CN111440723A - Intestinal microorganism culture medium for stimulating Th17 cell proliferation activation and culture method - Google Patents

Abstract

The invention relates to the technical field of microbial culture methods, in particular to an intestinal microbial culture medium for stimulating Th17 cell proliferation and activation and a culture method. The culture medium A and the culture medium B are used for separating and culturing microorganisms capable of stimulating Th17 cell proliferation and activation from feces containing complex microorganism populations, and a method is provided for improving host pathogenic microorganism infection resistance by using a specific intestinal microorganism strain transplanting method. The culture method comprises the following steps: 1) collecting microorganisms in excrement; 2) separating target strains; 3) carrying out amplification culture on a target strain; 4) and (4) collecting target strains.

Description

Intestinal microorganism culture medium for stimulating Th17 cell proliferation activation and culture method

The technical field is as follows:

the invention relates to the technical field of microbial culture methods, in particular to an intestinal microbial culture medium for stimulating Th17 cell proliferation and activation and a culture method.

Secondly, background art:

immunotherapy refers to the use of immunological techniques to deliver directionally activated immune cells/molecules in vitro to receptors to improve the state of low or disturbed immunity of the receptors, thereby achieving the final goal of treating diseases of the body. Compared with other treatment technologies, immunotherapy has incomparable specificity, accuracy and targeting. Therefore, immunotherapy is favored and has become one of the most rapidly developing disciplines in the field of applied immunology today.

One of the indispensable conditions for achieving and accomplishing the goal of immunotherapy is the material of immune cells required for therapy, especially immune cells with extremely high precision. In the case of current disease treatment, the most predominant cell required for immunotherapy is also T lymphocytes (T cells for short). From an immunological point of view, T cells are not a homogeneous cell subset, consisting of at least 5 subsets. These different sub-populations of T cells exert their respective immunological effects in the course of the body's anti-tumor, anti-viral, anti-bacterial infection, etc.

Th17 cell is a newly discovered T cell subgroup, because it secretes interleukin-17 (I L-17) and gets its name, the comprehensive and intensive research results carried out on Th17 cell in recent years reveal, Th17 cell plays a key role in resisting infection of extracellular parasitic bacteria in organism, Th17 cell exerts its immune effect mainly through secreting cytokine I L-17, there have been abundant research data to prove I L-17 plays an irreplaceable core role in mediating and chemotactic neutrophilic granulocyte, monocyte, macrophage, clearing pathogenic microorganism in vivo, Th17 cell quantity is insufficient in vivo, will cause the pathogenic microorganism infecting organism to obtain effective and sufficient clearance, input exogenous Th17 cell can make pathogenic microorganism get rid of rapidly, therefore, the cell amplification technique of 17 cell has very important theoretical and application value to laboratory and clinical anti-infective immunity.

The amplification technology of Th17 cells is only a technology researched in scientific research laboratories at present, in the technology, complex and expensive test materials are needed for preparing Th17 cells, such as 4 mouse-resistant monoclonal antibodies (anti-CD 3, CD28, IFN-gamma, I L-4 monoclonal antibodies), 3 mouse cytokines (I L-6, I L-23, TGF- β 1) and other stimulators (such as Brefeldin A and Monensin Solution) and the like.

Therefore, the innovative development of the Th17 cell amplification technology from a new perspective, by adopting a new technology and by adopting conventional materials has very important value for clinical and laboratory scientific researches.

Third, the invention

The invention provides an intestinal microorganism culture medium and a culture method for stimulating Th17 cell proliferation activation, wherein a culture medium A and a culture medium B are used for separating and culturing microorganisms capable of stimulating Th17 cell proliferation activation from excrement containing complex microorganism populations, and a method for improving host anti-pathogenic microorganism infection by using a specific intestinal microorganism strain transplanting method is provided.

In order to achieve the purpose, the invention adopts the technical scheme that: an intestinal microorganism culture medium for stimulating the proliferation and activation of Th17 cells, which is characterized in that: comprises a separation culture medium A and a separation culture medium B;

the separation culture medium A is prepared from 4mM-5mM glutamine, 4000 mg/L-4800 mg/L glucose, 1mM-3mM pyruvate, 7 mu g/m L0-15 mu g/m L1 ciprofloxacin hydrochloride, 8 mu g/m L-12 mu g/m L neomycin sulfate, 8,000IU/m L-11,000 IU/m L penicillin, 9 mu g/m L-12 mu g/m L streptomycin and 20 mu g/m L-30 mu g/m L amphotericin B;

the separation culture medium B is prepared from 2mM-6mM glutamine, 4000 mg/L-5500 mg/L glucose and 2mM-3mM pyruvate.

The method for culturing the intestinal microorganisms for stimulating the proliferation and activation of Th17 cells is characterized in that: the culture method comprises the following steps:

1) collecting microorganisms in excrement: aseptically taking the rectal contents of the organism into PBS solution, centrifuging for 3min-5min at 4000r/min-6000r/min, discarding the supernatant, and repeating twice;

2) separating target strains: after 50 ml of culture medium A is used for resuspending the sediment, adding two ml of the sediment into a 24-hole polystyrene plate, and culturing for 6d-8d under standard anaerobic culture conditions;

3) and (3) amplification culture of the target strain: collecting culture in 24-well polystyrene plate, centrifuging at 3000-5000 r/min for 3-5 min, and discarding supernatant; resuspending and precipitating 50 ml of the culture medium B, adding the suspension precipitate into a new 24-hole polystyrene plate, culturing for 5-8 days under the standard anaerobic culture condition, and replacing the new culture medium B;

4) collecting target strain, culturing for 5-7 days, collecting culture in 24-well polystyrene plate, centrifuging for 3-6 min at 200 × -400 × g, discarding supernatant, and resuspending the precipitate with 1.5m L-2 m L sterile water, and freezing at-80 deg.C in refrigerator.

Compared with the prior art, the invention has the following advantages and effects:

the invention utilizes the culture medium A and the culture medium B to separate and culture microorganisms in an enlarged way from the excrement containing complex microorganism populations, the technical operation is simple, and the purity of the separated strains stimulates the Th17 cells to have obvious differentiation and proliferation effects.

Fourthly, explanation of the attached drawings:

FIG. 1 shows real-time PCR results of Th17 cell activation stimulated by intestinal microorganisms isolated after transplantation into the body;

FIG. 2 shows the results of flow cytometry for stimulating the activation of Th17 cells after transplantation of isolated intestinal microorganisms into the body.

The fifth embodiment is as follows:

an intestinal microorganism culture medium for stimulating Th17 cell proliferation activation comprises isolation medium A and isolation medium B;

the separation culture medium A is prepared from 4mM-5mM glutamine, 4000 mg/L-4500 mg/L glucose and 1mM-3mM pyruvate, 7 mu g/m L0-15 mu g/m L1 ciprofloxacin hydrochloride, 8 mu g/m L-12 mu g/m L neomycin sulfate, 8,000IU/m L-11,000 IU/m L penicillin, 9g/m L-12 mu g/m L streptomycin and 20 mu g/m L-30 mu g/m L amphotericin B;

the separation culture medium B is prepared from 2mM-6mM glutamine, 4000 mg/L-5500 mg/L glucose and 2mM-3mM pyruvate.

A method for culturing intestinal microorganisms capable of stimulating Th17 cell proliferation activation comprises the following steps:

1) collecting microorganisms in excrement: aseptically taking the rectal contents of the organism into PBS solution, centrifuging for 3min-5min at 4000r/min-6000r/min, discarding the supernatant, and repeating twice;

2) separating target strains: after 50 ml of culture medium A is used for resuspending the sediment, adding two ml of the sediment into a 24-hole polystyrene plate, and culturing for 6d-8d under standard anaerobic culture conditions;

3) and (3) amplification culture of the target strain: collecting culture in 24-well polystyrene plate, centrifuging at 3000-5000 r/min for 3-5 min, and discarding supernatant; resuspending and precipitating 50 ml of the culture medium B, adding the suspension precipitate into a new 24-hole polystyrene plate, culturing for 5-8 days under the standard anaerobic culture condition, and replacing the new culture medium B;

4) collecting target strain, culturing for 5-7 days, collecting culture in 24-well polystyrene plate, centrifuging for 3-6 min at 200 × -400 × g, discarding supernatant, and resuspending the precipitate with 1.5m L-2 m L sterile water, and freezing at-80 deg.C in refrigerator.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

Example 1:

a method for culturing intestinal microorganisms capable of stimulating Th17 cell proliferation activation comprises:

1) collecting microorganisms in excrement: selecting a sheep farm which strictly executes the latest requirements of national related antibiotic use, wherein the sample collection object is a young healthy sheep, no relevant pathogen exists through specific antigen detection, cecal contents of the sample collection object are aseptically collected in a PBS solution, the sample collection object is centrifuged at 4000r/min for 5 minutes, supernatant is discarded, and the steps are repeated twice;

2) separating target strains: after resuspending the pellet with 50 ml of medium a, two ml per well were added to a 24-well polystyrene plate and cultured for 7d under standard anaerobic culture conditions;

the culture medium A is a sterile high-sugar DMEM culture medium, and the formula of the culture medium A is 4.5mM glutamine, 4000 mg/L glucose and 2mM pyruvate, 7 mu g/m L ciprofloxacin hydrochloride, 10 mu g/m L neomycin sulfate, 8000IU/m L penicillin, 11 mu g/m L streptomycin and 30 mu g/m L amphotericin B;

3) and (3) amplification culture of the target strain: the culture in 24-well polystyrene plates was collected, centrifuged at 3000r/min for 5 minutes, the supernatant was discarded, 50 ml of medium B was resuspended and precipitated, and added to new 24-well polystyrene plates. Culturing for 5 days under standard anaerobic culture condition, and replacing with new culture medium B;

the culture medium B is a sterile high-sugar DMEM culture medium, and the formula of the culture medium B is 2mM glutamine, 4000 mg/L glucose and 3mM pyruvate;

4) collecting target strains, namely collecting cultures in a 24-hole polystyrene plate after culturing for 5 days, centrifuging for 3min at 200 × g, removing supernatant, and resuspending the precipitate with 1.5 ml of sterile water and freezing and storing the precipitate in a refrigerator at minus 80 ℃ for later use.

Example 2:

a method for culturing intestinal microorganisms capable of stimulating Th17 cell proliferation activation comprises:

1) collecting microorganisms in excrement: selecting a sheep farm which strictly executes the latest requirements of national related antibiotic use, wherein the sample collection object is a young healthy sheep, no relevant pathogen exists through specific antigen detection, cecal contents of the sample collection object are aseptically collected in a PBS solution, the sample collection object is centrifuged at 6000r/min for 4 minutes, supernatant is discarded, and the steps are repeated twice;

2) separating target strains: after resuspending the pellet with 50 ml of medium a, two ml per well were added to a 24-well polystyrene plate and cultured for 6d under standard anaerobic culture conditions;

the culture medium A is a sterile high-sugar DMEM culture medium, and the formula of the culture medium A is 5mM glutamine, 4300 mg/L glucose and 1mM pyruvate, 10 mu g/m L ciprofloxacin hydrochloride, 8 mu g/m L neomycin sulfate, 11000IU/m L penicillin, 12 mu g/m L streptomycin and 20 mu g/m L amphotericin B;

3) and (3) amplification culture of the target strain: the culture in 24-well polystyrene plates was collected, centrifuged at 4000r/min for 4 minutes, the supernatant was discarded, 50 ml of medium B was resuspended and precipitated, and added to new 24-well polystyrene plates. Culturing for 8 days under standard anaerobic culture condition, and replacing with new culture medium B;

the culture medium B is a sterile high-sugar DMEM culture medium, and the formula of the culture medium B is 6mM glutamine, 5000 mg/L glucose and 2.5mM pyruvate;

4) collecting target strains, namely collecting cultures in a 24-hole polystyrene plate after culturing for 6 days, centrifuging for 6min at 400 × g, removing supernatant, and resuspending the precipitate with 2.0 ml of sterile water and freezing and storing the precipitate in a refrigerator at minus 80 ℃ for later use.

Example 3:

a method for culturing intestinal microorganisms capable of stimulating Th17 cell proliferation activation comprises:

1) collecting microorganisms in excrement: selecting a sheep farm which strictly executes the latest requirements of national related antibiotic use, wherein the sample collection object is a young healthy sheep, no relevant pathogen exists through specific antigen detection, the caecum content of the sheep farm is aseptically collected in a PBS solution, the sheep farm is centrifuged at 5000r/min for 3 minutes, the supernatant is discarded, and the steps are repeated twice;

2) separating target strains: after resuspending the pellet with 50 ml of medium a, two ml per well were added to a 24-well polystyrene plate and cultured for 7d under standard anaerobic culture conditions;

the culture medium A is a sterile high-sugar DMEM culture medium, and the formula of the culture medium A is 4.5mM glutamine, 4500 mg/L glucose and 2mM pyruvate, 15 mu g/m L ciprofloxacin hydrochloride, 10 mu g/m L neomycin sulfate, 8000IU/m L penicillin, 11 mu g/m L streptomycin and 30 mu g/m L amphotericin B;

3) and (3) amplification culture of the target strain: the culture in 24-well polystyrene plates was collected, centrifuged at 5000r/min for 3 minutes, the supernatant was discarded, 50 ml of medium B was resuspended and precipitated, and added to new 24-well polystyrene plates. Culturing for 7 days under standard anaerobic culture condition, and replacing with new culture medium B;

the culture medium B is a sterile high-sugar DMEM culture medium, and the formula of the culture medium B is 5mM glutamine, 5500 mg/L glucose and 2mM pyruvate;

4) collecting target strains, namely collecting cultures in a 24-hole polystyrene plate after culturing for 7 days, centrifuging for 5min at 300 × g, removing supernatant, and resuspending the precipitate with 1.8 ml of sterile water and freezing in a refrigerator at-80 ℃ for later use.

The strain obtained in example 3 was used for feeding:

rejuvenating before feeding separated strain, collecting strain stored at low temperature, culturing under conventional anaerobic condition for 7d, collecting culture, adjusting bacteria concentration to 10⁸About/m L for goat feeding test;

selecting 10 young goats which are healthy and do not have related pathogens through detection, dividing the goats into a test group and a control group, wherein each group comprises 5 goats, feeding separated microbial strains to the goats in the test group, adding 15m L of prepared bacterial liquid to warm water and feeding the goats after the prepared bacterial liquid is fed each time, and the goat is fed to the goats without treatment in the control group, wherein the control group is not treated normally, and after continuous treatment for one month, collecting peripheral anticoagulation blood of two groups of goats, separating peripheral blood mononuclear cells to detect the change condition of Th17 cell activation of the test group and the control group through flow cytometry, extracting total RNA, and detecting the change condition of Th17 cell activation of the test group and the control group by using a real-time PCR method, so that the separated intestinal microorganisms can stimulate the proliferation activation of Th17 cells as shown in figures 1 and 2.

Claims (2)

1. An intestinal microorganism culture medium for stimulating the proliferation and activation of Th17 cells, which is characterized in that: comprises a separation culture medium A and a separation culture medium B;

the separation culture medium A is prepared from 4mM-5mM glutamine, 4000 mg/L-4800 mg/L glucose, 1mM-3mM pyruvate, 7 mu g/m L0-15 mu g/m L1 ciprofloxacin hydrochloride, 8 mu g/m L-12 mu g/m L neomycin sulfate, 8,000IU/m L-11,000 IU/m L penicillin, 9 mu g/m L-12 mu g/m L streptomycin and 20 mu g/m L-30 mu g/m L amphotericin B;

the separation culture medium B is prepared from 2mM-6mM glutamine, 4000 mg/L-5500 mg/L glucose and 2mM-3mM pyruvate.

2. The method for culturing the intestinal microorganism activating Th17 cell proliferation according to claim 1, wherein: the culture method comprises the following steps:

1) collecting microorganisms in excrement: aseptically taking the rectal contents of the organism into PBS solution, centrifuging for 3min-5min at 4000r/min-6000r/min, discarding the supernatant, and repeating twice;

2) separating target strains: after 50 ml of culture medium A is used for resuspending the sediment, adding two ml of the sediment into a 24-hole polystyrene plate, and culturing for 6d-8d under standard anaerobic culture conditions;

3) and (3) amplification culture of the target strain: collecting culture in 24-well polystyrene plate, centrifuging at 3000-5000 r/min for 3-5 min, and discarding supernatant; resuspending and precipitating 50 ml of the culture medium B, adding the suspension precipitate into a new 24-hole polystyrene plate, culturing for 5-8 days under the standard anaerobic culture condition, and replacing the new culture medium B;

4) collecting target strain, culturing for 5-7 days, collecting culture in 24-well polystyrene plate, centrifuging for 3-6 min at 200 × -400 × g, discarding supernatant, and resuspending the precipitate with 1.5m L-2 m L sterile water, and freezing at-80 deg.C in refrigerator.

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