CN114350604A - MC38-N4/OT-I co-culture system screening method and application of screened polysaccharide formula and polysaccharide composition thereof - Google Patents

Abstract

The invention discloses a screening method for an MC38-N4/OT-I co-cultivation system and the application of the screened polysaccharide formula and polysaccharide composition. The steps of obtaining a polysaccharide composition based on the screening method include: obtaining tumor target cells containing specific antigens and fluorescent proteins, isolating immune effector cells, co-culturing tumor target cells and immune effector cells, and evaluating the immune regulation function of the polysaccharide formula. The co-culture screening method provided by the present invention has the advantages that the interaction process between tumor cells and immune cells is simulated based on the characteristics of the tumor microenvironment, and has the characteristics of wide application range, visualization of experimental results, high-throughput screening and the like. The polysaccharide composition provided by the invention comprises raw materials of Agaricus blazei, Grifola frondosa and dried tangerine peel. The composition has the functions of improving immune function of the body, enhancing anti-tumor efficacy, etc., and can be used for the development of immune-enhancing related products. It provides experimental basis and scientific basis for the application of the prescription to clinical immune-enhancing drugs.

Description

Screening method of MC38-N4/OT-I co-culture system and application of screened polysaccharide formula and polysaccharide composition

technical field

The invention belongs to the technical field of functional foods, and particularly relates to a screening method of an MC38-N4/OT-I co-cultivation system and the application of the screened polysaccharide formula and polysaccharide composition.

Background technique

The occurrence, growth and metastasis of tumors are closely related to the internal and external environment of tumor cells. Tumor micro-environment (TME) refers to the surrounding microenvironment in which tumor cells exist, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived cells, various signaling molecules and extracellular matrix. In recent years, immunotherapy has developed into the fourth anti-tumor therapy after surgery, chemotherapy and radiotherapy in cancer treatment because it can effectively stimulate the host immune system and promote immune cells in the tumor microenvironment to kill cancer cells. Studies have found that natural polysaccharides have strong anti-tumor and tumor-inhibiting effects, and compared with traditional tumor treatment methods, natural polysaccharides have the advantages of low toxicity and immune regulation. Food-derived natural polysaccharides not only have no obvious toxicity to most body cells, but also can stimulate the proliferation, differentiation and maturation of various immune active cells, so that the body's immune system can be restored and strengthened, so it is recognized as an important Biological effect modifier. Therefore, it is of great significance for cancer patients to find food-derived active polysaccharides that can be eaten for a long time and have less side effects.

At present, animal models for screening immunomodulatory active substances mainly include: xenograft models, syngeneic transplantation models, gene knockout models, genetically engineered mouse models, and human gene knock-in models. Compared with animal models, in vitro cell models have the advantages of short experimental period and high screening throughput. The models for in vitro screening of immunomodulatory active substances mainly include RAW264.7 cell polarization model, 2D co-culture model, organoid and microfluidic cell model. The ideal in vitro test for polysaccharide screening should closely simulate the tumor microenvironment in vivo, and perform high-throughput screening of immunotherapy with high sensitivity and specificity, so that the expected effective drugs can be further tested. The RAW264.7 cell polarization method cannot realistically simulate the complex interaction process between cells in the tumor microenvironment; although organoid models are widely used in cancer biopsy tissue cultures, they usually contain only tumor cells and not immune cells. Compared with single-cell in vitro screening models, co-culture cell models are more complex, biologically relevant, and predictive cell screening methods. Co-culture models can better simulate the in vivo environment and response to drug treatment. Suitable for compound screening. Based on the multicellular nature of the tumor microenvironment, the co-culture model of tumor cells and immune cells can simulate the interaction between cells in the tumor microenvironment and can be used to test the efficacy of immunotherapy and immunotherapy in vitro.

At present, it is also necessary to develop more effective in vitro cytotoxicity detection methods to evaluate the immunomodulatory efficacy of screened substances in the tumor microenvironment by quantifying the killing effect of immune cells in the tumor microenvironment on tumor cells. Among them, the ⁵¹ Cr release assay is the most common in vitro assay, which is usually used to measure T cell-mediated cytotoxicity, but this assay has radiotoxicity and its application is limited. Pimentel et al. proposed the use of firefly luciferase assay to evaluate the targeted killing mortality of tumor cells. The experimental process of this method is cumbersome, not only requires the addition of reaction substrates, but also requires rapid detection due to the short reaction time, and the experimental results cannot be visualized, so it is difficult to satisfy The need for experimental large-scale screening (Olivo Pimentel V, Yaromina A, Marcus D, Dubois LJ, Lambin PA novel co-culture assay to assess anti-tumor CD8+T cell cytotoxicity via luminescence and multicolor flowcytometry. J Immunol Methods. 2020 Dec;487:112899 .doi:10.1016/j.jim.2020.112899.Epub 2020Oct 15.PMID:33068606.). Transfecting tumor cells with fluorescent proteins, while ensuring no radiotoxicity, automatically image tumor target cells with microscopic imaging and high-content, high-throughput analysis tools, so as to realize the visualization of tumor cells in co-culture, and visualize the detection of co-culture killing of immune cells.

In summary, it is particularly necessary to develop a rapid screening visualization system with high throughput, good reproducibility, and can simulate the tumor microenvironment. Moreover, it is urgent for immunocompromised patients to screen out compounds that can improve the immune function of the body. .

SUMMARY OF THE INVENTION

In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a screening method for the MC38-N4/OT-I co-culture system and the application of the screened polysaccharide formulation and polysaccharide composition. The polysaccharide composition can promote immune cells to kill tumor cells, improve the viability and cell viability of CD8 ⁺ T cells, and promote the growth of CD8 ⁺ CD6L memory T cells, and can be used to prepare products for improving the immune function of the body.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

The present invention provides a high-throughput, rapid and repeatable screening method for MC38-N4/OT-I co-culture system for screening immunomodulatory active substances, comprising the following steps:

MC38-N4/OT-I co-culture system screening method, the MC38-N4/OT-I co-culture system screening method comprises the following steps:

(1) Obtain tumor target cells (Target, T) containing specific antigens and fluorescent proteins: Infect tumor target cells with the viral supernatant containing the recombinant plasmids containing specific antigens and fluorescent proteins, and add antibiotics to screen after virus infection, and continue the drug treatment. After screening, the fluorescent protein expression was observed under a fluorescence microscope. After monoclonal expansion and flow cytometry screening, visualized tumor target cells expressing specific antigens were obtained.

(2) Isolation of immune effector cells (Effector, E): Mice were sacrificed by cervical dislocation, the immune organs of the mice were taken aseptically, the immune organs of the mice were isolated, and lightly ground to obtain a single-cell suspension, cell strainer After filtration, erythrocytes were centrifuged and lysed, washed and resuspended to obtain a single-cell suspension of immune effector cells.

(3) Co-cultivation of tumor target cells and immune effector cells: The tumor target cells are plated, and after overnight adherence, the culture medium is discarded, immune effector cells are added in a certain proportion, and the compositions obtained in the examples to be screened are added at the same time, and co-cultured for a period of time Afterwards, the mortality of tumor target cells was detected.

Further, in step (1), the recombinant plasmid containing the specific antigen and fluorescent protein is the pLVX-IRES-tdTomato-N4 recombinant plasmid; the tumor target cells include: LLC mouse lung cancer cells, MC38 mouse colon cancer cells cells, B16 mouse melanoma cells or GL261 mouse glioblastoma cells; the antibiotics include more than one of puromycin, penicillin or streptomycin; the concentration of the antibiotics is 1 -10 μg/mL; the duration of the sieve is 4-15 days.

Further, in step (2), the mice include OT-I or OT-II mice, the CD8 ⁺ T cells of the OT-I mice have specificity for recognizing the N4 peptide of SIINFEKL, and the OT-II mice The CD4 ⁺ T cells have specificity for recognition of the N4 peptide of SIINFEKL.

Further, in step (2), the organ includes more than one of spleen, thymus or lymph node; the immune effector cells are OT-I whole spleen immune effector cells.

Further, the cell number ratio of the immune effector cells and tumor target cells in step (3) is (100-1):1, and the co-cultivation time in step (3) is 12-96 hours.

The present invention provides polysaccharide recipes screened by the MC38-N4/OT-I co-culture system screening method. The constituent raw materials of the recipes include: Agaricus blazei, Grifola frondosa, tangerine peel, Phellinus linteus, Ganoderma lucidum, Poria, More than one of Angelica and Lycium barbarum, the formula is more than one of Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, tangerine peel polysaccharide, Phellinus linteus polysaccharide, Ganoderma lucidum polysaccharide, Poria polysaccharide, Angelica polysaccharide and Lycium barbarum polysaccharide, the polysaccharide The immunomodulatory function screening evaluation of the prescription includes: (1) After co-culture of tumor cells and immune cells, aspirate the cell supernatant, wash the cells twice with phosphate-buffered saline (PBS), digest with trypsin, and collect the mixed cells by flow cytometry. In the tube, the cells were resuspended in staining buffer (FACS buffer) after centrifugation, and the number of tumor target cells was detected by a flow cytometer, and the polysaccharide composition was obtained by screening according to the mortality of tumor target cells; (2) using Cytation7 high The flux live cell imaging system performs high-throughput fluorescence imaging screening on the co-cultured cells, and the polysaccharide composition is obtained by screening the changes in the fluorescence quantity of tumor cells.

Further, the mass fraction ratio of the Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, tangerine peel polysaccharide and Phellinus linteus polysaccharide is (1-125) parts: (1-125) parts: (1-25) parts: (0- 25) servings.

Further, the Cytation7 high-throughput live cell imaging system is used to perform high-throughput fluorescence imaging screening on co-cultured cells for screening and evaluating substances with immunomodulatory activity; the detection is visualized by fluorescence imaging.

The present invention also provides the application of the polysaccharide composition in the preparation of a drug for enhancing the anti-tumor immune memory effect of CD8 ⁺ T cells, enhancing the body's immune activity and promoting anti-tumor efficacy.

Further, the enhancement of immune activity mainly lies in increasing the survival rate of CD8 ⁺ T cells and the ratio of CD8 ⁺ CD62L.

Further, the promotion of anti-tumor efficacy mainly lies in improving the killing effect of immune cells on tumor cells containing specific antigens.

Agaricus blazei, Grifola frondosa and tangerine peel in the composition of the present invention cooperate with each other to produce a synergistic effect, which can significantly improve the immune function of the body, and is a composition with immunoregulatory activity.

Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

1. The rapid screening method for co-culture immunomodulatory activity provided by the present invention includes four steps: construction of tumor target cells, isolation of immune effector cells, co-cultivation of tumor target cells-immune effector cells, and evaluation of immunomodulatory function. This method can simulate tumors. Different types of cells interact in the microenvironment, and compared with the detection method of ⁵¹ Cr, the detection process of this model does not generate radioactivity and has the advantages of visualization of tumor target cells.

2. The screening method of the MC38-N4/OT-I tumor cell and immune cell co-culture system provided by the present invention has a wide range of applications, can be used to screen various food-derived active substances, is simple and efficient, and is beneficial to the selection of immunomodulatory functional foods. Rapid development and promotion of applications.

3. The MC38-N4/OT-I co-culture system provided by the present invention is a multifunctional in vitro evaluation platform, which can be used to evaluate the killing effect of CD8 ⁺ T cells on tumor cells; it can also be used for high-throughput screening for in vivo models of immunomodulators. This method can shorten the experimental period and help to explore the molecular mechanism and target of immunotherapy drugs.

4. The polysaccharide composition provided by the present invention has the effect of improving the immune function of the body. The components of the composition cooperate with each other to significantly promote the killing effect of immune cells on tumor cells, and significantly improve the survival rate and cell viability of CD8 ⁺ T cells. It can promote the growth of CD8 ⁺ CD62L memory T cells and can be used to prepare products that improve the immune function of the body.

Description of drawings

FIG. 1 is a schematic diagram of the co-culture rapid screening method of MC38-N4/OT-I of the present invention.

Fig. 2 is a graph of bright field and red fluorescence field of MC38-tdTomato-N4 cells under a fluorescence microscope.

Figure 3 is a bar graph showing the effect of a single raw material on the proliferation toxicity of MC38-N4 cells.

FIG. 4 is a bar graph for evaluating the immune function-enhancing activity of Examples 1-3 and Comparative Examples 1-7 based on co-culture.

FIG. 5 is a gate graph of the polysaccharide composition of Example 1 on immune cells.

Figure 6 is a bar graph showing the effects of the polysaccharide composition of Example 1 and the control group on the survival rate of immune effector cells, the size of CD8 ⁺ T cells, the ratio of CD8 ⁺ T cells, and the ratio of CD8 ⁺ CD62L.

Detailed ways

The present invention provides a composition and its application in the preparation of a product for enhancing the immune function of the body, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

In the specific embodiments of the present invention, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available commodities.

In order to verify the feasibility of the rapid screening method for MC38-N4/OT-I co-culture immunomodulatory activity provided by the present invention, Examples 1-3 and Comparative Examples 1-7 are specifically listed, using Agaricus blazei, Grifola frondosa, tangerine peel respectively. , Phellinus linteus, Ganoderma lucidum, Poria, Angelica and Lycium barbarum extracts were composed of different formulas according to different proportions, and were added to the co-culture system for intervention treatment, and then the apoptosis rate of MC38-N4 cells was detected and statistically analyzed. Then screen and evaluate the immunomodulatory activity of different polysaccharide formulations. The specific experimental methods and test results are as follows:

Different raw material extract preparation methods include:

1. Preparation method of Agaricus blazei polysaccharide extract

Dry Agaricus mushrooms to constant weight, grind the dried raw materials into powder, and pass through a 200-mesh sieve. Distilled water was added at a ratio of 1:20 (m/m) of material to liquid, leached at 55°C for 2 hours, the times of leaching were 2 times, the two leaching solutions were combined, the leaching solution was centrifuged at 4 000 r/min for 10 min, and the supernatant was collected. The solution was concentrated under reduced pressure by rotary evaporation at 55°C. Add 3 times the volume of anhydrous ethanol of the concentrated solution, stand at 4°C for 24 hours, centrifuge at 4 000 r/min for 15 minutes, collect the precipitate, and vacuum freeze-dry to obtain Agaricus blazei crude polysaccharide.

2. Preparation method of Grifola frondosa polysaccharide extract

Dry the maitake to constant weight, grind the dried raw materials into powder, and pass through a 200-mesh sieve. Add distilled water at a ratio of 1:20 (m/m) of material to liquid, extract at 55°C for 2 hours, and the extraction times are 2 times. Combine the two extracts, centrifuge the extract at 4 000 r/min for 10 minutes, and then extract the supernatant. Concentrate under reduced pressure by rotary evaporation at 55°C. Add 3 times the volume of anhydrous ethanol of the concentrated solution, stand at 4°C for 24 hours, centrifuge at 4000 r/min for 15 minutes, collect the precipitate, and vacuum freeze-dry to obtain the crude polysaccharide of Grifola frondosa.

3. Preparation method of tangerine peel polysaccharide extract

Dry Agaricus mushrooms to constant weight, grind the dried raw materials into powder, and pass through a 200-mesh sieve. Distilled water was added at a ratio of 1:20 (m/m) of material to liquid, leached at 55°C for 2 hours, the times of leaching were 2 times, the two leaching solutions were combined, the leaching solution was centrifuged at 4 000 r/min for 10 min, and the supernatant was collected. The solution was concentrated under reduced pressure by rotary evaporation at 55°C. Add 3 times the volume of absolute ethanol to the crude polysaccharide solution, let stand at 4°C for 24 h, centrifuge at 4 000 r/min for 15 min, collect the precipitate, and vacuum freeze-dry to obtain the crude polysaccharide of tangerine peel.

4. Preparation method of Phellinus linteus polysaccharide extract

Dry Phellinus linteus to constant weight, grind the dried raw materials into powder, and pass through a 200-mesh sieve. Distilled water was added according to the ratio of material to liquid ratio of 1:20, leaching at 55 °C for 2 hours, the number of leaching was 2 times, the two leaching solutions were combined, the leaching solution was centrifuged at 4 000 r/min for 10 min, and the supernatant was rotary evaporated at 55 °C Concentrate under reduced pressure. Add 3 volumes of absolute ethanol to the crude polysaccharide solution, stand at 4°C for 24 h, centrifuge at 4 000 r/min for 15 min, collect the precipitate, and vacuum freeze-dry to obtain the crude polysaccharide.

5. Preparation method of Ganoderma lucidum, Poria, Angelica and Lycium barbarum extracts

Ganoderma lucidum, Poria, Angelica and Lycium barbarum were dried to constant weight, 1 mass part of raw materials were added to 40 mass parts of ethanol with a volume percentage concentration of 70%, concentrated and extracted in a 45°C water bath, suction filtration, and vacuum freeze-dried, respectively. Each extract was prepared.

Agaricus blazei polysaccharide extract, Grifola frondosa polysaccharide extract, tangerine peel polysaccharide extract and Phellinus linteus polysaccharide extract are mixed according to the formula to prepare a polysaccharide composition.

Example 1

The polysaccharide composition was prepared according to the following ratio: Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, dried tangerine peel polysaccharide and Phellinus linteus polysaccharide in a ratio of 125 parts: 1 part: 1 part: 0 part.

Example 2 (Comparative Example 6)

The polysaccharide composition was prepared according to the following ratio: Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, Phellinus linteus polysaccharide and tangerine peel polysaccharide were in a ratio of 1 part: 1 part: 5 parts: 1 part.

Example 3 (Comparative Example 7)

The polysaccharide composition was prepared according to the following ratio: Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, Phellinus linteus polysaccharide and tangerine peel polysaccharide were in a ratio of 25 parts: 125 parts: 25 parts: 125 parts.

Comparative Examples 1-7

1. Prepare the mixture according to the formula in Table 1:

Table 1 Composition formula (parts by mass)

According to formula table 1, the respective extracts of Agaricus blazei, Grifola frondosa, Phellinus linteus, dried tangerine peel, Ganoderma lucidum, Poria cocos, Angelica sinensis and Lycium barbarum were prepared to prepare Comparative Examples 1-7.

Efficacy verification

FIG. 1 is a schematic diagram of the co-culture rapid screening method of MC38-N4/OT-I of the present invention. As can be seen from Figure 1, after the immune effector cells were isolated from the spleen of OT-I mice, they were co-cultured with tumor target cells transfected with specific antigens and fluorescent proteins for 48 hours. During the co-culture process, immune effector cells recognize the specific antigen peptide N4 of tumor cells through T cell receptor (TCR). After recognition, the effector cells will release inflammatory factors, thereby promoting tumor cell apoptosis. Microscopy can reflect the apoptosis of tumor target cells.

Based on the co-culture system, the polysaccharide compositions of Examples 1-3 and the polysaccharide compositions of Comparative Examples 1-7 of the present invention were tested for their activity of improving immune function.

1. Experimental animals

The animal experiments were OT-I transgenic mice, which were raised under SPF (specific pathogen-free) conditions, the ambient temperature was 23±2°C, the relative air humidity was 60%, and the light and dark were cycled for 12 hours each day. Before dissecting the spleen tissue, cut the toe to lyse the DNA to identify the genotype.

After the genotype of the OT-I mice was confirmed, the subsequent mouse dissection operation was performed on ice. First, the mice were sacrificed by cervical vertebrae. After the mice were completely immersed in 75% alcohol for disinfection, the spleen tissue was quickly taken out and placed in pre-cooled RPMI 1640 complete medium. Place a nylon sieve with a pore size of 70 μm on a 50 mL centrifuge tube, remove the spleen tissue from the culture medium and place it on a 70 μm nylon membrane, and gently grind the spleen tissue with the piston plane of the syringe. The single-cell suspension of the spleen was filtered through a 70 μm cell strainer. Centrifuge and discard the supernatant to obtain the cell pellet. The pellet was resuspended with 1x erythrocyte lysate, the cells were mixed, and the erythrocytes were lysed by standing at room temperature for 5 minutes. Then, the lysis was terminated with 10 mL of pre-cooled FACS buffer (flow staining buffer). The complete medium was resuspended so that the resuspended cell concentration was 300,000/mL, and the obtained cells were OT-I spleen immune effector cells.

2. Obtain tumor target cells containing specific antigens and fluorescent proteins

One day before transfection, 0.5×10 ⁶ MC38 tumor target cells in good condition were inoculated into a 6-well plate, and 3 mL of medium was added to each well and mixed, and cultured in a carbon dioxide incubator. Add 4 μg pLVX-IRES-tdTomato-N4 recombinant plasmid and 4 μg retrovirus packaging plasmid to 500 μL OPTI-MEM medium respectively; mix well. 20 μL of lipo 2000 (Lipofectamine ^™ 2000) was added to 500 μL of OPTI-MEM medium and mixed well. Incubate at room temperature for 5 min. Add the premix of lipo 2000 to the DNA premix, mix well, and incubate at room temperature for 20 min. Discard the original medium and add the transfection reagent mix. After 5 h, the original medium was discarded and fresh complete medium was added. 1.2 μg/mL of puromycin was added 24 h after transfection. Depending on the cell growth density, passage or medium change is selected. After continuous drug screening for 7 days, the expression of red fluorescent protein was observed under a fluorescence microscope; after monoclonal expansion and flow cytometry sorting, visualized tumor target cells that could express specific antigens were obtained. The observation results under the fluorescence microscope are shown in Figure 2. The cells grow well in bright field. At the same time, it is observed that MC38 cells express tdTomato red fluorescent protein, and the expression level is high, indicating that the plasmid transfection is successful.

3. Co-culture of tumor target cells and immune effector cells

MC38-N4 cells were plated in 96-well plates at 6 000 cells/well. After overnight adherence, the medium was discarded, and the ratio of immune effector cells to tumor target cells (E:T) was 5:1. Add 100 μL of the above concentration of OT-I spleen immune effector cells at the ratio. At the same time, 100 μL of the object to be screened at twice the concentration was added, and the cells were co-cultured for 48 h, and the death rate of MC38-N4 cells with red fluorescent protein was detected.

4. Evaluation of immune regulation function

During co-culture, MC38-N4 target cells could be targeted and killed by CD8 ⁺ T effector cells in OT-I spleen, and the killing effect of co-culture was reflected by counting the number of MC38-N4 red fluorescent cells. There are two evaluation methods: a) directly use the Cytation7 high-throughput live cell imaging system to perform fluorescence imaging on the 96-well plate, and count the number of fluorescent cells by Gen5 software (Biotek); b) Aspirate the cell supernatant and wash with PBS The cells were digested twice with trypsin, and the cells were collected in a flow tube. After centrifugation, the cells were resuspended in a DAPI solution containing 10 μg/mL, and the MC38-N4 red fluorescent cells were detected by a flow cytometer FACS LSRII (BD FACSverse). quantity.

5. MTT method to determine the concentration range of the extract without obvious toxicity to MC38-N4 cells

The death of MC38-N4 cells in the co-culture system may come from the killing effect of OT-I cells on the one hand, and the direct toxic effect of the extract on MC38-N4 cells on the other hand. In order to exclude the direct toxic effect of the extract on MC38-N4 cells in the co-culture system, it is necessary to determine the dose range of each extract without obvious toxic effect on MC38-N4 cells.

The MC38-N4 cells in the logarithmic growth phase were digested, resuspended and counted, and then seeded in a 96-well plate at a density of 6×10 ⁴ cells/mL, with 100 μL per well. After culturing in the incubator to adhere to the wall, the blank control group was added with 200 μL of fresh complete medium, and the experimental group was added with four crude extracts of Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, tangerine peel polysaccharide and Phellinus linteus polysaccharide, and 10 μg of each polysaccharide was added respectively. /mL, 50 μg/mL, 250 μg/mL, 1250 μg/mL to the corresponding complete medium. After culturing for 48 hours in a constant temperature incubator at 37°C, 20 μL of 5 mg/mL MTT solution was added to each well. After continuing to culture for 4 hours, the supernatant culture medium was aspirated, and 150 μL of DMSO solution was added to each well to dissolve the formazan crystals. Absorbance values were detected at 490 nm using a fully functional microplate detection microplate reader. The ratio of the absorbance value of the experimental group to the absorbance value of the blank control group was the survival rate of MC38-N4 cells.

6. Based on the MC38-N4 co-cultivation system to screen the formulas with different ratios

According to the results of the MTT experiment, after determining the dose concentration range of the extract with no obvious toxicity to MC38-N4, 9 different comparative examples were designed according to the formula in Table 1. MC38-N4 cells were plated in 96-well plates at 6 000 cells/well. After overnight adherence, the medium was discarded, and 100 μL of the above concentration was added according to the ratio of effector cells: target cells (5:1). effector cells. At the same time, 100 μL of twice the concentration of the object to be screened was added, and co-cultured for 48 h.

After co-culture, the cell supernatant was aspirated, the cells were washed twice with PBS, digested with trypsin, and the cells were collected in a flow tube. After centrifugation, cells were resuspended in 200 μL of DAPI solution containing 10 μg/mL, and the number of MC38-N4 red fluorescent cells was detected by flow cytometry analyzer FACS LSRII (BDFACSverse).

7. In vitro stimulation experiment of spleen cells with the polysaccharide composition of Example 1

The mice were sacrificed by de-neck, and the OT-I mice were dissected to obtain the whole spleen, which was ground into a single-cell suspension. Centrifuge and discard the supernatant to obtain the cell pellet. The pellet was resuspended with 1 mL of 1x erythrocyte lysate, and the erythrocytes were lysed by standing at room temperature for 5 min. Then, the lysis was terminated with 10 mL of pre-cooled FACS buffer. After mixing, the pellet was centrifuged to obtain a pellet. Antigen polypeptide fragment) peptide K medium was resuspended to 2 million/mL, and plated into 6-well plates, 1 mL per well or 2 million/well. The control group and the experimental group without the polysaccharide composition were added with 100IU/mL of IL-2 (interleukin-2), the experimental group was added with twice the concentration of the polysaccharide composition of Example 1, and the control group was supplemented with culture medium. to 2mL. After culturing for 48 h, the cells were collected in flow tubes, 1 mL of FACS buffer was added to each tube, and the supernatant was discarded by centrifugation. The Fc receptor blocker was diluted with FACS buffer, 50 μL was added to each tube, shaken and mixed, and let stand on ice for 15 min in the dark. Staining extracellular markers: Anti-CD8, Anti-TCRβ, Anti-CD62L; after the antibody was diluted with FACS buffer, the dilution ratio was 1:150 (v/v), add 50 μL to each tube for staining, shake evenly, and place on ice Protect from light for 30min. After staining, 1 mL of FACS buffer was added to each tube, washed once, and centrifuged to discard the supernatant. Add 200 μL of FACS buffer to each tube, vortex to mix cells, and then detect by flow cytometer.

Analysis of results

1. Analysis of MTT experimental results of four polysaccharide extracts of Agaricus blazei polysaccharide, Grifola frondosa polysaccharide, tangerine peel polysaccharide and Phellinus linteus polysaccharide on MC38-N4

In order to exclude the direct toxic effect of the extract on MC38-N4 cells in the co-culture system, it is necessary to determine the dose range of each extract without obvious toxic effect on MC38-N4 cells. As shown in Figure 3, Grifola frondosa has no obvious cytotoxic effect on MC38-N4 cells in the concentration range of 10-250 μg/mL, and Agaricus blazei has no obvious effect on MC38-N4 cells in the concentration range of 10-1250 μg/mL. The cytotoxic effect of tangerine peel was not obvious to MC38-N4 cells in the concentration range of 10-1250 μg/mL, and Phellinus linteus had no obvious cytotoxicity to MC38-N4 cells in the concentration range of 50-250 μg/mL. effect. Therefore, a concentration without obvious dose toxicity was selected for the next co-culture experiment.

2. Analysis of co-cultivation experiment results

According to the dose of raw material without obvious toxicity, different comparative examples are designed according to Table 1. The killing effect of OT-I spleen immune cells on MC38-N4 cells was detected by flow cytometry. The results are shown in Figure 4. Compared with the control group without immune effector cells (E:T=5:0), The control group (E:T=5:1) supplemented with immune effector cells significantly promoted the death of tumor target cells. Compared with the control group (E:T=5:1), the polysaccharide composition of Example 1 significantly promoted the activity of OT-I immune cells in the co-culture system, and the activity was increased by 22%. The formula can promote immune cells to exert better tumor killing function, and is used in the preparation of medicines for enhancing immune activity of the body and promoting anti-tumor efficacy.

3. Analysis of the results of the polysaccharide composition in Example 1 promoting the proliferation and activation of CD8 ⁺ T cells in vitro

In order to further clarify the regulation mode of the polysaccharide composition of Example 1 on immune enhancement, the effect of the polysaccharide composition of Example 1 on the proliferation and activation of immune cells was examined. Figure 5 shows the schematic diagram of the circle gate of the polysaccharide composition of Example 1 on the OT-I spleen immune cells. It can be seen from Figure 5 that the lymphocytes are circled from all cells, and the single lymphocytes are circled after removing the adherent cells. T cells were grouped and circled in the live single lymphocytes, and finally the CD62L+ positive cell group was circled. The statistical results of flow data are shown in Figure 6. The results show that compared with the control group (E:T=5:1) without the polysaccharide composition, the survival rate of spleen immune cells is 16.13%±0.41%. The survival rate of spleen immune cells of 1 polysaccharide composition was 32.33%±0.33%, and the polysaccharide composition of Example 1 could significantly enhance the survival rate of spleen immune cells; compared with the control group without the polysaccharide composition (E:T=5:1 ) compared with the proportion of CD8 ⁺ T cells (19.87% ± 0.75%), the proportion of CD8 ⁺ T cells added with the polysaccharide composition of Example 1 was 33.03% ± 1.64%, and the polysaccharide composition of Example 1 can also significantly increase CD8 ⁺ Proportion of T cells. At the same time, the polysaccharide composition of Example 1 can also promote the activation of CD8 ⁺ T cells, and the relative volume of CD8 ⁺ T cells in the control group without the polysaccharide composition (E:T=5:1) is 100.00%±0.10% In contrast, the relative volume of CD8 ⁺ T cells added with the polysaccharide composition of Example 1 was 102.40%±0.08%, and the polysaccharide composition of Example 1 resulted in a significant increase in the relative volume of CD8 ⁺ T cells by 2%; by further testing the polysaccharide of Example 1 The effect of the composition on CD8 ⁺ central memory T cells, compared with the ratio of CD8 ⁺ CD62L T cells in the control group without the polysaccharide composition (E:T=5:1) was 83.07%±2.62%, it was found that The proportion of CD8 ⁺ CD62L T cells added with the polysaccharide composition of Example 1 was 95.40% ± 0.23%. The polysaccharide composition of Example 1 significantly increased the proportion of CD8 ⁺ CD62L T cells and promoted the anti-tumor effect of CD8 ⁺ T cells, suggesting that the implementation of Example 1 The polysaccharide composition can be used in immunotherapy to improve the anti-tumor activity of CD8 ⁺ T immune cells, and the composition can be used in adoptive T cell-mediated tumor therapy.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principles of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be It is regarded as the protection scope of the present invention.

Claims (10)

1. The method for screening the MC38-N4/OT-I co-culture system is characterized by comprising the following steps of:

   (1) transfecting tumor target cells by using recombinant plasmids containing specific antigens and fluorescent proteins, adding antibiotics for screening after transfection, and observing the expression condition of the fluorescent proteins under a fluorescent microscope after continuous drug screening; obtaining a visual tumor target cell expressing a specific antigen through monoclonal propagation and flow cell sorting;

   (2) separating immune organ tissues of the mouse, grinding and cracking red blood cells to obtain a single cell suspension of immune effector cells;

   (3) after the tumor target cells are plated and adhered to the wall, immune effector cells are added, and simultaneously, the substances to be screened are added for co-culture.
2. 2. The MC38-N4/OT-I co-culture system screening method of claim 1, wherein in step (1), the recombinant plasmid containing specific antigen and fluorescent protein is pLVX-IRES-tdTomato-N4 recombinant plasmid; the tumor target cells include: one or more of LLC mouse lung cancer cell, MC38 mouse colon cancer cell, B16 mouse melanoma cell or GL261 mouse glioblastoma cell; the antibiotic comprises more than one of puromycin, penicillin or streptomycin; the concentration of the antibiotic is 1-10 mug/mL; the time of the medicine sieving is 4-15 days.
3. 3. The MC38-N4/OT-I co-culture system screening method of claim 1, wherein in step (2), the mice comprise OT-I or OT-II mice; the viscera comprises more than one of spleen, thymus or lymph node; the immune effector cell is OT-I whole spleen immune effector cell.
4. 4. The MC38-N4/OT-I co-culture system screening method as claimed in any one of claims 1-3, wherein the ratio of the number of immune effector cells to the number of tumor target cells in step (3) is (100-1): 1, the co-culture time in the step (3) is 12-96 h.
5. 5. The polysaccharide formulation screened by the screening method of the MC38-N4/OT-I co-culture system of claim 4, wherein the polysaccharide formulation comprises the following raw materials: the immune regulation function screening and evaluation method comprises the following steps of selecting and evaluating one or more of agaricus blazei murrill, grifola frondosa, dried orange peel, phellinus igniarius, ganoderma lucidum, poria cocos, angelica sinensis and lycium barbarum, wherein the polysaccharide formula is one or more of agaricus blazei murrill polysaccharide, grifola frondosa polysaccharide, dried orange peel polysaccharide, phellinus igniarius polysaccharide, ganoderma lucidum polysaccharide, pachyman, angelica sinensis polysaccharide and lycium barbarum polysaccharide, and the immune regulation function screening and evaluation of the polysaccharide formula comprises the following steps: (1) after co-culturing the tumor cells and the immune cells, carrying out pancreatin digestion, collecting the mixed cells in a flow tube, carrying out centrifugation, then carrying out heavy suspension on the cells, detecting the number of the tumor target cells, and screening according to the death rate of the tumor target cells to obtain a polysaccharide composition; (2) and (3) carrying out high-throughput fluorescence imaging screening on the co-cultured cells by using a rotation 7 high-throughput living cell imaging system, and screening according to the change of the fluorescence quantity of the tumor cells to obtain the polysaccharide composition.
6. 6. The MC38-N4/OT-I co-culture system screening method of claim 5, wherein the polysaccharide composition comprises (1-125) Agaricus blazei Murill polysaccharide, Grifola frondosa polysaccharide, pericarpium Citri Tangerinae polysaccharide and Phellinus linteus polysaccharide in parts by weight: (1-125) parts: (1-25) parts of: (0-25).
7. 7. The polysaccharide composition screened by the MC38-N4/OT-I co-culture system screening method according to claim 5, wherein the co-culture cells are subjected to high-throughput fluorescence imaging screening by using a rotation 7 high-throughput living cell imaging system for screening and evaluating substances with immunoregulatory activity; the detection is visualized by fluorescence imaging.
8. 8. Use of the polysaccharide composition of claim 5 for the preparation of a CD 8-enhancing composition⁺T cell anti-tumor immunological memory effect, and can be used for enhancing organism immunocompetence and promoting anti-tumor effect.
9. 9. Use of the polysaccharide composition according to claim 8, wherein the enhancement of the immune activity is mainly due to the increase of CD8⁺Survival of T cells and CD8⁺Proportion of CD 62L.
10. 10. Use of a polysaccharide composition according to claim 8, wherein the anti-tumor promoting effect is mainly characterized by an increased killing effect of immune cells against tumor cells containing specific antigens.

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