Application of bifidobacterium longum BL21 in preparation of tumor treatment synergist

Abstract

The invention relates to a brand new application of bifidobacterium longum BL21, in particular to an application of the bifidobacterium longum BL21 in preparation of a tumor treatment synergist. The BL21 strain provided by the invention can effectively improve the treatment effect of conventional tumor treatment means such as radiotherapy and / or alphaPD-L1 antibody intervention. Specifically, compared with pure radiotherapy and / or alphaPD-L1 antibody intervention, the intervention of the bifidobacterium longum BL21 can further prolong the survival time of a patient, reduce the tumor growth speed, regulate intestinal microbiota, regulate the cholesterol metabolism level and change the tumor immune microenvironment, and has a prevention effect on cancer cell proliferation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and relates to a brand-new use of Bifidobacterium longum BL21, in particular to application of Bifidobacterium longum BL21 in preparation of a tumor treatment synergist. BACKGROUND

[0002] In recent years, tumor immunotherapy has become an important strategy for clinical treatment and has significantly changed the traditional tumor treatment mode. However, its clinical application still faces the bottleneck problem of low response rate, which leads to poor treatment effect on various cancers and seriously limits the wide application and development of immunotherapy.

[0003] Studies have shown that cholesterol, as a key regulatory molecule for human health, is closely related to the occurrence, development and metastasis of tumors. The cholesterol level in the tumor microenvironment can significantly affect the cell surface expression of immune checkpoint molecules, programmed cell death-ligand 1 (PD-L1) and programmed cell death-1 (PD-1), and further regulate the anti-tumor effect of immunotherapy.

[0004] At the same time, with the progress of genomics, high-throughput sequencing and microbial culture technology, the role of intestinal microbiome in maintaining host immune homeostasis and disease regulation has become increasingly prominent. Intestinal flora can affect cholesterol metabolism through direct metabolic regulation or indirect immune regulation, thereby locally or systematically changing the host immune microenvironment and ultimately affecting the response efficiency of tumor immunotherapy.

[0005] Intestinal microorganisms are closely related to the regulation of the host immune system. In addition, the synergistic potential of intestinal microorganisms and mainstream treatment methods has attracted widespread attention and become a new breakthrough for improving anti-tumor efficacy.

[0006] Therefore, developing an intestinal microbial preparation capable of enhancing the efficacy of tumor treatment not only has important scientific significance but also has significant clinical translation value. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a brand-new use of Bifidobacterium longum BL21, in particular to application of Bifidobacterium longum BL21 in preparation of a tumor treatment synergist.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides application of Bifidobacterium longum BL21 in preparation of a tumor treatment synergist.

[0010] The Bifidobacterium longum BL21 is a Bifidobacterium longum strain with a preservation number of CGMCC No.10452.

[0011] The BL21 strain involved in the present application is named Bifidobacterium longum, and is preserved in China General Microbiological Culture Collection Center on January 27, 2015, with a preservation number of CGMCC No.10452, and an address of No.3, Beichen West Road, Chaoyang District, Beijing.

[0012] Preferably, the tumor treatment includes tumor radiotherapy, tumor immunotherapy or tumor radioimmunotherapy combined treatment.

[0013] Preferably, the tumor immunotherapy includes αPD-L1 antibody treatment.

[0014] Preferably, the tumor includes melanoma, colorectal cancer or liver cancer.

[0015] Preferably, the number of the Bifidobacterium longum BL21 in the synergist is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g, for example, 1×10 10 CFU / mL (CFU / g), 2×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc.; other specific point values in the numerical range can be selected, and thus will not be described here.

[0016] Preferably, the dosage form of the synergist includes powder, tablet, granule or solution.

[0017] Preferably, the dosage of the synergist is (1×10 11 -1×10 14 )CFU / 60kg person / day, for example, 1×10 11 CFU / 60kg person / day, 5×10 11 CFU / 60kg person / day, 1×10 12 CFU / 60kg person / day, 5×10 12 CFU / 60kg person / day, 1×10 13 CFU / 60kg person / day, 5×1013 CFU / 60kg human / day, etc. Other specific point values within this range can be selected, and are not listed here.

[0018] In the present application, the Bifidobacterium longum BL21 inhibits tumor growth and prolongs the survival of tumor patients.

[0019] In the present application, the Bifidobacterium longum BL21 reduces the cholesterol level in serum and tumor.

[0020] In the present application, the Bifidobacterium longum BL21 reduces the PD-L1 content in serum and tumor.

[0021] In a second aspect, the present application provides the use of Bifidobacterium longum BL21 in the preparation of a microbial preparation for enhancing the therapeutic effect of tumor treatment.

[0022] The Bifidobacterium longum BL21 is the Bifidobacterium longum BL21 strain with the preservation number of CGMCC No. 10452.

[0023] Preferably, the tumor treatment includes tumor radiotherapy, tumor immunotherapy, or tumor radioimmunotherapy combined treatment.

[0024] Preferably, the tumor immunotherapy includes αPD-L1 antibody treatment.

[0025] Preferably, the tumor includes melanoma, colorectal cancer, or liver cancer.

[0026] Preferably, the number of Bifidobacterium longum BL21 in the microbial preparation is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g, for example, 1×10 10 CFU / mL (CFU / g), 2×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc. Other specific point values within this range can be selected, and are not listed here.

[0027] Preferably, the dosage form of the microbial preparation includes powder, tablet, granule, or solution.

[0028] Preferably, the amount of the microbial preparation is (1×10 11 -1×1014 ) 1 x 10 11 CFU / 60kg human / day, 5 x 10 11 CFU / 60kg human / day, 1 x 10 12 CFU / 60kg human / day, 5 x 10 12 CFU / 60kg human / day, 1 x 10 13 CFU / 60kg human / day, 5 x 10 13 CFU / 60kg human / day, 1 x 10

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The BL21 strain involved in the present application can effectively improve the therapeutic effect of conventional tumor treatment methods such as radiotherapy and / or αPD-L1 antibody intervention. Specifically, compared with simple radiotherapy and / or αPD-L1 antibody intervention, the intervention of Bifidobacterium BL21 can further prolong the survival time of patients, reduce the growth rate of tumors, regulate intestinal microbiota, regulate cholesterol metabolism levels, change tumor immune microenvironments, and have a preventive effect on cancer cell proliferation. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a tumor growth curve graph of each group of mice in Example 1;

[0032] Figure 2 is a survival curve graph of each group of mice in Example 1;

[0033] Figure 3 is a tumor growth curve graph of each group of mice in Example 2;

[0034] Figure 4 is a survival curve graph of each group of mice in Example 2;

[0035] Figure 5 is a total cholesterol content statistical result graph in the serum of each group of mice in Example 3;

[0036] Figure 6 is a total cholesterol content statistical result graph in the tumor of each group of mice in Example 3;

[0037] Figure 7 is a PD-L1 content statistical result graph in the serum of each group of mice in Example 3;

[0038] Figure 8 is a PD-L1 content statistical result graph in the tumor of each group of mice in Example 3;

[0039] Figure 9 is a graph of the expression level of tumor cell surface PD-L1 of each group of mice in Example 3;

[0040] Figure 10 is a graph of the liver cancer infiltration area of each group of rabbits in Example 4;

[0041] Figure 11 is a graph of the survival curve of each group of rabbits in Example 4. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0043] The Bifidobacterium longum subsp. longum BL21 freeze-dried powder involved in the following content is purchased from Weikang Probiotics (Suzhou) Co., Ltd. (1.0 x 10 12 CFU / g); the antibody aPD-L1 is InVivoMAb anti-mouse PD-L1 (B7-H1), brand: BioXcell; the B16F10 melanoma cells are purchased from the China General Microbiological Culture Collection Center; the MC38 colorectal cancer cells are purchased from the China General Microbiological Culture Collection Center; the VX2 cells (rabbit anaplastic epidermal squamous carcinoma strain) are purchased from the China General Microbiological Culture Collection Center.

[0044] All animal experiments in the following content meet the requirements of national regulations and are approved by the Experimental Animal Ethics Committee of Suzhou University.

[0045] Example 1

[0046] Intervention effect of BL21 on melanoma mice:

[0047] (1) Experimental animals: 6-8 week old female C57BL / 6J mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were placed in a pathogen-free environment and had free access to food and water during the experiment. The light / dark cycle was maintained for 12 hours, the temperature was 20-25℃, and the humidity was 60%-70%.

[0048] (2) Modeling method: The in vitro cultured B16F10 melanoma cells were trypsinized and peeled off, counted, and then resuspended in sterile PBS to adjust the cell concentration to 1 x 10 7

[0049] (3) Experimental grouping and intervention method:

[0050] ​(3.1) Ctrl group (control group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, no treatment + 0.2 mL of normal saline by gavage per mouse per day;

[0051] (3.2) BL21 group (probiotic intervention group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, no treatment + 0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) by gavage per mouse per day;

[0052] (3.3) RT group (radiotherapy group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, radiotherapy + 0.2 mL of normal saline by gavage per mouse per day; wherein the specific mode of radiotherapy is that the tumor of the mouse is locally irradiated once with X-rays (21 Gy / time, dose rate of 1.1 Gy / min) on the 7th day of the experiment.

[0053] (3.4) aPD-L1 group (intraperitoneal injection of aPD-L1 antibody group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, intraperitoneally inject 200 pg of aPD-L1 antibody per mouse on the 7th, 11th, 15th, and 19th days of the experiment + 0.2 mL of normal saline by gavage per mouse per day;

[0054] (3.5) BL21+RT group (BL21+radiotherapy group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, radiotherapy + 0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) by gavage per mouse per day; wherein the specific mode of radiotherapy is the same as above;

[0055] (3.6) BL21+aPD-L1 group (BL21+intraperitoneal injection of aPD-L1 antibody group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, intraperitoneally inject 200 pg of aPD-L1 antibody per mouse on the 7th, 11th, 15th, and 19th days of the experiment + 0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) by gavage per mouse per day;

[0056] (3.7) RT+aPD-L1 group (radiotherapy+intraperitoneal injection of aPD-L1 antibody group, n=5): subcutaneously inoculate B16F10 melanoma cells in mice, radiotherapy + intraperitoneally inject 200 pg of aPD-L1 antibody per mouse on the 7th, 11th, 15th, and 19th days of the experiment + 0.2 mL of normal saline by gavage per mouse per day; wherein the specific mode of radiotherapy is the same as above;

[0057] (3.8) BL21+RT+αPD-L1 group (BL21+radiotherapy+intraperitoneal injection of αPD-L1 antibody group, n=5): B16F10 melanoma cells were inoculated subcutaneously in mice, and radiotherapy+intraperitoneal injection of 200 μg of αPD-L1 antibody in each mouse on the 7th, 11th, 15th and 19th day of the experiment+0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) was administered by gavage to each mouse per day; wherein the specific method of radiotherapy is the same as above.

[0058] The probiotic dose of the above probiotic intervention group is equivalent to 2.4x10 11 CFU BL21 per day for a 60 kg adult according to the body surface area conversion formula.

[0059] The experiment lasted for 30 days, and if the tumor volume reached 1500 mm 3 , the individual experiment endpoint was considered, and the mice were euthanized.

[0060] (4) Index determination:

[0061] (4.1) Tumor growth curve:

[0062] The tumor growth curves of mice in each group are shown in Figure 1 From the figure, it can be seen that the tumor growth rate of each group of gavage BL21 (BL21+αPD-L1, BL21+RT and BL21+RT+αPD-L1 groups) is significantly lower than that of the group without gavage BL21 (RT, αPD-L1, RT+αPD-L1 groups). At the same time, the gavage BL21 group alone does not affect the tumor growth rate compared with the Ctrl group.

[0063] (4.2) Survival curve:

[0064] The experiment took the tumor of the mouse reaching 1500 mm 3 as the individual experiment endpoint, and the survival rate of each group during the experiment was calculated. The survival curves of mice in each group are shown in Figure 2 From the figure, it can be seen that the survival time of mice in the gavage BL21 group (BL21+αPD-L1, BL21+RT and BL21+RT+αPD-L1 groups) is significantly increased compared with the group without gavage BL21 (RT, αPD-L1, RT+αPD-L1 groups); and by the 30th day of the experiment, the BL21+RT+αPD-L1 group still had a survival rate of 100%.

[0065] Example 2

[0066] Intervention effect of BL21 on colorectal cancer mice:

[0067] (1) Experimental animals: 6-8 week-old female C57BL / 6J mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were placed in a pathogen-free environment, and free access to food and water during the experiment, 12 hours light / dark cycle, temperature 20-25℃, humidity 60%-70%.

[0068] (2) Modeling method: The in vitro cultured MC38 colorectal cancer cells were trypsinized and stripped, counted and resuspended in sterile PBS, and the cell concentration was adjusted to 0.5x10 7 CFU / mL. 0.1 mL of the above cell suspension was taken with a 1 mL syringe and slowly injected subcutaneously into the right lower back of C57BL / 6J female mice.

[0069] (3) Experimental grouping and intervention method:

[0070] (3.1) Ctrl group (control group, n=6): subcutaneously inoculate MC38 colorectal cancer cells in mice, no treatment + 0.2 mL of physiological saline per day per oral administration;

[0071] (3.2) BL21 group (probiotic intervention group, n=6): subcutaneously inoculate MC38 colorectal cancer cells in mice, no treatment + 0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) per day per oral administration;

[0072] (3.3) RT group (radiotherapy group, n=6): subcutaneously inoculate MC38 colorectal cancer cells in mice, radiotherapy + 0.2 mL of physiological saline per day per oral administration; The specific method of radiotherapy is as follows: on the 9th, 13th, 17th and 21st day of the experiment, the tumor of the mice was locally irradiated with X-rays (5Gy / time, dose rate 1.1Gy / min) once.

[0073] (3.4) αPD-L1 group (intraperitoneal injection of αPD-L1 antibody group, n=6): subcutaneously inoculate MC38 colorectal cancer cells in mice, intraperitoneally inject 200 μg of αPD-L1 antibody per mouse on the 9th, 13th, 17th and 21st day of the experiment + 0.2 mL of physiological saline per day per oral administration;

[0074] (3.5) BL21+RT group (BL21+radiotherapy group, n=6): subcutaneously inoculate MC38 colorectal cancer cells in mice, radiotherapy + 0.2 mL of BL21 bacterial solution (5x10 9 CFU / mL) per day per oral administration; The specific method of radiotherapy is as follows:

[0075] (3.6) BL21 + aPD-L1 group (BL21 + intraperitoneal injection of aPD-L1 antibody group, n = 6): subcutaneous inoculation of MC38 colorectal cancer cell mice, intraperitoneal injection of 200 ug aPD-L1 antibody in each mouse on the 9th, 13th, 17th, 21st day of the experiment + 0.2 mL BL21 bacterial solution (5 x 10 9 CFU / mL) per day per mouse by gavage; wherein the specific mode of radiotherapy is the same as above.

[0076] (3.7) RT + aPD-L1 group (radiotherapy + intraperitoneal injection of aPD-L1 antibody group, n = 6): subcutaneous inoculation of MC38 colorectal cancer cell mice, radiotherapy + intraperitoneal injection of 200 ug aPD-L1 antibody in each mouse on the 9th, 13th, 17th, 21st day of the experiment + 0.2 mL physiological saline per mouse per day by gavage; wherein the specific mode of radiotherapy is the same as above.

[0077] (3.8) BL21 + RT + aPD-L1 group (BL21 + radiotherapy + intraperitoneal injection of aPD-L1 antibody group, n = 6): subcutaneous inoculation of MC38 colorectal cancer cell mice, radiotherapy + intraperitoneal injection of 200 ug aPD-L1 antibody in each mouse on the 9th, 13th, 17th, 21st day of the experiment + 0.2 mL BL21 bacterial solution (5 x 10 9 CFU / mL) per day per mouse by gavage; wherein the specific mode of radiotherapy is the same as above.

[0078] The probiotic dose of the above probiotic intervention group is equivalent to 2.4 x 10 11 CFU BL21 per day for a 60 kg adult human according to the species body surface area conversion formula.

[0079] The experiment lasted for 50 days, during which if the tumor volume reached 1500 mm 3 , the individual experiment endpoint was considered, and the mice were euthanized.

[0080] (4) Index determination:

[0081] (4.1) Tumor growth curve:

[0082] The tumor growth curves of mice in each group are shown in Figure 3 The results show that the tumor growth rate of each group of mice gavaged with BL21 (BL21 + aPD-L1, BL21 + RT and BL21 + RT + aPD-L1 groups) is significantly lower than that of the group without gavage BL21 (RT, aPD-L1, RT + aPD-L1 groups). At the same time, the gavage BL21 group alone does not affect the tumor growth rate compared with the Ctrl group.

[0083] (4.2) Survival curve:

[0084] The experiment reached 1500 mm3 As the experimental endpoint for each individual mouse, euthanasia was assumed, and the survival rate of each group during the experiment was calculated. The survival curves for each group of mice are shown below. Figure 4 As shown in the figure, the survival time of mice in the BL21 gavage groups (BL21+αPD-L1, BL21+RT, and BL21+RT+αPD-L1 groups) was significantly increased compared with the BL21-free groups (RT, αPD-L1, and RT+αPD-L1 groups); and by day 50 of the experiment, the BL21+RT+αPD-L1 group still had a survival rate of over 60%.

[0085] Example 3

[0086] The intervention effects of BL21 on serum and tumor cholesterol and PD-L1 levels:

[0087] (1) Experimental animals: 6-8 week old female C57BL / 6J mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The mice were placed in a pathogen-free environment with free access to food and water during the experiment, and a 12-hour light / dark cycle was maintained at a temperature of 20-25℃ and a humidity of 60%-70%.

[0088] (2) Modeling method: MC38 colorectal cancer cells cultured in vitro were digested and excised with trypsin, counted, and then resuspended in sterile PBS to adjust the cell concentration to 0.5 × 10⁻⁶ cells / mL. 7 Cells / mL. Using a 1mL syringe, draw 0.1mL of the above cell suspension and slowly inject it subcutaneously into the right lower back of a female C57BL / 6J mouse.

[0089] (3) Experimental grouping and intervention methods:

[0090] (3.1) Ctrl group (control group, n=5): Mice subcutaneously inoculated with MC38 colorectal cancer cells, with no treatment and 0.2 mL of physiological saline per mouse per day by gavage, and euthanized on day 14 after inoculation;

[0091] (3.2) BL21 group (probiotic intervention group, n=5): Mice subcutaneously inoculated with MC38 colorectal cancer cells, without any treatment, and administered 0.2 mL of BL21 bacterial solution (5 × 10⁻⁶) per mouse by gavage daily from 14 days before inoculation to 14 days after inoculation. 9 (CFU / mL)

[0092] (4) Index Measurement:

[0093] (4.1) Total cholesterol (TC) content:

[0094] After euthanasia, tumor tissue was collected from each group of mice. The tumor tissue was rinsed thoroughly with pre-chilled PBS and then minced. 100 mg of the tissue fragment was weighed and added to approximately 1 mL of pre-chilled physiological saline. The tissue was then homogenized using a KZ-III high-speed tissue homogenizer (60 Hz, 45 s homogenate, 15 s pause, repeated twice). The homogenate was centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was gently aspirated and transferred to a new, pre-chilled microcentrifuge tube placed on ice; this was the tumor sample. Serum samples were obtained from each group of mice and placed in an anticoagulant tube. The blood was centrifuged at 4°C and 3000 rpm for 10 min, and the supernatant was collected. The total cholesterol levels in both tumor and blood samples were determined using a total cholesterol assay kit.

[0095] The results are as follows Figure 5 (serum total cholesterol level) and Figure 6 As shown in the (total cholesterol content in tumors) data, compared to the Ctrl group, the BL21 group showed a significant decrease in serum total cholesterol, approximately 2.24 times lower, and a 1.6-fold decrease in total cholesterol (TC) in the tumor. This indicates that BL21 alters the gut microbiota structure, thereby affecting cholesterol metabolism and reducing cholesterol levels in both the body and tumors.

[0096] (4.2) PD-L1 content:

[0097] The content of tumor and blood samples from the previous step was detected using a PD-L1 kit.

[0098] The results are as follows Figure 7 (serum PD-L1 level) and Figure 8 As shown in the results (PD-L1 content in tumors), compared with the Ctrl group, the BL21 group showed a significant decrease in PD-L1 levels in both the tumor and serum.

[0099] (4.3) In addition, the expression of PD-L1 on the surface of tumor cells was detected by flow cytometry.

[0100] like Figure 9 As shown, compared with the Ctrl group, the BL21 group resulted in a 1.52-fold decrease in the PD-L1 content on the surface of tumor cells (Figure a), and a reduction of approximately 1.5-fold in the immunosuppressive molecule PD-1 on the surface of CD3+ T cells and CD8+ T cells (Figure b and c).

[0101] Example 4

[0102] The intervention effect of BL21 on in situ hepatocellular carcinoma:

[0103] (1) Experimental animals: New Zealand rabbits were placed in a pathogen-free environment and had free access to food and water during the experiment. They were kept in a 12-hour light / dark cycle with a temperature of 20-25℃ and a humidity of 60%-70%.

[0104] (2) Modeling method: VX2 cells cultured in vitro were digested and detached with trypsin, counted, and then resuspended in sterile PBS to adjust the cell concentration to 0.5 × 10⁻⁶. 7 VX2 tumors were implanted in the left and right legs of rabbits using a 1 mL syringe at a concentration of 0.2 mL per mL. Three weeks later, the VX2 tumors were removed from the subcutaneous tissue and cut into pieces of 1-2 mm in size. 3 Small tissue blocks, for later use.

[0105] Before in situ liver tumor implantation, the rabbit must fast for 8 hours. After anesthesia, following aseptic procedures, the rabbit is fixed in a supine position on a rabbit table, and hair is removed from the upper abdomen. Following aseptic procedures, the skin of the surgical area is disinfected, and a midline incision of approximately 3 cm is made below the xiphoid process. Next, the skin and muscle are incised layer by layer to expose the left or midline lobe of the liver. A single layer of gauze, cut into small pieces with a small hole in the middle, is placed on the surface of the liver (to prevent the tumor from implanting along the liver capsule). Using ophthalmic forceps, a tunnel is formed by inserting the forceps through the gauze hole and into the liver parenchyma about 0.5-1.0 cm deep along the liver capsule. Two to three small tumor fragments are placed deep into the tunnel, and the tunnel is then filled with gelatin to stop bleeding. After local treatment, 2-3 mL of gentamicin is sprinkled on the liver capsule, and the incision is sutured. The wound is disinfected, gauze is applied, and the rabbit is observed for 20 minutes before being returned to its cage.

[0106] (3) Experimental grouping and intervention methods:

[0107] (3.1) Ctrl group (control group, n=3): rabbits with VX2 tumor blocks inoculated in the liver, no treatment + 20mL of physiological saline per rabbit by gavage every day;

[0108] (3.2) BL21 group (probiotic intervention group, n=3): Rabbits with VX2 tumor blocks inoculated in the liver, no treatment + 20mL of BL21 bacterial solution (5×10) per rabbit by gavage daily. 10 CFU / mL);

[0109] (3.3) RT group (radiotherapy group, n=3): Rabbits with VX2 tumor blocks inoculated in the liver were given radiotherapy plus 20 mL of physiological saline by gavage per rabbit per day; the specific method of radiotherapy was: the tumors of mice were irradiated locally with X-rays (15 Gy / time, dose rate 1.5 Gy / min) once on days 18, 25 and 32 of the experiment.

[0110] (3.4) BL21+RT group (BL21+radiotherapy group, n=3): Rabbits with VX2 tumor blocks inoculated into the liver, radiotherapy + 20mL of BL21 bacterial solution (5×10) per rabbit by gavage daily. 10 CFU / mL); the specific method of radiotherapy is the same as above;

[0111] The probiotic dosage in the above probiotic intervention group, calculated using the species body surface area conversion formula, is equivalent to a daily intake of 1.08 × 10⁸ probiotics for a 60kg adult. 13 CFU BL21.

[0112] This experiment lasted for 35 days. During this period, if the tumor volume reached 8cm... 3 If the result is not achieved, the individual experimental endpoint is considered reached, and the New Zealand rabbit is euthanized.

[0113] (4) Index Measurement:

[0114] (4.1) Curve of liver cancer infiltration area:

[0115] After performing magnetic resonance imaging on New Zealand rabbits, the area of ​​in situ VX2 hepatocellular carcinoma was delineated and statistically analyzed. Tumor growth curves for each group of mice are shown below. Figure 10 As shown in the figure, compared with the Ctrl group, the BL21 group significantly reduced the growth rate and infiltrative area of ​​rabbit orthotopic VX2 hepatocellular carcinoma. Simultaneously, compared with the RT group, the BL21+RT group also significantly reduced the growth rate and infiltrative area of ​​rabbit orthotopic VX2 hepatocellular carcinoma.

[0116] (4.2) Survival curve:

[0117] The experiment used rabbits with tumors reaching 8cm. 3 As the endpoint of the experiment for each individual rabbit, euthanasia was assumed, and the survival rate of each group during the experiment was calculated. The survival curves of the rabbits in each group are shown below. Figure 11 As shown in the figure, the survival rate of rabbits in the BL21 and BL21+RT groups was significantly higher than that in the Ctrl and RT groups; on day 35, the BL21+RT group still had a 100% survival rate. Furthermore, during the experiment, rabbits in the Ctrl and RT groups experienced natural deaths, but this did not occur in the BL21 and BL21+RT groups.

[0118] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0119] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0120] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of Bifidobacterium longum BL21 in the preparation of tumor therapeutic enhancers; The Bifidobacterium longum BL21 strain is the Bifidobacterium longum strain with accession number CGMCC No.10452.

2. The application according to claim 1, characterized in that, The tumor treatment includes tumor radiotherapy, tumor immunotherapy, or a combination of tumor radioimmunotherapy.

3. The application according to claim 2, characterized in that, Tumor immunotherapy includes αPD-L1 antibody therapy.

4. The application according to claim 1, characterized in that, The tumors include melanoma, colorectal cancer, or liver cancer.

5. The application according to claim 1, characterized in that, The number of Bifidobacterium longum BL21 in the synergist is not less than 1×10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

6. The application according to claim 1, characterized in that, The dosage form of the synergist includes powder, tablet, granule or solution.

7. The application according to claim 1, characterized in that, The dosage of the synergist is (1×10) 11 -1×10 14 CFU / 60kg person / day.

8. The application according to claim 1, characterized in that, The Bifidobacterium longum BL21 inhibits tumor growth and prolongs the survival of cancer patients.

9. The application according to claim 1, characterized in that, The Bifidobacterium longum BL21 reduces cholesterol levels in serum and tumors.

10. The application according to claim 1, characterized in that, The Bifidobacterium longum BL21 reduces PD-L1 levels in serum and tumors.

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