and incubate at 37°C for 24 h in 5% CO2; S2, Stem Cell Inoculation: Add 200 μL of anti-tumor stem cell preparation to each Transwell upper chamber and incubate at 37°C and 5% CO2 for 48 h. S3 and MTT cell proliferation assays: a. Four hours before the end of the culture, add 20 μL of 5 mg / ml thiazolyl blue solution to each well, put the culture plate back into the incubator, and continue incubation for another 4 hours; b. Carefully aspirate and discard the culture supernatant from the wells. Add 150 μL of dimethyl sulfoxide to each well and shake on a shaker at 100 rpm for 10 min to fully dissolve the crystals. Measure the absorbance of each well at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The proliferation inhibition rate of Y-1 cells is expressed by the formula: Among them, OD 给药组 and OD 对照组 These are the OD values ​​measured for Y-1 cells co-cultured with and without anti-tumor stem cells, respectively. S4. The expression level of NADH oxidase after co-culture was determined using an ELISA kit.

[0045] To more accurately verify the efficacy of the anti-tumor stem cell preparation prepared in this invention, stem cells without plasmid transfection were set as the control group, and stem cells transfected with the blank pLVX-EF1α-IRES-puro plasmid were set as the empty plasmid group. The Y-1 cell proliferation inhibition rate and NADH oxidase expression level of mice in the control group, empty plasmid group, examples, and each comparative group are shown in Table 3.

[0046] Table 3. Y-1 cell proliferation inhibition rate and NADH oxidase expression levels in each group of mice.

[0047] The experimental results showed that group A1 exhibited the highest NADH oxidase expression level, reaching 124.6 ng / mg total protein. Compared with group A1, the NADH oxidase expression level in group B1 decreased by 45.2%, indicating that the NTRK1 promoter mediates the specific expression of the NADH oxidase gene, and also demonstrating that the specific high expression of the NADH oxidase gene depends on the synergistic effect of the NTRK1 promoter and the VEGFA-1 enhancer gene.

[0048] Compared with stem cells without plasmid transfection (control group), the Y-1 cell proliferation inhibition rate in the empty plasmid group was slightly increased, but the difference was not significant. Compared with the control group, the Y-1 cell proliferation inhibition rates in Example 1 and Comparative Examples 1-3 increased by 1533.3%, 789.6%, 633.3%, and 1114.5%, respectively, indicating that the anti-tumor stem cell preparations prepared in Example 1 and Comparative Examples 1-3 all showed good Y-1 cell proliferation inhibition rates, with umbilical cord mesenchymal stem cells transfected with the intact pLVX-VEGFA-NTRK1-NOX plasmid showing the best performance. Compared with group A1, the Y-1 cell proliferation inhibition rate in group B1 decreased by 45.5%, indicating that the NTRK1 promoter and VEGFA-1 enhancer have a synergistic effect on the specific expression of the NADH oxidase gene. The Y-1 cell proliferation inhibition rate in the B3 group, where umbilical cord mesenchymal stem cells were directly loaded with mitotane, was 58.3%, which was higher than that in the B1 and B2 groups with incomplete plasmids, but lower than that in the example group. This indicates that the dual regulatory system of genetically modified stem cells mediating the expression of NADH oxidase is crucial for the killing efficiency of adrenocortical tumors.

[0049] Furthermore, group B2 showed higher NADH oxidase expression levels than group B1, but exhibited the lowest Y-1 cell proliferation inhibition rate among the experimental groups. This may be because EF-1α, as a non-specific strong promoter, may overexpress NADH oxidase. However, due to constitutive expression leading to excessive ROS production by NADH oxidase, stem cells in the co-culture system have short survival times and poor functional status, resulting in the lowest actual total ROS acting on Y-1 cells. This indirectly confirms that the dual regulation of the VEGFA-1 enhancer and NTRK1 promoter genes in this invention not only achieves targeted expression of the target gene but also protects the functional integrity of the stem cell vector, ensuring therapeutic efficiency.

[0050] Test Example 4 Mouse adrenocortical tumor modeling To verify the therapeutic effect of the anti-tumor stem cell preparation prepared in this invention on adrenocortical tumors, mice were used as model animals to establish an adrenocortical tumor model. The modeling process included the following steps: S1. Take Y-1 cells in the logarithmic growth phase and resuspend them in phosphate buffer (pH 7.2-7.4) to a concentration of 1×10⁻⁶. 7 One cell / mL, to obtain Y-1 cell suspension; S2 male BALB / c-nu nude mice aged 4-6 weeks were anesthetized intraperitoneally, and the left back was cut open to expose the adrenal region. 10 μL of Y-1 cell suspension was injected into the adrenal cortex region of the mice using a microsyringe. The skin was sutured after the operation and antibiotics were given to prevent infection. S3. Eight weeks after modeling, tumor volume was measured using a small animal in vivo imaging system. Tumor volume was expressed as follows: Calculation; where L and W are the major and minor axes of the tumor, respectively; tumor volume is 150-300 mm. 3 The model is considered successfully created; S4. In the second week after successful modeling, the mice were randomly divided into 4 groups and injected with the anti-tumor stem cell preparations prepared in the example and each comparative example. 100 μL was injected into each mouse via the tail vein. The injection was given once every two weeks for a total of 8 weeks. S5. One week after the last injection, the tumor volume of the mice was measured again, and the mice were sacrificed to collect tumor tissue. The expression level of NADH oxidase in the tumor was measured by ELISA.

[0051] To more accurately verify the efficacy of the anti-tumor stem cell preparation prepared in this invention, stem cells without plasmid transfection were set as the control group, and stem cells transfected with the blank pLVX-EF1α-IRES-puro plasmid were set as the empty plasmid group. The tumor volume and NADH oxidase expression level in the tumor tissues of mice in each group are shown in Table 4.

[0052] Table 4. Tumor volume and NADH oxidase expression levels in tumor tissues of mice in each group.

[0053] It can be seen that the tumor volume in the model group mice was 247.3 mm. 3 This indicates that the model was successfully created.

[0054] The injection therapy results showed that the tumor volume and NADH oxidase expression levels in the control group and the empty plasmid group were similar to those in the model group, indicating that umbilical cord mesenchymal stem cells and the blank pLVX-EF1α-IRES-puro plasmid vector had no therapeutic effect on mouse adrenocortical tumors. The A1 group, with its complete dual regulatory system, exhibited the smallest tumor volume and the highest NADH oxidase expression level, at 58.4 mm. 3The tumor volume of the B1 group was 76.4% lower than that of the model group, and the NADH oxidase expression level was increased by 6494.4%, indicating that NADH oxidase was successfully expressed in mice and showed a good therapeutic effect on adrenocortical tumors. The three comparative groups also showed good therapeutic effects on adrenocortical tumors and high NADH oxidase expression levels. However, compared with group A1, group B1 showed a 144.0% increase in tumor volume and a 46.8% decrease in NADH oxidase expression level, indicating that the VEGFA-1 enhancer plays an important role in gene expression activation under hypoxic microenvironment, and also demonstrating a synergistic effect between the VEGFA-1 enhancer and the NTRK1 promoter on the specific expression of NADH oxidase. Group B2, which used the EF-1α promoter to replace the VEGFA-1 enhancer and the NTRK1 promoter to regulate NADH oxidase expression, showed a higher NADH oxidase expression level than group B1, but also exhibited the largest tumor volume among the four treatment groups, consistent with the lower cell viability observed in test case 2. The study confirmed that the EF-1α promoter, acting as a constitutive strong promoter, leads to non-specific overexpression of NADH oxidase, resulting in a large amount of ROS-damaging stem cell vectors and reducing drug efficacy at the tumor site. Only the B3 group, loaded with mitotane on umbilical cord mesenchymal stem cells, showed better tumor-suppressive effects than the B1 and B2 groups, indicating that the therapeutic effect of NADH oxidase on adrenocortical tumors depends on the specific overexpression regulated by both the VEGFA-1 enhancer and the NTRK1 promoter.

[0055] Test Example 5 To verify the specific expression of the anti-tumor stem cell preparation prepared in this invention, the expression level of NADH oxidase in non-target organs of model mice was detected, specifically including the following steps: The mice used in test example 4 were sacrificed one week after the last injection, and lung, liver, kidney, brain tissue, and arterial blood were collected. The expression level of NADH oxidase was determined by ELISA. The expression level of NADH oxidase in non-target organs of mice is shown in Table 5.

[0056] Table 5. NADH oxidase expression levels in non-target organs and arterial blood of mice.

[0057] It can be seen that the expression levels of NADH oxidase in non-target organs and arterial blood of groups A1, B3, the control group, and the empty plasmid group were not significantly different from those of the model group. This indicates that umbilical cord mesenchymal stem cells transfected with empty plasmids and those not transfected with plasmids have no effect on NADH oxidase expression, and that the dual regulatory system constructed by the VEGFA-1 enhancer and the NTRK1 promoter can achieve specific expression of NADH oxidase in the adrenocortical tumor microenvironment. Compared with group A1, the expression levels of NADH oxidase in lung, liver, kidney, brain tissue, and arterial blood of groups B1 and B2 were significantly increased. Among them, group B2 showed the highest NADH oxidase expression level, which was nearly 20 times higher than that of group A1 in various organs and blood. This indicates that the VEGFA-1 enhancer and NTRK1 promoter have a synergistic effect on the regulation of the specific expression of the NADH oxidase-encoding gene in the adrenocortical tumor environment, while the EF-1α promoter, as a constitutive strong promoter, has high activity in various tissues, further verifying that the dual regulatory system of the present invention can effectively mediate the specific and efficient expression of the NADH oxidase-encoding gene.

[0058] The above results indicate that the dual regulatory system of VEGFA-1 enhancer and NTRK1 promoter constructed in this invention can achieve specific expression of NADH oxidase in the adrenocortical tumor microenvironment, effectively killing tumor cells while protecting the functional integrity of stem cell carriers, and has significant in vivo anti-tumor effects.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An anti-tumor stem cell preparation, characterized in that, The formulation includes genetically modified stem cells, which are dispersed in a medically acceptable carrier to obtain an anti-tumor stem cell formulation.

2. The anti-tumor stem cell preparation according to claim 1, characterized in that, The concentration of the genetically modified stem cells is 1×10⁻⁶. 5 -2×10 5 per mL.

3. The anti-tumor stem cell preparation according to claim 1, characterized in that, The genome of the genetically modified stem cells contains an introduced target gene, a hypoxia response element, and a regulatory element. The target gene is located downstream of the regulatory element, and its expression is regulated by the regulatory element. The regulatory element is located downstream of the hypoxia response element, and the three are operatively connected.

4. The anti-tumor stem cell preparation according to claim 3, characterized in that, The target gene is the NADH oxidase encoding gene, the hypoxia response element is the VEGFA-1 enhancer, and the regulatory element is the NTRK1 promoter.

5. The anti-tumor stem cell preparation according to claim 4, characterized in that, The sequences of the NADH oxidase encoding gene, NTRK1 promoter, and VEGFA-1 enhancer are SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, respectively.

6. The anti-tumor stem cell preparation according to claim 1, characterized in that, The genetically modified stem cells are umbilical cord mesenchymal stem cells.

7. The anti-tumor stem cell preparation according to claim 1, characterized in that, The tumor is an adrenocortical tumor.

8. The anti-tumor stem cell preparation according to claim 1, characterized in that, The medically acceptable carrier is phosphate buffer or LB liquid culture medium.

9. A method for preparing an anti-tumor stem cell preparation, used to prepare the anti-tumor stem cell preparation according to any one of claims 1-8, characterized in that, Includes the following steps: S1, Plasmid Construction: Obtain the required gene sequences from the database and construct plasmids; S2, Lentiviral Packaging: Lentiviral packaging was performed on the plasmid constructed in step S1 using a liposome transfection kit. S3, Stem Cell Culture: a. Mouse umbilical cord mesenchymal stem cells were injected at a rate of 5 × 10⁻⁶. 3 cells / cm 2 The culture flasks were inoculated at a density of 1% penicillin-streptomycin and cultured and passaged in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics. S4. Cell transfection: After incubating 4th generation umbilical cord mesenchymal stem cells in 12-well culture plates for 12 hours, lentiviral particles were mixed with umbilical cord mesenchymal stem cells, polybrene was added, and transfection was carried out for 3-4 days to obtain VEGFA-NTRK1-NOX-MSCs. S5. Cell screening: The transfected VEGFA-NTRK1-NOX-MSCs were screened with puromycin to obtain cell clones that stably expressed the target gene. After expansion culture, the anti-tumor stem cell preparation was obtained. Flow cytometry was used to detect the cells to confirm their stem cell characteristics.

10. Application of anti-tumor stem cell preparations in the preparation of drugs for the treatment of adrenocortical tumors.

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