CN113999808A - Method for regulating and controlling immunity in cow mammary gland epithelial cells by using escherichia coli - Google Patents
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Abstract
The invention provides a method for regulating and controlling immune function in milk cow mammary gland epithelial cells by using escherichia coli, which comprises the following steps: cell culture and cell treatment with E.coli. The method provided by the invention can use wild A-type escherichia coli to construct an inflammation model, and uses a specific escherichia coli to regulate and control the mammary epithelial inflammatory reaction of the dairy cow.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a method for regulating and controlling immune function in mammary epithelial cells of a milk cow by using escherichia coli.
Background
Enterobacteria are environmental pathogens that are ubiquitous in nature and can cause infections of humans and animals to a variety of degrees. The causes of the cow mastitis are complex and various, pathogenic bacteria infection is a main biological cause of the cow mastitis, and Escherichia coli is a main environmental pathogenic bacteria causing the cow mastitis. Clermont and the like perform phylogenetic typing on escherichia coli simply and quickly by a triple PCR mode, and the method performs PCR tests by taking genes chuA, yjaA and TspE4.C2 with evolution characteristics as target genes and then performs typing according to different gene sister ways. Coli can be classified into 4 main types from a phylogenetic point of view, i.e., A, B1, B2, and D. Wherein, the A type is mostly environment symbiotic bacteria, and the separation rate in the cow mastitis pathological material is higher.
In view of the wide source of A type Escherichia coli in nature, the A type Escherichia coli has great advantages and advantages if being put into related application of cow mastitis.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or other problems associated with the prior art methods of modulating immunity in the mammary epithelial cells of a dairy cow.
Therefore, one of the purposes of the present invention is to overcome the shortcomings of the existing products and provide a method for regulating the immune function in the mammary epithelial cells of the dairy cow by using escherichia coli.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a method for modulating immunity in cow mammary gland epithelial cells by Escherichia coli, which comprises the following steps:
cell culture: culturing the milk cow mammary gland epithelial cells by using a culture medium to obtain cultured milk cow mammary gland epithelial cells;
cell treatment: the culture medium containing the specific microorganism is replaced in the middle of the culture of the mammary epithelial cells of the dairy cows to carry out the culture.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in cell culture, the culture medium is RM [ I1640 culture medium.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in cell culture, the complete medium comprises 90% of culture medium and 10% of fetal bovine serum in proportion.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in cell culture, the temperature of culture is 37 ℃, and CO is2The concentration was 5%.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in the cell treatment, the medium was changed when the cells were cultured to 70%.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in cell processing, the specific microorganisms include Escherichia coli and Staphylococcus aureus.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in cell processing, the specific microorganism comprises wild type A Escherichia coli, wild type B1 Escherichia coli, standard Escherichia coli ATCC25922, and standard Staphylococcus aureus ATCC 25923.
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in the cell treatment, the specific microorganism is wild type A Escherichia coli.
As a preferable embodiment of the method for regulating and controlling the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method isThe method comprises the following steps: in cell processing, the concentration of a specific microorganism in the culture medium is 1 × 107CFU/mL。
As a preferable embodiment of the method for regulating the immune function in the mammary epithelial cells of the milk cow by using the Escherichia coli, the method comprises the following steps: in the cell treatment, the time for culturing after replacing the specific microorganism was 3 hours.
Research finds that Escherichia coli can activate genes which play a 30% role in inflammatory reaction in the mammary epithelial cells of the dairy cattle, and the genes are more than the gene expression changes after mammary tissue infection, so that the mammary epithelial cells of the dairy cattle have strong immunity, can accurately reflect the pathological changes after the mastitis infection of the dairy cattle, and serve as an important model for in-vitro research of the mastitis of the dairy cattle. No model of the induction effect of clinically isolated A-type escherichia coli on the inflammatory response of the mammary epithelial cells of the dairy cows is reported at present. The research is carried out on a wild A-type escherichia coli separated from a pasture to induce a milk cow mammary epithelial cell inflammatory reaction construction model.
The clinical separated wild A-type escherichia coli utilized by the method performs induction reaction on mammary epithelial cells of the dairy cow and constructs an inflammation model. Therefore, relevant experiments including a flow apoptosis experiment, an EdU cell proliferation experiment, a Zeiss field emission scanning electron microscope experiment, a fluorescence quantitative PCR experiment and the like are carried out according to research needs. Finally, it was demonstrated that wild isolated type A E.coli was present at a concentration of 1x107And when the concentration of the CFU is reduced to CFU/mL, the mammary epithelial cells of the dairy cow can be induced to generate inflammatory reaction, and the inflammatory model is successfully constructed.
The invention achieves the following beneficial effects:
1) the invention proves the application of wild A-type escherichia coli in inducing the inflammatory reaction of the mammary epithelial cells of the dairy cow for the first time. Compared with the existing research on the construction of a mammary epithelial cell inflammatory response model, the invention proves the immunoregulation effect of clinically separated wild-type escherichia coli on the mammary epithelial cells of the dairy cow from a cell level, improves the gene expression quantity of cell inflammatory factors, reduces the cell proliferation quantity, and destroys the normal forms of the cells, thereby having higher accuracy and reliability.
2) The invention proves that the wild separated A-type escherichia coli isConcentration of 1x107And when the concentration of the CFU is reduced to CFU/mL, the mammary epithelial cells of the dairy cow can be induced to generate inflammatory reaction, and the inflammatory model is successfully constructed. The flow cytometry is used for detecting the increase of the apoptosis rate and the increase of the gene expression quantity of the immune factor, thereby having higher feasibility.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 shows the results of detecting apoptosis rate of milk cow mammary gland epithelial cells by flow cytometry after wild type A Escherichia coli, wild type B1 Escherichia coli, standard Escherichia coli ATCC25922 and standard Staphylococcus aureus ATCC25923 are treated;
FIG. 2 shows the result of EDU cell proliferation of bovine mammary epithelial cells infected with wild type A E.coli;
FIG. 3 shows the results of cell scanning electron microscopy after infection of bovine mammary epithelial cells with wild type A Escherichia coli;
FIG. 4 shows the fluorescent quantitative PCR results of IL1 beta and TNF alpha genes of bovine mammary epithelial cells infected by wild type A Escherichia coli.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. The following examples are set forth to enable those skilled in the art to practice the invention.
Example 1
In the embodiment, effect screening is carried out on milk cow mammary epithelial cells infected by wild type A escherichia coli, wild type B1 escherichia coli, standard escherichia coli ATCC25922 and standard staphylococcus aureus ATCC25923 respectively, and comprises the following steps:
(1) cell culture: the mammary epithelial cells of the milk cow are cultured in RMPI 1640 culture medium with complete culture medium of 90% culture medium and 10% fetal calf serum at 37 deg.C in the presence of CO2The initial number of cells was about 5X 10 at a concentration of 5% and a medium volume of 2mL per well in a six-well plate3one/mL.
(2) Cell treatment: the cow mammary gland epithelial cells were divided into 5 groups, group 1 was a control group, and groups 2-5 were treatment groups. Culturing cells in a six-hole plate, and when the cells are cultured to 70%, respectively replacing the treatment groups 2, 3, 4 and 5 with wild type A escherichia coli, wild type B1 escherichia coli, standard escherichia coli ATCC25922 and standard staphylococcus aureus ATCC25923 with the concentration of 1x107CFU/mL of the medium, and culturing was continued for 3 hours.
(3) Sample preparation: after 3 hours, the medium was discarded and washed 2 times with PBS buffer, 2mL of trypsin was added to each well, digested with trypsin and centrifuged at low speed, and the cells were collected at about 5X 10 cell count5one/mL. Resuspend the cells with 100. mu.l of 1 XBinding Buffer; add 5. mu.L Annexin V and 5. mu.L PI, protect from light for 20 minutes at room temperature; adding 500. mu.L buffer solution, and detecting on a machine.
(4) Image analysis: annexin V-FITC and PI are most commonly used staining agents for detecting apoptosis, the two fluorescein can be excited by a laser with the wavelength of 488nm, signal receiving channels are respectively a first channel (FITC) and a third channel (ECD), therefore, the first graph of an apoptosis detection scheme is FSC/SSC, and the proportion of each apoptosis period in a cell population can be analyzed by taking the second graph of the cell population as a scatter graph of the first channel and the third channel in the whole circle, and the result is shown in figure 1, and the early apoptosis rate (Q1-UR) and the late apoptosis rate (Q1-LR) of the mammary epithelial cells of the dairy cattle treated by wild type A escherichia coli are increased. The wild type A escherichia coli can induce the apoptosis of the mammary epithelial cells of the dairy cow to cause the damage of the cells. The apoptosis detection of each group of cells is carried out by a flow cytometer, and the result is shown in figure 1, and the treatment effect of the wild A-type escherichia coli is superior to that of other treatment modes.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Example 2
In this example, cells were treated with an EdU kit, and the proliferation of cells was observed with an immunofluorescence microscope.
(1) Cell culture: the same as in example 1.
(2) Sample preparation: 1) the complete cell culture medium of example 1 was used as a 1000: 1 (reagent A) to prepare an appropriate amount of 50. mu.M EdU medium; adding 100 mu L of 50 mu M EdU culture medium into each hole, incubating for 2 hours, and discarding the culture medium; cells were washed 2 times with PBS for 5 minutes each. 2) Adding 50 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating at room temperature for 30 min, and discarding the fixing solution; adding 50 mu L of 2mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution; adding 100 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS; adding 100 μ L of penetrant (0.5% TritonX-100 PBS) into each well, decolorizing and shaking for 10 min; PBS wash 1 times, 5 minutes. 3) Add 100. mu.L of 1X per well
Incubating the dyeing reaction liquid for 30 minutes in a dark place at room temperature by using a decoloring shaker, and then discarding the dyeing reaction liquid; adding 100 μ L of penetrant (0.5% TritonX-10)0 PBS) was washed 2-3 times each for 10 minutes, and the penetrant was discarded. 4) Deionized water was added according to a 100: 1, diluting a reagent Hoechst33342 reaction solution according to the proportion of 1, preparing a proper amount of 1X Hoechst33342 reaction solution, and storing in a dark place; adding 100 mu L of 1X Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the dyeing reaction solution; each well was washed 3 times with 100. mu.L PBS per well.
(3) And (3) image observation: observing immediately after dyeing is finished; if the conditions are limited, the sample should be stored in a dark place at 4 ℃ for more than 3 days. When debugging the instrument, please adjust the exposure time to about 30ms, and do not need to adjust the exposure time as much as possible>1 s. The results are shown in FIG. 2, at a concentration of 1X107The proliferation amount of the milk cow mammary gland epithelial cells treated by the CFU/mL wild type A escherichia coli is obviously reduced, which indicates that the wild type A escherichia coli can promote the proliferation of the milk cow mammary gland epithelial cells.
Example 3
In this example, the morphology change of the cells after the treatment of escherichia coli was observed by using a GeminiSEM 300 zeiss field emission scanning electron microscope.
(1) Cell culture: the same as in example 1 (1).
(2) Preparing a cell climbing sheet: cells were cultured to 70%, counted after digesting the cells with 2ML of trypsin and resuspending the cells in the complete cell culture in example 1. And selecting proper cell density according to the size of the slide to be planted in the slide. After the cells were adherent, the experimental groups removed the excess complete medium and replaced the type a escherichia coli concentration of 1x107The culture was continued for 3 hours in CFU/mL of the medium. Before dropping 2.5% glutaraldehyde, the slide was submerged completely and pre-fixed at 4 ℃ overnight.
(3) Rinsing the sample: washed 4 times with 0.1M PBS or double distilled water. With 1% OSO4Post-fixation was performed and washed 3-4 times with 0.1M PBS or double distilled water. Each washing was carried out at 4 ℃ for 15 min.
(4) Sample dehydration: gradient concentration ethanol dehydration (30% → 50% → 70% → 80% → 90% → 95% → 100% → 100% anhydrous sodium sulfate), the sample was not touched when the liquid was changed, and stayed for 15min per step.
(5) Conducting and observing the appearance of the sample: drying the sample by a CDP-300 type critical point drying instrument, sticking the sample to a sample table with the surface to be observed facing upwards, spraying gold on the surface of the sample by using an SCD 500 type ion sputtering instrument, and observing the cell morphology by using a GeminiSEM 300 Zeiss field emission scanning electron microscope within one day. The results are shown in fig. 3, the morphology of the cow mammary epithelial cells treated by wild type A escherichia coli is obviously changed, and the cells are subjected to apoptosis lysis. Cell surface phagosome activity was impaired after treatment with wild type a e.
Example 5
In this example, changes in gene expression levels of IL-1. beta. and TNF. alpha. in cells treated with wild type A E.coli were detected by fluorescent quantitative PCR.
(1) Cell culture: the same as in example 2.
(2) RNA extraction: after 30h, the medium was discarded and washed 2 times with PBS buffer, 1ml ltrnzol was added to each well, and collected and frozen in tubes after sufficient pipetting. The lysed cells were transferred to new EP tubes, 200uL chloroform was added per tube (0.2 mL chloroform per 1mL of LTRNzol), shaken vigorously for 15s, allowed to stand at room temperature for 5min, and centrifuged at 12000r at 4 ℃ for 15 min. Gently take out, aspirate supernatant and transfer to a new EP tube, add 500mL isopropanol (equal to the supernatant volume), mix gently by inverting, and centrifuge at 12000r for 10min at 4 ℃. The supernatant was slowly discarded and a white precipitate formed at the bottom of the tube. Each tube was washed by adding 1mL of 75% ethanol, centrifuged at 7500r for 5min at 4 ℃ and the supernatant was discarded, and the above procedure was repeated. Air-drying at room temperature for 5min, adding 20uL deionized water, repeatedly blowing, and fully dissolving RNA. The RNA concentration and OD value (OD value range is 1.9-2.0 is optimal) are detected by using a spectrophotometer.
(3) Designing a primer: the nucleotide sequences of primers using IL1 beta, TNF alpha gene are as follows:
the IL1 beta upstream primer is: 5'-ATTCTCTCCAGCCAACCTTCATT-3', respectively;
the IL1 beta downstream primer is: 5'-TTCTCGTCACTGTAGTAAGCCATCA-3'
The TNF alpha upstream primer is: 5'-TCTCAAGCCTCAAGTAACAAGCC-3', respectively;
the TNF alpha downstream primer is: 5'-CCATGAGGGCATTGGCATAC-3'
(4) Reverse transcription: 1) genomic DNA was removed, 4 XgDNA wiper Mix 4uL was added, the RNA concentration was adjusted to 1ug/uL, the OD was 1.93 as detected, the template RNA was added in 1uL, and RNase free ddH2O to 16uL were added. Gently pipetting and beating with a pipette, and mixing uniformly at 42 ℃ for 2 min. 1) Preparing a reverse transcription reaction system, directly adding 5 XNo RT Control Mix 4uL into a reaction tube 1), and gently blowing and stirring the mixture evenly by a pipette, wherein the temperature is 50 ℃ for 15min, and the temperature is 85 ℃ for 2 min.
(5) qRCR: preparing a mixed solution as follows: 2 × AceQ qPCR SYBR Green Master Mix 10.0uL, Primer1(10uM)0.4uL, Primer2(10uM)0.4uL, 50 × ROX Rerenew Dye20.4 uL, cDNA 0.2uL, ddH2O8.8 uL. The amplification procedure was: pre-denaturation: 5min at 95 ℃; and (3) cyclic reaction: 10s at 95 ℃, 30s at 60 ℃ and 40 cycles; melting curve: 95 ℃ for 15s,60 ℃ for 60s,95 ℃ for 15 s. The results are shown in fig. 4, the expression levels of IL1 beta and TNF alpha of the cow mammary epithelial cell inflammatory genes after wild type A escherichia coli treatment are obviously up-regulated, and are respectively 2.17 times and 1.95 times.
Claims (10)
1. A method for regulating and controlling immunity in milk cow mammary gland epithelial cells by using Escherichia coli is characterized in that: the method comprises the following steps:
cell culture: culturing the milk cow mammary gland epithelial cells by using a culture medium to obtain cultured milk cow mammary gland epithelial cells;
cell treatment: the culture medium containing the specific microorganism is replaced in the middle of the culture of the mammary epithelial cells of the dairy cows to carry out the culture.
2. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell culture, the culture medium is RM [ I1640 culture medium.
3. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell culture, the complete culture medium comprises a culture medium with a proportion of 90% and fetal bovine serum with a proportion of 10%.
4. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell culture, the culture temperature is 37 ℃, and CO is adopted2The concentration was 5%.
5. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell treatment, when the cells are cultured to 70%, the culture medium is replaced.
6. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell treatment, the specific microorganisms include Escherichia coli and Staphylococcus aureus.
7. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 7, wherein the immune response is selected from the group consisting of: in the cell treatment, the specific microorganism includes wild type A Escherichia coli, wild type B1 Escherichia coli, standard Escherichia coli ATCC25922, and standard Staphylococcus aureus ATCC 25923.
8. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 7, wherein the immune response is selected from the group consisting of: in the cell treatment, the specific microorganism is wild type A Escherichia coli.
9. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell treatment, the concentration of a specific microorganism in the culture medium is 1x107CFU/mL。
10. The method of modulating immunity in the mammary epithelial cells of a dairy cow according to claim 1, wherein the immune response is selected from the group consisting of: in the cell treatment, the time for culturing after replacing the specific microorganism was 3 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717180A (en) * | 2022-04-06 | 2022-07-08 | 中国农业科学院兰州畜牧与兽药研究所 | In-vitro inflammation model of cow mammary epithelial cells and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559759A (en) * | 2011-12-19 | 2012-07-11 | 西安交通大学医学院第一附属医院 | HIP/PAP recombinant adenovirus and application thereof in resisting ulcerative colitis |
| WO2017053845A1 (en) * | 2015-09-24 | 2017-03-30 | The Regents Of The University Of California | Media for culturing epithelial cells |
| CN110205283A (en) * | 2018-04-18 | 2019-09-06 | 浙江大学 | A kind of method and its application that induction human amnion membrane breaks up to retinal pigment epithelium |
| CN111454879A (en) * | 2020-03-23 | 2020-07-28 | 中国农业大学 | Construction method of mammary epithelial cell model of escherichia coli infected single-layer compact dairy cow |
-
2021
- 2021-10-29 CN CN202111280651.7A patent/CN113999808A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559759A (en) * | 2011-12-19 | 2012-07-11 | 西安交通大学医学院第一附属医院 | HIP/PAP recombinant adenovirus and application thereof in resisting ulcerative colitis |
| WO2017053845A1 (en) * | 2015-09-24 | 2017-03-30 | The Regents Of The University Of California | Media for culturing epithelial cells |
| CN110205283A (en) * | 2018-04-18 | 2019-09-06 | 浙江大学 | A kind of method and its application that induction human amnion membrane breaks up to retinal pigment epithelium |
| CN111454879A (en) * | 2020-03-23 | 2020-07-28 | 中国农业大学 | Construction method of mammary epithelial cell model of escherichia coli infected single-layer compact dairy cow |
Non-Patent Citations (1)
| Title |
|---|
| 吴媛媛;李键;杨栎;毕琳;田甜;: "牦牛乳腺上皮细胞的体外培养的研究", 西南民族大学学报(自然科学版), no. 04 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717180A (en) * | 2022-04-06 | 2022-07-08 | 中国农业科学院兰州畜牧与兽药研究所 | In-vitro inflammation model of cow mammary epithelial cells and construction method and application thereof |
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