CN108866145B - Detection steps and application of interaction between Saccharomyces cerevisiae and DON to reduce apoptosis of IPEC-J2 cells - Google Patents
Detection steps and application of interaction between Saccharomyces cerevisiae and DON to reduce apoptosis of IPEC-J2 cells Download PDFInfo
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Abstract
本发明提出了酿酒酵母与DON作降低IPEC‑J2细胞凋亡的检测步骤及其应用，利用酿酒酵母对猪肠上皮细胞损伤进行修复的问题。所述的酿酒酵母与DON（呕吐毒素）互作降低IPEC‑J2细胞凋亡的应用，对DON诱导的猪肠上皮细胞炎症和坏死具有减缓作用。本申请将酿酒酵母（MOI=1）与DON共培养，在一定程度上促进了细胞增殖，可缓解DON对猪肠道上皮细胞的损伤，保护了肠道细胞膜的完整性；当DON浓度为1.2μg/mL时，酿酒酵母的添加可显著地提高猪肠上皮细胞的增殖率及IL‑10抗炎因子的表达量，显著地降低细胞的凋亡率和坏死率，说明酿酒酵母对DON诱导的细胞病变有一定的缓解作用。
The invention proposes the detection steps and application of Saccharomyces cerevisiae and DON for reducing the apoptosis of IPEC-J2 cells, and the problem of using Saccharomyces cerevisiae to repair the damage of porcine intestinal epithelial cells. The application of the interaction between Saccharomyces cerevisiae and DON (vomitoxin) to reduce the apoptosis of IPEC-J2 cells has a slowing effect on DON-induced inflammation and necrosis of porcine intestinal epithelial cells. In this application, Saccharomyces cerevisiae (MOI=1) was co-cultured with DON, which promoted cell proliferation to a certain extent, alleviated the damage of DON to porcine intestinal epithelial cells, and protected the integrity of intestinal cell membranes; when the concentration of DON was 1.2 At μg/mL, the addition of Saccharomyces cerevisiae can significantly increase the proliferation rate of porcine intestinal epithelial cells and the expression of IL-10 anti-inflammatory factors, and significantly reduce the apoptosis rate and necrosis rate of cells. Cytopathies have a certain alleviation effect.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a detection step for reducing IPEC-J2 cell apoptosis by saccharomyces cerevisiae and DON interaction and application thereof.
Background
Vomitoxin (vomotoxin), also known as Deoxynivalenol (DON), is one of the most common mycotoxins in cereals and foods worldwide. People and animals ingest food containing DON and cause a series of toxic symptoms.
Pig intestinal epithelial cells (IPEC-J2) are the first physical barrier for defending against external stimuli, and DON is the first accepted toxin with proinflammatory response and immunoregulation effect on the intestinal epithelial cells, so the experiment aims to screen out strains capable of degrading DON, construct a DON-IPEC-J2 cell model, and study the degradation effect of the screened microorganisms on DON and relieve the influence of the toxin on cell proliferation.
DON is the first recognized toxin that has both a pro-inflammatory response and an immunomodulatory effect on intestinal epithelial cells. The toxic effect of DON on cells mainly changes cell morphology, DNA damage, inhibits protein synthesis, promotes apoptosis and the like. The action mechanism of DON on intestinal epithelial cells is mainly to up-regulate tight junction protein, so that the expression quantity of RNA expressing related genes is up-regulated as a compensation mechanism. Intestinal tests prove that DON passes through the surface of the intestinal tract, is rapidly absorbed by intestinal epithelial cells, destroys the tight connection of the intestinal epithelial cells, combines RNA peptide transferase and inhibits the synthesis of protein. In addition, DON has a promoting effect on apoptosis.
Probiotics such as yeast and the like are widely applied as feed additives, mainly for regulating microbial flora in animal intestinal tracts, protecting intestinal health, regulating immune function of organisms, playing a direct intervention role on pathogenic bacteria to prevent the organisms from being infected, degrading pathogenic toxins and the like. The probiotics can also enhance the barrier function of the intestinal epithelium by means of promoting the formation of mucus layers, secreting antibacterial factors, enhancing the formation of tight junctions and the like.
According to the invention, on the cellular level, the harm effect of saccharomyces cerevisiae on DON on intestinal epithelial cell proliferation is researched, and a basis is provided for actual production.
Disclosure of Invention
The invention provides a detection step for reducing IPEC-J2 cell apoptosis by saccharomyces cerevisiae and DON interaction and application thereof, and solves the problem of repairing porcine intestinal epithelial cell injury by saccharomyces cerevisiae.
The technical scheme of the invention is realized as follows:
the detection step for reducing IPEC-J2 cell apoptosis by saccharomyces cerevisiae and DON interaction comprises the following steps:
(1) taking IPEC-J2 cells in logarithmic phase, digesting, counting, inoculating to 6-well plate, 2 mL/well, and the number of cells per well is 5 × 105After the cells are cultured to be adherent, the original culture medium is discarded, and the cells are washed once by PBS;
(2) the detection test is provided with four groups, namely a blank control group, a saccharomyces cerevisiae preparation group, a DON preparation group and a saccharomyces cerevisiae preparation + DON preparation group, wherein each group is repeated by three;
(3) adding the four groups of preparations obtained in the step (2) into a 6-well plate filled with IPEC-J2 cells obtained in the step (1) respectively;
(4) placing the 6-hole plate treated in the step (3) at 30-35 ℃, co-culturing for 8h, discarding the supernatant, washing with PBS for 2 times, centrifuging at 2000rpm for 5min, and collecting cells;
(5) and (3) adding 500 mu L of Binding Buffer suspension cells into the cells collected in the step (4), adding 5 mu L of Annexin V-FITC, uniformly mixing, adding 5 mu L of Propidium Iodide, uniformly mixing, reacting at room temperature in a dark place for 15min, and then carrying out cell activity detection.
The yeast is Saccharomyces cerevisiae with preservation number of CGMCC No. 2.3866.
The concentration of the DON in the step (2) is 0.075-0.15 mu g/mL, and the cell number of the saccharomyces cerevisiae is the same as that of IPEC-J2 cells.
The treatment of the saccharomyces cerevisiae preparation in the step (2) comprises the following steps:
a. activating the stored saccharomyces cerevisiae in a YPD liquid culture medium for 24 hours, inoculating the activated bacterium liquid into a fresh YPD liquid culture medium according to the inoculation amount of 2-5%, continuously culturing for 16-24 hours, coating plates for counting, and storing in a refrigerator at 4 ℃;
b. taking the saccharomyces cerevisiae culture medium stored in the step a, centrifuging for 5min at 8000rpm/min, removing supernate, sterilizing through a 0.22-micron filter membrane to obtain thalli cells, and storing at 4 ℃ for later use;
c. and re-suspending the centrifuged thallus cells by using a DMEM/F-12 culture medium, centrifuging at 8000rpm/min for 5min, washing twice, suspending the thallus by using the DMEM/F-12 culture medium according to the equal volume to obtain a saccharomyces cerevisiae preparation, and storing the saccharomyces cerevisiae preparation in a refrigerator at 4 ℃ for later use.
The application of the interaction of the saccharomyces cerevisiae and the DON for reducing the IPEC-J2 cell apoptosis has the effect of relieving the pig intestinal epithelial cells with inflammation induced by the DON.
Each 2mL of the mixture of Saccharomyces cerevisiae and DON contains 5X 105One Saccharomyces cerevisiae cell, 5X 105Individual pig intestinal epithelial cells and 1.2 mugmL DON。
5-10mL of the mixture of the saccharomyces cerevisiae and the DON is added into each Kg of the feed.
The invention has the beneficial effects that:
(1) according to the application, saccharomyces cerevisiae (MOI ═ 1) and DON are co-cultured, so that cell proliferation is promoted to a certain extent, the damage of DON to porcine intestinal epithelial cells can be relieved, and the integrity of intestinal cell membranes is protected; compared with the DON group, the release of LDH is obviously reduced, and the degradation of DON is well acted; when the DON concentration is 1.2 mug/mL, the addition of the saccharomyces cerevisiae can obviously improve the proliferation rate of porcine intestinal epithelial cells and the expression quantity of IL-10 anti-inflammatory factors, obviously reduce the apoptosis rate and the necrosis rate of the cells, and simultaneously reduce the expression quantities of Tight Junction Protein (TJP) and occludin (Occuldin) genes to a certain extent, which indicates that the saccharomyces cerevisiae has a certain relieving effect on DON-induced cytopathic effect.
(2) Compared with a control group, the DON group early apoptosis cell and the DON group late apoptosis cell are respectively increased by 2.67 times and 4.53 times (P is less than 0.05); the comparison between the yeast group and the control group increased the number of early and late apoptotic cells, and the number of viable cells was reduced, but the difference was not significant (P > 0.05).
(3) The proliferation inhibition of vomitoxin (DON) on porcine intestinal epithelial cells (IPEC-J2) shows a dose-time dependent effect, the proliferation inhibition rate of PEC-J2 cells is gradually increased (P <0.05) along with the increase of DON concentration and the prolongation of action time, the release amount of LDH (lactate dehydrogenase) is increased (P <0.05), and the DON destroys the cell membrane of IPEC-J2, increases the cell permeability and has certain damage effect on the porcine intestinal epithelial cells.
(4) When the saccharomyces cerevisiae is co-cultured with DON and porcine intestinal epithelial cells, the saccharomyces cerevisiae can relieve the inhibition effect of the DON on the proliferation of the intestinal epithelial cells, reduce the release amount (P <0.05) of Lactate Dehydrogenase (LDH), improve the degradation rate of the DON and obviously increase the expression amount (P <0.05) of interleukin IL-8; especially in the high dose DON addition group (1.2 mu g/mL), the addition of Saccharomyces cerevisiae significantly increased the expression level of IL-6, IL-8, IL-10 genes (P <0.05), but had no significant effect on the expression level of the Tight Junction Protein (TJP) and occludin (Occuldin) genes.
(5) Compared with the DON group, the yeast and DON co-culture group respectively reduces early and late apoptotic cells by 44.78% and 46.37% (P <0.05), increases the number of living cells by 2.35% (P <0.05), and reduces the number of necrotic cells by 38.05% (P > 0.05).
Drawings
FIG. 1 is a graph showing the effect of different concentrations of DON on IPEC-J2 cell viability and cell proliferation rate for 24 h.
FIG. 2 shows the expression level of mRNA for cytokine IL-6 in the co-culture of Saccharomyces cerevisiae and DON at different concentrations.
FIG. 3 shows the expression level of mRNA for cytokine IL-8 in the co-culture of Saccharomyces cerevisiae and DON at different concentrations.
FIG. 4 shows the expression level of mRNA for cytokine IL-10 in the co-culture of Saccharomyces cerevisiae and DON at various concentrations.
FIG. 5 shows the expression level of mRNA of cytokine TJP by co-culture of Saccharomyces cerevisiae and DON at various concentrations.
FIG. 6 shows the expression level of mRNA of cytokine Ocplus in the co-culture of Saccharomyces cerevisiae and DON at various concentrations.
FIG. 7 is a fluorescent double-stained two-dimensional dot plot of the effect of Saccharomyces cerevisiae and DON co-culture on apoptosis, wherein 7-1 is the control group, 7-2 is the DON group, 7-3 is the Saccharomyces cerevisiae group, 7-4 is the Saccharomyces cerevisiae + DON group, and the four quadrants represent the area ratios of the cells; the abscissa represents the number of Annexin V (phospholipid-binding protein) stained and FITC (fluorescein isothiocyanate) fluorescently labeled IPEC-J2 cells (number of fluorescent channels); the ordinate is the number of IPEC-J2 cells (number of fluorescent tracks) stained with PI (propidium iodide), fluorescently labeled with PerCP (chlorophyll protein); wherein Q1 represents necrotic cells, Q2 represents late apoptotic cells, Q3 represents live cells, and Q4 represents early apoptotic cells.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
1 materials and methods
1.1 test materials
Porcine intestinal epithelial cells (IPEC-J2): is a gift from animal-derived food safety key research laboratory of Henan province of Henan university of Henan agriculture. The saccharomyces cerevisiae is a strain preserved in the laboratory.
1.1.1 reagents
DMEM/F-12 medium (Hyclone, USA); fetal Bovine Serum (FBS) (hangzhou sijiqing bioengineering materials ltd); streptomycin mixed solution, MTT, PBS buffer solution, 0.25% pancreatin protease-EDTA digestive juice, 0.25% pancreatin protease (Beijing Solibao science and technology Co., Ltd.); DMSO (dimethyl sulfoxide) (Sigma company, usa); trizol, RNase-free water, reverse transcription kit, real-time fluorescence quantification kit (Takara, Japan).
1.1.2 Main apparatus (see Table 1)
TABLE 1 Main instrumentation
1.1.3 preparation of reagents
(1) Cell culture medium: fully and uniformly mixing 10% of fetal calf serum, 1% of the mixed solution of the cyanothecine and 90% of DMEM/F-12 culture solution (the fetal calf serum is stored at the temperature of-20 ℃, is firstly melted in a refrigerator at the temperature of 4 ℃ and then is inactivated in a water bath at the temperature of 56 ℃ for 30min when in use), and is stored at the temperature of 4 ℃ for later use.
(2) Cell cryopreservation solution: the preparation method comprises the steps of uniformly mixing 40% fetal calf serum, 10% DMSO and 50% DMEM/F-12 culture solution, and preparing the preparation for use.
(3) YPD medium was prepared as described above.
(4) Preparing a DON storage solution: dissolving DON pure product with ethanol completely to obtain 5mg/mL mother liquor, and storing at-20 deg.C in dark. When in use, the solution is diluted to 100 mu g/mL by using DMEM/F-12 culture solution and stored at 4 ℃ for later use.
(5) Preparing DON working solution: 100 ug/mL DON stock solution was prepared into 0, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6, and 19.2 ug/mL working solutions with DMEM/F-12 medium, and stored at 4 deg.C in dark for use.
1.2 culture of cells
1.2.1 cell Resuscitation
Taking out IPEC-J2 cell strain preserved in liquid nitrogen, rapidly dissolving in water bath at 37 deg.C, transferring into cell culture medium containing 5 times of volume, centrifuging at 1000r/min for 5min, discarding supernatant, uniformly blowing with 1mL complete culture medium containing 10% serum and 1% double antibody, transferring into 25cm2The culture flask of (3) was charged with 5mL of fresh medium at 37 ℃ and 5% CO2The culture is carried out under the conditions of (1), the liquid changing time is determined according to the growth condition of cells, and if the culture medium turns yellow or dead cells are excessive, the fresh culture medium is immediately changed. When the cells grow to 80% -90% of the bottle wall, digesting and passaging by using 0.25% trypsin protease-EDTA digestive juice.
1.2.2 cell culture and passage
Discarding the old culture solution in the culture bottle, washing with 1mL PBS buffer solution for 2-3 times, adding 1mL 0.25% pancreatin-EDTA digestive fluid, digesting in a cell culture box for 3-5min, observing the cell shape under an inverted microscope, and adding 1mL culture medium to terminate digestion when the cell becomes round, the gap becomes larger or the cell falls down like fine sand from the bottle wall facing to the place with light; repeatedly blowing and beating cells on the bottle wall by using a pipette to form a cell suspension, centrifuging the suspension for 5min at the speed of 1000r/min, removing supernatant, adding culture medium to suspend the cells, carrying out passage according to the ratio of 1:2 or 1:3, supplementing the culture medium to 5mL, carrying out passage at the temperature of 37 ℃ with 5% CO2Culturing under the conditions of (1), changing the culture medium every other day, and observing the morphology of the cells.
1.2.3 cell cryopreservation
Discarding the old culture solution in the culture bottle, washing with 1mL PBS buffer solution for 2-3 times, adding 1mL 0.25% pancreatin-EDTA digestive solution, digesting in cell culture box for 3-5min, adding 1mL complete culture medium to stop digestion, repeatedly blowing cells on the bottle wall with a pipette to form cell suspension, centrifuging the suspension at 1000r/min for 5min, discarding the supernatant, adding freezing medium, counting with a blood counting plate, and adjusting the cell concentration to 1-3 × 106And (3) transferring the seeds/m L into a freezing storage tube, marking, precooling for 20min at 4 ℃, putting the seeds at-20 ℃ for 1h, then transferring the seeds to be frozen at-80 ℃ overnight, and transferring the seeds to liquid nitrogen for long-term storage the next day.
1.3 activation and culture of the Strain
Respectively activating the stored saccharomyces cerevisiae in a YPD liquid culture medium for 24 hours, respectively inoculating the activated bacterial liquid into a fresh culture medium according to the inoculum sizes of 5% and 2%, continuously culturing for 16 hours and 24 hours respectively, coating plates, counting, and storing in a refrigerator at 4 ℃ for later use.
1.4 establishment of IPEC-J2-DON cell proliferation model
1.4.1 Effect of DON on IPEC-J2 cell proliferation
Taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate after conventional digestion counting, wherein each well has the number of cells of 1 multiplied by 10 and each well has the concentration of 100 mu L/well4After 24h of cell culture, the original culture medium is discarded after the cell is attached to the wall, the cell is washed once by PBS, serum-free and double-antibody-free DMEM/F-12 culture media with different concentrations of DON (0, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.6 and 19.2 mu g/mL) are respectively added, 6 holes are made for each concentration, the 3 times are repeated, a control group contains ethanol with the same volume as that of the DON group, a zero adjustment group containing only the culture medium and no cell is used for respectively culturing the cells for 12h, 24h and 48h, the morphology of the cells is observed under a microscope, and then 10 mu L of MTT (the concentration is 5mg/mL) is added into each hole, the temperature is 37 ℃, and the CO content is 5 percent2After incubating for 4 hours in the incubator, the culture solution was carefully aspirated, 150 μ L of DMSO was added and shaken on a shaker for 10min to sufficiently dissolve formazan, and absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader, and cell viability was calculated as OD value, and the cell viability was calculated as follows:
the cell survival rate (test group OD value-zero-adjusted group OD value)/(control group OD value-zero-adjusted group OD value) × 100%
1.4.2 Effect of different DON concentrations at different times on IPEC-J2 cell proliferation
Taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate after conventional digestion counting, wherein each well has the number of cells of 1 multiplied by 10 and each well has the concentration of 100 mu L/well4After 24h of cell culture and adherence, the original culture medium is discarded, washed once by PBS, and DON (0, 0.075, 0.15, 0.3, 0.6, 1.2 mu g/mL) with different concentrations is respectively addedThe serum-free and double-antibody-free DMEM/F-12 culture medium is prepared into 6 duplicate wells at each concentration, the duplicate wells are repeated for 3 times, a control group contains ethanol with the same volume as that of a DON group, cells are observed under a microscope when the cells are cultured for 4h, 8h, 16h and 32h by using a zero-setting well containing only the culture medium and no cells, and then 10 mu LMTT (5mg/mL) is added into each well to detect the cell viability.
1.5 Effect of Saccharomyces cerevisiae on IPEC-J2 cell proliferation
1.5.1 treatment of Saccharomyces cerevisiae
Centrifuging the cultured yeast at 8000rpm/min for 5min, collecting supernatant, sterilizing with 0.22 μm filter membrane, and storing at 4 deg.C; and (3) resuspending the centrifuged somatic cells by using a DMEM/F-12 culture medium, centrifuging at 8000rpm/min for 5min, washing twice, suspending the cells by using the DMEM/F-12 culture medium according to the equal volume, and storing in a refrigerator at 4 ℃ for later use.
1.5.2 Effect of Saccharomyces cerevisiae on the viability of IPEC-J2 cells
Taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate after conventional digestion counting, wherein each well has the number of cells of 1 multiplied by 10 and each well has the concentration of 100 mu L/well4After 24h of cell culture and adherence, the original culture medium is discarded and washed once with PBS, and the test groups are as follows:
(1) control group: DMEM/F-12 medium group;
(2) yeast fermentation broth (S-FL group): adding 5 μ L of yeast fermentation liquid into each well to make MOI ratio of yeast to cell 0.1, 1, 10 respectively, i.e. yeast in each well is 1 × 103、1×104、1×105The remaining 95. mu.L were supplemented with DMEM/F-12 medium;
(3) yeast supernatant (S-CFS) group: adding 5 μ L of supernatant of yeast culture solution with the same viable count as the previous step into each well, and supplementing the rest 95 μ L with DMEM/F-12 culture medium;
(4) yeast cell (S-C) group: adding 100 μ L of yeast diluted with DMEM/F-12 to obtain yeast cells with different viable counts in each well, respectively, to obtain yeast cells with a cell size of 1 × 103、1×104、1×105The MOI ratios are 0.1, 1 and 10 respectively.
Are divided into groups according to the above, each group is divided intoRepeating for 3 times for 1 hr, 2 hr, 4 hr and 8 hr, washing with PBS buffer solution for 2-3 times to remove added thallus, adding 100 μ L DMEM/F-12 medium with final concentration of 0.5mg/mL MTT, 37 deg.C and 5% CO2Incubations were performed in an incubator for 4h, after 4h the culture was carefully aspirated, 150. mu.L of DMSO was added and shaken on a shaker for 10min to dissolve formazan sufficiently, and the OD value was measured with a microplate reader at a wavelength of 450 nm.
1.5.3 Effect of Saccharomyces cerevisiae supernatant on the viability of IPEC-J2 cells
And centrifuging fermentation liquor of the saccharomyces cerevisiae cultured for 24 hours at 8000rpm/min for 5min, collecting supernatant, diluting the supernatant with DMEM/F-12 culture medium in gradient manner to 2, 4, 8, 16, 32, 64 and 128 times of the original solution respectively. Taking cells in logarithmic growth phase, inoculating the cells to a 96-well plate after conventional digestion counting, wherein each well has the number of cells of 1 multiplied by 10 and each well has the concentration of 100 mu L/well4And (3) removing the original culture medium after the cells are cultured for 24 hours and adhere to the wall, washing the cells once by using PBS (phosphate buffer solution), adding 100 mu L of the supernatant diluent into each hole, repeating the treatment for 3 times for 6 times, and culturing the cells until the cell activity is measured by using MTT (methyl thiazolyl tetrazolium) for 1 hour, 2 hours, 4 hours and 8 hours respectively.
1.6 Effect of Saccharomyces cerevisiae Co-culture with DON on IPEC-J2 cell proliferation
1.6.1 Effect of Saccharomyces cerevisiae Co-culture with DON on IPEC-J2 cell viability
Cells in logarithmic phase are taken, inoculated to a 96-well plate after conventional digestion counting, the number of the cells in each well is 1 multiplied by 104, the original culture medium is discarded after the cells are cultured for 24h and attached to the wall, and washed once by PBS, and the experimental groups are 1.7. Repeating for 3 times each group for 6 multiple wells, culturing for 4h and 8h, respectively, washing with PBS buffer solution for 2-3 times until the added thallus is washed off, adding 100 μ L DMEM/F-12 medium with final concentration of 0.5mg/mL MTT into each well, incubating at 37 deg.C with 5% CO2Incubations were performed in an incubator for 4h, after 4h the culture was carefully aspirated, 150. mu.L of DMSO was added and shaken on a shaker for 10min to dissolve formazan sufficiently, and the OD value was measured with a microplate reader at a wavelength of 450 nm.
1.6.2 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 cell lactate dehydrogenase release and DON residual amount
Taking cells in logarithmic growth phase, and removingAfter the cells are counted, the cells are inoculated into a 6-well plate with 2 mL/well and the number of cells per well is 2 multiplied by 105And after the cells are cultured to be adherent, discarding the original culture medium, washing the cells once by PBS (phosphate buffer solution), grouping the cells into 1.8.1 groups, repeating each group for 3 times, after the cells are cultured for 8 hours, sucking the culture solution of each hole into a 2mL centrifuge tube, centrifuging the cells at 3000rpm/min for 5min, sucking 120 mu L of supernatant again, placing the supernatant into a new centrifuge tube, measuring the release amount of LDH according to the steps of a Lactate Dehydrogenase (LDH) kit, taking 500 mu L of the supernatant to measure the content of DON, and calculating the degradation rate of the DON.
1.7 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 cytokine Gene expression level
Taking cells in logarithmic growth phase, inoculating the cells to a 6-well plate after conventional digestion counting, wherein each well has 2 mL/well and the number of the cells per well is 2 multiplied by 105And (3) culturing the cells until the cells are attached to the wall, discarding the original culture medium, washing the cells once by using PBS (phosphate buffer solution), grouping the cells into 1.8.1 groups, repeating the groups for 3 times, after culturing for 8 hours, sucking the supernatant, washing the cells for 2-3 times by using PBS, and collecting the cells.
1.7.1 cellular RNA extraction
(1) Adding 1mL trizol into each hole, blowing and beating for 3-5 times by a pipette gun to fully crack cells, and standing for 5min at room temperature;
(2) adding 200 μ L chloroform into the lysate, shaking vigorously by hand for 15s, standing at room temperature for 3min, centrifuging at 12000 Xg for 15min at 4 deg.C;
(3) carefully transferring the supernatant (about 300. mu.L) to a new 1.5mL centrifuge tube, adding 600. mu.L isopropanol, vortex mixing, standing at room temperature for 10min, centrifuging at 12000 Xg for 10min to precipitate RNA;
(4) discarding the supernatant, adding 1mL 75% ethanol, mixing by vortex, centrifuging at 4 deg.C and 7500 Xg for 5 min;
(5) discarding supernatant, reversely buckling the centrifuge tube on clean absorbent paper to absorb residual liquid, and air-drying for 10-15 min.
(6) Adding 20 μ L RNase-free water into RNA precipitate, vortex resuspending RNA precipitate, standing on ice for 10-30min to dissolve RNA, and storing at-80 deg.C;
(7) mu.L of RNA stock solution was taken to detect its concentration.
1.7.2 Synthesis of cDNA
Reverse transcribing the extracted RNA to cDNA, invertingRecording operation according to Bao bioengineering (Dalian) Co Ltd
RT reagent Kit reverse transcription Kit instruction, reverse transcription cDNA at-20 ℃ storage.
1.7.3 real-time fluorescent quantitative PCR detection
(1) Primer design
Based on the mRNA sequence of the pig gene available on Genbank, the Primier Premier 5.0 software was used to design quantitative primers (Table 2) synthesized by Shanghai bioengineering technology, Inc.
(2) Reaction system
The reaction system is shown in Table 3, following the instructions of the Probiotics PrimeScript RT reagent Kit Perfect Real Time Kit.
(3) Reaction procedure: the reaction system was performed on a Bio-Rad iQ5 fluorescent quantitative PCR instrument, and the reaction procedure is shown in Table 4.
TABLE 2 fluorescent quantitation primers
TABLE 3 RT-PCR reaction System (. mu.L)
TABLE 4 RT-PCR reaction procedure
1.8 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 apoptosis
Taking cells in logarithmic growth phase, inoculating the cells to a 6-pore plate after conventional digestion and counting2 mL/well, 5X 10 cells/well5After the cells were cultured to the adherent surface, the original medium was discarded and washed once with PBS. The assay was divided into control, Saccharomyces cerevisiae (and cell ratio 1:1), DON addition (1.2. mu.g/mL), Saccharomyces cerevisiae + DON, 3 replicates per group. After the co-culture for 8 hours, the supernatant was aspirated, washed with PBS 2 to 3 times, digested with EDTA-free pancreatin for 3min, centrifuged at 1000rpm for 5min, washed with PBS 2 times, centrifuged at 2000rpm for 5min, and the cells were collected. Adding 500 mu L Binding Buffer suspension cells, adding 5 mu L Annexin V-FITC, mixing, adding 5 mu L Propidium Iodide, mixing, reacting at room temperature in dark place for 15min, and detecting.
1.9 data analysis
The results are expressed as mean. + -. standard error, analyzed by variance and multiplicity using SPSS 20.0, and compared by significance using Turkey method, as P<0.05 indicated significant difference. Data for qPCR assay results comparison of ct with Bio-Rad CFX96 software (2)-△△CtMethod) method for analytical calculation.
2 analysis of results
2.1 Effect of different DON concentrations on IPEC-J2 cell viability
The effect of different DON concentrations on the cell viability of IPEC-J2 cells after 24h is shown in FIG. 1. As can be seen from fig. 1, the effect of DON on IPEC-J2 cells is a dose-dependent effect, with cell viability decreasing significantly with increasing DON concentration (P < 0.05). When the concentration of DON is more than 0.075 μ g/mL, the cell proliferation rate is significantly reduced (P < 0.05). The inhibition rates of cell proliferation are respectively 9.49% (P >0.05) and 16.19%, 22.74%, 36.18%, 38.96%, 40.46%, 43.00%, 46.70% and 50.94% (P <0.05) when the concentration of DON is 0.075 μ g/mL-19.2 μ g/mL.
2.2 Effect of different DON concentrations at different times on the viability of IPEC-J2 cells
As can be seen from Table 5, at 2h, all DON concentrations were not significantly different compared to the control cell viability (P > 0.05). At 4h, the DON concentration is 0.075 μ g/mL and 0.15 μ g/mL, the difference with the control group is not significant (P > 0.05); the DON concentrations of 0.3. mu.g/mL, 0.6. mu.g/mL and 1.2. mu.g/mL were significantly different from the control group (P <0.05), and the difference between the respective DON concentrations was not significant (P > 0.05). At 8h, the DON concentration of 0.075 μ g/mL was not significantly different from the control group (P >0.05), and the remaining four concentrations were significantly different from the control group (P < 0.05). Cell viability was significantly reduced at both 16h and 32h with 5 DON concentrations (P <0.05), with DON concentrations of 0.15 μ g/mL, 0.3 μ g/mL, 0.6 μ g/mL, and 1.2 μ g/mL differing significantly at 32h (P < 0.05). The cell growth is dose-time dependent, and the cell proliferation rate shows a descending trend along with the increase of time under the same toxin concentration; at the same time, the cell proliferation rate decreased with time. Compared with a control group, the cell proliferation rates of different concentrations of DON under the action of 4h are 89.31% (P >0.05), 87.83% (P >0.05) and 86.53%, 85.00% and 85.78% (P < 0.05); the cell proliferation rate at 8h is respectively 95.25% (P >0.05) and 82.27%, 89.01%, 82.12% and 78.11% (P < 0.05); the 16h cell proliferation rate is 88.42% (P <0.05), 91.14% (P >0.05) and 77.74%, 69.62% and 69.64% (P <0.05) respectively; the 32h cell proliferation rate is 94.45%, 88.83%, 80.42%, 72.10% and 64.56% (P <0.05), respectively.
Table 5 effect of different DON concentrations and different times on IPEC-J2 cell viability (OD value) (n ═ 6)
Note: capital English letters indicate the difference in activity of IPEC-J2 cells when different DON concentrations are acted on for the same time; lower case letters indicate the difference in activity of IPEC-J2 cells over time for the same DON concentration. The same applies below.
2.3 Effect of Saccharomyces cerevisiae on IPEC-J2 cell proliferation
2.3.1 Effect of different constituents of Saccharomyces cerevisiae on IPEC-J2 cell proliferation
As can be seen from table 6, the MOI of 0.1 and 1, yeast supernatant and broth had no significant effect on cell proliferation at each time period (P > 0.05); at 8h, the difference between the yeast cell addition groups with MOI of 1 and 10 respectively is not significant (P >0.05), but the cell viability is increased by 25.71% (P >0.05) and 37.14% (P <0.05) compared with the control group respectively.
2.3.2 Effect of supernatants from Saccharomyces cerevisiae cultures on IPEC-J2 cell proliferation
As can be seen from Table 7, the differences between the dilutions of the Saccharomyces cerevisiae supernatant and the control group were not significant (P >0.05) at 1h, 2h, and 4h, and the cell proliferation was significantly inhibited (P <0.05) at 8h by 2-fold dilution of the supernatant, with the cell proliferation inhibition rate being 30.77% (P < 0.05).
TABLE 6 Effect of different Saccharomyces cerevisiae components on the viability (OD value) of IPEC-J2 cells (n ═ 6)
TABLE 7 Effect of the supernatants of Saccharomyces cerevisiae cultures on the viability (OD value) of IPEC-J2 cells (n ═ 6)
2.3.3 Effect of Saccharomyces cerevisiae interfering with DON on IPEC-J2 cell proliferation
As shown in Table 8, the cell viability of the Saccharomyces cerevisiae-added group was increased by 7.69% (P >0.05) at 4h compared to the control group, and the cell viability of the yeast-added group was increased by 21.05% (P <0.05) compared to the yeast-not-added group at a DON concentration of 1.2. mu.g/mL; at 8h, the cell viability of the yeast-added group is increased by 44.83% (P <0.05) compared with that of the control group, and when the DON concentration is 0.075 μ g/mL, 0.15 μ g/mL, 0.3 μ g/mL, 0.6 μ g/mL, or 1.2 μ g/mL, the cell viability of the yeast-added group is increased by 29.63%, 11.11%, 20.83%, 4%, or 4.17% respectively compared with the yeast-not-added group, but the difference is not significant (P > 0.05).
TABLE 8 Effect of Saccharomyces cerevisiae and DON coculture on the viability (OD value) of IPEC-J2 cells (n ═ 6)
2.3.4 Effect of Saccharomyces cerevisiae and DON Co-culture on cellular LDH Release and DON residual amount
As can be seen from table 9, when the DON concentration is greater than 0.075 μ g/mL, the LDH release amount is significantly increased (P <0.05), compared with the control group, the LDH release is increased by 7.14% (P >0.05) and 146.43%, 160.71%, 182.14%, 167.86% (P <0.05), respectively. Saccharomyces cerevisiae decreased LDH release compared to control but did not differ significantly (P > 0.05); LDH release was reduced by 26.67% (P >0.05) and 66.67%, 69.86%, 72.15%, 68.00% (P <0.05) for the yeast and DON groups, respectively, compared to the DON group. The concentration difference of DON in yeast and the DON addition group and the corresponding DON addition group is not significant (P >0.05), but the DON concentration is reduced to a certain extent, and the degradation rates are 57.14%, 46.15%, 42.31%, 26.67% and 13.51% (P >0.05), respectively.
TABLE 9 Effect of Saccharomyces cerevisiae coculture with DON at various concentrations on cellular LDH release and DON retention (n. 3)
2.3.5 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 cell-associated Gene expression levels
The expression of cytokine mRNA by Saccharomyces cerevisiae co-cultured with DON at various concentrations is shown in FIG. 2. As shown in FIG. 2, the gene expression level of IL-6 was up-regulated by 67% (P <0.05) compared to the control group at a DON concentration of 1.2. mu.g/mL, and the expression level of IL-6 was not significantly affected by DON at other concentrations (P > 0.05); the expression level of IL-6 in yeast and 0.3, 0.6 and 1.2. mu.g/mL DON co-addition groups was respectively up-regulated by 50.89%, 123.88% and 110.78% (P < 0.05). As shown in FIG. 3, the expression level of IL-8 was decreased by 46% (P <0.05) and 36% (P <0.05) at DON concentrations of 0.075. mu.g/mL and 0.3. mu.g/mL, respectively; after yeast and DON are added, the expression level of IL-8 is remarkably increased compared with that of a DON group, and the increasing rates are 83.33%, 73.44%, 59.22%, 153.33% and 221.55% (P is less than 0.05). As can be seen from FIG. 4, the DON at different concentrations had no significant effect on the expression of IL-10 compared to the control group (P > 0.05); the yeast plus DON (1.2. mu.g/mL) group up-regulated the expression of IL-10 by 36.81(P <0.05) compared to the DON (1.2. mu.g/mL) group. As can be seen from fig. 5, the expression level of TJP showed an increasing trend with increasing DON concentration, and the expression up-regulation rates of TJP were 18% (P >0.05) and 55%, 91%, 80%, 116% (P <0.05), respectively, compared to the control group; the expression level of TJP was adjusted in the co-cultured group of yeast and DON as compared with the DON group, and the down-regulation rate of TJP was 36.13% (P <0.05) at a DON concentration of 0.3. mu.g/mL. As can be seen from FIG. 6, the expression level of Ocplus was up-regulated in all the DON-added groups, and the upregulation rates of Ocplus were 48% (P >0.05), 44% (P >0.05), 175% (P <0.05), 100% (P <0.05), and 150% (P <0.05), respectively, as compared with the control group; the yeast and DON co-culture groups down-regulated the expression level of Ocplus compared with the DON group, and the down-regulation rates were 35.81% (P <0.05), 27.78% (P >0.05), 50.18% (P <0.05), 19%, and 18.4% (P >0.05), respectively.
2.3.6 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 apoptosis
As can be seen from fig. 7 and table 10, the DON-added group significantly increased the proportion of late apoptotic cells (P <0.05) compared to the control group, which was 4.53 times that of the control group, early apoptotic cells were 2.23 times that of the control group (P <0.05), and the number of dead cells was 2.83 times that of the control group (P < 0.05); compared with the DON group, the yeast and DON co-culture group respectively reduces early and late apoptotic cells by 44.78% and 46.37% (P <0.05), increases the number of living cells by 2.35% (P <0.05), and reduces the number of necrotic cells by 38.05% (P > 0.05); the yeast group and the control group have no significant difference in each parameter (P > 0.05).
TABLE 10 Effect of Saccharomyces cerevisiae and DON Co-culture on IPEC-J2 proliferation and apoptosis (%)
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (4)
1. A method for detecting non-disease diagnosis and treatment purposes of reducing IPEC-J2 cell apoptosis by saccharomyces cerevisiae and DON interaction is characterized by comprising the following steps:
(1) taking IPEC-J2 cells in logarithmic phase, digesting, counting, inoculating to 6-well plate, 2 mL/well, and the number of cells per well is 5 × 105After the cells are cultured to be adherent, the original culture medium is discarded, and the cells are washed once by PBS;
(2) the detection test is provided with four groups, namely a blank control group, a saccharomyces cerevisiae preparation group, a DON preparation group and a saccharomyces cerevisiae preparation + DON preparation group, wherein each group is repeated by three;
(3) adding the four groups of preparations obtained in the step (2) into a 6-well plate filled with IPEC-J2 cells obtained in the step (1) respectively;
(4) placing the 6-hole plate treated in the step (3) at 30-35 ℃, co-culturing for 8h, discarding the supernatant, washing with PBS for 2 times, centrifuging at 2000rpm for 5min, and collecting cells;
(5) adding 500 mu L of Binding Buffer suspension cells into the cells collected in the step (4), adding 5 mu L of Annexin V-FITC, uniformly mixing, adding 5 mu L of Propidium Iodide, uniformly mixing, reacting at room temperature in a dark place for 15min, and then carrying out cell activity detection; the saccharomyces cerevisiae has a preservation number of CGMCC No: 2.3866;
the concentration of DON in the step (2) is 0.075-0.15 mu g/mL, and the cell number of the saccharomyces cerevisiae is the same as that of IPEC-J2 cells;
the treatment of the saccharomyces cerevisiae preparation in the step (2) comprises the following steps:
a. activating the stored saccharomyces cerevisiae in a YPD liquid culture medium for 24 hours, inoculating the activated bacterium liquid into a fresh YPD liquid culture medium according to the inoculation amount of 2-5%, continuously culturing for 16-24 hours, coating plates for counting, and storing in a refrigerator at 4 ℃;
b. taking the saccharomyces cerevisiae culture medium stored in the step a, centrifuging for 5min at 8000rpm/min, removing supernate, sterilizing through a 0.22-micron filter membrane to obtain thalli cells, and storing at 4 ℃ for later use;
c. and re-suspending the centrifuged thallus cells by using a DMEM/F-12 culture medium, centrifuging at 8000rpm/min for 5min, washing twice, suspending the thallus by using the DMEM/F-12 culture medium according to the equal volume to obtain a saccharomyces cerevisiae preparation, and storing the saccharomyces cerevisiae preparation in a refrigerator at 4 ℃ for later use.
2. Use of the test method according to claim 1 for non-disease diagnostic and therapeutic purposes, characterized in that: saccharomyces cerevisiae was used to alleviate DON-induced apoptosis of porcine intestinal epithelial cells with inflammation.
3. Use according to claim 2 for non-disease diagnostic and therapeutic purposes, characterized in that: each 2mL of the mixture of Saccharomyces cerevisiae and DON contains 5X 105One Saccharomyces cerevisiae cell, 5X 105Individual porcine intestinal epithelial cells and 1.2. mu.g/mL DON.
4. Use according to claim 3 for non-disease diagnostic and therapeutic purposes, characterized in that: every Kg of feed is added with 1-10 mL of the mixture of saccharomyces cerevisiae and DON with the preservation number of CGMCC No:2.3866 as claimed in claim 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101437845A (en) * | 2006-02-15 | 2009-05-20 | 莫沃斯技术有限公司 | Apoptosis methods, genes and proteins |
| US9428758B2 (en) * | 2011-12-02 | 2016-08-30 | Rutgers, The State University Of New Jersey | Plant genes that confer resistance to trichothecene mycotoxins and fusarium head blight |
| CN107988271A (en) * | 2017-12-14 | 2018-05-04 | 安徽瑞福祥食品有限公司 | A kind of processing method of wheat containing vomitoxin and application |
| CN108070599A (en) * | 2018-01-30 | 2018-05-25 | 南京农业大学 | High efficient expression and its application of the Recombinant Swine interleukin-22 2 in Escherichia coli |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6537762B1 (en) * | 1999-07-30 | 2003-03-25 | Board Of Trustees Of Michigan State University | Peptide mimotope to mycotoxin deoxynivalenol and uses thereof |
| CN104388247B (en) * | 2014-11-27 | 2017-07-21 | 江苏洋河酒厂股份有限公司 | A kind of production method of white wine function enzyme preparation |
-
2018
- 2018-06-01 CN CN201810556994.3A patent/CN108866145B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101437845A (en) * | 2006-02-15 | 2009-05-20 | 莫沃斯技术有限公司 | Apoptosis methods, genes and proteins |
| US9428758B2 (en) * | 2011-12-02 | 2016-08-30 | Rutgers, The State University Of New Jersey | Plant genes that confer resistance to trichothecene mycotoxins and fusarium head blight |
| CN107988271A (en) * | 2017-12-14 | 2018-05-04 | 安徽瑞福祥食品有限公司 | A kind of processing method of wheat containing vomitoxin and application |
| CN108070599A (en) * | 2018-01-30 | 2018-05-25 | 南京农业大学 | High efficient expression and its application of the Recombinant Swine interleukin-22 2 in Escherichia coli |
Non-Patent Citations (4)
| Title |
|---|
| Efects of Saccharomyces cerevisiae on alleviating cytotoxicity of porcine jejunal epithelia cells induced by deoxynivalenol;Yang Liu et al.;《AMB Expr》;20190903;第9卷(第137期);第1-11页 * |
| Protective effects of mannan/β-glucans from yeast cell wall on the deoxyniyalenol-induced oxidative stress and autophagy in IPEC-J2 cells;Wenyan Guo et al.;《International Journal of Biological Macromolecules》;20190525;第135卷;第619–629页 * |
| S.boulardii预防DON诱导猪巨噬细胞凋亡的作用机理研究;王琨;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160615(第6期);摘要、第13-14页、第29页第3.2.1节、第45页第2段、第9页第2段 * |
| 脱氧血腐镰刀菌烯醇在IPEC-J2细胞凋亡中的作用及机制研究;张志岐等;《中国毒理学会第七次全国毒理学大会暨第八届湖北科技论坛论文集》;20151015;第274页 * |
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