Application of animal lactobacillus in the preparation of anti-tumor drugs

Abstract

The present invention relates to the field of microorganisms and tumor immunotherapy, and in particular to an application of animal lactobacillus in the preparation of anti-tumor drugs. The application of animal lactobacillus in the preparation of anti-tumor drugs is characterized in that the animal lactobacillus is animal lactobacillus ( Lactobacillus animalis )ATCC35046. The present invention surprisingly found that animal Lactobacillus ATCC35046 can inhibit tumors.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and tumor immunotherapy, and in particular to application of animal lactobacillus in the preparation of anti-tumor drugs. Background Art

[0002] Melanoma is a highly malignant skin tumor caused by gene mutations in melanocytes. Melanoma is prone to occur in young and middle-aged women and men over 55 years old. The external environmental factors that cause melanoma are mainly solar ultraviolet UV-B and artificial ultraviolet UV-A, and the internal factors include genetic susceptibility and inheritance. Melanoma is the skin tumor with the highest mortality rate. At present, traditional treatments for melanoma mainly include surgical resection, radiotherapy and chemotherapy, but after treatment with these methods, the tumor is prone to recurrence and metastasis, and is also prone to drug resistance. New immunotherapies, such as monoclonal antibodies, nivolumab, pembrolizumab, ipilimumab, etc., have low response rates and high costs. Therefore, it is of great research significance to find new and effective drugs for the treatment of melanoma.

[0003] Probiotics are a type of microorganism that can have beneficial effects on the host and are closely related to human health. Studies have shown that probiotics can activate a variety of immune cells such as macrophages and NK cells, induce the production of effector factors such as tumor necrosis factor-α, β (TNF-α, β), interferon-γ (IFN-γ), interleukin-12 (IL-12), nitric oxide (NO), and have immunomodulatory effects. However, the use of one or more probiotics alone often has poor therapeutic effects on tumors. Therefore, there are currently two main application strategies: one is oral probiotics combined with chemotherapy or immunotherapy for the treatment of tumors. For example, there are literature reports that oral probiotics (such as Bifidobacterium, Lactobacillus rhamnosus or Enterococcus) can enhance the immunotherapy effect of immune checkpoint inhibitors (such as anti-PD1 therapy) or cytokines in melanoma. Another strategy is to introduce the gene of recombinant protein into probiotics, and express active protein factors with anti-tumor effects in vivo through engineered probiotics to exert anti-tumor effects. For example, there are literature reports: the probiotic Escherichia coli Nissle1917 (EcN) was transformed into an engineered bacterium that can produce cyclic di-AMP (CDA) for tumor treatment. Cyclic di-AMP is a stimulator of the STING (stimulator of interferon genes) pathway, which plays a key role in activating antigen presenting cells (APCs) and presenting tumor antigens to trigger anti-tumor immune responses.

[0004] Lactobacillus animalis is a probiotic found in dental plaque, intestinal tract or feces of animals. Lactobacillus animalis is widely used in dairy fermentation and has antibacterial activity, especially inhibiting multi-drug resistant pathogens. It also has good effects in inhibiting allergic attacks, improving obesity, reducing insulin resistance and lowering cholesterol. However, the anti-tumor effect of Lactobacillus animalis has not been reported yet. Summary of the invention

[0005] Technical issues to be solved:

[0006] The present invention screened a strain of animal lactobacillus that can effectively treat melanoma, which can strongly activate CD4 + and CD8 + T lymphocytes reverse the tumor immunosuppressive microenvironment and exert a good anti-tumor effect.

[0007] Technical solution:

[0008] The invention discloses an application of animal lactobacillus in preparing an anti-tumor drug, characterized in that the animal lactobacillus is animal lactobacillus (Lactobacillus animalis) ATCC35046.

[0009] Lactobacillus animalis ATCC35046 was purchased from Beina Biotech - Beijing Beina Chuanglian Biotechnology Research Institute. After the frozen Lactobacillus animalis ATCC35046 was revived in solid culture medium, a single colony was picked and activated in liquid culture medium, and then expanded and cultured until it grew to the logarithmic phase, that is, the OD value was about 0.8 (2×10 8 CFU / mL), the cells were obtained by centrifugation, washed 3 times with PBS, and resuspended in PBS for later use.

[0010] The tumor is melanoma.

[0011] Mechanism of action: Animal Lactobacillus ATCC35046 is administered subcutaneously through peritumoral tissue. The animal Lactobacillus ATCC35046 can strongly activate CD4 in the tumor microenvironment by promoting the maturation of dendritic cells. + and CD8 + T lymphocytes, thereby effectively killing tumor cells and achieving the effect of tumor immunotherapy.

[0012] Specifically:

[0013] In the present invention, animal lactobacillus ATCC35046 and mouse macrophage RAW 264.7 were co-cultured in vitro, and the RT-qPCR technology was used to verify that animal lactobacillus ATCC35046 could significantly promote the expression of cytokines TNF-α, IL-1β and IL-6 in macrophages at the mRNA level, indicating that animal lactobacillus can stimulate the activation of macrophages and secrete cytokines with anti-tumor effects.

[0014] The present invention co-incubated animal lactobacillus ATCC35046 with dendritic cells derived from mouse bone marrow in vitro, and verified through flow cytometry that animal lactobacillus ATCC35046 can promote the expression of MHC-Ⅱ and CD86 on the surface of dendritic cells, indicating that animal lactobacillus ATCC35046 can effectively promote the differentiation and maturation of dendritic cells. The maturation of dendritic cells helps to further activate T lymphocytes.

[0015] The present invention uses C57BL / 6 mice as experimental animals and establishes a mouse melanoma tumor model by subcutaneously implanting B16-F10 cells. When the average tumor volume reaches about 50 mm 3 At the same time, Lactobacillus animalis ATCC35046 (4×10 8 CFU / mouse), and an untreated PBS group and a Lactococcus lactis NZ9000-treated group (2×10 9 CFU / mouse) was used as the control, and the first administration was recorded as day 0. The changes in tumor volume began to be recorded. On day 12, the tumor volume exceeded 1500mm 3 The experimental mice were treated with Lactobacillus ATCC35046, so the treatment was terminated, the tumor tissue was weighed, photographed, and then fixed with formaldehyde for tissue section analysis. The experimental results showed that on the 12th day, the average tumor volume of the animal Lactobacillus ATCC35046 treated group was 561mm lower than that of the untreated PBS control group. 3, decreased by 78%; the average tumor weight of the animal Lactobacillus ATCC35046 treatment group was 0.66g lower than that of the PBS control group, a decrease of 75%, and in the animal Lactobacillus ATCC35046 treatment group, 1 / 3 of the mice had completely subsided their tumors. At the same time, on the 12th day, compared with the lactococcus lactis NZ9000 treatment group, the average tumor weight of the animal Lactobacillus ATCC35046 treatment group was 0.13g lower, a decrease of 37%. Further, the results of H&E and TUNEL staining showed that after animal Lactobacillus ATCC35046 treatment, large areas of necrosis appeared in the mouse tumor tissue, and a large number of tumor cells apoptosis. The above results show that animal Lactobacillus ATCC35046 can more effectively inhibit the growth of mouse melanoma, and its tumor inhibition activity is significantly better than that of Lactococcus lactis. At the same time, the effective dose of animal Lactobacillus ATCC35046 is 1 / 5 of that of Lactococcus lactis. It is particularly noteworthy that in the animal Lactobacillus ATCC35046 treatment group, 1 / 3 of the mice had complete tumor regression, which fully demonstrated that animal Lactobacillus ATCC35046 has the advantages of low dosage and good anti-tumor effect.

[0016] The present invention also observed the infiltration of T lymphocytes in melanoma tumor tissue after treatment with animal Lactobacillus ATCC35046 by immunofluorescence staining of tumor tissue paraffin sections. The results showed that compared with the blank control group, the CD4 + T and CD8 + The infiltration of T cells increased significantly, indicating that animal lactobacillus ATCC35046 can promote the infiltration of T lymphocytes in tumor tissue, reverse the tumor immunosuppressive microenvironment, enhance the anti-tumor immune response, and thus effectively inhibit tumor growth.

[0017] Beneficial effects:

[0018] 1. The present invention surprisingly discovered that animal Lactobacillus ATCC35046 can effectively inhibit the growth of melanoma, and 1 / 3 of the mice had completely regressed tumors. Melanoma is a skin tumor with the highest mortality rate, and current treatments have limited effects. Most probiotics cannot achieve effective anti-tumor effects when used alone. The present invention used Lactococcus lactis NZ9000, a probiotic reported to have certain anti-tumor effects, as a control group, and proved that the tumor inhibition effect of animal Lactobacillus ATCC35046 was better than that of Lactococcus lactis NZ9000 (with a significant difference), and its effective dose was 1 / 5 of that of Lactococcus lactis NZ9000. It is particularly noteworthy that, Figure 4As shown in D, in the group treated with Lactobacillus animalis ATCC35046, one-third of the mice had complete tumor regression, which is a major discovery in the field of using probiotics for tumor treatment, indicating that the specific Lactobacillus animalis ATCC35046 produced unexpected technical effects.

[0019] 2. The animal lactobacillus ATCC35046 of the present invention can be made into a lyophilized powder injection for peritumoral subcutaneous injection, or prepared into a transdermal dosage form for direct application on the tumor and the surrounding skin surface. The animal lactobacillus ATCC35046 used in the present invention is a probiotic that can be used for dairy fermentation and has the advantages of safety and non-toxicity.

[0020] 3. An outstanding advantage of the present invention is that it does not need to be combined with other immunotherapies. Animal Lactobacillus ATCC35046 alone can exert an efficient tumor inhibitory effect. Its therapeutic effect is consistent with the literature report (Griffin ME, Espinosa J, Becker JL, Luo JD, Carroll TS, Jha JK, Fanger GR, Hang HC. Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy. Science. 2021 Aug 27; 373 (6558): 1040-1046.) The effect of enterococci combined with anti-PD-1 antibodies for the treatment of mouse melanoma models (Fig. 1B, after 15 days of treatment, the average tumor volume of the enterococci combined with anti-PD-1 antibody treatment group was about 150mm 3 , compared with the untreated control group (about 550 mm 3 ) compared with the mean tumor volume of the control group (P<0.05).

[0021] the term

[0022] TNF-α: Tumor necrosis factor-α

[0023] IL-1β: interleukin-1β

[0024] IL-6: interleukin-6

[0025] MHC-Ⅱ: major histocompatibility complex-Ⅱ

[0026] CD86: Leukocyte differentiation antigen 86

[0027] H&E: Hematoxylin-eosin staining

[0028] TUNEL: terminal untransferase labeling BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Growth curve of Lactobacillus animalis ATCC35046

[0030] Figure 2 RT-qPCR results of cytokine production by macrophage RAW264.7 after stimulation

[0031] (A) Relative expression of IL-1β mRNA (B) Relative expression of TNF-α mRNA (C) Relative expression of IL-6 mRNA (compared with the control group, *p<0.05; **p<0.01; ***p<0.001)

[0032] Figure 3 Dendritic cells stimulated by flow cytometry

[0033] (A) CD11c + CD86 + Cell proportion (B) CD11c + The proportion of cells expressing MHC-Ⅱ in the cells (compared with the control group, *p<0.05; **p<0.01; ***p<0.001)

[0034] Figure 4 Treatment effect diagram of mouse melanoma model

[0035] (A) Mouse weight change (B) Mouse tumor growth curve (C) Mouse tumor weight on day 12 (D) Visual image of mouse tumor tissue on day 12 (compared with the control group, *p<0.05; **p<0.01; ***p<0.001, n=12)

[0036] Figure 5 Results of H&E staining and TUNEL staining of mouse melanoma tumor tissue

[0037] (A) H&E staining of mouse tumor tissue (magnification: 100×, scale bar: 500 μm), (B) TUNEL staining of mouse tumor tissue (magnification: 100×, scale bar: 100 μm)

[0038] Figure 6 CD4 in mouse melanoma tumor tissue + and CD8 + T cell immunofluorescence

[0039] (A) CD4 + T cell immunofluorescence image (B) CD8 + T cell immunofluorescence image (magnification: 100×, scale bar 100 μm).

[0040] The L. animalis in the figure is Lactobacillus animalis ATCC35046, and the L. lactis is Lactococcus lactis NZ9000. DETAILED DESCRIPTION

[0041] The following examples may help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0042] Lactobacillus animalis ATCC35046 and Lactococcus lactis NZ9000 were purchased from Beina Biotech - Beijing Beina Chuanglian Biotechnology Research Institute. Lipopolysaccharide (LPS) was purchased from Sigma.

[0043] Example 1: Determination of the growth curve of Lactobacillus animalis ATCC35046

[0044] Methods: The growth curve of animal lactobacillus ATCC35046 was determined by turbidimetry. The frozen animal lactobacillus ATCC35046 was taken out and inoculated on MRS solid culture medium by three-zone line method, and placed in a 30℃ constant temperature incubator for static culture. After the colony was formed in the culture dish, a single colony was picked into 10mL liquid culture medium and incubated at 30℃ for 14-16 hours as seed culture medium. 13 triangular flasks containing 20mL MRS liquid culture medium were taken and numbered as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12h, for a total of 13 time points. 200μL of seed culture medium was drawn with a sterile pipette and added to the numbered triangular flasks, incubated at 30℃, and then the triangular flasks were taken out at the corresponding time and stored in a 4℃ refrigerator. Finally, the OD value of the culture medium at all time points was measured at a wavelength of 600nm, and the growth curve was drawn.

[0045] Results: Figure 1 As shown, animal Lactobacillus ATCC35046 enters the logarithmic phase within 4-6 hours and gradually enters the plateau phase after 8 hours. The OD value corresponding to the logarithmic phase of growth of animal Lactobacillus ATCC35046 is about 0.8.

[0046] Example 2: Determination of the total number of colonies corresponding to the logarithmic phase of Lactobacillus animalis ATCC35046 and Lactococcus lactis NZ9000

[0047] Method: In order to determine the total number of colonies corresponding to the logarithmic phase of Lactobacillus animalis ATCC35046, 200 μL of the seed culture solution in Example 1 was taken into 20 mL of fresh liquid culture medium, i.e., it was expanded 100 times and continued to be cultured. After it grew to the logarithmic phase, i.e., the OD value was about 0.8, it was diluted 10 times continuously, and 10 -2 , 10-4 and 10 -6 Three serial dilutions of 1 mL of bacterial solution were plated, and the solid culture dish was incubated at 30°C. After the colonies grew, an appropriate culture dish was selected to calculate the average colony count. The culture medium of Lactococcus lactis NZ9000 (abbreviated as Lactococcus lactis (L. lactis)) was M17, and the method for determining the total colony count corresponding to the logarithmic phase of Lactococcus lactis (L. lactis) was the same as that of Lactobacillus animalis ATCC35046.

[0048] Results: When Lactobacillus animalis ATCC35046 grew to an OD value of 0.8, the total number of colonies was 2×10 8 CFU / mL. The OD value corresponding to the logarithmic growth phase of Lactococcus lactis NZ9000 is about 0.8, and the corresponding total colony count is 1×10 9 CFU / mL.

[0049] Example 3. Animal Lactobacillus ATCC35046 can stimulate macrophage activation and secrete related cytokines

[0050] Methods: Take the bacterial culture of animal Lactobacillus ATCC35046 in the logarithmic growth phase (2×10 8 CFU / mL), centrifuged and resuspended in DMEM cell culture medium without antibiotics for later use. Macrophage RAW 246.7 was cultured at 1×10 5 The concentration of 2 × 10 5 Cells / well), then the prepared animal lactobacillus ATCC35046 suspension was added to a 6-well plate (multiplicity of infection MOI = 25), and a negative control group (control) with only culture medium and a positive control group with LPS (100 ng / mL) were set up. After incubation at 37°C for 4 hours, the supernatant was discarded, and the cells were washed with PBS 3 times, and then replaced with fresh DMEM medium containing antibiotics. After incubation for another 6 hours, cell RNA was extracted, and the expression of IL-1β, IL-6 and TNF-α at the mRNA level was detected by RT-qRCR.

[0051] Results: Lipopolysaccharide (LPS) is a characteristic component of the cell wall of Gram-negative bacteria. LPS and its lipid A part can induce the activation of immune cells (such as macrophages and dendritic cells) by binding to Toll-like receptor 4 (TLR4). LPS was selected as a positive control, which can optimize the experimental conditions on the one hand, and serve as a reference for evaluating the intensity of immune cell activation in the experimental test group on the other hand. As shown in Table 1 and Figure 2As shown in the figure, compared with the negative control group, after stimulation by animal lactobacillus ATCC35046, the expression of pro-inflammatory cytokines mRNA such as IL-1β, TNF-α and IL-6 in macrophages increased significantly. At the same time, compared with the positive control drug (LPS), it increased significantly. This shows that animal lactobacillus ATCC35046 can stimulate the activation of macrophages and secrete cytokines with anti-tumor effects.

[0052] Table 1 Analysis of significant differences in related cytokines (P<0.05 indicates significant differences)

[0053] IL-1β TNF-α IL-6 control VS L.animalis P<0.001 P<0.001 ns control VS LPS P<0.5 P<0.5 P<0.5

[0054] Example 4. Lactobacillus animalis ATCC35046 can promote the maturation of dendritic cells

[0055] Methods: Take the bacterial culture of animal Lactobacillus ATCC35046 in the logarithmic growth phase (2×10 8 CFU / mL), centrifuged to obtain the bacteria, and resuspended in RPMI-1640 cell culture medium without antibiotics containing 10% fetal bovine serum for later use. Mouse bone marrow-derived dendritic cells were cultured as follows: 6-8 week old C57BL / 6 male mice were killed by cervical dislocation, the bone marrow in the tibia and femur of the mice was removed and the cells were resuspended in RPMI-1640 complete medium (containing L-glutamine (2mM), 2-mercaptoethanol (50μM), streptomycin (100μg / mL), penicillin (100U / mL), fetal bovine serum (10%) and GM-CSF (20ng / mL)). After counting, the cell suspension was adjusted to 2×10 5 The cells were plated at a concentration of 10 cells / mL in a 100 mm bacterial culture dish (medium volume was 10 mL), and the day was recorded as day 0. On the third day, 10 mL of fresh RPMI-1640 complete medium was added. On the sixth and eighth days, half of the medium was replaced. On the tenth day, the cells were collected by centrifugation and counted. Then, RPMI-1640 medium containing 10% fetal bovine serum was used at a concentration of 1×10 6 The cells were plated in a 48-well plate at a concentration of 5 × 10 5Cells / well), add the prepared animal lactobacillus ATCC35046 suspension to a 48-well plate (multiplicity of infection MOI = 25), and set up a negative control group (control) with only culture medium and a positive control group with LPS (1 μg / mL). After incubation at 37°C for 24 hours, remove the culture medium, wash it 3 times with PBS, replace it with fresh RPMI-1640 culture medium containing antibiotics and 10% fetal bovine serum, continue incubation for 12 hours, and collect the cells for flow cytometry analysis. The specific method for determining MHC-Ⅱ and CD86 by flow cytometry: add monoclonal antibodies of APC-antiCD11c, PE-antiMHC-Ⅱ and FITC-anti CD86 to the cell suspension, incubate it in the dark at 4°C for 30 minutes, centrifuge, wash to remove unbound free antibodies, resuspend the cells with 200 μL PBS, and detect them by flow cytometry.

[0056] Results: As shown in Table 2 and Figure 3 As shown, compared with the negative control group, animal Lactobacillus ATCC35046 can significantly promote the expression of MHC-Ⅱ and CD86 on the surface of dendritic cells; compared with LPS, animal Lactobacillus ATCC35046 significantly promotes the expression of MHC-Ⅱ and CD86 on the surface of dendritic cells; this shows that animal Lactobacillus ATCC35046 can effectively induce the maturation of dendritic cells, and dendritic cells, as professional antigen presenting cells, can further activate T lymphocytes.

[0057] Table 2 Analysis of significant differences in the expression of MHC-Ⅱ and CD86 on the surface of dendritic cells

[0058] MHC-Ⅱ CD86 control VS L.animalis P<0.001 P<0.001 control VS LPS P<0.001 P<0.001

[0059] Example 5. Animal Lactobacillus ATCC35046 can effectively inhibit the growth of mouse melanoma

[0060] Methods: Male C57BL / 6 mice weighing 22-24 g were implanted with B16-F10 melanoma cells subcutaneously on their backs. The average tumor volume was about 50 mm. 3 The mice were randomly divided into three groups: PBS group, Lactobacillus animalis ATCC35046 (L.animalis) treatment group and Lactococcus lactis NZ9000 (L.lactis) treatment group, with 12 mice in each group. The bacterial liquid of Lactobacillus animalis ATCC35046 (2×10 8 CFU / mL) and Lactococcus lactis NZ9000 culture medium (1×10 9 CFU / mL), the cells were centrifuged, resuspended in PBS, and injected subcutaneously around the tumor. The injection volume for each mouse was 100 μL each time, and the injection volume of animal Lactobacillus ATCC35046 was 4×10 8CFU / mouse, the injection volume of Lactococcus lactis NZ9000 was 2×10 9 CFU / mouse. The first administration was recorded as day 0, and then the administration was repeated on days 1, 7 and 8 respectively. The changes in the tumor growth volume of the mice were monitored regularly. When the tumor volume of the experimental mice reached 1500mm 3 , that is, the experiment was ended. After euthanizing the mice, the tumors were removed, weighed, and photographed.

[0061] Results: Most probiotics cannot achieve effective anti-tumor effects when used alone, and usually need to be used in combination with immunotherapy. For example, the literature (Griffin ME, Espinosa J, Becker JL, Luo JD, Carroll TS, Jha JK, Fanger GR, Hang HC. Enterococcus peptidoglycan remodeling promotes checkpoint inhibitor cancer immunotherapy. Science. 2021 Aug 27; 373(6558): 1040-1046.) reported the effect of Enterococcus combined with anti-PD-1 antibody in treating mouse melanoma: After 15 days of treatment, the average tumor volume of the Enterococcus combined with anti-PD-1 antibody treatment group was about 150 mm 3 , compared with the untreated control group (about 550 mm 3 ) compared with the mean tumor volume of the control group (Fig. 1B). In this study, as shown in Tables 3, 4 and Figure 4 As shown, there was no significant difference in the body weight of mice in the treated and untreated groups during the entire treatment process. On day 12, the average tumor volume in the Lactobacillus animalis ATCC35046-treated group was 561 mm lower than that in the untreated PBS control group. 3 , decreased by 78%; the average tumor weight of the animal lactobacillus ATCC35046 treatment group was 0.66g lower than that of the PBS control group, a decrease of 75%. This treatment effect is comparable to the effect reported in the above literature. At the same time, in the animal lactobacillus ATCC35046 treatment group, 1 / 3 of the mice had complete tumor regression ( Figure 4D). The above results fully illustrate that it is not necessary to combine with other immunotherapies, and animal lactobacillus ATCC35046 can exert an efficient tumor inhibition effect when used alone. At the same time, the present invention uses the reported probiotics-Lactococcus lactis NZ9000 with certain anti-tumor effects as the control group. On the 12th day, the average tumor weight of the animal lactobacillus ATCC35046 treatment group was 0.13g lower than that of the lactococcus lactis NZ9000 treatment group, which decreased by 37%. The above results show that animal lactobacillus ATCC35046 can more effectively inhibit the growth of mouse melanoma, and its tumor inhibition activity is significantly better than that of lactococcus lactis. At the same time, the effective dose of animal lactobacillus ATCC35046 is 1 / 5 of that of lactococcus lactis. It is particularly noteworthy that in the animal lactobacillus ATCC35046 treatment group, 1 / 3 of the mice had completely subsided their tumors. It fully illustrates that animal lactobacillus ATCC35046 has the advantages of low dosage and good anti-tumor effect.

[0062] Table 3 Average body weight, average tumor weight, average tumor volume and tumor regression rate of mice on day 12 (n=12)

[0063] Experimental groups PBS group Lactis group L.animalis group Weight (g) 24 24 25 Tumor weight (g) 0.88 0.35 0.22 <![CDATA[Tumor volume (mm 3 )]]> 716 156 155 Tumor regression rate 0 0 4 / 12

[0064] Table 4. Analysis of significant differences in average body weight, average tumor weight and average tumor volume of mice on day 12

[0065] weight Tumor weight Tumor volume control VS L.animalis ns P<0.01 P<0.001 control VS L.lactis ns P<0.5 P<0.001

[0066] Example 6. H&E staining and TUNEL staining analysis of mouse melanoma tumor tissue

[0067] Method: The tumor tissue removed from Example 3 was fixed with 10% paraformaldehyde for 24 hours, embedded in paraffin, made into paraffin sections, and then subjected to H&E staining and TUNEL staining.

[0068] Results: Figure 5 As shown in the figure, H&E results showed that in the animal Lactobacillus ATCC35046 treatment group, the tumor tissue had a large area of ​​necrosis, and the tumor cells showed swelling, necrosis, and nuclear fragmentation; TUNEL results showed that after animal Lactobacillus ATCC35046 treatment, the degree of tumor cell apoptosis was very serious. The results show that animal Lactobacillus ATCC35046 can promote the necrosis and apoptosis of tumor cells, thereby inhibiting tumor growth.

[0069] Example 7. Animal Lactobacillus ATCC35046 can significantly promote CD4 + and CD8 + Infiltration of T cells

[0070] Methods: After dewaxing and rehydrating the paraffin sections of the tumor tissue in Example 6, antigen retrieval was performed with a pH = 6 citrate buffer, and then incubated with specific anti-CD4 and CD8 primary antibodies at 4°C overnight, the primary antibodies were washed, and then incubated with PE-labeled anti-IgG (H+L) secondary antibodies at room temperature for 30 minutes, the secondary antibodies were washed, DAPI was counterstained, the sections were sealed, and observed under a fluorescence microscope.

[0071] Results: Figure 6 As shown, compared with the PBS group, the CD4 + and CD8 + The number of T cell infiltrations increased, indicating that animal lactobacillus ATCC35046 promotes the infiltration of T cells in tumor tissues, activates the immune response in tumors, and thus plays a role in killing tumor cells, achieving the effect of inhibiting tumor growth.

Claims

1. An application of animal lactobacillus in the preparation of anti-tumor drugs, characterized in that: Lactobacillus animalis is Lactobacillus animalis ( Lactobacillus animalis ) ATCC35046, and the tumor is melanoma.

2. The use according to claim 1, characterized in that: After the frozen animal Lactobacillus ATCC35046 was revived in solid culture medium, a single colony was picked and transferred to liquid culture medium for activation, and then expanded and cultured. When it grew to the logarithmic phase, the bacteria were obtained by centrifugation, washed 3 times with PBS, and resuspended in PBS for later use.

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