CN106754641A - A kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model and its application - Google Patents
A kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model and its application Download PDFInfo
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- CN106754641A CN106754641A CN201611184582.9A CN201611184582A CN106754641A CN 106754641 A CN106754641 A CN 106754641A CN 201611184582 A CN201611184582 A CN 201611184582A CN 106754641 A CN106754641 A CN 106754641A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention belongs to biological technical field, and in particular to a kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model and its application.Step is as follows：Escherichia coli O 157:The activation and culture of H7；Escherichia coli O 157:H7 infected pigs intestinal epithelial cell.The Escherichia coli O 157:H7 infected pigs intestinal epithelial cell model, as detection Escherichia coli O 157:H7 infected pigs intestinal epithelial cell sticks the application of index and Apoptosis.Using the cell model, Escherichia coli O 157 is studied:H7 adheres to the ability of IPEC J2 cells in vitro, and induction IPEC J2 apoptosis ability, be research Escherichia coli O 157:H7 lays a good foundation with the interaction relationship of host cell, is also Escherichia coli O 157:The prevention and treatment of H7 infection associated diseases provide new thinking and means.
Description
Technical field
The invention belongs to biological technical field, it is related to Escherichia coli O 157:A kind of H7, and in particular to Escherichia coli O 157:
The method for building up of H7 infected pigs intestinal epithelial cell model and its application.
Background technology
Escherichia coli O 157:H7 is a kind of important Arbo infectious disease pathogen, and it is anti-that the bacterium is mainly settled in some
In the enteron aisle of hay animal, the infection of people and animal is caused by the water source and food that pollute.Escherichia coli O 157:H7 infection animals
With main causing bleeding property enteritis, thrombotic thrombocytopenic purpura and kidney hemolytic uremic syndrome (HUS) after the mankind, with
And the damage of some other tract even results in death, case fatality rate is 10%, and case fatality rate is up to 30% after triggering HUS.By
In Escherichia coli O 157:H7 routes of transmission are wide, be easy to culture, infectious strong, and its virulence factor-lethal shiga toxin also has can
The structure of genetic weapon can be used for, therefore it is classified as notifiable infectious diseases by many countries, in U.S.'s prevention and control of diseases
The heart has been classified as B class bio-terrorism pathogen and has strictly been taken precautions against, and Escherichia coli are classified as 21 century and compatriots' health is good for by China
Health has one of 12 kinds of pathogenic microorganisms of significant impact.Because antibiotic can result in Escherichia coli O 157:H7 thalline are destroyed, and are led
Shiga toxin (Stx) release is caused, so as to promote the generation of HUS and the exacerbation of symptom, the clinically treatment to the disease is forbidden using anti-
Raw element, the at present infection for the bacterium still lacks effectively preventing means.And so far about Escherichia coli O 157:H7 infection is drawn
The immune mechanism of causing a disease for rising also is had little understanding, it is difficult to accomplish effectively to control Escherichia coli O 157:H7 infects.
Escherichia coli O 157:Pathogenic and bacterial adhesion, the toxin of H7 etc. are relevant.Adhesion factor adhesion target cell is large intestine
Bacillus O157:H7 pathogenic committed step, germ invades host's enteric cavity, by plasmid-mediated adhesion factor adhere to caecum and
The epithelial cell of colon, and produce adhesion and wiping to damage (attaching and under the synergy of other virulence factors
Effacinglesions, A/E are damaged).Bacterial colonization produces will to congratulate poison to causing A/E to damage behind enterocyte surface
Element, causes strong pathogenic to host cell.
The content of the invention
The present invention is solution Escherichia coli O 157:H7 (preserving numbers：ATCC 43895) there is no bio-adhesive model in vitro
(enteron aisle sticks and wiping damage model), for research Escherichia coli O 157:Interaction between H7 and host intestine cell
Technical barrier, disclose a kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell (IPEC-J2) model
And its application.
To solve above-mentioned technical barrier, the present invention uses following technical scheme：
A kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model, step is as follows：
(1) Escherichia coli O 157:The activation and culture of H7；
(2) Escherichia coli O 157:H7 infected pigs intestinal epithelial cell.
The concrete operations of the step (1) are：
A. -70 DEG C of Escherichia coli O 157s of preservation are taken:The μ L of H7 reference cultures 50 are placed in 37 DEG C of water in the liquid LB of 5mL
200~300r/min cultivates the Escherichia coli O 157 that 12~14h is activated in bath constant temperature oscillator:H7；
B. the Escherichia coli O 157 that will be activated:H7 rules on agar plate, in 37 DEG C of incubators be inverted culture 12~
16h, then in picking single bacterium colony under aseptic condition in 5mL LB culture mediums, 37 DEG C of 12~14h of water-bath shaken cultivation；
C. in picking step b under aseptic condition, the single bacterium colony on LB culture mediums in 5mL liquid LB test tubes, 37
12~14h is cultivated in DEG C constant incubator, Escherichia coli O 157 is obtained:H7 seed bacterium solutions.
The concrete operations of the step (2) are：
A. the cover glass that advance autoclaving is treated is put into Tissue Culture Dish, then by chitterlings epithelial cell with 2
×105U/ holes are inoculated in Tissue Culture Dish, put 37 DEG C of 5%CO2Incubator in overnight incubation；
B. RPMI-1640s of the 1mL containing 10% hyclone, the 1st are added afterwards with aseptic D-HanK ' s liquid washed cell three times
Hole left blank control, the 2nd hole adds standby Escherichia coli O 157:The μ L of H7 bacterium solutions 15, put 37 DEG C, 5%CO2Incubator in
It is incubated 3h, the Escherichia coli O 157:The concentration of H7 bacterium solutions is 2 × 109cfu/mL；
C. after being incubated, with aseptic D-HanK ' s liquid washed cell three times, wash nonadherent bacterium off, trained in cell immediately
Support and added in ware the methyl alcohol of 500 μ L 70% fixation 15min, take out slide, absolute methanol treatment 3s uses Gram's stain respectively
With the dyeing of Giemsa staining method, treat that slide is fully dried, mounting treatment is carried out with dimethylbenzene and resin, complete Escherichia coli
O157:The foundation of H7 infected pigs intestinal epithelial cell model.
A kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, the Escherichia coli O 157:H7 feels
Dye chitterlings epithelial cell model, as detection Escherichia coli O 157:H7 infected pigs intestinal epithelial cell sticks the application of index.
The applying step is as follows：
A. chitterlings epithelial cell is with 2 × 105Individual/hole is inoculated in Tissue Culture Dish, is placed in 37 DEG C of 5%CO2It is incubated
Overnight incubation in case；
B. washed 3 times with aseptic D-HanK ' s, blanc cell is compareed, standby large intestine is separately added into other culture dishes
Bacillus O157:The μ L of H7 bacterium solutions 15, are placed in 37 DEG C of 5%CO2Cultivated in constant incubator, after being incubated 1h, with aseptic D-HanK ' s liquid
Wash 3 times, remove nonadherent bacterium；
C. to through step b treat culture dish in, add 500 μ L 0.25%Triton X-100 cell lysis
15min, discharges the bacterium living for sticking to cell surface, and then 8000r/min centrifugation 5min precipitums are washed with D-HanK ' s
The bacterial solution living sticked for 2 times；
D. the bacterial solution that will live is with 10 times of doubling dilutions to 10-4, each dilution factor takes 100 μ L and is coated on solid LB culture plates
In, each dilution factor is coated with three flat boards；
E. LB culture plates are placed in 37 DEG C of constant incubators and cultivate 12h, while using solid medium LB flat boards as sky
White control, chooses flat board of the bacterium colony between 30~300 and is counted, and then seeks its average value, its adherence rate=attach to carefully
Total number of bacteria/the total number of bacteria of born of the same parents, sticks index=adherent bacteria sum/cell number.
A kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, the Escherichia coli O 157:H7 feels
Dye chitterlings epithelial cell model, as detection by Escherichia coli O 157:The metainfective chitterlings Epithelial Cell Apoptosis of H7 should
With.
It is described detection chitterlings Epithelial Cell Apoptosis application, the detection method for using for：Hoechst33258 decoration methods
Or Annexin V-FITC/PI double labelling flow cytometries.
The beneficial effects of the present invention are：
1. the invention discloses setting up Escherichia coli O 157:The method of H7 infected pigs intestinal epithelial cell model, demonstrate,proves first
Real Escherichia coli O 157:H7 can in vitro adhere to swine intestinal epithelium cells IPEC-J2, be research Escherichia coli O 157:H7 feels
The enteron aisle A/E damage process and its mechanism that dye causes are laid a good foundation.
2. Escherichia coli O 157 is applied:H7 infected pigs intestinal epithelial cell model, studies Escherichia coli O 157:H7 glues in vitro
The ability of attached IPEC-J2 cells, and the ability of IPEC-J2 apoptosis is induced, it is research Escherichia coli O 157:H7 and host cell
Interaction relationship lay a good foundation, be also Escherichia coli O 157:The prevention and treatment of H7 infection associated diseases provide new
Thinking and means.
Brief description of the drawings
Fig. 1 is Escherichia coli O 157:The growth curve of H7.
Fig. 2 is that Gram's stain detects Escherichia coli O 157:H7 sticks IPEC-J2 cell microscopics；Wherein A is normal
IPEC cells, B is type strain and IPEC cells.
Fig. 3 is that Giemsa staining method detects Escherichia coli O 157:H7 sticks IPEC-J2 cell microscopics；Wherein A is normal
IPEC cells, B is type strain and IPEC cells.
Fig. 4 is Hoechst33258 coloration result figures；Wherein A：Normal cell；B:Escherichia coli O 157 infection causes IPEC
Apoptosis.
Fig. 5 is that Annexin V-FITC/PI methods detect Escherichia coli O 157:H7 induces IPEC-J2 Apoptosis figures, wherein
A:IPEC-J2 blanc cells；B-E：It is respectively Escherichia coli O 157:30min, 1h, 2h, 3h draw after H7 infection IPEC-J2 cells
The Apoptosis situation for rising；B1 in figure:Annexin V-FITC-/PI+ damage fields, B2:Annexin V-FITC+/PI+ are damaged
Hinder region, B3:Annexin V-FITC-/PI- regions, B4:Annexin V-FITC+/PI- regions.
Specific embodiment
A kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model, step is as follows：
(1) Escherichia coli O 157:The activation and culture of H7；
(2) Escherichia coli O 157:H7 infected pigs intestinal epithelial cell.
The concrete operations of the step (1) are：
A. -70 DEG C of Escherichia coli O 157s of preservation are taken:The μ L of H7 reference cultures 50 are placed in 37 DEG C of water in the liquid LB of 5mL
200~300r/min cultivates the Escherichia coli O 157 that 12~14h is activated in bath constant temperature oscillator:H7；
B. the Escherichia coli O 157 that will be activated:H7 rules on agar plate, in 37 DEG C of incubators be inverted culture 12~
16h, then in picking single bacterium colony under aseptic condition in 5mL LB culture mediums, 37 DEG C of 12~14h of water-bath shaken cultivation；
C. in picking step b under aseptic condition, the single bacterium colony on LB culture mediums in 5mL liquid LB test tubes, 37
12~14h is cultivated in DEG C constant incubator, Escherichia coli O 157 is obtained:H7 seed bacterium solutions.
The concrete operations of the step (2) are：
A. the cover glass that advance autoclaving is treated is put into Tissue Culture Dish, then by chitterlings epithelial cell with 2
×105U/ holes are inoculated in Tissue Culture Dish, put 37 DEG C of 5%CO2Incubator in overnight incubation；
B. RPMI-1640s of the 1mL containing 10% hyclone, the 1st are added afterwards with aseptic D-HanK ' s liquid washed cell three times
Hole left blank control, the 2nd hole adds standby Escherichia coli O 157:The μ L of H7 bacterium solutions 15, put 37 DEG C, 5%CO2Incubator in
It is incubated 3h, the Escherichia coli O 157:The concentration of H7 bacterium solutions is 2 × 109cfu/mL；
C. after being incubated, with aseptic D-HanK ' s liquid washed cell three times, wash nonadherent bacterium off, trained in cell immediately
Support and added in ware the methyl alcohol of 500 μ L 70% fixation 15min, take out slide, absolute methanol treatment 3s uses Gram's stain respectively
With the dyeing of Giemsa staining method, treat that slide is fully dried, mounting treatment is carried out with dimethylbenzene and resin, complete Escherichia coli
O157:The foundation of H7 infected pigs intestinal epithelial cell model.
A kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, the Escherichia coli O 157:H7 feels
Dye chitterlings epithelial cell model, as detection Escherichia coli O 157:H7 infected pigs intestinal epithelial cell sticks the application of index.
The applying step is as follows：
A. chitterlings epithelial cell is with 2 × 105Individual/hole is inoculated in Tissue Culture Dish, is placed in 37 DEG C of 5%CO2It is incubated
Overnight incubation in case；
B. washed 3 times with aseptic D-HanK ' s, blanc cell is compareed, standby large intestine is separately added into other culture dishes
Bacillus O157:The μ L of H7 bacterium solutions 15, are placed in 37 DEG C of 5%CO2Cultivated in constant incubator, after being incubated 1h, with aseptic D-HanK ' s liquid
Wash 3 times, remove nonadherent bacterium；
C. to through step b treat culture dish in, add 500 μ L 0.25%Triton X-100 cell lysis
15min, discharges the bacterium living for sticking to cell surface, and then 8000r/min centrifugation 5min precipitums are washed with D-HanK ' s
The bacterial solution living sticked for 2 times；
D. the bacterial solution that will live is with 10 times of doubling dilutions to 10-4, each dilution factor takes 100 μ L and is coated on solid LB culture plates
In, each dilution factor is coated with three flat boards；
E. LB culture plates are placed in 37 DEG C of constant incubators and cultivate 12h, while using solid medium LB flat boards as sky
White control, chooses flat board of the bacterium colony between 30~300 and is counted, and then seeks its average value, its adherence rate=attach to carefully
Total number of bacteria/the total number of bacteria of born of the same parents, sticks index=adherent bacteria sum/cell number.
A kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, the Escherichia coli O 157:H7 feels
Dye chitterlings epithelial cell model, as detection by Escherichia coli O 157:The metainfective chitterlings Epithelial Cell Apoptosis of H7 should
With.
It is described detection chitterlings Epithelial Cell Apoptosis application, the detection method for using for：Hoechst33258 decoration methods
Or AnnexinV-FITC/PI double labelling flow cytometries.
With reference to specific embodiment, explanation is further explained to the present invention：
1 material
1.1 strains and cell
Escherichia coli O157 reference culture (ATCC 43895) and swine intestinal epithelium cells (IPEC-J2) are this experiment
Room preserves.
1.2 main agents
Cell culture examination 1640, serum used, trypsase Deng Jungou Hyclone companies；Tryptone and yeast are extracted
Thing is purchased from OXOID Co., Ltds of Britain；Gram staining liquid and Giemsa staining liquid are Sigma Products；Cell culture six
Orifice plate is Corning Products；20mm × 20mm cover glasses are purchased from BOSTER companies；Other AR are by this experiment
Room provides.
1.3 key instruments
Inverted microscope (Leica Microsystems Wetzlar GmbH)；CO2 constant incubators (Thermo)；754
Ultraviolet-uisible spectrophotometer (Shanghai essence tech equipment Co., Ltd)；(Ningbo mechanotronics is designed conventional constant incubator
Institute)；Superclean bench (safe and sound company of Su Jing groups)；Desk centrifuge (SIGMA)；It is flow cytometer (U.S. company BD), glimmering
Light microscope (Japanese Nikon company), CFX96Real-Time PCR instruments (BioRad companies) etc..
2 methods
2.1 Escherichia coli O 157s:The activation and culture of H7
Take -70 DEG C of Escherichia coli O 157s of preservation:The μ L of H7 reference cultures 50 are placed in 37 DEG C of water-baths in the liquid LB of 5mL
200~300r/min cultivates 12~14h in constant temperature oscillator.
By the Escherichia coli O 157 after activation:H7 is inverted culture in the flat lining out of maconkey agar in 37 DEG C of incubators
12-16h.Then in picking single bacterium colony under aseptic condition in 5mL LB culture mediums, 37 DEG C of water-bath shaken cultivation 12-14h.
Then in picking single bacterium colony under aseptic condition in 5mL liquid LB test tubes, in 37 DEG C of heat-insulating type constant incubators
Middle culture 12-14h, obtains Escherichia coli O 157:H7 seed bacterium solutions.
2.2 Escherichia coli O 157s:The measure of H7 growth curves and drafting
By Escherichia coli O 157:H7 seeds bacterium solution is inoculated into sterilizing, 37 DEG C of preheated 150mL by 1% inoculum concentration
In LB culture mediums, 42 Boiling tubes are distributed into after shaking up, often pipe 5mL, 37 DEG C of water-bath constant temperature oscillator 300r/min are cultivated,
3 pipe bacterium (parallel as 3 times) are taken out every 1h, after 10 times of doubling dilutions, is counted with the method for plate culture count, record each time
Three average values of point draw growth curve as final result.Time with above-mentioned each measuring point, as abscissa, measures
Bacterial population logarithm be ordinate, draw Escherichia coli O 157 growth curve.
2.3 bacterial adhesions are invented
The cover glass of 20mm × 20mm that advance autoclaving is crossed is put into Tissue Culture Dish, then by IPEC-J2 cells
With 2 × 105/ hole is inoculated in Tissue Culture Dish, puts 37 DEG C of 5%CO2Incubator in overnight incubation.With aseptic D-HanK ' s
Liquid washed cell adds for three times RPMI-1640s (without dual anti-) of the 1mL containing 10% hyclone, the 1st hole left blank to compare afterwards,
2nd hole adds standby Escherichia coli O 157:The μ L (2 × 10 of H7 bacterium solutions 159Cfu/mL), 37 DEG C, 5%CO are put2Incubator in
It is incubated 3h.After incubation, with aseptic D-HanK ' s liquid washed cell three times, nonadherent bacterium is washed off.Immediately in cell culture
Add in ware the methyl alcohol of 500 μ L 70% to fix 15min, take out slide, absolute methanol treatment 3s, respectively with Gram's stain and
Giemsa staining method is dyeed, and treats that slide is fully dried, and mounting treatment is carried out with dimethylbenzene and resin.After cover glass is fixed,
MOTIC imaging microscopes are observed.
2.4 stick index and adhesion rate calculating
IPEC-J2 cells are with 2 × 105Individual/hole is inoculated in Tissue Culture Dish, puts 37 DEG C of 5%CO2Incubator in cultivate
Overnight.Washed 3 times with aseptic D-HanK ' s, blanc cell is compareed, 3 plants of standby large intestine bars are separately added into other culture dishes
The μ L of O157: H7 bacterium solution of bacterium 15, are placed in 37 DEG C of 5%CO2Cultivated in incubator, after being incubated 1h, washed 3 times with aseptic D-HanK ' s liquid,
After the nonadherent bacterium of removal, 500 μ L 0.25%Triton X-100 cell lysis 15min are added, discharge and stick to cell
The bacterium living on surface, 8000r/min centrifugation 5min precipitums, is then washed 2 times, doubling dilution to 10 with D-HanK ' s-4, by its
100 μ L are drawn by different dilution factors to be coated in solid medium LB, each dilution factor is coated with three flat boards.It is heat-insulated at 37 DEG C
12h is cultivated in formula electro-heating standing-temperature cultivator, while with a solid medium LB flat board as blank.Bacterium colony is chosen 30
Flat board between~300 is counted, and then seeks its average value, calculates the total number of bacteria of its adherence rate=attach to cell/thin
Bacterium sum, sticks index=adherent bacteria sum/cell number.
2.5 Escherichia coli O 157s:The apoptosis detection of IPEC-J2 cells is induced after H7 infection
2.5.1Hoechst33258 decoration method detects the apoptosis situation of IPEC-J2 cells
IPEC-J2 cell (cells about 2 × 10 are inoculated with Tissue Culture Dish5Individual/hole), culture to cell density is more than
90%, washed 3 times with aseptic D-HanK ' s, by 1:100 amount in Tissue Culture Dish to adding Escherichia coli O 157:H7, in 37
DEG C 5%CO2Cultivated in incubator.The IPEC-J2 cells and Escherichia coli O 157 of control group are taken respectively:H7 bacterium infections 3h's
IPEC-J2 cells, grasp do according to the following steps：
1. supernatant is abandoned, the methyl alcohol of 0.5mL is added, 10 minutes are fixed；
2. go fixer, D-HanK ' s to wash 3 times, exhaust liquid.Hand is gently rocked for several times during washing；
3. the dyeing liquors of 1mL Hoechst 33258 are added, lucifuge is dyeed 5 minutes.
4. washed with D-HanK ' s 3 times, every time 3 minutes.
5. anti-fluorescent quenching mounting liquid is dripped in Tissue Culture Dish；
6. in fluorescence microscopy Microscopic observation.
2.5.2Annexin V-FITC/PI double labellings Apoptosis by Flow Cytometry situation
IPEC-J2 cells are inoculated into (2 × 10 in Tissue Culture Dish5Individual/hole), cultivate to individual layer, with aseptic D-HanK '
S is washed 3 times, by 1:100 amount in Tissue Culture Dish to adding Escherichia coli O 157:H7, is placed in 37 DEG C of 5%CO2In incubator
Culture.0,0.5,1, the 2 and 3h after bacterium is added, after being digested with pancreatin, collects cell, and 3 000r/min centrifugation 10min use D-
HanK ' s are washed 3 times.According to the double transfection reagent box specifications of Annexin V-FITC/PI, 500 μ L combination buffers are added to hang again
Floating cell, the then PI mixings of the Annexin V-FITC and 10 μ L of 5 μ L of each addition, after room temperature lucifuge dyeing 15min, uses streaming
Cytometric Analysis, Cellquest software analysis results.
3 results
3.1 Escherichia coli O 157s:The drafting of H7 growth curves
It is ordinate with the bacterial population logarithm for measuring, with the time as abscissa, draws the growth curve of Escherichia coli O 157
(Fig. 1), from figure 1 it appears that initial 2h Escherichia coli O 157s growth is in lag phase, Escherichia coli O 157 growth after 2h
Into exponential phase, Escherichia coli O 157 grows into stationary phase after 5h.
3.2 bacterial adhesion experimental results
Escherichia coli O 157:After H7 and IPEC-J2 cell adhesions 3h, contaminated by the Gram's stain and Jim Sa that improve
Color method is dyeed, and adhesion situation is observed under MOTIC imaging microscopes：Normal IPEC-J2 iuntercellulars have space, it is seen that intracellular
Grain impurity, and it is clear-cut, cell is in polygonal or fusiform；Metainfective cell week is with substantial amounts of negative rod-shaped bacteria and sticks
(see Fig. 2 and 3).
3.3 Escherichia coli O 157s:H7 is to the adherence rate of IPEC-J2 cells and sticks index
Escherichia coli O 157:After H7 infects IPEC-J2 cells, 500 μ L 0.25%Triton X-100 cell lysis are added
15min, discharges and sticks to cell surface and intracellular bacterium living.100 μ L are drawn after 10 times of doubling dilutions to be spread evenly across
Solid LB flat boards, the dilution factor for choosing bacterium colony number 30~300 carries out colony counting, obtains intraor extracellular total viable count, and
Calculate adherence rate and stick index.As shown in table 1, Escherichia coli O 157 strains expressed goes out stronger Adhering capacity to result, right
The Adhering capacity of IPEC-J2 cells is 30.4%, and it is 14.8 to stick index.
The Escherichia coli O 157 of table 1:The adhesion rate of H7
3.4 Escherichia coli O 157s:H7 causes the apoptosis situation of IPEC-J2 cells
3.4.1 Hoechst33258 coloration results
With Hoechst33258 to cell dyeing after, observe living cells core in disperse, uniform glimmering under fluorescence microscope
Light, the cell of necrosis is not dyeed by Hoechst, and the cell of apoptosis, in its nucleus or cytoplasm visible dense dye it is fine and close
The block blue-fluorescence of grain and the change of obvious nuclear morphology, if the DNA fluorescence fragments for seeing 3 or more than 3 are considered as apoptosis
Cell.With Hoechst33258 to normal IPEC-J2 cells and ehec infection O157:IPEC-J2 cells after H7 3h
Dyeed, as a result as shown in Figure 4.The core of compared with control cells is in the uniform fluorescence of disperse, and Escherichia coli O 157：The portion of H7 infected groups
The particle bulk fluorescence for dividing visible dense dye in nucleus fine and close occurs, and illustrates apoptosis (scheming place shown in B arrows).
3.4.2 Annexin V-FITC/PI are double contaminates flow cytometer results
According to the double transfection reagent box specifications of Annexin V-FITC/PI, in Escherichia coli O 157:Different time after H7 infection
(0,0.5,1,2 and 3h) collects IPEC-J2 cells and is dyeed, and with flow cytomery, obtains the cell at corresponding time point
Apoptosis rate, is as a result shown in Fig. 5 (A-E).The viable apoptotic cell of B4 region representations cell mass in figure, B2 region representations withered for late period
Die and non-viable non-apoptotic cell, B3 Regional Representative's is normal cell.Late apoptic and non-viable non-apoptotic cell by this stage of early apoptosis,
Therefore the apoptosis rate total with expression of B2 and B4.IPEC-J2 cells are in Escherichia coli O 157 as seen from the figure:Small half after H7 infection
When just have an Apoptosis, and apoptosis rate afterwards gradually increases.With the extension of infection time, total apoptosis rate also increases therewith
Plus.
4. discuss
4.1 Escherichia coli O 157s:The measure of H7 growth curves
The growth curve of bacterium reflects unicellular microorganism and is shown when under the conditions of certain environment in Liquid Culture
Population growth rule.According to the difference of its growth rate, typically growth curve can be divided into lag phase, logarithmic phase, stationary phase
And decline phase.This four length in period are changed because of the difference of the heredity of strain, inoculum concentration and condition of culture.It is different
The bacterium in source has its each different growth rhythm, and the different phenotype of same bacterium also has different growth rhythms.
Therefore by determining Escherichia coli O 157:The growth curve of H7, it may be appreciated that Escherichia coli O 157:The growth rhythm of H7.This research
Middle Escherichia coli O 157:The measurement result of H7 growth curves shows, Escherichia coli O 157:The lag phase of H7 is 2h or so, more early
Entrance exponential phase；Enter stationary phase after 5h, the data carry out cell adherence invention research to next step certain guidance
Meaning.
4.2 cell adherences are invented
Further investigation pathogen is thoroughly to solve to infect pathogenetic basic place with the interaction mechanism of host cell.
Escherichia coli O 157:Although H7 is extracellular bacteria, but can be with enterocyte tight adhesion, produce the adhesion of characteristic and de-
Fall (A/E) damage.The present invention have studied Escherichia coli O 157 with swine intestinal epithelium cells IPEC-J2 as external model:H7 is to pig
The adhesion activity of intestinal epithelial cell IPEC-J2, as a result shows that substantial amounts of Escherichia coli O 157 is sticked on IPEC-J2 cells,
And cause loose part cellular contraction, connection, the change of intracellular vacuole sample, endochylema projection to reduce or disappear, or even cell karyorhexis.It is viscous
Attached ability illustrates that by force the ability of the bacterium invasion cell is strong, and cell is easier to sustain damage, and is exactly that the bacterium is easier for body
Allow body to cause a disease and show corresponding symptom, can also be observed that part cell death, cellular morphology occur in course of infection
Obvious change.
It is reported that Escherichia coli O 157:H7 can adhere to the cells of Henle 407 (from people's small intestine), HEp-2 cells
(people's larynx cancer cell), T84 (colon cancer cell) cell, HCT8 (people's ileocecal) cell, HEL (human embryonic lung) cells and
HeLa cells, and its adhesion is often focal type or microcolony type.The present invention confirms Escherichia coli O 157 first:H7 can be external
Adhesion swine intestinal epithelium cells IPEC-J2, is next step research Escherichia coli O 157:The adhesive mechanism of H7, it is particularly initial
Adhesive mechanism establishes certain basis.
4.3 Escherichia coli O 157s:H7 induces IPEC-J2 Apoptosis
Apoptosis refers to that under many physiology and pathological conditions, it is procedural that cell occurs in terms of morphology and biochemistry
It is dead.It is main with the cell DNA specific degraded of generation, shows being formed as karyopycnosis, after birth foaming and apoptotic body
Feature.Research report, many bacteriums can cause Apoptosis.It is existing it has proven convenient that staphylococcus aureus, pseudomonas aeruginosa,
Salmonella, helicobacter pylori etc. can occur or exacerbation apoptosis with inducing host cell.After body is subject to bacterium infection, a side
Face pathogenic bacteria inducing host cell apoptosis, contributes to the invasion of bacterium, the release of toxin or kills macrophage and lymphocyte；
On the other hand, it is also a kind of defense reaction that host cell occurs apoptosis, can eliminate or limit the growth of pathogenic bacteria.
The present invention dyes observation of cell morphological change and the double dye streams of Annexin-V FITC/PI with Hoechst 33258
Two methods of formula Cytometric Analysis, study Escherichia coli O 157:Inducing actions of the H7 to IPEC-J2 Apoptosis.Result shows,
Escherichia coli O 157:H7 can induce IPEC-J2 apoptosis, and apoptotic cell morphologic change is all apparent.
Hoechst33258 dyes consistent with the double dye both approaches acquired results of Annexin-V FITC/PI.Escherichia coli O 157:H7
30min just has Apoptosis after infection IPEC-J2 cells, with Escherichia coli O 157:The extension of H7 stimulation times, apoptotic cell
Rate is with increasing.Research discovery, Escherichia coli O 157:The B subunits of major virulent factor class shiga toxin (STX) of H7 can be lured
The apoptosis of expression Gb3 acceptors are led, therefore to Apoptosis and Escherichia coli O 157:H7 infects further grinding for relation
Study carefully, will be Escherichia coli O 157:The prevention and treatment of H7 infection associated diseases provide new thinking and means.
Claims (7)
1. a kind of Escherichia coli O 157:The method for building up of H7 infected pigs intestinal epithelial cell model, it is characterised in that step is such as
Under：
（1）Escherichia coli O 157:The activation and culture of H7；
（2）Escherichia coli O 157:H7 infected pigs intestinal epithelial cell.
2. Escherichia coli O 157 as claimed in claim 1:The method for building up of H7 infected pigs intestinal epithelial cell model, its feature
It is, the step（1）Concrete operations be：
A. -70 DEG C of Escherichia coli O 157s of preservation are taken:The μ L of H7 reference cultures 50 are placed in 37 DEG C of water-baths in the liquid LB of 5 mL
200~300 r/min cultivate the Escherichia coli O 157 that 12~14 h are activated in constant temperature oscillator:H7；
B. the Escherichia coli O 157 that will be activated:H7 rules on agar plate, 12 ~ 16h of culture is inverted in 37 DEG C of incubators, so
After picking single bacterium colony under aseptic condition in 5mL LB culture mediums, 37 DEG C of 12 ~ 14h of water-bath shaken cultivation；
C. in picking step b under aseptic condition, the single bacterium colony on LB culture mediums in 5 mL liquid LB test tubes, at 37 DEG C
12 ~ 14 h are cultivated in constant incubator, Escherichia coli O 157 is obtained:H7 seed bacterium solutions.
3. Escherichia coli O 157 as claimed in claim 1:The method for building up of H7 infected pigs intestinal epithelial cell model, its feature
It is, the step（2）Concrete operations be：
A. the cover glass that advance autoclaving is treated is put into Tissue Culture Dish, then by chitterlings epithelial cell with 2 ×
105U/ holes are inoculated in Tissue Culture Dish, put 37 DEG C of 5% CO2Incubator in overnight incubation；
B. RPMI-1640s of 1 mL containing 10% hyclone, the 1st hole are added for three times afterwards with aseptic D-HanK ' s liquid washed cell
Left blank is compareed, and the 2nd hole adds standby Escherichia coli O 157:The μ L of H7 bacterium solutions 15, put 37 DEG C, 5% CO2Incubator in
It is incubated 3 h, the Escherichia coli O 157:The concentration of H7 bacterium solutions is 2 × 109 cfu/mL；
C. after being incubated, with aseptic D-HanK ' s liquid washed cell three times, nonadherent bacterium is washed off, immediately in cell culture
Add the methyl alcohol of 500 μ L 70% to fix 15 min in ware, take out slide, absolute methanol processes 3 s, Gram's stain is used respectively
With the dyeing of Giemsa staining method, treat that slide is fully dried, mounting treatment is carried out with dimethylbenzene and resin, complete Escherichia coli
O157:The foundation of H7 infected pigs intestinal epithelial cell model.
4. a kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, it is characterised in that：The Escherichia coli
O157:H7 infected pigs intestinal epithelial cell model, as detection Escherichia coli O 157:H7 infected pigs intestinal epithelial cell sticks finger
Several applications.
5. Escherichia coli O 157 as claimed in claim 4:The application of H7 infected pigs intestinal epithelial cell model, its feature exists
In the applying step is as follows：
A. chitterlings epithelial cell is with 2 × 105Individual/hole is inoculated in Tissue Culture Dish, is placed in 37 DEG C of 5% CO2Constant incubator
Middle culture to cell density is more than 90%；
B. washed 3 times with aseptic D-HanK ' s, blanc cell is compareed, standby large intestine bar is separately added into other culture dishes
Bacterium O157:The μ L of H7 bacterium solutions 15, are placed in 37 DEG C of 5 % CO2Cultivated in constant incubator, after being incubated 1 h, with aseptic D-HanK ' s
Liquid is washed 3 times, removes nonadherent bacterium；
C. to through step b treat culture dish in, add the % Triton X-100 cell lysis 15 of 500 μ L 0. 25
Min, discharges the bacterium living for sticking to cell surface, and 8000 r/min are centrifuged 5 min precipitums, are then washed with D-HanK ' s
The bacterial solution living sticked for 2 times；
D. the bacterial solution that will live is with 10 times of doubling dilutions to 10-4, it is flat that 100 μ L bacterium solutions of each dilution factor absorption are coated on solid LB
In plate, each dilution factor is coated with three flat boards；
E. flat board is placed in 37 DEG C of constant incubators and cultivates 12 h, while using solid LB flat boards as blank, choosing bacterium
The flat board fallen between 30 ~ 300 is counted, and then seeks its average value, the total number of bacteria of its adherence rate=attach to cell/thin
Bacterium sum, sticks index=adherent bacteria sum/cell number.
6. a kind of Escherichia coli O 157:The application of H7 infected pigs intestinal epithelial cell model, it is characterised in that：The Escherichia coli
O157:H7 infected pigs intestinal epithelial cell model, as detection by Escherichia coli O 157:The metainfective chitterlings epithelial cells of H7
The application of apoptosis.
7. Escherichia coli O 157 as claimed in claim 6:The application of H7 infected pigs intestinal epithelial cell model, its feature exists
In, it is described detection chitterlings Epithelial Cell Apoptosis application, the detection method for using for：Hoechst33258 decoration methods or
Annexin V-FITC/PI double labelling flow cytometries.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261371A (en) * | 2019-06-27 | 2019-09-20 | 广州天科生物科技有限公司 | The method of quality control of trace mineral supplement |
| 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 |
-
2016
- 2016-12-20 CN CN201611184582.9A patent/CN106754641A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| XIANHUA YIN等: "Adherence of Escherichia coli O157:H7 Mutants In Vitro and inLigated Pig Intestines", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| XIANHUA YIN等: "Adherence of Escherichia coli O157:H7 to epithelial cells in vitro and in pig gut loops is affected by bacterial culture conditions", 《THE CANADIAN JOURNAL OF VETERINARY RESEARCH》 * |
| XIANHUA YIN等: "Contributions of O Island 48 to Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Epithelial Cells In Vitro andin Ligated Pig Ileal Loops", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110261371A (en) * | 2019-06-27 | 2019-09-20 | 广州天科生物科技有限公司 | The method of quality control of trace mineral supplement |
| 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 |
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