Application of lactobacillus in treatment of gastric cancer

Abstract

The invention discloses an application of lactobacillus in treatment of gastric cancer. The lactobacillus is at least one of lactobacillus salivarius with the preservation number of CICC23175 and lactobacillus crispatus with the preservation number of CGMCC1.2743. The lactobacillus is at least one of lactobacillus salivarius with the preservation number of CICC23175 and lactobacillus crispatus with the preservation number of CGMCC1.2743. According to the present invention, the lactobacillus salivarius CICC23175 or the lactobacillus crispatus CGMCC1.2743, which has the excellent inhibition effect on the proliferation of SGC7901, AGS and MKN45 gastric cancer cells, is found; a mouse solid tumor model experiment shows that the lactobacillus salivarius CICC23175 or the lactobacillus crispatus CGMCC1.2743 can be used for inhibiting the growth of gastric cancer transplanted tumors. According to the invention, viable bacteria of lactobacillus salivarius CICC23175 or lactobacillus crispatus CGMCC1.2743 in lactobacillus are directly applied to treatment of gastric cancer for the first time, and a new direction and basis can be provided for treatment of gastric cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of lactobacillus in treatment of gastric cancer. BACKGROUND

[0002] Gastric cancer (GC) is one of the most common malignant tumors worldwide. The pathogenesis of gastric cancer is complex, involving multiple factors such as genetic mutations, environmental pollution, dietary habits, and changes in gastrointestinal microorganisms. Although certain progress has been made in recent years in treatment methods such as surgery, chemotherapy, and targeted therapy, due to the heterogeneity of tumors and the difficulty in early diagnosis, the high mortality rate of gastric cancer has not been effectively controlled, and traditional treatment methods such as chemotherapy and radiotherapy, while killing cancer cells, also cause damage to normal cells, resulting in serious side effects such as nausea, vomiting, hair loss, and decreased immunity, greatly affecting the quality of life of patients, and some patients also have drug resistance problems, resulting in a significant reduction in treatment effectiveness. Therefore, finding new targeted treatment strategies and improving treatment effectiveness have become a hot spot in current research on the treatment of gastric cancer.

[0003] Recent studies have shown that lactobacillus not only plays an important role in maintaining the balance of host microbial communities, but is also closely related to the occurrence, development, and immune regulation of cancer. The distribution difference of lactobacillus in normal tissues and cancer tissues may provide new clues for the diagnosis, treatment, and prevention of cancer. Lactobacillus can maintain the acidic environment of the intestinal tract through its metabolic products (such as lactic acid), inhibit the growth of pathogenic bacteria, maintain the integrity of the intestinal barrier, and interact with the host immune system, enhance the immune response in the intestinal tract, regulate immune cells in the intestinal tract, and play an anti-inflammatory role. However, there is currently no research on the direct application of lactobacillus in the treatment of gastric cancer. SUMMARY

[0004] Based on the above background, the present application provides the application of lactobacillus in the treatment of gastric cancer. By detecting the distribution of lactobacillus in the gastric cancer tissue, its paired paracancer tissue, and the distal normal tissue of a gastric cancer patient, it is found that saliva lactobacillus and crimple lactobacillus have a potential inhibitory effect on gastric cancer. Through in-depth research, a saliva lactobacillus and a crimple lactobacillus that are excellent in inhibiting the proliferation of gastric cancer cells and the growth of cancer transplanted tumors are found, which can provide a new direction and basis for the treatment of gastric cancer.

[0005] The technical scheme provided by the present application is as follows:

[0006] The application of lactobacillus in the preparation of a drug for treating gastric cancer, wherein the lactobacillus is at least one of the following 1) or 2) biological bacteria:

[0007] 1) Lactobacillus salivarius, preservation number CICC23175, preserved in China Industrial Microorganism Bacteria Preservation Management Center;

[0008] 2) Lactobacillus crispatus, preservation number CGMCC1.2743, preserved in China General Microorganism Preservation Management Center.

[0009] Based on the same inventive concept, the present application provides an application of the bacteria agent in preparation of a stomach cancer cell inhibitor and / or a stomach cancer treatment drug, wherein the bacteria agent contains the Lactobacillus, and the Lactobacillus is one of live Lactobacillus and heat-inactivated Lactobacillus.

[0010] Preferably, the bacteria agent is a suspension of Lactobacillus.

[0011] Preferably, the suspension is prepared by resuspending the Lactobacillus in physiological saline.

[0012] Based on the same inventive concept, the present application further provides an application of Lactobacillus and bacteria agent in a non-diagnostic and treatment-related stomach cancer occurrence or treatment mechanism research experiment, wherein the Lactobacillus is at least one of the following 1) or 2):

[0013] 1) Lactobacillus salivarius, preservation number CICC23175, preserved in China Industrial Microorganism Bacteria Preservation Management Center;

[0014] 2) Lactobacillus crispatus, preservation number CGMCC1.2743, preserved in China General Microorganism Preservation Management Center.

[0015] Further, the stomach cancer is a stomach cancer based on one of SGC7901 stomach cancer cell line, AGS stomach cancer cell line and MKN45 stomach cancer cell line.

[0016] Based on the same inventive concept, the present application further provides a stomach cancer treatment drug, which contains an effective amount of Lactobacillus; and the Lactobacillus is at least one of the following 1) or 2):

[0017] 1) Lactobacillus salivarius, preservation number CICC23175, preserved in China Industrial Microorganism Bacteria Preservation Management Center;

[0018] 2) Lactobacillus crispatus, preservation number CGMCC1.2743, preserved in China General Microorganism Preservation Management Center.

[0019] Preferably, the Lactobacillus is one of live Lactobacillus or heat-killed Lactobacillus.

[0020] Preferably, the medicine comprises a Lactobacillus suspension.

[0021] Preferably, the suspension is prepared by resuspending Lactobacillus in physiological saline.

[0022] Preferably, the live Lactobacillus is the only active ingredient.

[0023] Preferably, the concentration of live Lactobacillus is 1x10 8 CFU / 20 μL.

[0024] The present application has the following beneficial effects:

[0025] (1) The present application detects the distribution of Lactobacillus in gastric cancer tissues of gastric cancer patients, their paired paracancer tissues and distal normal tissues, and finds five Lactobacillus with high distribution in the paired paracancer tissues and distal normal tissues. Through further research, it is found that Lactobacillus salivarius CICC23175 or Lactobacillus crispatus CGMCC1.2743 has particularly excellent inhibitory effect on the proliferation of SGC7901, AGS and MKN45 gastric cancer cells.

[0026] Through solid tumor model experiments in mice, it is found that Lactobacillus salivarius CICC23175 or Lactobacillus crispatus CGMCC1.2743 can inhibit the growth of gastric cancer transplanted tumors.

[0027] (2) The present application first directly applies live Lactobacillus salivarius CICC23175 or Lactobacillus crispatus CGMCC1.2743 in the treatment of gastric cancer, which can provide a new direction and basis for the treatment of gastric cancer.

[0028] Drawings of the specification

[0029] Figure 1 The drawings of the specification are the results of detecting the distribution differences of five Lactobacillus in gastric cancer tissues of gastric cancer patients, their paired paracancer tissues and distal normal tissues in Example 1 of the present application.

[0030] Figure 2 The present application is Lactobacillus salivarius CICC23175 and Lactobacillus crispatus CGMCC1.2743 inhibiting the proliferation of gastric cancer cells in Example 2 of the present application: wherein Figure 2 A is the effect of five Lactobacillus on gastric cancer cells AGS; Figure 2B. The effect of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 on the proliferation of MKN45. Figure 2 C. The effect of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 on the proliferation of SGC7901. WN: Normal gastric mucosa, WP: Para-carcinoma, WC: Gastric cancer tissue. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 3 B. The effect of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 on the proliferation of MKN45. Figure 3 A. The effect of L. salivarius CICC23175 on the proliferation of gastric cancer cell line AGS; Figure 3 B. The effect of L. salivarius CICC23175 on the proliferation of gastric cancer cell line SGC7901; Figure 3 C. The effect of L. crispatus CGMCC1.2743 on the proliferation of gastric cancer cell line AGS. Figure 3 D. The effect of L. crispatus CGMCC1.2743 on the proliferation of gastric cancer cell line SGC7901. HK: Heat Killed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 4 B. The effect of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 on the proliferation of MKN45. Figure 4 A. The volume of the excised tumor of 615 mice; Figure 4 B. The weight of the excised tumor of 615 mice; Figure 4 C. The growth curve of the tumor of 615 mice. n=8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0033] Figure 5The results of immunohistochemical detection of the expression of Ki67 in the intratumor injection of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 in the gastric cancer cell transplanted tumor of 615 mice in Example 3 of the present application are as follows: Figure 5 A is the expression of Ki67 protein in the transplanted tumor tissue of BALB / c nude mice detected by immunohistochemistry; Figure 5 B is the statistical rate of Ki67 positive cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0034] Figure 6 The effects of intratumor injection of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 on the subcutaneous tumor formation of immunodeficient BALB / c nude mice in Example 3 of the present application are as follows: Figure 6 A is the volume of the transplanted tumor stripped from BALB / c nude mice; Figure 6 B is the weight of the transplanted tumor stripped from BALB / c nude mice; Figure 6 C is the growth curve of the transplanted tumor of BALB / c nude mice. n=6, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0035] Figure 7 The results of immunohistochemical detection of the expression of Ki67 in the intratumor injection of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 in the gastric cancer cell transplanted tumor of BALB / c nude mice in Example 3 of the present application are as follows: Figure 7 A is the expression of Ki67 protein in the transplanted tumor tissue of BALB / c nude mice detected by immunohistochemistry; Figure 7 B is the statistical rate of Ki67 positive cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] The reagents, kits and instruments used in the following embodiments can be obtained from the market, and the methods used in the embodiments are consistent with the commonly used methods unless otherwise specified.

[0038] The cell lines used in the following examples are SGC7901, MKN45, AGS 3 human gastric cancer cell lines, purchased from the Chinese Academy of Sciences Cell Bank. The cells were identified by STR, and mycoplasma detection showed no mycoplasma contamination. The SCG-7901, MKN45, AGS gastric cancer cell lines were cultured in 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a 5% CO2, 37°C constant temperature incubator. The mouse gastric cancer cell line MFC was purchased from Wuhan Punsai Life Science and Technology Co., Ltd. The cells were identified by STR, and mycoplasma detection showed no mycoplasma contamination. Cultured in 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a 5% CO2, 37°C constant temperature incubator.

[0039] Example 1: Distribution differences of lactobacillus in gastric cancer tissues of gastric cancer patients, their paired cancer-adjacent tissues, and distal normal tissues

[0040] Gastric cancer (WC), cancer-adjacent cancer (WP), and distal normal gastric mucosa tissue (WN) collected 5 cm from the matched tumor tissue (WC) of 20 gastric cancer patients who underwent surgical treatment at the Fifth Affiliated Hospital of Zhengzhou University (Zhengzhou, China) from January 2018 to August 2019. Samples were collected from the gastric mucosa tissue of 10 non-gastric cancer individuals as non-cancer controls (NC). Patients who used antibiotics within 3 months, were addicted to drugs or alcohol, or had a history of abdominal tumor were excluded from the samples. The sampling and experimental protocols of this example were approved by the Ethics Committee of the Fifth Affiliated Hospital of Medical College of Zhengzhou University and the Medical Research Ethics Committee of the Fifth Affiliated Hospital of Zhengzhou University (KY2020029). Written informed consent was obtained from all patients before recruitment, and the study strictly followed the guidelines of the 1964 Helsinki Declaration and its latest amendments.

[0041] (1) 16S rRNA gene full-length amplicon sequencing

[0042] 1) Total DNA extraction: First, cut the gastric cancer tissue and non-cancer tissue stored in liquid nitrogen into 1-2 mm small pieces, put them into a homogenizer tube, add buffer, homogenize and break, and release bacteria. Then incubate at 55-65°C for 1-3h, digest the protein with proteinase K, and occasionally invert the centrifuge tube during the process until the solution becomes clear or the viscosity is significantly reduced. Next, add an equal volume of chloroform and mix well, centrifuge at 12000-15000 rpm for 10-15 min to separate the aqueous phase, and take the supernatant. Add an equal volume of isopropanol, incubate at -20°C, then centrifuge at 12000-15000 rpm for 10-15 min to collect the DNA precipitate. Wash the DNA precipitate with 70% ethanol, centrifuge at 12000-15000 rpm for 2 min to remove ethanol and dry. Dissolve the DNA with an appropriate amount of TE buffer and detect the DNA quality and concentration using gel electrophoresis or spectrophotometer.

[0043] 2) Full-length amplification of 16S rRNA gene: Extract and purify DNA from bacterial samples, ensuring a concentration of 10-100 ng / μL. Use primer pairs targeting the conserved region of 16S rRNA gene for amplification, with forward primer 27F (5'-AGGTTYGATYMTGGCTAG-3') and reverse primer 1492R (5'-RGYTACCTTGTTACGACTT-3'). The 25 μL PCR reaction system includes: 12.5 μL 2x Taq PCR Master Mix, 1 μL of each upstream and downstream primer, 2 μL of DNA template, and 8.5 μL of sterile deionized water to make up the total volume of 25 μL. The PCR amplification conditions are set as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55-60°C annealing for 30 s (adjust according to the Tm value of the primer), 72°C extension for 1 min, a total of 30-35 cycles, and finally 72°C extension for 10 min. Subsequently, the PCR product is detected by 1% agarose gel electrophoresis to confirm the size of the amplified band and the purity of the band. The amplified product is purified using a PCR purification kit and quantified using a DNA quantification kit. Use the DNA / polymerase binding kit to sequence the library on the PacBio platform.

[0044] (2) Through 16S rDNA sequencing of 20 gastric cancer samples and paired adjacent and normal tissues collected in the clinic, it was found that Lactobacillus salivarius, Lactobacillus agilis, Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus paracasei were enriched in adjacent and normal tissues, but were not detected in gastric cancer tissues Figure 1

[0045] Example 2: In vitro cancer inhibition experiment verification of L. salivarius CICC23175 and L. crispatus CGMCC1.2743

[0046] In order to further verify the effect of the five kinds of Lactobacillus screened in Example 1 on inhibiting gastric cancer, L. salivarius CICC23175, L. paracasei CGMCC1.9089, L. johnsonii CGMCC1.10778, L. crispatus CGMCC1.2743, and L. agilis CGMCC1.3914 standard strains were purchased for verification.

[0047] Lactobacillus salivarius CICC23175 lyophilized powder was purchased from China Industrial Microbial Strain Preservation and Management Center. Lactobacillus paracasei CGMCC1.9089 lyophilized powder, Lactobacillus johnsonii CGMCC1.10778 lyophilized powder, Lactobacillus crispatus CGMCC1.2743 lyophilized powder, and Lactobacillus agilis CGMCC1.3914 lyophilized powder were purchased from China General Microorganism Preservation and Management Center. The Lactobacillus strains were cultured in MRS liquid medium in a 37°C saturated humidity anaerobic incubator, and subcultured every 24 hours.

[0048] (1) Experiment on the effect of Lactobacillus on the proliferation activity of gastric cancer cells.

[0049] Gastric cancer cells AGS, SGC7901, and MKN45 were inoculated in 96-well plates, 100 μL of cell suspension was added to each well, and the cell density was 5×10 4 ​The cells were incubated at 37°C in 5% CO2 for 24 h in 1640 medium containing 10% fetal bovine serum.

[0050] Each group of gastric cancer cells was divided into an intervention group and a control group. Different MOIs of non-inactivated lactobacilli were added to each group of gastric cancer cells, and the cells were cultured for 24 h. The cell supernatant was then discarded, and the cells were washed with PBS buffer. Then, 100 μL of cell culture medium containing a CCK8 detection reagent (10 μL of CCK-8 reagent and 90 μL of cell culture medium) was added to each well, and the cells were incubated for another 2 h. Then, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The OD values of each group reflected the cell proliferation activity. The control group was cultured with blank cell culture medium, and the detection was performed after the intervention group was cultured. Results: The effects of five strains of lactobacilli on the proliferation of different gastric cancer cell lines were verified by treating the gastric cancer cell lines with different MOIs of lactobacilli, respectively. The results are shown in Table 1. Figure 2 As shown in Table 1, L. salivarius CICC23175 and L. crispatus CGMCC1.2743 had better inhibitory effects on the proliferation of different gastric cancer cell lines compared to other strains. L. salivarius CICC23175 and L. crispatus CGMCC1.2743 both had inhibitory effects on the proliferation of gastric cancer cells.

[0051] (2) Effect of the activity of lactobacilli on the proliferation activity of gastric cancer cells AGS and SGC7901

[0052] L. salivarius CICC23175 and L. crispatus CGMCC1.2743 were grown in MRS broth at 37°C under micro-aeration conditions for 24 h. Then, the culture solution was adjusted to a concentration of 1.5 x 10 10 CFU / mL, and centrifuged at 5000 rpm for 15 min. The bacterial pellet was washed and centrifuged with PBS, resuspended with PBS, and heated in a metal bath at 100°C for 30 min to inactivate the lactobacilli. The inactivated lactobacilli were obtained and labeled as L. salivarius CICC23175 HK and L. crispatus CGMCC1.2743 HK, respectively. Then, gastric cancer cells AGS and SGC7901 were intervened with different MOIs of non-inactivated / heat-inactivated L. salivarius and non-inactivated / heat-inactivated L. crispatus, respectively. The proliferation activity of the gastric cancer cells was detected after 24 h of incubation in a 5% CO2, 37°C incubator.

[0053] The results show that the heat-inactivated L. salivarius CICC23175 and L. crispatus CGMCC1.2743 have weaker inhibitory effect on the proliferation of gastric cancer cells AGS and SGC7901 than the un-inactivated L. salivarius CICC23175 and L. crispatus CGMCC1.2743. Figure 3 )。

[0054] Example 3: In-vivo cancer inhibition experiment verification of L. salivarius CICC23175 and L. crispatus CGMCC1.2743

[0055] 1. In this example, 4-week-old male BALB / c nude mice (immunodeficient) and 615 mice (wild type) with a body weight of about 20 g were first normally raised in a separate air-supplied cage for 1 week to adapt to the environment, and then subsequent experiments were carried out.

[0056] The MFC mouse gastric cancer cell suspension (purchased from Wuhan Punsai Life Science and Technology Co., Ltd. The cells were identified by STR, and mycoplasma detection showed no mycoplasma contamination. The cells were cultured in 1640 complete culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a 5% CO2, 37°C constant temperature incubator.) was adjusted to an appropriate concentration (200,000 cells / 100 μL), and was injected subcutaneously into the right forelimb of the two types of mice through a 1 mL syringe. After injection, the inoculation site was gently pressed to avoid cell liquid overflow. After inoculation, the mice were continued to be raised under standard feeding conditions, and the growth of the tumor was regularly monitored. After 4-5 days, the tumor size was observed, and when the tumor grew to the size of a soybean, the mice were grouped into a control group (Saline), a L. salivarius CICC23175 group, a L. crispatus CGMCC1.2743 group, a L. salivarius CICC23175 group was injected intratumorally with a physiological saline suspension of live bacteria with a concentration of 1x10 8 CFU / 20 μL of L. salivarius CICC23175 lactobacillus suspension (20 μL), and a L. crispatus CGMCC1.2743 group was injected intratumorally with a physiological saline suspension of live bacteria with a concentration of 1x10 8 CFU / 20 μL of L. crispatus CGMCC1.2743 lactobacillus suspension. During the operation, intratumoral injection was avoided to avoid bleeding and to avoid subcutaneous blood vessels. After injection, the needle was rotated and pressed with a cotton swab for a few moments to avoid leakage. Intratumoral injection was performed every 2-3 days, and the long diameter (L) and short diameter (W) were measured according to the formula V = (L x W2 During the experiment, the health status of the mice was checked regularly, and after 2 weeks, the mice were sacrificed under anesthesia, and the tumor tissues were immediately photographed and weighed. The tumor tissues were divided into small pieces according to the experimental needs. Part of the tumor tissues was fixed in 4% paraformaldehyde for paraffin section analysis, and another part was immediately frozen for molecular biology detection.

[0057] 2. Immunohistochemistry

[0058] 1) Tissue fixation: The tumor tissues were removed and fixed in 4% paraformaldehyde at room temperature for 24 h.

[0059] 2) Dehydration and embedding: The fixed tissues were sequentially placed in different concentrations of alcohol, from 50%, 60%, 70%, 80%, 90% to 100% alcohol, with each concentration gradient treated for 30 min to replace the water in the tissues with alcohol. Then, the tissues were transparentized by placing them in xylene for 2-3 times, 20-30 min each time, to further transparentize the tissues and facilitate paraffin infiltration. Subsequently, the tissues were infiltrated with paraffin by placing them in melted paraffin in a constant temperature oven for 2-3 times, 1 h each time, to allow the paraffin to fully infiltrate the tissues. After completing the paraffin infiltration, the tissues were embedded into paraffin blocks using an embedding machine.

[0060] 3) Tissue sectioning: After embedding, the paraffin blocks were sectioned using a microtome to produce 4 μm thick sections, which were placed on glass slides and then placed in a slide warmer at 65°C for 2 h to firmly adhere the sections.

[0061] 4) De-paraffinization and hydration: The sections were sequentially placed in xylene for 2 times, 10 min each time, and then hydrated through a gradient of alcohol, from 100%, 95%, 85% to 70% alcohol, 5 min each, and finally rinsed with distilled water for 2 times, 5 min each.

[0062] 5) Antigen retrieval: The sections were heated in a microwave oven using sodium citrate buffer (pH 6.0) for 15-30 min. Then, the sections were cooled to room temperature and washed with PBS for 3 times, 5 min each. Next, 3% hydrogen peroxide solution was added for 10 min to remove endogenous peroxidase activity.

[0063] 6) Immunohistochemical staining: Circle the tissue around the section with an immunohistochemical pen, and drop 5% goat serum blocking solution to reduce non-specific binding. After the blocking solution is poured out, the primary antibody (specific antibody for the target protein) is diluted to the appropriate concentration and added to the section, and incubated overnight at 4°C. The next day, take it out to room temperature, wash with PBS buffer for 3 times, 5 min each time. Then drop the secondary antibody and incubate at room temperature for 60 min, and then wash with PBS buffer for 3 times, 5 min each time. Prepare DAB developing solution according to the instructions and drop it on the section, observe the color development under the microscope, and wash with distilled water to stop the color development in time. Then put the section into hematoxylin staining solution for 1-2 min, rinse with tap water, and then differentiate with hydrochloric acid alcohol for a few seconds, and then rinse with tap water to return to blue. Finally, dehydrate and transparently mount the section, dehydrate the section with gradient alcohol, 70%, 85%, 95% to 100% alcohol, 5 min each, then put it into xylene for transparency 2 times, 5 min each time, drop neutral gum and cover with a cover glass. After completing the mounting, observe the section under the microscope and take pictures, and use ImageJ software to count the percentage of positive cells.

[0064] 3. Results

[0065] The above examples have verified that L. salivarius CICC23175 and L. crispatus CGMCC1.2743 have obvious inhibitory effect on gastric cancer cell lines in vitro experiments.

[0066] And in the mouse model, compared with the control group, the volume and weight of the tumors of the two groups of mice intervened by L. salivarius CICC23175 and L. crispatus CGMCC1.2743 were significantly smaller than those of the control group Figure 4 A- Figure 4 C). Immunohistochemical experiments detected the expression of cell proliferation marker Ki67, and the results showed that the positive expression rate of Ki67 in the implanted tumors of mice in the L. salivarius CICC23175 and L. crispatus CGMCC1.2743 groups was significantly lower than that of the control group Figure 5 ).

[0067] 4. The above experiments have verified that intratumoral injection of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 has obvious inhibitory effect on the subcutaneous tumor formation of 615 mice with immune ability. The following experiments will further intervene in the same way in the immune-deficient mice, and the experimental operation is the same as above. The results show that in the immune-deficient BALB / c-nude mice, intratumoral injection of L. salivarius CICC23175 and L. crispatus CGMCC1.2743 can also effectively inhibit the growth rate of transplanted gastric cancer tumor (C), tumor volume (A) and tumor weight (B). Figure 6 C), tumor volume ( Figure 6 A) and tumor weight ( Figure 6 B). Similarly, the expression of Ki67 detected by immunohistochemistry is lower in the L. salivarius CICC23175 and L. crispatus CGMCC1.2743 intervention group than in the control group ( Figure 7 ).

[0068] The above describes the present application and its embodiments, which is not limited, and the examples shown are only one of the embodiments of the present application, and the actual structure is not limited thereto. In general, if a person skilled in the art is inspired, without departing from the purpose of the present application, without creative design, similar structure and examples of the technical solution should belong to the protection scope of the present application.

Claims

1. The application of Lactobacillus in the preparation of drugs for treating gastric cancer, characterized in that, The lactobacillus is at least one of the following 1) or 2): 1) Lactobacillus salivarius, accession number CICC23175, is deposited at the China Industrial Microbial Culture Collection Center; 2) Lactobacillus crispatus, accession number CGMCC1.2743, is deposited at the China General Microbiological Culture Collection Center.

2. The application of microbial agents in the preparation of gastric cancer cell inhibitors and / or drugs for treating gastric cancer, characterized in that, The microbial agent contains the Lactobacillus as described in claim 1, wherein the Lactobacillus is either a live Lactobacillus or a heat-inactivated Lactobacillus.

3. The application according to claim 2, characterized in that, The bacterial agent is a suspension of Lactobacillus.

4. The application according to claim 3, characterized in that, The suspension was prepared by resuspending the lactobacillus in physiological saline.

5. The application of the Lactobacillus of claim 1 and the bacterial agent of any one of claims 2 to 4 in experiments related to the study of the occurrence and / or treatment mechanisms of gastric cancer for non-diagnostic and non-therapeutic purposes, characterized in that, The lactobacillus is at least one of the following 1) or 2): 1) Lactobacillus salivarius, accession number CICC23175, is deposited at the China Industrial Microbial Culture Collection Center; 2) Lactobacillus crispatus, accession number CGMCC1.2743, is deposited at the China General Microbiological Culture Collection Center.

6. The application according to claim 5, characterized in that, The gastric cancer mentioned is gastric cancer based on one of the following cell lines: SGC7901, AGS, and MKN45.

7. A drug for treating gastric cancer, characterized in that, It includes an effective amount of lactobacilli; said lactobacilli are at least one of the following 1) or 2): 1) Lactobacillus salivarius, accession number CICC23175, is deposited at the China Industrial Microbial Culture Collection Center; 2) Lactobacillus crispatus, accession number CGMCC1.2743, is deposited at the China General Microbiological Culture Collection Center.

8. The drug for treating gastric cancer according to claim 7, characterized in that, The lactobacillus is either a live lactobacillus or a heat-inactivated lactobacillus.

9. A drug for treating gastric cancer according to claim 8, characterized in that, The drug includes a lactobacillus suspension.

10. A drug for treating gastric cancer according to any one of claims 7 to 9, characterized in that, The lactobacillus is the only active ingredient.

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