CN113583956A - Construction method and application of cell scorch model - Google Patents
Construction method and application of cell scorch model Download PDFInfo
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Abstract
The invention discloses a construction method and application of a cell apoptosis model. The construction method of the cell apoptosis model comprises the following steps: 1) pathogenic bacteria activation; 2) activated pathogen-treated cells; 3) collecting a cell sample and detecting the construction effect; the pathogenic bacteria is Escherichia coli K88, and the concentration used for treating cells is OD600= 0.4-0.6, and the number of viable bacteria contained is 1-3 × 107CFU/mL, adding 2 mM of trimethyl adenosine ATP after treating the cells for 2 hours, wherein the treatment time is 2.5 hours in total; the cell is mouse-derived mononuclear macrophage J774A.1. Application to mice, including 1) E.coli K88 activation; 2) the activated bacterium liquid is used for intragastric administration of the mice; 3) collecting mouse samples and detecting the construction effect. The invention is close to the actual production, the effect is equivalent to LPS, and the use cost is greatly reduced. The invention provides a basis for the subsequent research of the function and mechanism of cell apoptosis in the process of resisting pathogenic bacteria infection of a body, andlays a foundation for treating common pathogenic bacteria infection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a construction method of a cell apoptosis model and application thereof.
Background
Apoptosis is a programmed death mode different from apoptosis and is a natural immune defense mechanism of the body against pathogen infection, which is concerned in recent years. The specific characteristic is that when the pattern recognition receptor of the cell is stimulated by pathogen, the inflammatory corpuscle is activated, and then a large amount of proinflammatory factors are released, so that the immune cell is recruited to the infection focus.
Because the occurrence and regulation mechanism of the cell apoptosis are different from other cell death modes such as apoptosis, necrosis and the like, the construction of the cell apoptosis model is favorable for exploring the function of the immune response of the organism in various diseases. For example, in animal husbandry production, pathogenic bacteria infection often causes inflammation, diarrhea and even death of livestock and poultry, and in the process, intestinal tracts are stimulated by Lipopolysaccharide (LPS) secreted by the pathogenic bacteria, so that cell scorching occurs, and therefore, the construction of a cell scorching model is helpful for further development of a method for resisting the pathogenic bacteria infection.
The existing common method for constructing the cell apoptosis model comprises the steps of transfecting cell apoptosis sensitive gene plasmids, processing Lipopolysaccharide (LPS) and Adenosine Triphosphate (ATP), and the like. However, the price of the cell apoptosis sensitive gene plasmid is high, and the defects of low plasmid transfection efficiency, complex operation steps, easily influenced effect by cell state and the like exist. The LPS is used as a chemical product artificially purified from the cell wall of gram-negative bacteria, the application effect of the LPS-containing pathogenic bacteria can be simulated only partially, and the LPS-containing pathogenic bacteria has greater difference and limitation with the condition that the pathogenic bacteria infect livestock and poultry in actual production.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a construction method of a cell apoptosis model and application thereof.
A method for constructing a cell apoptosis model comprises the following steps:
1) pathogenic bacteria activation;
2) activated pathogen-treated cells;
3) collecting a cell sample and detecting the construction effect;
the pathogenic bacteria is Escherichia coli K88, and the concentration used for treating cells is OD600= 0.4-0.6, and the number of viable bacteria contained is 1-3 × 107CFU/mL, adding 2 mM of trimethyl adenosine ATP after treating the cells for 2 hours, wherein the treatment time is 2.5 hours in total;
the cell is mouse-derived mononuclear macrophage J774A.1.
According to the construction method, the Escherichia coli K88 is treated to improve the IL-1 beta and IL-18 protein contents in the supernatant of the culture medium J774A.1 of the murine monocyte macrophage, and the expression level of genes and proteins related to intracellular apoptosis.
The application of the cell apoptosis model comprises the following steps:
1) activating Escherichia coli K88;
2) the activated bacterium liquid is used for intragastric administration of the mice;
3) collecting mouse samples and detecting the construction effect.
The mice are male C57BL/6 mice with 3 weeks of age, and are randomly divided into 3 groups, namely a control group, a K88 group and an LPS group.
The control group of mice is intragastrically injected with 100-200 mu L phosphate buffer solution PBS and intraperitoneally injected with 100-200 mu L PBS, and the K88 group of mice is intragastrically injected with 1-2 multiplied by 109100-200 mu L of CFU/mL escherichia coli K88 bacterial liquid and 100-200 mu L of PBS are intraperitoneally injected, 100-200 mu L of PBS are intragastrically injected to mice in an LPS group, and 0.3-0.6 mg/kg of LPS of the mice body weight is intraperitoneally injected.
The application of the Escherichia coli K88 for intragastric administration can improve the IL-1 beta and IL-18 protein content in the lavage liquid of the abdominal cavity of the mouse, improve the expression of genes related to scorching of colon cells of the mouse, cause infiltration of the colon inflammatory cells, and has the effect similar to the scorching effect of the cells induced by the intraperitoneal injection of LPS.
The invention has the beneficial effects that:
1) the invention discloses a method for constructing a cell apoptosis model by using Escherichia coli K88 for the first time. Compared with Lipopolysaccharide (LPS) used in the prior art, the Escherichia coli K88 capable of inducing cell apoptosis is screened and determined to be susceptible pathogenic bacteria of livestock and poultry, the use of the Escherichia coli K88 is closer to actual production, the effect of constructing a cell apoptosis model is equivalent to that of the LPS, and the use cost is greatly reduced.
2) The cell apoptosis model disclosed by the invention is successfully applied to mouse-derived macrophages J774A.1 and mice, provides a basis for the subsequent research of the function and mechanism of the cell apoptosis in the process of resisting pathogenic bacteria infection of a body, and lays a foundation for the treatment of common pathogenic bacteria infection in production practice.
Drawings
FIG. 1 shows the results of the content of apoptosis-related proteins in the supernatant after treatment of J774A.1 with pathogenic bacteria.
FIG. 2 shows the expression result of protein related to J774A.1 cell apoptosis.
FIG. 3 shows the expression result of J774A.1 cell apoptosis-related genes.
FIG. 4 shows the content of proteins associated with apoptosis in mouse peritoneal lavage fluid.
FIG. 5 shows the results of the expression of genes associated with mouse colon cell apoptosis.
FIG. 6 is the H & E staining results of mouse colon.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
Example 1: screening of common pathogenic bacteria capable of inducing cell scorching
1) Preparing a culture medium: LB plate medium: 10g of sodium chloride, 10g of tryptone, 5g of yeast extract, 25g of agar and 1L of deionized water, and sterilizing at 121 ℃ for 21 min; LB liquid medium: 10g of sodium chloride, 10g of tryptone, 5g of yeast extract and 1L of deionized water, and sterilizing at 121 ℃ for 21 min.
2) Activating strains: thawing Escherichia coli K88, Escherichia coli O157 and Salmonella STM frozen at-80 deg.C at normal temperature, streaking each loop on LB plate culture medium, culturing at 37 deg.C for 12 hr, selecting single colony, inoculating in LB liquid culture medium, and culturing at 37 deg.C and 250rpm for 12 hr to obtain seed solution.
3) Bacterial liquid dilution and cell treatment: diluting the activated three bacterial liquids to OD by PBS respectively600And (4) = 0.4-0.6, centrifuging at 600rpm for 5 minutes, then removing the supernatant, resuspending the supernatant in an isovolumetric DMEM basic culture medium, and treating the murine mononuclear macrophage J774A.1. After 2 hours of treatment, ATP was added to a final concentration of 2 mM for 0.5 hours of treatment. Pure DMEM basic culture medium without Escherichia coli K88 bacterial liquid is used as a negative control, and LPS is used as a positive control. LPS treatment was carried out at a concentration of 0.5. mu.g/mL for 4 hours, and ATP (final concentration: 2 mM) was added thereto for 0.5 hour.
4) Sample collection and index determination: after ATP is added for 0.5 hour of treatment, namely bacterial liquid treatment for 2.5 hours, culture medium supernatant is collected, and the contents of cytokines IL-1 beta and IL18 are measured by adopting an ELISA kit.
5) And (4) analyzing results: the content of IL-1 beta and IL18 in the culture medium supernatant is remarkably improved by the treatment of escherichia coli K88 (see figure 1), the effect is superior to that of escherichia coli O157 and salmonella STM, and the treatment is close to that of LPS, so that the fact that escherichia coli K88 can induce cell apoptosis is preliminarily determined.
Example 2: influence of Escherichia coli K88 on expression of J774A.1 cell apoptosis-related protein
1) Same as in step 1 of example 1).
2) Activating strains: thawing Escherichia coli K88 strain frozen at-80 deg.C at normal temperature, streaking on LB plate culture medium, culturing at 37 deg.C for 12 hr, selecting single colony, inoculating in LB liquid culture medium, and culturing at 37 deg.C and 250rpm for 12 hr to obtain seed solution.
3) Bacterial liquid dilution and cell treatment: activated E.coli K88 was diluted to OD with PBS600And (4) = 0.4-0.6, centrifuging at 600rpm for 5 minutes, then removing the supernatant, resuspending the supernatant in an isovolumetric DMEM basic culture medium, and infecting mouse-derived mononuclear macrophage J774A.1. After 2 hours of infection, ATP was added at a final concentration of 2 mM for 0.5 hour. Pure DMEM basic culture medium without Escherichia coli K88 bacterial liquid is used as a negative control, and LPS is used as a positive control. LPS treatment was carried out at a concentration of 0.5. mu.g/mL for 4 hours, and ATP (final concentration: 2 mM) was added thereto for 0.5 hour.
4) Sample collection and protein expression level determination: the expression levels of the endogenous reference protein beta-actin, the target protein NLRP3, the caspase1, the ASC and the cytokine protein IL-1 beta are measured by using a Western Blot technique.
5) And (4) analyzing results: the results were statistically analyzed by Image J software, and the relative expression amounts of the target protein and the target protein were calculated. Coli K88 treatment significantly increased the expression of NLRP3 protein (2.0 fold) and ASC (1.9 fold) in the cells compared to the negative control (see figure 2), with effects close to LPS. This indicates that Escherichia coli K88 can activate the expression of inflammatory body protein in macrophage, and further activate cell apoptosis.
Example 3 Effect of E.coli K88 on expression of J774A.1 cell apoptosis-related genes
1) Strain activation and cell treatment: same as in steps 1), 2), 3) of example 2.
2) Sample collection and gene expression level determination: cells were harvested and RNA was extracted using TRIzol (ex Invitrogen), cDNA was synthesized by Reverse Transcription using the RevertAID RT Reverse Transcription Kit (ex Thermo), and cell apoptosis-related gene expression was determined using RT-PCR technology.
3) And (4) analyzing results: by using 2-△△CtCalculating the expression of the gene related to the apoptosis of the cells by the method. Compared with a negative control, the Escherichia coli K88 obviously improves the gene expression (1.7 times) of the cell NLRP3, and is obviously higher than that of LPS treatment. Coli K88 significantly increased IL-1 β gene expression (175.4 fold) without significant effect on caspase1 gene and GSDMD gene expression (see fig. 3). This shows that Escherichia coli K88 can activate the expression of macrophage inflammatory body and cytokine related gene, thereby activating cell apoptosis.
In the preparation method of the light-operated tumor cell apoptosis system disclosed in CN111514313A, a mesoporous silica template, near-infrared excited up-conversion long-afterglow nanoparticles, light-operated precursor genes Cib-GSDMDCAAX, Cry2-Caspase-1 plasmids and the like need to be prepared, the steps are complicated, and the cell apoptosis effect generated by induction is not evaluated.
Example 4 Effect of E.coli K88 on secretion of proteins involved in apoptosis of mouse intestinal cells
1) Establishing a mouse infection model: taking male mice of 3-5 weeks old, and intragastrically administering the mice to a patient of 1-2 × 109The bacterial solution of CFU/mL escherichia coli K88 is 100-200 mu L, and 100-200 mu L phosphate buffer PBS is injected into the abdominal cavity. The negative control was achieved by intragastric gavage and intraperitoneal injection of an equal volume of Phosphate Buffered Saline (PBS), and the positive control was achieved by intraperitoneal injection of an equal volume of Lipopolysaccharide (LPS) solution. The LPS concentration is 0.3-0.6 mg/kg mouse weight.
2) Sample collection and determination: after 6 hours of treatment, alum was taken in 5 mg: dissolving the mixture in a ratio of 1 mL to obtain a mixed solution, and performing intraperitoneal injection on the mouse. After 12 hours of treatment, i.e., 6 hours after intraperitoneal injection of the mixture, the mice were sacrificed, the peritoneal cavity was perfused with PBS buffer and the perfusate was collected and the cytokine content was determined by ELISA.
3) And (4) analyzing results: coli K88 treatment significantly increased the secretion of IL-18 protein in the mouse intestinal tract compared to the negative control, with an effect comparable to LPS, but with no significant effect on IL-1 β secretion (see figure 4).
Example 5 Effect of E.coli K88 on expression of genes associated with apoptosis in mouse Colon cells
1) Establishing a mouse infection model: same as in step 1 of example 4).
2) Sample collection and determination: colon tissue from mice was collected 12 hours after treatment. RNA was extracted using TRIzol (purchased from Invitrogen), cDNA was synthesized by Reverse Transcription using the RevertAID RT Reverse Transcription Kit (purchased from Thermo), and the expression of apoptosis-related genes was determined using RT-PCR.
3) And (4) analyzing results: by adopting 2-△△CtCalculating the expression of the gene related to the apoptosis of the cells by the method. Compared with negative control, Escherichia coli K88 remarkably improves cell NLRP3 gene expression (1.7 times), ASC gene expression (4.1 times), GSDMD gene expression (2.7 times), IL-1 beta gene expression (3.2 times) and IL-18 gene expression (2.1 times), and has no remarkable influence on caspase1 gene expression (see figure 5). This shows that Escherichia coli K88 can activate mouse colon inflammasome and cytokine related geneExpression, thereby activating apoptosis of the cell.
Example 6 Effect of E.coli K88 on Colon morphology in mice
1) Establishing a mouse infection model: same as in step 1 of example 4).
2) Sample collection and processing observation: after 12 hours of treatment, mouse colons were harvested and fixed with paraformaldehyde overnight. The embedded sections were then stained with H & E and observed for colon morphology under an optical microscope.
3) And (4) analyzing results: coli K88 infection and LPS treatment both caused infiltration of colon immune cells (see fig. 6), and e.coli K88 was shown to be similar in effect to LPS, compared to the negative control.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is clear that the invention is not limited to the embodiments described above, but that many variations and equivalents are possible. All modifications and equivalents that may be directly derived or suggested to one of ordinary skill in the art from the disclosure are to be included within the scope of this invention. The scope of the invention is given by the appended claims and any equivalents thereof.
Claims (6)
1. A method for constructing a cell apoptosis model is characterized by comprising the following steps:
1) pathogenic bacteria activation;
2) activated pathogen-treated cells;
3) collecting a cell sample and detecting the construction effect;
the pathogenic bacteria is Escherichia coli K88, and the concentration used for treating cells is OD600= 0.4-0.6, and the number of viable bacteria contained is 1-3 × 107CFU/mL, adding 2 mM of trimethyl adenosine ATP after treating the cells for 2 hours, wherein the treatment time is 2.5 hours in total;
the cell is mouse-derived mononuclear macrophage J774A.1.
2. The constructing method according to claim 1, wherein the Escherichia coli K88 treatment increases IL-1 beta and IL-18 protein contents in the supernatant of the culture medium J774A.1 of murine monocyte macrophage, and intracellular apoptosis-related gene and protein expression levels.
3. Use of a model for cell apoptosis as defined in claim 1, comprising the steps of:
1) activating Escherichia coli K88;
2) the activated bacterium liquid is used for intragastric administration of the mice;
3) collecting mouse samples and detecting the construction effect.
4. The use of claim 3, wherein the mice are 3 week old male C57BL/6 mice randomized into 3 groups, control, K88 and LPS groups, respectively.
5. The use of claim 4, wherein the control mice are gavaged with 100-200 μ L PBS phosphate buffer solution and injected with 100-200 μ L PBS intraperitoneally, and the K88 mice are gavaged with 1-2 x 109100-200 mu L of CFU/mL escherichia coli K88 bacterial liquid and 100-200 mu L of PBS are intraperitoneally injected, 100-200 mu L of PBS are intragastrically injected to mice in an LPS group, and 0.3-0.6 mg/kg of LPS of the mice body weight is intraperitoneally injected.
6. The use of claim 3, wherein the Escherichia coli K88 for gastric lavage can increase IL-1 β and IL-18 protein content in mouse peritoneal lavage fluid, increase mouse colon cell apoptosis-related gene expression, and cause infiltration of colon inflammatory cells, with effect similar to that of cell apoptosis induced by intraperitoneal injection of LPS.
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