CN114456978B - Co-culture method for culturable anaerobic strain and pig intestine epithelial cells - Google Patents
Co-culture method for culturable anaerobic strain and pig intestine epithelial cells Download PDFInfo
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Abstract
The invention discloses a co-culture method of a culturable anaerobic strain and pig intestine epithelial cells, which comprises the steps of subculturing pig intestine epithelial cells and anaerobic bacteriaAkkermansia muciniphilaCo-culturing under anaerobic condition. The invention can obviously improve the culture speed of the akkermansia muciniphila.
Description
Technical Field
The invention belongs to the field of microecological nutrition and physiology, and in particular relates to a co-culture method for an anaerobic strain capable of being cultured and pig intestine epithelial cells.
Background
The animal intestinal canal inhabits a huge, complex and various microbial flora, and a great deal of researches show that intestinal canal microorganisms are closely related to host nutrient metabolism, intestinal canal health and physiological conditions. At present, amplicon sequencing or metagenome sequencing methods are generally adopted to study the microbial composition and functions of animal gastrointestinal tracts, but the methods depend on bioinformatics means to a great extent, the accuracy and precision of analysis results are determined by the perfection degree of a reference database, and the specific effect of certain bacteria cannot be deeply studied in the aspects of physiology, metabolism, signal transduction and the like.
Based on this, CN201780077207.0 discloses a culture system for co-culturing a 1 st cell group consisting of 1 or more cells and a cell layer or tissue formed of a 2 nd cell group consisting of 1 or more cells different from the 1 st cell group, characterized by having: a 1 st culture tank in which a 1 st cell group composed of 1 or more cells such as anaerobic bacteria and a cell layer or tissue composed of a 2 nd cell group composed of 1 or more cells such as epithelial cells are co-cultured under anaerobic conditions; a 2 nd culture tank for storing a culture solution in an aerobic state; 1 or more substance exchange structures arranged so as to connect the 1 st culture tank and the 2 nd culture tank; and the cell layer or tissue provided so as to cover the surface of the substance exchange structure on the 1 st culture tank side.
In paragraph 6 of the specification of this document: intestinal bacteria are often absolutely anaerobic, and more than half of them are difficult to culture, so many bacteria identification and characteristic confirmation have not been performed. Therefore, the relationship between the type and ratio of the intestinal bacteria and the external and internal factors has not been accurately grasped.
The contribution of this scheme lies in that proposes a comparatively suitable co-cultivation equipment.
Everard et al, PNAS 110 (2013) 9066-71; the publication by renanaen et al, appl Environ Microbiol, 3/20/2015, believes that the intestinal mucosal barrier has evolved a complex "intestinal mucosal immune system" to distinguish symbiotic bacteria (i.e., beneficial bacteria) from pathogenic bacteria and other deleterious agents. The intestinal mucosal immune system is an integral part of the intestinal mucosal barrier, including lymphoid tissues and specialized immune cells (i.e., lymphocytes and plasma cells), which are widely distributed throughout the intestinal mucosal barrier. One of the microorganisms naturally occupying the mucosa of healthy subjects is mucin-degrading akkermansia muciniphila (Akkermansia muciniphila), which has been shown to increase intestinal barrier function, affecting diseases associated with impaired intestinal barrier function.
In addition, CN201910865365.3 discloses a method for culturing akkermansia muciniphila in low oxygen, which mainly creates a low oxygen gas environment, and the volume ratio of the components of the mixed gas is: 70-94% N 2 、1～5％O 2 、5～25％CO 2 The method comprises the steps of carrying out a first treatment on the surface of the The culture time is 24-36 h.
The examples and comparative examples of this protocol demonstrate that the culture rate in a hypoxic environment is higher than in an anaerobic environment.
Based on the above, the technical problems to be solved by the invention are as follows: how to increase the culture speed of the mucin-philin Acremonium.
Disclosure of Invention
In view of the above, the present invention provides a co-culture method for an anaerobic strain and pig intestinal epithelial cells, which can significantly increase the culture speed of akkermansia muciniphila.
In order to solve the technical problems, the invention discloses a co-culture method for a culturable anaerobic strain and pig intestine epithelial cells, which is characterized in that the pig intestine epithelial cells subjected to subculture and anaerobic bacteria Akkermansia muciniphila are co-cultured in an anaerobic environment.
In the method for co-culturing the culturable anaerobic strain and the pig intestine epithelial cells, the anaerobic strain is commercial strain Akkermansia muciniphila DSM 22959; the pig epithelial cells are IPEC-J2 pig intestine epithelial cells.
In the above-described method of co-culturing a culturable anaerobic strain with porcine intestinal epithelial cells, anaerobic bacteria Akkermansia muciniphila are inoculated to porcine intestinal epithelial cells at a ratio of multiplicity of infection moi=10.
In the above-described method for co-culturing a culturable anaerobic strain with porcine intestinal epithelial cells, the culture temperature was 37 ℃.
In the method for co-culturing the culturable anaerobic strain and the pig intestinal epithelial cells, the culture time is 18-48 hours;
as a further optimization of the above method, the method of the present invention comprises the steps of:
step 2, measuring the concentration of the bacterial liquid by adopting a plate counting method;
step 3, rapidly determining the number of bacteria through association analysis of the bacteria and the bacteria;
step 4, inoculating the accurately counted microorganisms into the intestinal epithelial cell line cultured in advance.
Optionally, the spectrophotometry in the step 1 is used for measuring the optical density value of the bacterial liquid, which specifically includes:
step 1.1, anaerobic water preparation: 1L of distilled water is measured and poured into an conical flask, 1mL of resazurin solution with the mass fraction of 0.1% is added, and the liquid is blue at the moment; after heating and boiling, rapidly cooling by running water, and sealing the bottle mouth of the conical flask by using disposable PE gloves and rubber bands during cooling; then CO is introduced 2 Or N 2 0.5-1 h, adding 1g of L-cysteine or L-cysteine hydrochloride, wherein the liquid is pink or yellow; finally, ventilation is carried out again for 10min, and the culture medium is heated by sealing small fire or kept stand for 10-12h until the culture medium turns colorless or yellowish; packaging the prepared anaerobic water into glass bottles with good tightness for preservation;
step 1.2, preparation of anaerobic culture medium: 38.5g of BHI culture medium is weighed into 1L of anaerobic water, and CO is continuously introduced into the anaerobic water 2 Or N 2 Maintaining its anaerobic state; rapidly stirring with a glass rod to completely dissolve the BHI culture medium; taking anaerobic rolling pipe and introducing CO 2 Or N 2 Taking 9mL of BHI culture medium into an anaerobic tube by using a 5mL pipette after discharging air, maintaining ventilation for 30s, rapidly plugging a butyl rubber plug, and punching an aluminum cover to prepare the anaerobic BHI liquid culture medium; at the position ofAdding 0.75-1.5% agar into the prepared BHI liquid culture medium to prepare a BHI solid culture medium, and sub-packaging into an anaerobic bottle; sterilizing the packaged anaerobic culture medium at 121 ℃ for 20min to prepare an anaerobic BHI solid culture medium;
step 1.3, reviving culture of the culturable anaerobic strain: taking out the strain stored in the freezing tube or anaerobic tube from the refrigerator at-80 ℃, inserting the strain into ice for thawing, and then inoculating the strain into the prepared anaerobic BHI liquid culture medium according to the proportion of 20%, and shake culturing for 18-24h at 37 ℃ and 180 r/m;
step 1.4, bacterial growth curve measurement: absorbing 10mL of the resuscitating bacterial liquid, inoculating the resuscitating bacterial liquid into 90mL of an anaerobic bottle filled with anaerobic BHI liquid culture medium, and shake culturing at 37 ℃ and 180 r/m; OD was measured every 2h from 0h using unvaccinated anaerobic BHI broth as a blank 600nm Sampling for 48 hours, wherein 3 parallels are made for each sampling; at OD 600nm The value is the ordinate, the culturing time is the abscissa, and a smooth S-shaped growth curve is drawn.
Optionally, the BHI medium comprises the following composition: 1L solid BHI culture medium comprises 10g tryptone, 17.5g bovine heart extract powder, 5g sodium chloride, 2g glucose, 2.5g disodium hydrogen phosphate, pH value of 7.4+ -0.2, 25 deg.C, distilled water to constant volume to 1L, 121 deg.C, and sterilizing for 20 min.
Optionally, the concentration of the bacterial liquid is measured by adopting a plate counting method in the step 2, which specifically comprises the following steps:
step 2.1, preparation of a plate culture medium: cooling the sterilized anaerobic bottle filled with the anaerobic BHI liquid culture medium in the step 1.2 to 45-50 ℃, spraying or wiping with alcohol, placing the anaerobic bottle and the plate with the cover in an anaerobic workstation, and opening an ultraviolet lamp in the station for sterilization for 30min; slightly opening a cover of the plate, pouring a culture medium, covering the cover, slightly shaking the cover, preventing the cover from being stained, standing for 1h, completely solidifying the culture medium and dispersing dry water, and judging that the plate is flat, has no bulge and has no wall hanging;
step 2.2, measuring the optical density of the bacterial liquid: in an anaerobic workstation, taking bacterial liquid in a growth platform stage, taking the anaerobic BHI liquid culture medium prepared in the step 1.2 as a diluent, and diluting the bacterial liquid by 2 timesReleasing to obtain bacterial solutions of 1/2, 1/4, 1/8, 1/16 and 1/32, after each sampling, vibrating for 20s by a vortex instrument to fully mix, then rapidly adding samples by a pipetting gun sterilized under high pressure, and adding BHI culture medium with corresponding volume after adding the stock solution; the OD of the 6 different dilution gradient bacterial solutions is measured by a spectrophotometer 600nm Values, 3 replicates for each dilution gradient;
step 2.3, measuring the concentration of the bacterial liquid by a dilution coating method: in an anaerobic workstation, taking stock solution and bacterial solutions with dilution gradients of 1/2, 1/4, 1/8, 1/16 and 1/32, and carrying out gradient dilution by 10 times; the dilution gradient for inoculation is typically 10 -6 ～10 -8 3 per dilution gradient were made in parallel; after full oscillation by a vortex instrument, rapidly taking 100 mu L of bacterial liquid, inoculating the bacterial liquid onto the BHI flat plate prepared in the step 2.1, and uniformly coating by a coating rod; after inoculation is finished, placing for 10-30min, after bacterial liquid is completely absorbed, inverting a flat plate, placing into an anaerobic tank and an anaerobic gas generating bag or an anaerobic workstation, culturing at 37 ℃ for 24-48h, and counting bacterial colonies; if the anaerobic tank is not arranged, a PP sealing box is used or a flat plate is placed in a vacuum bag, and a food vacuum machine is used for pumping out redundant gas to form an anaerobic environment;
step 2.4, colony count: colony count: selecting a flat plate with the colony number of 30-300, and using Windows self-contained drawing software and mouse counter software Winomiter V1.5 after photographing.
Optionally, the relationship between the concentration of the bacterial liquid and the optical density value determined in the step 3 is specifically:
at OD 600nm Drawing an XY scatter diagram by Excel or Origin8, obtaining regression curves of the Excel or Origin8 by linear fitting, observing whether a linear relationship exists between the Excel and the Origin, and R 2 And the correlation is strong and is more than or equal to 0.8.
Alternatively, the step 4 of inoculating the accurately counted microorganisms into the pre-cultured intestinal epithelial cell line is specifically:
step 4.1, subculturing pig intestinal epithelial cells: the pig jejunal cell line was resuscitated at 37℃using DMEM/F12 complete medium at 37℃with 5% CO 2 Culturing in a cell culture box for 48h, and normally passaging for 3 times and then laterA formal experiment can be performed;
step 4.2 after obtaining activated IPEC-J2 cells, IPEC-J2 in the flask was digested with pancreatin first, followed by 1X 10 5 Cell/well dose cells were seeded in 12-well plates and incubated with DMEM/F12 complete medium without antibiotics at 37 ℃ for 24h for subsequent bacterial co-cultivation experiments;
step 4.3, taking out the frozen culturable anaerobic strain at the temperature of minus 80 ℃ in advance for carrying out expansion culture, measuring the OD value of the anaerobic strain after 18 hours, and calculating the bacterial number by utilizing the standard curve established in the step 3;
step 4.4, centrifuging the culture solution containing the culturable anaerobic strain at 3000-5000rpm for 5 minutes, removing the supernatant, and re-suspending the culture solution with a DMEM/F12 culture medium without antibiotics for later use;
step 4.5, the culturable anaerobic strain was inoculated into IPEC-J2 cells in step 4.2 at a dose of moi=10 and incubated at 37 ℃.
Optionally, the DMEM/F12 complete medium has the following composition: 10% FBS,5ng/mL EGF,10Um HEPES,1% diabody was added on the basis of DMEM/F12 basal medium.
Compared with the prior art, the invention can obtain the following technical effects:
the co-culture method can remarkably improve the culture speed of the akkermansia muciniphila, and can enter a platform stage in 24 hours under an anaerobic environment.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a value-added curve versus a standard curve for Akkermansia muciniphila of the present invention; wherein, a graph a is a growth curve of Akkermansia muciniphila; panel B shows the OD values of Akkermansia muciniphila at various dilution gradients; panel C shows a linear fit of Akkermansia muciniphila OD values to colony count;
FIG. 2 is a co-culture morphology (X200) of the invention Akkermansia muciniphila with porcine jejunal epithelial cells (IPEC-J2); wherein, the left graph shows the morphology of normal IPEC-J2 cells; the right panel shows the morphology of Akkermansia muciniphila after 24h co-culture with IPEC-J2 cells;
FIG. 3 shows the effect of Akkermansia muciniphila of the present invention on the expression level of the gene involved in tight junctions of IPEC-J2 cells; wherein CON is control group cells; a-24h represents IPEC-J2 cells after 24h co-culture with akkermansia muciniphila. * Indicating significant differences (P < 0.05); * Represents that the difference was very significant (P < 0.01).
FIG. 4 is an inverted microscopic pattern of cells of comparative example 1 of the present invention before co-culturing with Eggerthella lenta ATCC 25559 (CON) and after co-culturing for 24 hours (E-24H);
FIG. 5 shows the expression of the claudin gene in cells of comparative example 1 of the present invention before co-culturing with Eggerthella lenta ATCC 25559 (CON) and after co-culturing for 24 hours (E-24H).
Detailed Description
The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.
Example 1
A co-culture method of a culturable anaerobic strain and pig intestine epithelial cells, comprising the following steps:
step 1.1, anaerobic water preparation: 1L of distilled water is measured and poured into an conical flask, 1mL of resazurin solution with the mass fraction of 0.1% is added, and the liquid is blue at the moment; after heating and boiling, rapidly cooling by running water, and sealing the bottle mouth of the conical flask by using disposable PE gloves and rubber bands during cooling; then CO is introduced 2 Or N 2 0.5-1 h, adding 1g of L-cysteine or L-cysteine hydrochloride, wherein the liquid is pink or yellow; finally, ventilation is carried out again for 10min, and the culture medium is heated by sealing with small fire (without boiling) or is kept stand for 10-12h in a sealing way, so that the color of the culture medium is observed to be colorless or faint yellow; packaging the prepared anaerobic water into glass bottles with good tightness for preservation.
The preparation method of the resazurin solution comprises the following steps: weighing 0.1g of resazurin powder, adding distilled water to a constant volume to 100m L4 ℃ and storing; resazurin is an oxidation-reduction indicator which changes from pink to colorless in an anoxic environment, and can visually display whether the bacterial culture process reaches the anaerobic standard; l-cysteine or L-cysteine hydrochloride. L-cysteine has reducibility and oxidation resistance, can remove oxygen molecules in water, reduce oxidation-reduction potential in a culture medium, and provide an anaerobic environment for anaerobic bacteria.
Step 1.2, preparation of anaerobic culture medium: 38.5g of BHI culture medium is weighed into 1L of anaerobic water, and CO is continuously introduced into the anaerobic water 2 Or N 2 Maintaining its anaerobic state; rapidly stirring with a glass rod to completely dissolve the BHI culture medium; taking anaerobic rolling pipe and introducing CO 2 Or N 2 Taking 9mL of BHI culture medium into an anaerobic tube by using a 5mL pipette after discharging air, maintaining ventilation for 30s, rapidly plugging a butyl rubber plug, and punching an aluminum cover to prepare the anaerobic BHI liquid culture medium; adding 0.75-1.5% agar into the prepared BHI liquid culture medium to prepare a BHI solid culture medium, and sub-packaging into an anaerobic bottle; sterilizing the packaged anaerobic culture medium at 121 ℃ for 20min to prepare an anaerobic BHI solid culture medium;
wherein, the composition of the BHI culture medium is as follows: 1L solid BHI culture medium comprises 10g tryptone, 17.5g bovine heart extract powder, 5g sodium chloride, 2g glucose, 2.5g disodium hydrogen phosphate (Na 2 HPO 4), pH 7.4+ -0.2, 25deg.C, adding distilled water to constant volume to 1L, 121deg.C, and sterilizing for 20 min.
Step 1.3, akkermansia muciniphila, reviving culture: the strain stored in the freezing tube or the anaerobic tube is taken out from the refrigerator at the temperature of minus 80 ℃, is inserted into ice for thawing, and is inoculated into the prepared anaerobic BHI liquid culture medium according to the proportion of 20 percent, and is subjected to shake culture for 18 to 24 hours at the temperature of 37 ℃ and 180 r/m.
Akkermansia muciniphila used in the present invention is selected from commercial bacteria Akkermansia muciniphila DSM 22959.
Step 1.4, bacterial growth curve measurement: absorbing 10mL of the resuscitating bacterial liquid, inoculating the resuscitating bacterial liquid into 90mL of an anaerobic bottle filled with anaerobic BHI liquid culture medium, and shake culturing at 37 ℃ and 180 r/m; by no means ofInoculated anaerobic BHI liquid culture medium is used as blank control, and OD is measured every 2h from 0h 600nm Sampling for 48 hours, wherein 3 parallels are made for each sampling; at OD 600nm The value is the ordinate, the culture time is the abscissa, and a smooth S-shaped growth curve is drawn, as shown in FIG. 1A;
step 2, measuring the concentration of the bacterial liquid by adopting a plate counting method:
step 2.1, preparation of a plate culture medium: cooling the sterilized anaerobic bottle filled with the anaerobic BHI solid culture medium in the step 1.2 to 45-50 ℃, spraying or wiping with alcohol, placing the anaerobic bottle and the plate with the cover in an anaerobic workstation, and opening an ultraviolet lamp in the station for sterilization for 30min; slightly opening the cover of the plate, pouring the culture medium, covering the cover, slightly shaking, preventing the culture medium from being adhered to the cover, standing for 1h, completely solidifying the culture medium and dispersing the water, and judging the culture medium to be flat, free of protrusions and free of wall hanging. Two methods for solidifying the plate culture medium exist, one is to spread the plates one by one for solidification; and secondly, stacking a plurality of flat plates together for solidification. The former has higher solidification speed and is adopted when the room temperature is higher; the latter has a slower solidification rate, can be used at a lower room temperature, has the advantage of less formed solidification water, and is particularly suitable for flat scribing and the like.
Step 2.2, measuring the optical density of the bacterial liquid: in an anaerobic workstation, taking bacterial liquid in a growth platform stage, taking the anaerobic BHI liquid culture medium prepared in the step 1.2 as a diluent, diluting the bacterial liquid by 2 times to obtain bacterial liquid of 1/2, 1/4, 1/8, 1/16 and 1/32, after sampling each time, vibrating the bacterial liquid for 20 seconds by a vortex instrument to fully and uniformly mix the bacterial liquid, then rapidly adding samples by a pipetting gun sterilized under high pressure, and then adding a BHI culture medium with a corresponding volume after adding the stock solution; the OD of the 6 different dilution gradient bacterial solutions is measured by a spectrophotometer 600nm Values, 3 per dilution gradient, are made in parallel, as shown in fig. 1B.
Step 2.3, measuring the concentration of the bacterial liquid by a dilution coating method: in an anaerobic workstation, taking stock solution and bacterial solutions with dilution gradients of 1/2, 1/4, 1/8, 1/16 and 1/32, and carrying out gradient dilution by 10 times; the dilution gradient for inoculation is typically 10 -6 ～10 -8 Each dilution gradient was run in 3 replicates. After full oscillation by vortex instrument, the device is quickInoculating 100 mu L of bacterial liquid to the BHI flat plate prepared in the step 2.1, and uniformly coating by using a coating rod; after inoculation is finished, placing for 10-30min, after bacterial liquid is completely absorbed, inverting a flat plate, placing into an anaerobic tank and an anaerobic gas generating bag or an anaerobic workstation, culturing at 37 ℃ for 24-48h, and counting bacterial colonies; if the anaerobic tank is not arranged, a PP sealing box is used or a flat plate is placed in a vacuum bag, and the food vacuum machine is used for pumping out redundant gas to form an anaerobic environment.
Step 2.4, colony count: plates with colony numbers between 30 and 300 were selected, manually counted using Windows self-contained drawing software and mouse counter software Winometer V1.5 (Zhang Zhe, yang Feng, li Xinpu, et al (2016) based on Photoshop and novel methods of counting colonies on plates with accurate counting software. Microbiological notification, 7:1646-1648.) after photographing, and the colony numbers per milliliter of bacterial solutions were calculated.
The plating method does not greatly affect the colony count, but the placing time of the plate culture medium can affect the colony count, and the placing time exceeding 12 hours can lead to larger deviation of the colony count.
Step 3, determining the relation between the concentration of the bacterial liquid and the optical density value: at OD 600nm Drawing an XY scatter diagram by Excel or Origin8, obtaining a regression curve of the Excel or Origin8 by linear fitting, and observing whether a linear relationship exists between the Excel and the Origin 8; as shown in fig. 1C; a linear equation of Akkermansia muciniphila OD value and colony number was obtained as a basis for determining bacterial addition for subsequent cell experiments.
Step 4, inoculating the accurately counted microorganisms into a pre-cultured intestinal epithelial cell line:
step 4.1, subculturing pig intestinal epithelial cells: pig jejunal cell line (IPEC-J2) was resuscitated at 37℃using DMEM/F12 complete medium at 37℃with 5% CO 2 After normal passage for 3 times, the final experiment can be carried out after culturing for 48 hours in the cell culture incubator.
Wherein, the composition of the DMEM/F12 complete medium is as follows: 10% FBS,5ng/mL EGF,10Um HEPES,1% diabody was added on the basis of DMEM/F12 basal medium.
Step 4.2After obtaining usable IPEC-J2 cells, IPEC-J2 in the flask was digested first with pancreatin and then in accordance with 1X 10 5 Individual cell/well dose cells were seeded in 12-well plates and incubated with DMEM/F12 complete medium without antibiotic (diabody) at 37 ℃ for 24h for subsequent bacterial co-culture experiments.
And 4.3, taking out Akkermansia muciniphila frozen at the temperature of minus 80 ℃ in advance for expansion culture, measuring the OD value of the strain after 18 hours, and calculating the bacterial number by using the standard curve established in the step 3.
Step 4.4, centrifuging the culture solution containing Akkermansia muciniphila at 3000-5000rpm for 5 minutes, removing the supernatant, and re-suspending the culture solution with the DMEM/F12 medium without antibiotics for later use.
Step 4.5, akkermansia muciniphila was inoculated into IPEC-J2 cells in step 4.2 at a dose of moi=10, incubated at 37 ℃, sampling and incubation time as the case may be.
And 5, morphological observation is carried out by using an inverted microscope, the co-culture condition of Akkermansia muciniphila and IPEC-J2 is judged, whether bacteria and cells grow normally or not is detected, and as shown in figure 2, after Akkermansia muciniphila and IPEC-J2 cells are co-cultured for 24 hours, connection among the cells is more compact, and cavitation among the cells is less.
And 6, detecting the gene expression of the zona pellucida ZO-1, occidin and Claudin-1 after co-culturing IPEC-J2 and Akkermansia muciniphila by using qPCR technology, and detecting whether Akkermansia muciniphila has an influence on the tight junction of intestinal epithelial cells.
As shown in fig. 3, after IPEC-J2 cells were co-cultured with Akkermansia muciniphila for 24h by inverted microscopy, the cell-cell connection was tighter and the cell-cell vacuoles were fewer; meanwhile, the gene expression level of the tight junction protein is detected by adopting a real-time PCR method, and Akkermansia muciniphila is found to obviously improve the mRNA expression level (P < 0.05) of the tight junction ZO-1 in IPEC-J2 cells. Therefore, the established co-culture method proves that Akkermansia muciniphila can enhance the physical barrier function of the pig jejunum epithelial cells, thereby reducing the cell permeability and being beneficial to the intestinal health of pigs.
In the method, for anaerobic bacteria, anaerobic and aseptic are highly important in all operation processes, and the ultraviolet lamp is started 0.5-1 h in advance; for the articles needing to be put into the anaerobic working station in the experimental operation process, 75% alcohol is sprayed on the surface in advance and then is put into the station quickly, so that the sterilization effect can be enhanced.
By measuring the growth rate of bacteria in the well plate in step 4.5 of the invention, the time for entering the platform phase is about 24 hours.
The significance of this test is: 1. co-culture with Akkermansia muciniphila and IPEC-J2 cells is a significant aid to the proliferation of Akkermansia muciniphila bacteria;
2. the anaerobic environment is adopted in the scheme, and compared with the low-oxygen environment of CN201910865365.3, the culture environment is easier to control; the culture of Akkermansia muciniphila bacteria of the protocol of the present invention also entered the plateau at a faster rate than the anaerobic environment of CN 201910865365.3.
3. The scheme provides potential prospect and possibility for the Akkermansia muciniphila strain based on IPEC-J2 pig intestine epithelial cell co-culture.
Comparative example 1
As a comparative verification, this comparative example uses anaerobic bacterial standard strain Eggerthella lenta ATCC 25559 applied to examples 1, 4.3-4.5, in place of Akkermansia muciniphila.
Experiments show that the co-culture result of the strain and IPEC-J2 cells shows that: after 24h co-culture of IPEC-J2 cells with Eggerthella lenta ATCC 25559, there was no significant improvement in the intercellular junctions and no significant change in the intercellular vacuole number compared to the starting time point (fig. 4); meanwhile, real-time PCR results (FIG. 5) showed that Eggerthella lenta ATCC 25559 significantly reduced the mRNA expression level of the zona-1 in IPEC-J2 cells (P < 0.05) and had a tendency to reduce the mRNA expression level of the Occludin protein (0.05 < P < 0.01).
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (9)
1. A co-culture method of a culturable anaerobic strain and pig intestine epithelial cells is characterized in that the pig intestine epithelial cells and the anaerobic strain after subculture are usedAkkermansia muciniphilaCo-culturing in anaerobic environment; the pig epithelial cells are IPEC-J2 pig intestine epithelial cells; the pig intestine epithelial cells need to be subjected to subculture before use, and specifically comprise the following steps: IPEC-J2 pig intestine epithelial cells were resuscitated at 37℃using DMEM/F12 complete medium at 37℃with 5% CO 2 48 and h, and performing formal experiments after normal passage for 3 times; after obtaining activated IPEC-J2 cells, IPEC-J2 in the flask was first digested with pancreatin and then purified according to 1X 10 5 Cell/well dose cells were seeded in 12-well plates and cultured 24h using antibiotic-free DMEM/F12 complete medium at 37 ℃ for subsequent bacterial co-culture experiments; the anaerobic bacteria are commercial bacteriaAkkermansia muciniphila DSM 22959。
2. The method for co-culturing a culturable anaerobic strain and pig intestinal epithelial cells according to claim 1, wherein the anaerobic strain is inoculated at a ratio of multiplicity of infection moi=10Akkermansia muciniphilaTo porcine intestinal epithelial cells.
3. The method for co-culturing a culturable anaerobic strain and pig intestinal epithelial cells according to claim 1, wherein the culturing temperature is 37 ℃.
4. The co-culture method of a culturable anaerobic strain and pig intestine epithelial cells according to claim 1, comprising the steps of:
step 1, adopting light splittingDetermination of anaerobe content by a degree methodAkkermansia muciniphilaThe optical density value of the bacterial liquid;
step 2, measuring the concentration of the bacterial liquid by adopting a plate counting method;
step 3, rapidly determining the number of bacteria through association analysis of the bacteria and the bacteria;
step 4, inoculating the accurately counted microorganisms into the intestinal epithelial cell line cultured in advance.
5. The method for co-culturing a culturable anaerobic strain and pig intestinal epithelial cells according to claim 4, wherein the spectrophotometrically measuring the optical density value of the bacterial liquid in step 1 comprises the following steps:
step 1.1, anaerobic water preparation: 1, L distilled water is measured and poured into an conical flask, and 1mL of resazurin solution with the mass fraction of 0.1% is added, so that the liquid is blue; after heating and boiling, rapidly cooling by running water, and sealing the bottle mouth of the conical flask by using disposable PE gloves and rubber bands during cooling; then CO is introduced 2 Or N 2 Adding L-cysteine or L-cysteine hydrochloride 1g for 0.5-1 h, wherein the liquid is pink or yellow; finally, ventilation is carried out again for 10min, and the culture medium is heated by sealing small fire or kept stand for 10-12h until the color of the culture medium is colorless or yellowish; packaging the prepared anaerobic water into glass bottles with good tightness for preservation;
step 1.2, preparation of anaerobic culture medium: 38.5g of BHI culture medium is weighed into 1L of anaerobic water, and CO is continuously introduced into the anaerobic water 2 Or N 2 Maintaining its anaerobic state; rapidly stirring with a glass rod to completely dissolve the BHI culture medium; taking anaerobic rolling pipe and introducing CO 2 Or N 2 Taking 9mL of BHI culture medium into an anaerobic tube by using a 5mL pipette after discharging air, maintaining ventilation for 30s, rapidly plugging a butyl rubber plug, and punching an aluminum cover to prepare the anaerobic BHI liquid culture medium; adding 0.75-1.5% agar into the prepared BHI liquid culture medium to prepare a BHI solid culture medium, and subpackaging into anaerobic bottles; autoclaving the packaged anaerobic culture medium at 121 ℃ for 20min to obtain an anaerobic BHI solid culture medium;
step 1.3, reviving culture of the culturable anaerobic strain:to be stored in freezing or anaerobic tubesAkkermansia muciniphilaThe strain is taken out from a refrigerator at the temperature of-80 ℃, is inserted into ice for thawing, and is inoculated into the prepared anaerobic BHI liquid culture medium according to the proportion of 20 percent, and is subjected to shake culture at the temperature of 37 ℃ and 180r/m for 18-24h;
step 1.4, bacterial growth curve measurement: absorbing 10mL of the anaerobic strain bacterial liquid of the reviving culture, inoculating the strain bacterial liquid into an anaerobic bottle with 90mL of anaerobic BHI liquid culture medium, and shake culturing at 37 ℃ and 180 r/m; OD was measured at intervals of 2h from 0h using unvaccinated anaerobic BHI broth as a blank 600nm Values were sampled to 48h, 3 replicates were made for each sample; at OD 600nm The value is the ordinate, the culturing time is the abscissa, and a smooth S-shaped growth curve is drawn.
6. The method for co-culturing a culturable anaerobic strain and porcine intestinal epithelial cells according to claim 5, wherein said BHI medium comprises the following composition: the 1L solid BHI culture medium comprises 10g tryptone, 17.5g beef heart infusion powder, 5g sodium chloride, 2g glucose, 2.5g disodium hydrogen phosphate, pH value of 7.4+ -0.2, 25 ℃, distilled water, constant volume to 1L, 121 ℃ and 20min, and sterilizing.
7. The method for co-culturing the culturable anaerobic strain and the pig intestinal epithelial cells according to claim 5, wherein the concentration of the bacterial liquid measured by adopting a plate counting method in the step 2 is specifically as follows:
step 2.1, preparation of a plate culture medium: cooling the sterilized anaerobic bottle filled with the anaerobic BHI liquid culture medium in the step 1.2 to 45-50 ℃, spraying or wiping with alcohol, placing the anaerobic bottle and the plate with the cover in an anaerobic workstation, and sterilizing for 30min by turning on an ultraviolet lamp in the station; slightly opening the cover of the plate, pouring the culture medium, covering the cover, slightly shaking, preventing the culture medium from being adhered to the cover, standing for 1h to enable the culture medium to be completely solidified and dry, and judging that the plate is flat, has no bulge and no wall hanging;
step 2.2, measuring the optical density of the bacterial liquid: in an anaerobic workstation, taking bacterial liquid in a growth platform stage, using the steps of1.2, taking the prepared anaerobic BHI liquid culture medium as a diluent, diluting by 2 times to obtain bacterial solutions of 1/2, 1/4, 1/8, 1/16 and 1/32, shaking 20/s by a vortex instrument after each sampling to fully mix, rapidly adding the sample by a pipetting gun sterilized under high pressure, and adding the BHI culture medium with corresponding volume after the addition of the stock solution; measuring the OD of the different diluted gradient bacteria liquid by using a spectrophotometer 600nm Values, 3 replicates for each dilution gradient;
step 2.3, measuring the concentration of the bacterial liquid by a dilution coating method: in an anaerobic workstation, taking stock solution and bacterial solutions with dilution gradients of 1/2, 1/4, 1/8, 1/16 and 1/32, and carrying out gradient dilution by 10 times; the dilution gradient for inoculation is typically 10 -6 ~10 -8 3 per dilution gradient were made in parallel; after full oscillation by a vortex instrument, rapidly taking 100 mu L of bacterial liquid, inoculating the bacterial liquid onto the BHI flat plate prepared in the step 2.1, and uniformly coating by a coating rod; after inoculation is finished, placing for 10-30min, after bacterial liquid is completely absorbed, inverting a flat plate, placing into an anaerobic tank and an anaerobic gas generating bag or an anaerobic workstation, culturing at 37 ℃ for 24-48h, and counting bacterial colonies; if the anaerobic tank is not arranged, a PP sealing box is used or a flat plate is placed in a vacuum bag, and a food vacuum machine is used for pumping out redundant gas to form an anaerobic environment;
step 2.4, colony count: selecting a flat plate with the colony number of 30-300, and using Windows self-contained drawing software and mouse counter software Winomiter V1.5 after photographing.
8. The method according to claim 5, wherein the step 4 of inoculating accurately counted microorganisms into the intestinal epithelial cell line cultured in advance is specifically:
step 4.1, subculturing pig intestine epithelial cells;
step 4.2 after obtaining activated IPEC-J2 cells, IPEC-J2 in the flask was digested with pancreatin first, followed by 1X 10 5 Cell/well dose cells were seeded in 12-well plates and cultured 24h using antibiotic-free DMEM/F12 complete medium at 37 ℃ for subsequent bacterial co-culture experiments;
step 4.3, taking out the frozen-stored culturable anaerobic strain at the temperature of-80 ℃ in advance for carrying out expansion culture, measuring the OD value of the anaerobic strain after 18h, and calculating the bacterial number by utilizing the standard curve established in the step 3;
step 4.4, centrifuging the culture solution containing the culturable anaerobic strain at 3000-5000rpm for 5 minutes, removing the supernatant, and re-suspending the culture solution with a DMEM/F12 culture medium without antibiotics for later use;
step 4.5, the culturable anaerobic strain was inoculated into IPEC-J2 cells in step 4.2 at a dose of moi=10 and placed in culture at 37 ℃.
9. The method for co-culturing an anaerobic strain and a pig intestinal epithelial cell according to claim 8, wherein said DMEM/F12 complete medium has the following composition: 10% FBS,5ng/mL EGF,10Um HEPES,1% diabody were added to DMEM/F12 basal medium.
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