CN114350604A - MC38-N4/OT-I co-culture system screening method and application of screened polysaccharide formula and polysaccharide composition thereof - Google Patents
MC38-N4/OT-I co-culture system screening method and application of screened polysaccharide formula and polysaccharide composition thereof Download PDFInfo
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Abstract
The invention discloses a screening method of an MC38-N4/OT-I co-culture system, a screened polysaccharide formula and application of a polysaccharide composition. The steps of obtaining the polysaccharide composition based on the screening method comprise: obtaining tumor target cells containing specific antigens and fluorescent proteins, separating immune effector cells, co-culturing the tumor target cells and the immune effector cells, and evaluating the immune regulation function of a polysaccharide formula. The co-culture screening method provided by the invention has the advantages that: the method simulates the interaction process of tumor cells and immune cells based on the characteristics of a tumor microenvironment, and has the characteristics of wide application range, visual experimental results, high-throughput screening and the like. The polysaccharide composition provided by the invention comprises the raw materials of agaricus blazei, grifola frondosa and dried orange peel, and the formula has the effects of improving the body immunity, enhancing the anti-tumor efficacy and the like, can be used for developing products related to immunity enhancement, and provides experimental basis and scientific basis for the application of the food-borne polysaccharide formula to clinical immunity enhancement medicaments.
Description
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-culture system, a screened polysaccharide formula and an application of a polysaccharide composition.
Background
The occurrence, growth and metastasis of tumors are closely related to the internal and external environment of tumor cells. Tumor Microenvironment (TME) refers to the surrounding microenvironment in which Tumor cells exist, including surrounding blood vessels, immune cells, fibroblasts, myeloid-derived cells, various signaling molecules, and extracellular matrix. In recent years, immunotherapy has been developed as a fourth anti-tumor treatment means following surgery, chemotherapy, and radiotherapy in cancer treatment because it can effectively stimulate host immune system and promote immune cells in tumor microenvironment to kill cancer cells. Researches show that the natural polysaccharide has stronger effects of preventing tumor occurrence and inhibiting tumor, and compared with the traditional tumor treatment method, the natural polysaccharide has the advantages of low toxicity and immunity regulation. The food-derived natural polysaccharide has no obvious toxicity to most of body cells, and can stimulate the proliferation, differentiation and maturation of various immunocompetent cells to recover and enhance the immune system of the body, so that the food-derived natural polysaccharide is recognized as an important biological effect regulator. Therefore, the search for food-borne active polysaccharide which can be eaten for a long time and has less side effect is of great significance for tumor patients.
The current animal models for screening immunomodulatory active substances mainly include: a xenograft model, a homologous transplantation model, a gene knockout model, a genetically engineered mouse model, a human gene knock-in model, and the like. Compared with an animal model, the in vitro cell model has the advantages of short experimental period, high screening flux and the like. The model for in vitro screening of the immunoregulatory active substance mainly comprises a RAW264.7 cell polarization model, a 2D co-culture model, an organoid and microfluidic cell model and the like. An ideal in vitro polysaccharide screening assay would more closely mimic the in vivo tumor microenvironment for high throughput screening of immunotherapeutics with high sensitivity and specificity for further testing of prospective effective drugs. The RAW264.7 cell polarization method cannot truly simulate the complex interaction process between cells in a tumor microenvironment; organoid models, while widely used in cancer biopsy tissue culture, typically also contain only tumor cells and no immune cells. Compared with a single cell in vitro screening model, the co-culture cell model is a more complex, more biologically relevant and predictive cell screening method, and is more suitable for screening compounds because the co-culture model can better simulate the in vivo environment and the response to drug treatment. Based on the property of multiple cells in the tumor microenvironment, the tumor cell and immune cell co-culture model can simulate the interaction between cells in the tumor microenvironment, and can be used for testing the efficacy of immunotherapy and immune preparations in vitro.
There is also a need to develop more effective in vitro cytotoxicity assays to assess immunomodulatory efficacy of screenings in the tumor microenvironment by quantifying the killing of tumor cells by immune cells in the tumor microenvironment. Wherein,51the Cr release assay is the most common in vitro assay, commonly used to measure T cell mediated cytotoxic effects, but this assay is radiotoxic and limited in application. Pimentel et al have proposed that firefly luciferase assay is used to evaluate the targeted killing mortality of tumor cells, and this method is complicated in experimental procedures, not only requires the addition of reaction substrates, but also requires rapid detection due to short reaction time, and the experimental results are not visualized, thus it is difficult to meet the requirements for experimental large-scale screening (Olivo Pimentel V, Yaromina A, Marcus D, Dubois LJ, Lambin P.A novel co-culture assay to assay anti-tumor CD8+ T cell cytotoxin virus luminescence and multi-color flow assay. J Immunol methods.2020 Dec; 487:112899.doi:10.1016/j. jim.2020.2020.2020.899.Epub 11215. PMID: 33068606.). The tumor cells are transfected with fluorescent protein, and the tumor target cells are automatically imaged by microscopic imaging and high-content and high-throughput analysis tools while the absence of radioactive toxicity is ensured, so that the tumor cells in co-culture are realizedAnd the killing effect of the immune cells in the co-culture is visually detected.
In conclusion, it is necessary to research a rapid screening visualization system which has high throughput and good repeatability and can simulate a tumor microenvironment, and the screening of the compound with the function of improving the immune function of the organism is not slow for patients with low immunity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a screening method of an MC38-N4/OT-I co-culture system, a screened polysaccharide formula and an application of a polysaccharide composition. The polysaccharide composition can promote killing of tumor cells by immunocytes, and increase CD8+Survival rate and cell viability of T cells and promotion of CD8+The growth of CD6L memory T cell can be used for preparing products for improving organism immunity.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a high-throughput, rapid and repeatable screening method of an MC38-N4/OT-I co-culture system for screening immunomodulatory active substances, which comprises the following steps:
the screening method of the MC38-N4/OT-I co-culture system comprises the following steps:
(1) obtaining tumor Target cells (Target, T) containing specific antigens and fluorescent proteins: infecting the virus supernatant of recombinant plasmid containing specific antigen and fluorescent protein with tumor target cell, adding antibiotic after virus infection, screening, observing the fluorescent protein expression under a fluorescence microscope after continuous drug screening, and obtaining the visual tumor target cell expressing the specific antigen through monoclonal amplification and flow cell screening.
(2) Isolation of immune Effector cells (Effector, E): killing the mouse by breaking the neck, taking the immune organ tissues of the mouse by aseptic operation, separating the immune organ tissues of the mouse, slightly grinding to obtain single cell suspension, filtering by a cell filter, centrifugally treating and cracking red blood cells, washing and then re-suspending to obtain the single cell suspension of immune effector cells.
(3) Co-culturing tumor target cells with immune effector cells: and (3) paving the tumor target cells, after the cells adhere to the wall overnight, removing the culture medium, adding immune effector cells according to a certain proportion, simultaneously adding the composition obtained in the embodiment to be screened, and after co-culturing for a period of time, detecting the death rate of the tumor target cells.
Further, in the step (1), the recombinant plasmid containing the specific antigen and the fluorescent protein is a 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.
Further, in the step (2), the mice include OT-I or OT-II mice, CD8 of OT-I mice+T cells having recognized specificity for the N4 peptide of SIINFEKL, CD4 of OT-II mice+T cells have recognized specificity for the N4 peptide of SIINFEKL.
Further, the viscera in the step (2) comprise more than one of spleen, thymus or lymph node; the immune effector cell is OT-I whole spleen immune effector cell.
Further, the ratio of the number of the immune effector cells to the number of the tumor target cells in the step (3) is (100-1): 1, the co-culture time in the step (3) is 12-96 h.
The invention provides a polysaccharide formula screened by the screening method of the MC38-N4/OT-I co-culture system, which comprises the following raw materials: the immune regulation function screening and evaluation method comprises the following steps of selecting more than one of agaricus blazei murrill, grifola frondosa, dried orange peel, phellinus igniarius, ganoderma lucidum, poria cocos, angelica sinensis and lycium barbarum, wherein the formula is more than one 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 tumor cells and immune cells, sucking cell supernatant out, washing the cells twice with Phosphate Buffered Saline (PBS), digesting with pancreatin, collecting mixed cells in a flow tube, centrifuging, then re-suspending the cells with a staining buffer (FACS buffer), detecting the number of tumor target cells by using a flow cytometry, 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.
Further, the mass part ratio of the agaricus blazei polysaccharide, the grifola frondosa polysaccharide, the dried orange peel polysaccharide and the phellinus igniarius polysaccharide is (1-125): (1-125) parts: (1-25) parts of: (0-25).
Further, the co-cultured cells are subjected to high-throughput fluorescence imaging screening by using a rotation 7 high-throughput living cell imaging system, and the screening and evaluation are used for screening and evaluating substances with immunoregulatory activity; the detection is visualized by fluorescence imaging.
The invention also provides application of the polysaccharide composition in preparing CD8 enhancing products+T cell anti-tumor immunological memory effect, and can be used for enhancing organism immunocompetence and promoting anti-tumor effect.
Further, the enhancement of the body immunity mainly aims at improving CD8+Survival of T cells and CD8+Proportion of CD 62L.
Further, the anti-tumor promoting effect mainly aims at improving the killing effect of immune cells on tumor cells containing specific antigens.
The components of the agaricus blazei murill, the grifola frondosa and the dried orange peel in the composition are matched with each other to generate a synergistic effect, so that the composition has the effect of remarkably improving the immune function of the organism, and is a composition with the immunoregulation activity.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
1. the method for rapidly screening the co-culture immunoregulation activity comprises four steps of constructing tumor target cells, separating immune effector cells, co-culturing the tumor target cells and the immune effector cells and evaluating the immunoregulation function, can simulate the interaction of different types of cells in a tumor microenvironment, and is compared with the method for rapidly screening the co-culture immunoregulation activity51Method for detecting Cr, model thereforThe detection process has no radioactivity generation and has the advantage of tumor target cell visualization.
2. The MC38-N4/OT-I tumor cell and immune cell co-culture system screening method provided by the invention has a wide application range, can be used for screening various food-borne active substances, is simple and efficient, and is beneficial to rapid research, development, popularization and application of immunoregulation functional foods.
3. The MC38-N4/OT-I co-culture system provided by the invention is a multifunctional in-vitro evaluation platform and can be used for evaluating CD8+Killing effect of T cells on tumor cells; it is also useful for high throughput screening of immunomodulators for in vivo models. The method can shorten experimental period, and is helpful for exploring molecular mechanism and action target of immunotherapy medicine.
4. The polysaccharide composition provided by the invention has the function of improving the immune function of an organism, and the components of the composition are matched with each other, so that the killing effect of immune cells on tumor cells is remarkably promoted, and the CD8 is remarkably improved+Survival and cell viability of T cells, promotion of CD8+The growth of CD62L memory T cell can be used for preparing products for improving body immunity.
Drawings
FIG. 1 is a schematic diagram of the MC38-N4/OT-I co-culture rapid screening method of the present invention.
FIG. 2 is a graph of the bright field and red fluorescence field of MC38-tdTomato-N4 cells under a fluorescence microscope.
FIG. 3 is a bar graph of the effect of individual starting materials on the proliferative toxicity of MC38-N4 cells.
FIG. 4 is a bar graph showing the enhancement of the immune function activity of examples 1 to 3 and comparative examples 1 to 7 based on the co-culture evaluation.
FIG. 5 is a histogram of the polysaccharide composition of example 1 against immune cells.
FIG. 6 shows the survival rate of immune effector cells, CD8, for polysaccharide compositions and control groups of example 1+Size of T cells, CD8+Proportion of T cells, CD8+The effect of the proportion of CD62L is histogram.
Detailed Description
The invention provides a composition and application thereof in preparing products for enhancing body immunity, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the composition. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the embodiments of the present invention, unless otherwise specified, technical means used in the examples are conventional means well known to those skilled in the art, and materials, reagents and the like used are commercially available.
In order to verify the feasibility of the rapid screening method of MC38-N4/OT-I co-culture immunoregulatory activity provided by the invention, examples 1-3 and comparative examples 1-7 are specifically listed, extracts of agaricus blazei murrill, grifola frondosa, dried orange peel, phellinus igniarius, lucid ganoderma, poria cocos, angelica sinensis and medlar are respectively adopted, different formulas are formed according to different proportions, a co-culture system is respectively added for intervention treatment, then the apoptosis rate of MC38-N4 cells is detected and statistical analysis is carried out, and then the immunoregulatory activity of different polysaccharide formulas is screened and evaluated, and the specific experimental method and detection results are as follows:
the preparation method of the extracts of different raw materials comprises the following steps:
1. preparation method of Agaricus blazei Murill polysaccharide extract
Oven drying Agaricus blazei Murill to constant weight, grinding the dried raw materials into powder, and sieving with 200 mesh sieve. Adding distilled water according to a feed-liquid ratio of 1:20(m/m), leaching at 55 deg.C for 2 hr for 2 times, mixing the two leaching solutions, centrifuging at 4000 r/min for 10min, collecting supernatant, and rotary evaporating at 55 deg.C for vacuum concentration. Adding 3 times volume of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 24 hr, centrifuging at 4000 r/min for 15min, collecting precipitate, and vacuum freeze drying to obtain crude polysaccharide of Agaricus Blazei Murr.
2. Preparation method of grifola frondosa polysaccharide extract
Oven drying Grifola frondosa to constant weight, grinding the dried raw materials into powder, and sieving with 200 mesh sieve. Adding distilled water according to a feed-liquid ratio of 1:20(m/m), leaching at 55 deg.C for 2 hr for 2 times, mixing the two leaching solutions, centrifuging at 4000 r/min for 10min, and rotary evaporating the supernatant at 55 deg.C for vacuum concentration. Adding anhydrous ethanol with volume 3 times of the concentrated solution, standing at 4 deg.C for 24 hr, centrifuging at 4000 r/min for 15min, collecting precipitate, and vacuum freeze drying to obtain crude polysaccharide of Grifola frondosa.
3. Preparation method of dried orange peel polysaccharide extract
Oven drying Agaricus blazei Murill to constant weight, grinding the dried raw materials into powder, and sieving with 200 mesh sieve. Adding distilled water according to a feed-liquid ratio of 1:20(m/m), leaching at 55 deg.C for 2 hr for 2 times, mixing the two leaching solutions, centrifuging at 4000 r/min for 10min, collecting supernatant, rotary evaporating at 55 deg.C, and concentrating under reduced pressure. Adding 3 times volume of anhydrous ethanol into the crude polysaccharide solution, standing at 4 ℃ for 24h, centrifuging at 4000 r/min for 15min, collecting precipitate, and freeze-drying in vacuum to obtain pericarpium Citri Tangerinae crude polysaccharide.
4. Preparation method of phellinus igniarius polysaccharide extract
Oven drying Phellinus Linteus to constant weight, grinding the dried materials into powder, and sieving with 200 mesh sieve. Adding distilled water according to the ratio of 1:20, leaching at 55 deg.C for 2 hr for 2 times, mixing the two leaching solutions, centrifuging at 4000 r/min for 10min, and rotary evaporating the supernatant at 55 deg.C for vacuum concentration. Adding 3 times volume of anhydrous ethanol into the crude polysaccharide solution, standing at 4 deg.C for 24h, centrifuging at 4000 r/min for 15min, collecting precipitate, and vacuum freeze drying to obtain crude polysaccharide.
5. Preparation method of extracts of ganoderma lucidum, poria cocos, angelica sinensis and wolfberry fruits
Drying Ganoderma, Poria, radix Angelicae sinensis and fructus Lycii to constant weight, respectively adding 1 part by weight of the raw materials into 40 parts by weight of 70% ethanol, concentrating and extracting in 45 deg.C water bath, vacuum filtering, and vacuum freeze drying to obtain the extracts.
Mixing Agaricus blazei polysaccharide extract, Grifola frondosa polysaccharide extract, pericarpium Citri Tangerinae polysaccharide extract and Phellinus linteus polysaccharide extract according to formula to obtain polysaccharide composition.
Example 1
The polysaccharide composition was prepared as follows: the mass part ratio of the agaricus blazei polysaccharide to the grifola frondosa polysaccharide to the dried orange peel polysaccharide to the phellinus igniarius polysaccharide is 125: 1 part of: 1 part of: 0 part of (A).
Example 2 (comparative example 6)
The polysaccharide composition was prepared as follows: the mass portion ratio of agaricus blazei polysaccharide, grifola frondosa polysaccharide, phellinus igniarius polysaccharide and dried orange peel polysaccharide is 1:1 part of: 5 parts of: 1 part.
Example 3 (comparative example 7)
The polysaccharide composition was prepared as follows: the mass portion ratio of the agaricus blazei polysaccharide to the grifola frondosa polysaccharide to the phellinus igniarius polysaccharide to the dried orange peel polysaccharide is 25: 125 parts of: 25 parts of: and 125 parts.
Comparative examples 1 to 7
1. The mixtures were prepared according to the formulation of table 1:
table 1 composition (parts by mass)
According to the formula table 1, extracts of Agaricus blazei, Grifola frondosa, Phellinus igniarius, pericarpium citri reticulatae, Ganoderma lucidum, Poria cocos, Angelica sinensis and Lycium barbarum are prepared in comparative examples 1-7.
Efficacy verification
FIG. 1 is a schematic diagram of the MC38-N4/OT-I co-culture rapid screening method of the present invention. As shown in FIG. 1, after 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. In the co-culture process, the immune effector cells recognize specific antigen peptide N4 of the tumor cells through a T Cell Receptor (TCR), and the recognized effector cells release inflammatory factors, so that the apoptosis of the tumor cells is promoted, and the apoptosis condition of tumor target cells can be reflected through a flow cytometer and a fluorescence microscope.
The polysaccharide compositions of examples 1 to 3 of the present invention and the polysaccharide compositions of comparative examples 1 to 7 were tested for their activity to enhance the immune function of the body based on a co-culture system.
1. Laboratory animal
The animal experimental subject is OT-I transgenic mouse, and is bred under SPF (specific pathogen free) grade condition with the ambient temperature of 23 + -2 ℃, the relative air humidity of 60 percent, and the daily circulating light and dark for 12h respectively. Before spleen tissues are dissected and taken, toes are cut to crack DNA to identify the genotype.
After the genotype of the OT-I mouse is confirmed, subsequent mouse dissection operation is carried out, and the operation process is carried out on ice. Firstly, cervical vertebra is removed to kill a mouse, the mouse is completely soaked in alcohol with the volume percentage concentration of 75% for disinfection, spleen tissues are quickly taken out and placed in a precooled RPMI 1640 complete culture medium. A nylon sieve with a pore size of 70 μm was placed on a 50mL centrifuge tube, spleen tissue was taken out of the medium and placed on a 70 μm nylon membrane, and the spleen tissue was gently abraded with the piston plane of a syringe while rinsing the spleen tissue with complete medium, and the single cell suspension of the abraded spleen was filtered through a 70 μm cell filter. Centrifuging, and removing supernatant to obtain cell precipitate. And (3) resuspending the precipitate with 1x erythrocyte lysate, mixing the cells uniformly, standing at room temperature for 5min to lyse the erythrocytes, terminating lysis with 10mL of precooled FACS buffer (flow-type staining buffer), reversing and mixing uniformly, centrifuging to obtain the precipitate, resuspending the precipitate in an RPMI 1640 complete culture medium to ensure that the concentration of the resuspended cells is 30 ten thousand/mL, and obtaining the OT-I spleen immune effector cells.
2. Obtaining tumor target cells containing specific antigens and fluorescent proteins
The day before transfection, 0.5X 106The well-conditioned MC38 tumor target cells were inoculated into 6-well plates, 3mL of the medium was added to each well and mixed well, and cultured in a carbon dioxide incubator. Separately, 4. mu.g of pLVX-IRES-tdTomato-N4 recombinant plasmid was added to 500. mu.L of OPTI-MEM medium; 4 μ g of retroviral packaging plasmid; mixing well. To 500. mu.L of LOPTI-MEM medium was added 20. mu.L of lipo 2000 (Lipofectamine)TM2000) And mixing the mixture completely. Incubate each at room temperature for 5 min. Add a pre-mix of lipo 2000 to the DNA pre-mix, mix well and incubate at room temperature for 20 min. The original medium was discarded and the transfection reagent mixture was added. After 5h the original medium was discarded and fresh complete medium was added. After 24h of transfection, 1.2. mu.g/mL puromycin was added. And selecting to passage or change culture medium according to the cell growth density. After the drug screening is continued for 7 days, the expression condition of the red fluorescent protein is observed under a fluorescence microscope; obtaining expressive specific antigen through monoclonal propagation and flow cell sortingVisualizing the tumor target cell. The observation result under the fluorescence microscope is shown in fig. 2, the cell growth state under the bright field is good, and meanwhile, the MC38 cells are observed to express tdTomato red fluorescent protein, and the expression level is high, which indicates that the plasmid transfection is successful.
3. Co-culture of tumor target cells and immune effector cells
MC38-N4 cells were plated in a 96-well plate at 6000 cells/well, overnight adherence was performed, the medium was discarded, and 100. mu.L of OT-I spleen immune effector cells at the above concentration were added at a ratio of 5:1 in terms of the number ratio (E: T) of immune effector cells to tumor target cells. At the same time, 100. mu.L of the double-concentration substance to be screened was added and cultured for 48 hours, and the mortality of MC38-N4 cells with red fluorescent protein was examined.
4. Evaluation of immunoregulatory function
During co-culture, MC38-N4 target cells can be detected by OT-I spleen CD8+The target killing of T effector cells reflects the killing effect of co-culture by counting the number of MC38-N4 red fluorescent cells. There are two evaluation methods: a) directly using a rotation 7 high-flux living cell imaging system to perform fluorescence imaging on a 96-well plate, and counting the number of fluorescent cells by Gen5 software (Biotek); b) the cell supernatant was aspirated, the cells were washed twice with PBS, trypsinized, collected in a flow tube, centrifuged, resuspended in a DAPI solution containing 10. mu.g/mL, and the number of MC38-N4 red fluorescent cells was measured using a flow cytometer FACS LSRII (BD FACStersery).
5. MTT method for determining concentration range of extract without obvious toxicity to MC38-N4 cells
The death of MC38-N4 cells in the co-culture system may result 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. To exclude direct toxic effects of the extracts on MC38-N4 cells in a co-culture system, it was necessary to determine the dose range within which each extract had no significant toxic effect on MC38-N4 cells.
MC38-N4 cells in log-extended phase were digested, resuspended, and counted at 6X 104The cells were plated at density of 100. mu.L/mL in 96-well plates. Culture in incubatorAfter culturing the anchorage-dependent bacteria, 200 mu L of fresh complete culture medium is added into a blank control group, four crude extracts of agaricus blazei murrill polysaccharide, grifola frondosa polysaccharide, dried orange peel polysaccharide and phellinus igniarius polysaccharide are respectively added into experimental groups, and each polysaccharide is respectively added into corresponding complete culture medium by 10 mu g/mL, 50 mu g/mL, 250 mu g/mL and 1250 mu g/mL. After culturing in a 37 ℃ constant temperature incubator for 48h, 20. mu.L of 5mg/mL MTT solution is added to each well, after further culturing for 4h, the supernatant culture solution is aspirated, 150. mu.L of DMSO solution is added to each well to dissolve formazan crystals, and after shaking uniformly, the absorbance value is detected at 490nm by using a full-function microplate detection microplate reader. The ratio of the absorbance value of the experimental group to the absorbance value of the blank control group is the survival rate of the MC38-N4 cells.
6. Formula screening with different proportions based on MC38-N4 co-culture system
According to the MTT experiment result, after the concentration range of the extract on the MC38-N4 without obvious toxicity is determined, 9 different comparative examples are designed according to the formula proportion in the table 1.MC38-N4 cells were plated in 96-well plates at 6000 cells/well and after overnight adherence, the medium was discarded, following effector cell: the number ratio of target cells (5:1) was adjusted to 100. mu.L of effector cells at the above concentrations. At the same time, 100. mu.L of the double-concentrated material to be screened was added and incubated for 48 hours.
After co-culture, cell supernatants were aspirated, cells were washed twice with PBS, trypsinized, and cells were collected in flow tubes. After centrifugation, the cells were resuspended in 200. mu.L of a solution containing 10. mu.g/mL of DAPI, and the number of MC38-N4 red fluorescent cells was measured by a flow cytometer FACS LSRII (BD FACSterse).
7. Example 1 in vitro stimulation experiment of spleen cells with polysaccharide composition
The mice were sacrificed by decapitation, and the intact spleen was removed from OT-I mice and ground to prepare a single cell suspension. Centrifuging, and discarding the supernatant to obtain cell precipitate. The pellet was resuspended in 1mL of 1 × erythrocyte lysate, lysed for 5min at room temperature, then lysed with 10mL of pre-cooled FACS buffer, mixed well, centrifuged to obtain a pellet, resuspended to 2 millions/mL with 1nM of K medium containing N4(SIINFEKL, OVA257-264 antigen polypeptide fragment) peptide, and plated in 6-well plates, 1mL per well, i.e., 2 millions/well. Both the control and experimental groups without the polysaccharide composition were supplemented with 100IU/mL IL-2 (interleukin-2), the experimental group was supplemented with the polysaccharide composition of example 1 at twice the concentration, and the control group was supplemented with medium to a volume of 2 mL. After 48h incubation, cells were collected in flow tubes, 1mL FACS buffer was added to each tube, and the supernatant was discarded by centrifugation. The Fc receptor blocking agent was diluted with FACS buffer, 50. mu.L of each tube was added, mixed well with shaking, and allowed to stand on ice for 15min in the dark. Extracellular marker staining: Anti-CD8, Anti-TCR β, Anti-CD 62L; after the antibody was diluted with FACS buffer at a dilution ratio of 1:150(v/v), 50. mu.L of the antibody was added to each tube for staining, followed by shaking and light shielding on ice for 30 min. After staining was completed, 1mL of FACS buffer was added to each tube, washed once, centrifuged, and the supernatant discarded. Add 200. mu.L of FACS buffer to each tube, vortex and mix the cells, and then detect them with a flow cytometer.
Analysis of Experimental results
1. MTT (methanol to Top test) experimental result analysis of four polysaccharide extracts of agaricus blazei murrill polysaccharide, grifola frondosa polysaccharide, dried orange peel polysaccharide and phellinus igniarius polysaccharide on MC38-N4
To rule out the direct toxic effect of the extracts on MC38-N4 cells in the co-culture system, it was therefore necessary to determine the dose range of each extract that did not have a significant toxic effect on MC38-N4 cells. As shown in FIG. 3, Grifola frondosa has no significant cytotoxic effect on MC38-N4 cells in the concentration range of 10-250 μ g/mL, Agaricus blazei has no significant cytotoxic effect on MC38-N4 cells in the concentration range of 10-1250 μ g/mL, pericarpium citri reticulatae has no significant cytotoxic effect on MC38-N4 cells in the concentration range of 10-1250 μ g/mL, and Phellinus linteus has no significant cytotoxic effect on MC38-N4 cells in the concentration range of 50-250 μ g/mL. Therefore, concentrations without significant dose toxicity were selected for the next co-culture experiment.
2. Analysis of Co-culture Experimental results
Different comparative examples are designed according to table 1, depending on the dosage of the starting material which is not significantly toxic. As shown in fig. 4, the killing effect of OT-I spleen immune cells on MC38-N4 cells was detected by a flow cytometer, and the control group (E: T ═ 5:1) to which immune effector cells were added significantly promoted the death of tumor target cells, as compared to the control group (E: T ═ 5:0) to which no immune effector cells were added. Compared with a control group (E: T ═ 5:1), the polysaccharide composition in example 1 remarkably promotes the activity of OT-I immune cells in a co-culture system, and the activity is improved by 22%. The composition can promote immunocyte to exert better tumor killing function, and can be used for preparing medicine for enhancing organism immunocompetence and promoting anti-tumor effect.
3. Example 1 polysaccharide composition promotes CD8 in vitro+Proliferation and activation outcome analysis of T cells
To further clarify the manner in which the polysaccharide composition of example 1 modulates immunopotentiation, the polysaccharide composition of example 1 was tested for its effect on immune cell proliferation and activation. FIG. 5 shows a schematic representation of the cycle of OT-I spleen immune cells with the polysaccharide composition of example 1, as can be seen in FIG. 5, lymphocytes are circled from all cells, monocytes are circled after removal of adherent cells, T cells are then clustered from live lymphocytes, and finally a CD62L + positive cell population is circled. Flow data statistics are shown in fig. 6, and the results show that the spleen immune cell survival rate is 32.33% ± 0.33% when the polysaccharide composition of example 1 is added, compared with the spleen immune cell survival rate of a control group (E: T ═ 5:1) without the polysaccharide composition, which is 16.13% ± 0.41%, the spleen immune cell survival rate is significantly enhanced by the polysaccharide composition of example 1; CD8 with control group (E: T ═ 5:1) without polysaccharide composition+Proportion of T cells (19.87% + -0.75%) compared to CD8 added to the polysaccharide composition of example 1+The proportion of T cells is 33.03% + -1.64%, and the polysaccharide composition of example 1 can also significantly improve CD8+Proportion of T cells. Also, the polysaccharide composition of example 1 promotes CD8+Activation of T cells, and CD8 of control group (E: T ═ 5:1) without polysaccharide composition+CD8 added to the polysaccharide composition of example 1 at a relative volume of T cells of 100.00% + -0.10%+Relative volume of T cells 102.40% + -0.08%, example 1 polysaccharide composition resulted in CD8+The relative volume of T cells was significantly increased by 2%; CD8 was tested by further testing the polysaccharide composition of example 1 for CD8+Effect of Central memory T cells on CD8 of a control group (E: T ═ 5:1) without the addition of a polysaccharide composition+The proportion of CD62L T cells was 83.07% + -2.62% compared to the results found by the addition of realgarExample 1 CD8 of polysaccharide composition+The proportion of CD62L T cells was 95.40% ± 0.23%, the example 1 polysaccharide composition significantly increased CD8+CD62L T cell ratio, CD8+Anti-tumor effect of T cells, suggesting that the polysaccharide composition of example 1 can be used to increase CD8 in immunotherapy+The anti-tumor activity of T immune cells and the formula can be used in adoptive T cell mediated tumor therapy.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.
Claims (10)
- 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. 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. 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. 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. 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. 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. 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. 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. 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. 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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