Mesenchymal stem cell loaded with attenuated salmonella as well as preparation method and application of mesenchymal stem cell

Abstract

The invention discloses a mesenchymal stem cell loaded with attenuated salmonella, which is prepared by the following steps: loading attenuated salmonella VNP by using a mesenchymal stem cell (MSC) to obtain an MSC-VNP cell; the attenuated salmonella is attenuated salmonella typhimurium VNP20009 and a derivative or gene modified strain thereof, and the strain is an expression plasmid which does not carry or carries an expression therapeutic gene and a tracer protein exogenous gene; the therapeutic gene is a coding gene of a protein which can be expressed in attenuated salmonella and has a therapeutic effect, and comprises a cell apoptosis anti-tumor gene, an angiogenesis inhibitor gene or an immune checkpoint blocker antibody gene, and the foreign gene of the tracer protein is Dsred; the expression plasmid is provided with a promoter lac which is commonly used in attenuated salmonella; and the plasmid contains an anti-plasmid loss element AT. The mesenchymal stem cell can effectively load the attenuated salmonella, can also effectively release the attenuated salmonella, and can be used as a universal cell therapy carrier, so that the therapy cost is saved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a mesenchymal stem cell loaded with attenuated Salmonella, and a preparation method and application thereof. Background Art

[0002] VNP20009 (VNP) is an attenuated strain of Salmonella typhimurium, which has attracted widespread attention due to its lower toxicity and good preclinical anti-tumor effect. Although the toxicity of VNP has been greatly reduced compared with the original Salmonella by deleting two genes (purI and msbB), in preclinical mouse experiments, anti-tumor therapy with VNP20009 via intravenous or intraperitoneal injection still causes a certain degree of toxic side effects due to the presence and accumulation of the strain in normal organs, such as liver damage, splenomegaly, and sudden weight loss in mice. Although intratumoral injection can reduce the occurrence of this damage, it undoubtedly greatly limits the scope of application of microbial therapy. In clinical phase I experiments, VNP showed a gratifying safety profile, but poor efficacy. Therefore, how to further improve the tumor targeting and tumor inhibition effect of oncolytic bacteria such as VNP in vivo is a very challenging problem.

[0003] In recent years, various types of white blood cells, including macrophages, neutrophils, T cells, etc., have been used as effective tumor drug delivery carriers due to their unique homing effect on tumor areas. Mesenchymal stem cells (MSCs), as a type of stem cell, are widely present in most tissues of the body and are relatively easy to obtain. In addition to self-renewal and multidirectional differentiation potential, MSCs also have immunomodulatory effects and the characteristics of migrating to the site of injury. Since tumors are considered "wounds that never heal", MSCs have become an ideal carrier for tumor targeted therapy. Encapsulating anticancer drugs in stem cells makes it easier for them to enter or approach tumors, avoid being cleared by the immune system, and also reduce the systemic toxicity of anticancer drugs. At present, mesenchymal stem cells are widely used to deliver chemotherapeutic drugs, apoptosis inducers or oncolytic viruses for tumor treatment. At present, there are no studies reporting the use of mesenchymal stem cells as carriers of oncolytic bacteria for tumor treatment. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a mesenchymal stem cell capable of loading and releasing attenuated Salmonella, and a preparation method and application thereof.

[0005] On the one hand, the present invention provides a mesenchymal stem cell loaded with attenuated Salmonella, wherein attenuated Salmonella VNP is loaded with mesenchymal stem cells MSC to obtain MSC-VNP cells; the attenuated Salmonella VNP is attenuated Salmonella typhimurium VNP20009 and its derivative or genetically modified strains, and the strain is an expression plasmid that does not carry or carries an expression plasmid for expressing a therapeutic gene or a tracer protein exogenous gene; the therapeutic gene is a gene encoding a protein with a therapeutic effect that can be expressed in the attenuated Salmonella VNP, including an apoptosis anti-tumor gene, an angiogenesis inhibitor gene or an immune checkpoint blocker antibody gene, and the tracer protein exogenous gene is Dsred; the expression plasmid carries a promoter lac commonly used in attenuated Salmonella; and the plasmid contains an anti-plasmid loss element AT.

[0006] In a second aspect, the present invention provides a method for preparing MSC-VNP cells, comprising the following steps: after serum-free culturing of mesenchymal stem cells MSC, co-incubating the mesenchymal stem cells MSC with attenuated Salmonella VNP, treating the cells with gentamicin to kill extracellular free or adherent attenuated Salmonella, and preparing MSC-VNP cells.

[0007] Preferably, mesenchymal stem cells MSC are co-cultured with attenuated Salmonella VNP-Dsred expressing an exogenous gene of a tracer protein to obtain MSC-VNP-Dsred cells expressing the tracer protein.

[0008] In a third aspect, the present invention provides a method for evaluating the loading efficiency of attenuated Salmonella and the activity of mesenchymal stem cells in MSC-VNP cells. The MSC-VNP cells are transferred to 0.3% by volume TritonX100-PBS and allowed to stand for 15 minutes. The remaining cells are cultured overnight, and the loading efficiency of the MSC-VNP cells is evaluated by the total number of live bacteria loaded in the cells.

[0009] In a fourth aspect, the present invention provides a MSC-VNP system in which attenuated Salmonella expresses a tracer protein, wherein the attenuated Salmonella is attenuated Salmonella typhimurium VNP20009.

[0010] Preferably, the exogenous gene of the tracer protein is Dsred.

[0011] In a fifth aspect, the present invention provides a use of MSC-VNP cells in the preparation of anti-tumor drugs, wherein the MSC-VNP cells are used to inhibit tumor growth.

[0012] Preferably, the MSC-VNP cells are used to be enriched in tumor tissues.

[0013] Preferably, the MSC-VNP cells are used to reduce tissue damage.

[0014] In a sixth aspect, the present invention provides an application of MSC-VNP cells in combination with other anti-tumor substances, wherein the MSC-VNP cells in combination with other anti-tumor substances are used to prepare anti-tumor drugs.

[0015] Beneficial effects: 1. The mesenchymal stem cells of the present invention can both effectively load attenuated Salmonella and effectively release attenuated Salmonella. Compared with cell carriers such as macrophages and neutrophils, the mesenchymal stem cells of the present invention can be allogeneically infused and easily expanded and cultured in vitro. They can be used as universal cell therapy carriers, saving treatment costs.

[0016] 2. The present invention can effectively avoid the toxic side effects of oncolytic bacteria therapy, reduce the distribution and titer of bacteria in normal tissues and organs, has higher tumor targeting, and effectively improves the tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a picture of MSC-VNP cells under a confocal microscope in Example 2 of the present invention.

[0018] Figure 2 This is a graph showing the plate growth results of MSC-encapsulated VNPs at different co-culture ratios in Example 2 of the present invention.

[0019] Figure 3 This is a graph showing the plate growth results of MSC-encapsulated VNPs under different co-culture times and culture conditions in Example 2 of the present invention.

[0020] Figure 4 The results are statistical results of the viability of MSCs after co-culturing MSCs with VNPs for different time periods in Example 2 of the present invention.

[0021] Figure 5 This is a statistical diagram of the sustained release of intracellular VNPs by the MSC-VNP cells of the present invention in Example 3 of the present invention.

[0022] Figure 6 This is a graph showing the chemotaxis ability of mesenchymal stem cells loaded with VNPs in Example 3 of the present invention.

[0023] Figure 7 This is a graph showing the indirect killing ability of VNP on tumor cells in Example 3 of the present invention.

[0024] Figure 8 This is a comparative chart of the therapeutic effects of VNP, MSC, and MSC-VNP cells on tumor-bearing mice in Example 4 of the present invention.

[0025] Fig. 9 This is a diagram of the VNP-Dsred vector construction in Example 5 of the present invention.

[0026] Fig.10 This is a diagram showing the distribution of VNPs in various tissues on a plate in Example 5 of the present invention.

[0027] Fig.11 This is a graph showing the detection of serum ALT and AST concentrations in Example 5 of the present invention.

[0028] Fig.12 This is an H&E stained section image of the organ in Example 5 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments, and the implementation cases provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The embodiments provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0030] Example 1: Preparation of mesenchymal stem cells loaded with attenuated Salmonella (1) Preparation of electroporated competent VNPs: S1. Inoculate the recipient bacteria (VNP) the night before, pick a single colony and culture it in LB medium at 37℃ in a shaking incubator overnight; S2. Transfer 2 ml of overnight culture to 200 ml of LB medium and culture vigorously on a shaker at 37°C until OD 600 =0.6 (about 2.5-3 hours); S3. Cool the bacterial solution on ice for 10 min, centrifuge at 3000 g for 5 min at 4°C, discard the supernatant, and obtain the first precipitate. S4. Add 1500 μl of pre-cooled 10% (V / V) glycerol to the first precipitate, and suspend the competent cells by pipetting to obtain the first suspension; S5. Centrifuge the first suspension at 3000 g for 5 min at 4°C, discard the supernatant, and obtain the second precipitate; S6. Add 750 μl of pre-cooled 10% (V / V) glycerol to the first precipitate, and suspend the competent cells by pipetting to obtain the second suspension; S7. Centrifuge the second suspension at 3000g for 5 min at 4°C, discard the supernatant, add 20 μl of pre-cooled 10% (V / V) glycerol, and suspend the competent cells by pipetting to obtain competent cells. S8. Use the competent cells immediately or aliquot and store at -70℃.

[0031] (2) Preparation method of MSC-VNP cells: MSCs were pre-cultured in serum-free medium (1L low-glucose MEM with 100 U / mL penicillin and 100 g / mL streptomycin). After 24 hours, MSCs were co-incubated with attenuated Salmonella VNP20009 competent cells at a ratio of 1:100 for 1 hour. The cells were treated with 100 μg / mL gentamicin for 60 minutes to kill extracellular free or adherent VNP20009 to obtain MSC-VNP cells.

[0032] (3) Dsred plasmid electroporation competent VNP: S1. Take out the prepared competent VNP and put it on ice to melt; S2. Add 50-100 ng of Dsred plasmid to the competent cells, pipette evenly and place on ice; S3. Select the electroporation conditions of the electroporation instrument: 1600V, 200 ohms, 25uf; S4. Add the mixture to be transformed into a pre-cooled 1 mm electroporation cuvette for electroporation transformation; S5. Add 1 ml of liquid culture medium to the transformation cup to resuspend the cells; S6. Inoculate the cells into a liquid culture medium with kanamycin resistance and culture at 37°C with shaking for 1 h; S7. Pipette 100 μl of bacterial solution onto a Kanamycin-resistant plate and culture overnight at 37°C. S8. Use a fluorescence microscope to observe the expression of VNP red fluorescence (Dsred). If red fluorescence appears in the field of view, it means that Dsred has been successfully transferred into VNP cells.

[0033] (4) Preparation method of MSC-(VNP-Dsred) cells: MSCs were cultured in serum-free medium (1L low-glucose MEM with 100 U / mL penicillin and 100 g / mL streptomycin). After 24 hours, MSCs were co-incubated with attenuated Salmonella VNP20009 expressing the tracer protein (Dsred) at a ratio of 1:100 for 1 hour. The cells were treated with 100 μg / mL gentamicin for 60 minutes to kill extracellular free or adherent VNP20009 to obtain MSC-(VNP-Dsred) cells.

[0034] Example 2: Selection of VNP loading conditions for MSCs MSCs were co-cultured with VNPs at a ratio of 1:10-1:200 for 1, 3, and 5 hours, respectively. After treating the cells with 100 μg / mL gentamicin for 60 minutes, 0.3% (V / V) TritonX-100 (purchased from Sigma, catalog number: X100) prepared with PBS was added and allowed to stand for 15 minutes to perforate the MSCs and release the intracellular VNP strains. After gradient dilution, LB plates were coated to analyze the number of live VNP strains loaded by MSCs and the correlation between their loading efficiency and co-culture time. Trypan blue (purchased from Sigma, catalog number: 93595) was used to detect the activity of cells after 1, 2, and 3 hours of co-culture of MSCs and VNPs. To further verify that mesenchymal stem cells were effectively loaded with VNPs, after obtaining mesenchymal stem cells loaded with VNPs, the cells were fixed with 4% (V / V) paraformaldehyde (room temperature, protected from light, and allowed to stand for 30 min), washed 2-3 times with PBS, and MSC skeletons and cell nuclei were stained with phalloidin (purchased from Sigma, item number: P1951) and DAPI (purchased from Sigma, item number: D9542) according to the product instructions (room temperature, protected from light, and allowed to stand for 30 min), washed 2-3 times with PBS, and observed and photographed using an upright fluorescence microscope.

[0035] The results are as follows Figure 1-Figure 4 , Figure 1 Fluorescence microscopy was used to take pictures to indicate the loading of VNPs onto MSCs. Blue represents the MSC nucleus, red represents the MSC cell membrane, and green represents the VNPs. The results showed that the VNP strains were successfully loaded onto MSCs. Figure 2 It showed that the ability of MSCs to load VNPs tended to be saturated at a co-culture ratio of MSCs to VNPs of 1:100. Figure 3 The results showed that the number of MSC-VNP cells was proportional to the co-culture time. When the co-culture time was the same, the number of MSC-VNP cells in the starvation group was greater. The starvation group had a cell culture medium without serum, while the normal group had a cell culture medium with serum. Figure 4 The results showed that the viability of MSCs decreased significantly after 60 minutes of co-culture, while the cell viability after 60 minutes of co-culture was >85%, which was at a relatively high level. In summary, the treatment culture conditions selected by the present invention were MSC: VNP=100, serum-free co-culture for 60 minutes.

[0036] Example 3: Characteristic detection of MSC-VNP cells (1) Strain release of MSC-VNP: After obtaining MSC-VNP by the method of Example 1, MSC-VNP cells were cultured using a 3.0 mm Transwell plate (cells cannot pass through this pore size, but bacteria can still pass through effectively). After the Transwell plate was placed at 37°C and 5% (V / V) CO2 for 12 h, the supernatant in the lower chamber was collected for gradient dilution and then applied to LB plates for colony counting. The results are shown in Table 1. Figure 5 As shown, MSC-VNP cells were able to slowly release VNPs and showed obvious bacterial release effect at 9h.

[0037] (2) Detection of chemotaxis of MSC-VNPs: The Transwell experiment was used to compare the chemotactic ability of MSC and MSC-VNP. Tumor cells B16F10 were cultured in the lower chamber using an 8.0 mm Transwell plate. MSC-VNP cells were diluted with DMEM (containing 100 mg / mL gentamicin), and the diluted MSC-VNP cell suspension was added to the upper chamber. The Transwell plate was cultured at 37°C 5% (V / V) CO2 for 16 hours, and the upper chamber was removed. The upper cells of the upper chamber were wiped off, and the chamber was washed with PBS three times. The chamber was placed in 4% (M / V) paraformaldehyde for 35 minutes. After fixation, it was washed with PBS 2-3 times to remove residual paraformaldehyde. The chamber was immersed in crystal violet (purchased from Sigma, product number C6158-50G) and stained at room temperature in the dark for 30 minutes. The residual staining solution was washed with PBS to remove it. After drying, the cells in the lower layer of the chamber were photographed using an upright fluorescence microscope. The results are shown in Figure 6 The microscope fields were randomly selected to compare the chemotaxis of MSC-VNP cells. The purple one is the chemotactic MSC (control). The chemotactic ability of MSC-VNP on tumor cells is consistent with that of MSC, indicating that the chemotactic ability of MSC on tumor cells did not change after loading VNP.

[0038] (3) Detection of the anti-tumor ability of VNPs released by MSC-VNPs: Tumor cells B16F10 were cultured at 2.0x10 5 The cells were inoculated into a 12-well plate at a density of 100:1 and incubated for 8 hours until the cells adhered to the wall. The collected normal VNPs or released VNP strains were co-cultured with the cells at a ratio of 100:1 for 4 hours. All cells in the plate were collected, washed and resuspended in binding buffer, and stained with 1 μg Annexin V-APC protein (purchased from Solebo, catalog number: T2190). The cells were placed on ice in the dark for 30 minutes, and the apoptosis was analyzed by flow cytometry. Figure 7 As shown, the VNPs released by MSC-VNP cells were the same as those of control VNPs.

[0039] The present invention determines the anti-tumor activity of VNP released by MSC-VNP cells. Figure 7 As shown, the tumor-killing ability of VNPs released by MSC-VNP cells is the same as that of unloaded VNPs.

[0040] The above results indicate that MSC-VNP cells are able to release VNPs, and the independent functions of MSC and VNPs are not affected.

[0041] Example 4: Detection of tumor inhibition effect of MSC-VNP cells Experimental animals: 6-8 week old female C57BL / 6 mice were selected. The mice were randomly assigned to different groups and inoculated with 100 μl of 2×10 5 The density of melanoma cells B16F10 was such that the mice grew to 80-160 mm 3 Melanoma was detected in the mouse model of orthotopic melanoma. VNP, MSC, and MSC-VNP cells were diluted with PBS to a titer of 5×10 6 / ml, respectively, and injected intravenously, with an injection volume of 100μl. VNPs injected into the tail vein were activated twice before injection.

[0042] Tumor volume is calculated according to the formula V = length × width 2 ×0.52. The tumor size of mice in a specific group was measured at regular intervals and used to draw a tumor growth curve after calculation. The values ​​were expressed as Mean ± SEM. Figure 8 As shown, compared with VNP and MSC, MSC-VNP cells can significantly inhibit the growth of mouse melanoma.

[0043] Example 5: Detection of the attenuation effect of MSC-VNP cells (1) Construction of Dsred-expressing bacteria: Plasmids carrying exogenous genes expressing therapeutic genes and tracer genes were electroporated into attenuated Salmonella typhimurium VNP20009 to construct attenuated Salmonella that can express genes expressing therapeutic proteins and tracer proteins. The promoter used was the lac promoter commonly used in Escherichia coli, and the target gene was the Dsred fragment. Finally, VNP20009 bacteria (VNP-Dsred) expressing red fluorescent Dsred were constructed for VNP tracing in in vivo experiments. Fig. 9 As shown, VNP-Dsred emits red fluorescence under a fluorescence microscope. (Note: The VNPs used in the following in vivo experiments are all VNP-Dsred that can emit red fluorescence.) (2) Tissue distribution detection of VNP: MSC-(VNP-Dsred) cells were prepared according to the method of Example 1, and 100 μl of MSC-(VNP-Dsred) cells at a density of 2×10 5 VNP-Dsred and MSC-(VNP-Dsred) cells were injected, and 100μl PBS was injected as a control. After the mice were killed on the third day, the organs and tumor tissues of the mice were obtained, and 500μl 0.3% (V / V) TritonX-100 was added after crushing with a tissue grinder to obtain tissue grinding fluid, which was placed at room temperature for 20 minutes to perforate the cells and release VNPs. Then the tissue grinding fluid was diluted and applied to a kanamycin-resistant LB plate for bacterial culture to count the number of VNPs in various organs in the mouse body. The results are shown in Fig.10 As shown, the VNP used here is VNP-Dsred with red fluorescence. Fig.10 The results showed that compared with VNP, MSC-VNP was significantly enriched in mouse melanoma and had reduced distribution in other organs.

[0044] (3) Histopathology, blood routine and blood biochemical analysis: Blood was collected by removing the eyeball for routine blood tests, and the blood was allowed to stand at room temperature for 30 min, then centrifuged at 3000 rpm for 15 min at 4 °C, and the supernatant serum was drawn to measure blood biochemical indicators. Routine blood tests, blood biochemical analysis, H&E staining of tumor, heart, liver, spleen, lung and kidney sections, and fluorescent immunostaining of MSC in tumor sections were prepared by Wuhan Sewell Biotechnology Co., Ltd.

[0045] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are clinical indicators for evaluating liver function. If the liver is damaged, and it is a chronic and persistent damage, ALT and AST will increase. Therefore, the present invention detects the content of ALT and AST in mouse serum respectively. The results are as follows Figure 11-Figure 12 As shown, the VNPs used here are all VNP-Dsred with red fluorescence. Fig.11 It showed that after 3 days of treatment of mice with MSC-VNP, the concentrations of alanine aminotransferase ALT and aspartate aminotransferase AST in the serum dropped to 100U / L, while the concentration in the VNP group was 300U / L, indicating that MSC-VNP can significantly reduce the hepatotoxicity of VNP. Fig.12 The stained sections showed that compared with PBS, MSC, and VNP, MSC-VNP could reduce the degree of damage to various tissues.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mesenchymal stem cell loaded with attenuated Salmonella, characterized in that: Mesenchymal stem cells MSC are loaded with attenuated Salmonella VNP to obtain MSC-VNP cells; the attenuated Salmonella VNP is attenuated Salmonella typhimurium VNP20009 and its derivative or genetically modified strains, and the strain is an expression plasmid that does not carry or carries an expression plasmid for expressing therapeutic genes and exogenous genes for tracer proteins; the therapeutic gene is a gene encoding a protein with therapeutic effects that can be expressed in the attenuated Salmonella VNP, including apoptosis anti-tumor genes, angiogenesis inhibitor genes or immune checkpoint blocker antibody genes, and the exogenous tracer protein gene is Dsred; the expression plasmid contains the promoter lac commonly used in attenuated Salmonella; and the plasmid contains an anti-plasmid loss element AT.

2. The method for preparing MSC-VNP cells according to claim 1, characterized in that: The following steps are involved: After serum-free culture of mesenchymal stem cells MSC, the mesenchymal stem cells MSC were co-incubated with attenuated Salmonella VNP, and the cells were treated with gentamicin to kill extracellular free or adherent attenuated Salmonella to prepare MSC-VNP cells.

3. The preparation method according to claim 2, characterized in that: Mesenchymal stem cells MSC are co-cultured with attenuated Salmonella VNP-Dsred expressing an exogenous tracer protein gene to prepare MSC-VNP-Dsred cells expressing the tracer protein.

4. A method for evaluating the loading efficiency of attenuated Salmonella in MSC-VNP cells and the activity of mesenchymal stem cells, characterized in that: The MSC-VNP cells were transferred to 0.3% TritonX100-PBS and allowed to stand for 15 min. The remaining cells were cultured overnight, and the loading efficiency of the MSC-VNP cells was evaluated by the total number of live bacteria loaded into the cells.

5. The MSC-VNP system of attenuated Salmonella expressing a tracer protein according to claim 1, characterized in that: The attenuated Salmonella is attenuated Salmonella typhimurium VNP20009.

6. The MSC-VNP system according to claim 5, characterized in that: The exogenous gene of the tracer protein is Dsred.

7. The use of the MSC-VNP cells according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The MSC-VNP cells are used to inhibit tumor growth.

8. The use according to claim 7, characterized in that: The MSC-VNP cells are used to enrich in tumor tissues.

9. The use according to claim 7, characterized in that: The MSC-VNP cells are used to reduce tissue damage.

10. The use of the MSC-VNP cells according to claim 1 in combination with other anti-tumor substances, characterized in that: The MSC-VNP cells are combined with other anti-tumor substances to prepare anti-tumor drugs.

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