CN106947732A - A kind of method that helicobacter pylori and gastric epithelial cells are co-cultured - Google Patents
A kind of method that helicobacter pylori and gastric epithelial cells are co-cultured Download PDFInfo
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Abstract
The invention discloses a kind of method that helicobacter pylori and gastric epithelial cells are co-cultured, comprise the following steps：1）Prepare heiicobacter pylori cultivation base；2）Culture medium is added into steril cell blake bottle bevel；3）Gastric epithelial cells are cultivated adherent in the blake bottle in the slope of shape；4）Take helicobacter pylori 48h bacterium solutions resuspended with cell culture fluid after centrifuging；5）Resuspended bacterium solution is added into the blake bottle of existing attached cell and slant medium culture, viable bacteria amount and observation of cell morphological change are counted daily.Beneficial effects of the present invention are：The invention provides a kind of brand-new helicobacter pylori and gastric epithelial cells co-culture method.In new system, helicobacter pylori normal growth persistently stimulates attached cell to produce lesion, plays situation during fine simulated infection people stomach lining.Solve and helicobacter pylori be directly added into Traditional Method, because nutrient solution is unsuitable for its growth and caused by decline or dead, it is difficult to the problem of effective stimulus cell.
Description
Technical field
The present invention relates to bacterium and cell culture processes technical field, and in particular on a kind of helicobacter pylori and stomach lining
The method that chrotoplast is co-cultured.
Background technology
Helicobacter pylori is a kind of Grain-negative microaerobion of the screw shaped found in nineteen eighty-three.It can be in people's stomach lining
Epithelial cell is colonized, and causes a variety of diseases such as gastritis, peptic ulcer, stomach cancer, gastric mucosa-associated lymphoid tissue lymphoma.Due to
The animal model of helicobacter pylori infections is more difficult to be set up, therefore passes through gastric epithelial cells of the external helicobacter pylori to culture
Pathogenic effects research be inquire into helicobacter pylori pathogenicity mechanism important models.Set up scientific and effective helicobacter pylori and stomach
Mucosal epithelial cells co-culture model seems particularly significant.Because helicobacter pylori is longer-term to the pathogenic of cell in body
The result of effect, therefore it is required that helicobacter pylori and gastric epithelial cells long period are common in the external co-culture model set up
Deposit.Traditional co-culture method is mainly：First with cell culture fluid culture cell in Tissue Culture Flask, after after cell attachment again
Certain density helicobacter pylorus bacteria suspension is added in nutrient solution, follow-up correlation is carried out again after both interact a period of time
Research.But this method mainly has the disadvantage that：（1）After helicobacter pylori is added into cell culture fluid, because of cell culture
Liquid is unsuitable for the growth of helicobacter pylori, its physiological status is occurred significant changes, vigor is decreased obviously in the short time, extremely
All failed in 24h dead, this frequency is died on one's deathbed or dead helicobacter pylori is difficult to the pathogenic effects of attached cell
Under the situation for being reflected in infection people, the helicobacter pylori of people's stomach internal breeding active state is right to the pathogenic effects of Gastric Mucosal Cells
Result of study is produced a very large impact.（2）Needed every other day in attached cell, Traditional Method to maintain helicobacter pylori living to stimulate
Repeat to add helicobacter pylori into cultivating system, cause working procedure cumbersome, and the change of number of viable will certainly cause it
Fluctuation to cytosis, is equally unfavorable for experimental study.
The content of the invention
There is provided a kind of brand-new helicobacter pylori and stomach aiming at above-mentioned defect of the prior art for the purpose of the present invention
The method that mucosal epithelial cells are co-cultured, adds the inclined-plane solid of suitable growth of H. pylori in normal Tissue Culture Flask
Culture medium, ensures the existence of helicobacter pylori with this, while cell culture fluid can ensure the normal growth of Gastric Mucosal Cells, the two
Interaction is produced after culture, is solved in helicobacter pylori in vitro culture cytopathogenic effect experimental study because helicobacter pylori gives birth to
Reason state difference and the influence that is caused to experimental result of number of viable fluctuation, and need to repeatedly add the intricate operation of bacterium and ask
Topic.
To achieve these goals, the technical scheme that provides of the present invention is：A kind of helicobacter pylori and gastric epithelial are thin
The method that born of the same parents co-culture, comprises the following steps：
1）It is formulated for cultivating Colombia's Solid agar culture of helicobacter pylori and with HTHP moist heat sterilization, waits to train
When foster base is cooled to 50 DEG C -60 DEG C, glucose solution and newborn calf serum are added, is fully mixed, obtain helicobacter pylori training
Support base；
2）Take step 1）The heiicobacter pylori cultivation base 10ml of obtained ot-yet-hardened, is added in 50ml steril cell blake bottles
（If glass material class it is non-disposable the need for first carry out HTHP moist heat sterilization, the culture of disposable plastic material
Bottle then need not）, and the bottom surface for making Tissue Culture Flask treats that culture medium cooled and solidified is formed in bottom of bottle with the horizontal 30 degree of angles
Behind inclined-plane, blake bottle is horizontally or vertically placed standby；As shown in figure 1, the bottom of Tissue Culture Flask, which forms one, is specifically used to training
The solid slope of helicobacter pylori is supported, its area is about 20cm2, now Tissue Culture Flask bottom surface still residue about 18cm2Area is not yet
By solid slope all standing；
3）Take containing gastric epithelial cells 5 × 104Individual/ml cell suspension 3ml is added to step 2）Obtained Tissue Culture Flask
In（Fig. 2）, Tissue Culture Flask horizontal positioned is entered in cell culture incubator, 10%CO2, 37 DEG C of culture 24h, make gastric epithelial thin
Born of the same parents are adherent；
4）Take cultivated 48h be in stationary phase helicobacter pylori bacterium solution 1ml centrifuged, centrifugal rotational speed is 800rpm,
Centrifugation time is that to add cell culture fluid 1ml after 5min, supernatant discarded resuspended, obtains resuspended bacterium solution；
5）By step 4）Obtained resuspended bacterium solution is added to step 3）The adherent cell training of obtained gastric epithelial cells
Support in bottle, then Tissue Culture Flask is put into continuation in the incubator for be exclusively used in heiicobacter pylori cultivation and cultivate, condition of culture：37
DEG C, humidity 90%, micro- aerobic gaseous environment（5%O2、10%CO2And 85%N2）, helicobacter pylori in blake bottle liquid is counted daily
Cell and bacterium after the morphological change of viable bacteria amount and observation of cell, collecting action are used for follow-up correlative study.
Further, the method that a kind of above-mentioned helicobacter pylori and gastric epithelial cells are co-cultured, the step 1）
In, the preparation method of Colombia's Solid agar culture for cultivating helicobacter pylori is specially：Weigh Colombia's fine jade
Aliphatic radical plinth powder 4.25g adds ddH in triangular flask2O 85ml, fill in HTHP moist heat sterilization after bottle stopper, treat that solid is trained
When foster base is cooled to 50 DEG C -60 DEG C, the glucose injections of 5ml 10% are added, make the final concentration of glucose in terms of mass volume ratio
For 0.5%, 10ml newborn calf serum is added, makes the final concentration of newborn calf serum using volume basis as 10%, it is fully mixed
Heiicobacter pylori cultivation base is obtained after even.
Further, the method that a kind of above-mentioned helicobacter pylori and gastric epithelial cells are co-cultured, the step 1）
In, the temperature of HTHP moist heat sterilization is 121 DEG C, and the time is 15min.
Further, the method that a kind of above-mentioned helicobacter pylori and gastric epithelial cells are co-cultured, the step 3）
In, containing gastric epithelial cells 5 × 104The preparation method of individual/ml cell suspension is specially：Discard cultured adherent reach
The nutrient solution of 90% gastric epithelial cells, adds after 2ml PBSs 3 times, discards PBS, add 2ml 0.25% pancreas egg
The complete medium that 1ml is added after white digestive ferment, digestion 5min terminates digestion.The content of complete medium is matched according to volume ratio
The DMEM 90% of high glycoform is calculated as, the complete medium of newborn calf serum 10%, i.e. 100ml contains 90ml DMEM and 10ml
Newborn calf serum.Draw the mixed liquor of above-mentioned tryptic digestive juice and complete medium and 5min is centrifuged with rotating speed 800rpm
Afterwards, supernatant discarded, the complete medium piping and druming for drawing 1ml mixes precipitation, separately takes 5ml complete medium, mixed by having blown and beaten
Even celliferous nutrient solution is added dropwise, and the concentration of cell is reached 5 × 104Individual/ml, that is, obtain containing gastric epithelial cells
5×104Individual/ml cell suspension.
Beneficial effects of the present invention are：
A kind of method that the helicobacter pylori and gastric epithelial cells that the present invention is provided co-culture, adds pylorus in blake bottle
After helicobacter slant medium, the cell attachment of progress is it is demonstrated experimentally that the gastric epithelial cells added are after culture, observation
To cell in the still normal adherent growth of bottom surface of blake bottle, cellular morphology is normal, with the bottom surface in ordinary cells blake bottle
It is adherent to compare indifference.Illustrate that the no cell growth of addition of helicobacter pylori slant medium produces influence.Further, since
By cell anchoring factor is incompatible with solid slope etc. that factor is influenceed, gastric epithelial cells are not in inclined-plane culture primary surface
Form adherent growth, it is ensured that growth of H. pylori.
Count plate is found, in the helicobacter pylori and gastric epithelial cells co-culture system completely newly improved, pylorus
The sustainable survival of helicobacter, number of viable was undergone after the slow laundering period of 1 day, and growth metabolism is active, a large amount of propagation, from the 4th day
Start the relatively stable state of maintenance up to 6 days（Fig. 3）, lasting offer helicobacter pylori can be provided very well, it is and adherent thin
Born of the same parents' fully contact for a long time, plays pathogenic effects.Day by day the change of observation of cell finds that gastric epithelial cells are by pylorus spiral shell
After the effect of bacillus, significant cytopathic effect is generated, the change of vacuole sample is obvious, meets experiment and is expected.Moreover, with tradition
Method helicobacter pylori and gastric epithelial cells co-culture method and compared, and the method that the present invention is provided is in same time inner cell lesion
Effect becomes apparent from.
The stable existence of number of viable, it is to avoid repeat to add the cumbersome of the work of helicobacter pylori.
Brief description of the drawings
The solid for being used for cultivating helicobacter pylori formed in Tissue Culture Flask bottom that Fig. 1 is shown as this patent making is oblique
Face.
Wherein A：For ordinary cells blake bottle；B：For Tissue Culture Flask bottom formed be used for cultivate consolidating for helicobacter pylori
Body inclined-plane；C：The angle for representing inclined-plane at this is 30 °.
Fig. 2, which is shown as having been formed in the Tissue Culture Flask of solid slope, adds conventional cell nutrient solution.
Wherein A：For ordinary cells blake bottle；B：For Tissue Culture Flask bottom formed be used for cultivate consolidating for helicobacter pylori
Body inclined-plane；C：The angle for representing inclined-plane at this is 30 °；D：The cell culture fluid of addition.
Fig. 3 is shown as initial inoculation（Inoculum concentration is 103CFU/ml）Helicobacter pylori is to the only cell culture fluid containing inclined-plane
When middle, the viable count change in cell culture fluid（It is within 0-1 days the laundering period of helicobacter pylori；It is within 1.5-3 days logarithmic phase；4-9 days
For stationary phase；It is within 10-13 days decline phase）.
Fig. 4 is shown as when initial inoculum is different, viable count situation of change of the helicobacter pylori in nutrient solution.a、b、
C, d represent different inoculum concentrations, are followed successively by：107CFU/ml、105CFU/ml、103CFU/ml、102CFU/ml。
Embodiment
Embodiment 1：
A kind of method that helicobacter pylori and gastric epithelial cells are co-cultured, comprises the following steps：
1）It is formulated for cultivating Colombia's Solid agar culture of helicobacter pylori and with HTHP moist heat sterilization, treats solid
When body culture medium is cooled to 50 DEG C -60 DEG C, glucose solution and newborn calf serum are added, fully mixes, obtains helicobacter pylorus
Bacterium culture medium；The preparation method of Colombia's Solid agar culture for cultivating helicobacter pylori is specially：Weigh brother's human relations
Than sub- agar basis powder 4.25g in triangular flask, ddH is added2O 85ml, fill in HTHP moist heat sterilization after bottle stopper, treat
When solid medium is cooled to 50 DEG C -60 DEG C, the glucose injections of 5ml 10% are added, make the final concentration of glucose with mass body
Product ratio is calculated as 0.5%, adds 10ml newborn calf serum, makes the final concentration of newborn calf serum using volume basis as 10%,
Heiicobacter pylori cultivation base is obtained after fully mixing；The temperature of HTHP moist heat sterilization is 121 DEG C, and the time is 15min；
2）Take step 1）The heiicobacter pylori cultivation base 10ml of obtained ot-yet-hardened, is added in 50ml steril cell blake bottles
（If glass material class it is non-disposable the need for first carry out HTHP moist heat sterilization, the culture of disposable plastic material
Bottle then need not）, slant setting blake bottle makes its bottom surface with the horizontal 30 ° of angles, after after culture medium cooled and solidified, level
Or placement blake bottle is standby vertically；It can be used to cultivate helicobacter pylori as shown in figure 1, the bottom of Tissue Culture Flask forms one
Solid slope, its area is about 20cm2, the bottom surface of blake bottle is not by solid slope all standing, remaining bottom in addition
Face area is about 18cm2；
3）Take containing gastric epithelial cells 5 × 104Individual/ml cell suspension 3ml is added to step 2）Obtained Tissue Culture Flask
In, Tissue Culture Flask horizontal positioned is entered in cell culture incubator, 10%CO2, 37 DEG C of culture 24h, gastric epithelial cells are adherent；
Containing gastric epithelial cells 5 × 104The preparation method of individual/ml cell suspension is specially：Discard cultured adherent up to 90%
Gastric epithelial cells nutrient solution, add after 2ml PBSs 3 times, discard PBS, the tryptose for adding 2ml 0.25% disappears
Change the complete medium termination digestion that 1ml is added after enzyme, digestion 5min, the content proportioning of complete medium is according to volume basis
The DMEM 90% of high glycoform, the complete medium of newborn calf serum 10%, i.e. 100ml contains the new of 90ml DMEM and 10ml
Calve serum, after drawing the mixed liquor of the tryptic digestive juice and complete medium with rotating speed 800rpm centrifugations 5min,
Supernatant discarded, the complete medium piping and druming for drawing 1ml mixes precipitation, separately takes 5ml complete medium, will blow and beat mixing
Celliferous nutrient solution is added dropwise, and the concentration of cell is reached 5 × 104Individual/ml, that is, obtain containing gastric epithelial cells 5 ×
104Individual/ml cell suspension；
4）Take cultivated 48h be in stationary phase helicobacter pylori bacterium solution 1ml centrifuged, centrifugal rotational speed is 800rpm,
Centrifugation time is that to add cell culture fluid 1ml after 5min, supernatant discarded resuspended, obtains resuspended bacterium solution；
5）By step 4）Obtained resuspended bacterium solution is added to step 3）The adherent cell training of obtained gastric epithelial cells
Support in bottle, then Tissue Culture Flask is put into continuation in the incubator dedicated for heiicobacter pylori cultivation and cultivate, condition of culture：
37 DEG C, humidity 90%, micro- aerobic environment（5%O2、10%CO2And 85%N2）, the work of helicobacter pylori in blake bottle liquid is counted daily
Cell after bacterium amount and the morphological change for observing and recording gastric epithelial cells, collecting action is used for follow-up related to bacterium
Research.
Influence in the present invention to the angle on heiicobacter pylori cultivation base inclined-plane that is made in blake bottle to number of viable is entered
Discussion is gone, result when earlier figures 3 are 30 ° of medium slant angle.By experiment measurement, when inclined-plane gradient is 90 °,
The inclined-plane area of solid medium is about 12cm2.Now ensureing that the inoculum concentration of helicobacter pylori is 103In the case of CFU/ml,
Change curve when the number of viable change curve of helicobacter pylori is 30 ° with inclined-plane gradient in cell culture fluid has a little difference
Different, the laundering period for being mainly manifested in helicobacter pylori extends to the 3rd day, and exponential phase is time-consuming about 3 days, the ascent stage of curve
Compare slightly slow, and the duration of stationary phase and decline is then not different.The reason for causing is probably to be led because inclined-plane area is smaller
The apposition growth area of helicobacter pylori is caused to reduce, the time needed for reaching maximum growth amount has extended.When inclined-plane gradient
For 0 ° when, its surface area be 27cm2.Ensureing that the inoculum concentration of helicobacter pylori is 103In the case of CFU/ml, tilted with inclined-plane
Spend for 30 ° when compare, laundering period time of the helicobacter pylori in cell culture fluid slightly has shortening, exponential phase time-consuming about 2
My god, thalline quantity stationary phase is about 6 days, and the decline phase is about 4 days, and total time-to-live is about 11 days.Heiicobacter pylori cultivation basal plane
Long-pending increase contributes to it to grow, but because culture medium covers whole blake bottle bottom surface, causes the gastric epithelial cells can not
Form adherent growth.In summary, the angular dimension influence inclined-plane face on the heiicobacter pylori cultivation base inclined-plane made in the present invention
Product, so can be to viable bacteria number of variations in nutrient solution the certain influence of generation, therefore the specific angle person of needing to use is according to reality
Test demand determination.
In addition, the time that the inoculum concentration of initial helicobacter pylori reaches plateau on viable count in liquid produces influence, but
It is little for time-to-live and the process declined influence, as shown in Figure 4.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for a person skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or to which part technical characteristic progress equivalent.
Within the spirit and principles of the invention, any modifications, equivalent substitutions and improvements made etc., should be included in the present invention's
Within protection domain.
Claims (4)
1. a kind of method that helicobacter pylori and gastric epithelial cells are co-cultured, it is characterised in that comprise the following steps：
1）It is formulated for cultivating Colombia's Solid agar culture of helicobacter pylori and with HTHP moist heat sterilization, treats solid
When body culture medium is cooled to 50 DEG C -60 DEG C, glucose solution and newborn calf serum are added, fully mixes, obtains helicobacter pylorus
Bacterium culture medium；
2）Take step 1）Obtained heiicobacter pylori cultivation base 10ml, is added in 50ml steril cell blake bottles, and make cell
The bottom surface of blake bottle is horizontal or vertical to put after culture medium cooled and solidified behind bottom of bottle formation inclined-plane with the horizontal 30 degree of angles
Put blake bottle standby；
3）Take containing gastric epithelial cells 5 × 104Individual/ml cell suspension 3ml is added to step 2）Obtained Tissue Culture Flask,
Tissue Culture Flask horizontal positioned is entered in cell culture incubator, 10%CO2, 37 DEG C of culture 24h, make gastric epithelial cells adherent；
4）Take cultivated 48h be in stationary phase helicobacter pylori bacterium solution 1ml centrifuged, centrifugal rotational speed is 800rpm,
Centrifugation time is that to add cell culture fluid 1ml after 5min, supernatant discarded resuspended, obtains resuspended bacterium solution；
5）By step 4）Obtained 1ml resuspended bacterium solutions are added to step 3）The adherent cell of obtained gastric epithelial cells
In blake bottle, then by Tissue Culture Flask be put into the incubator dedicated for heiicobacter pylori cultivation continue cultivate, condition of culture：
37 DEG C, humidity 90%, micro- aerobic gaseous environment, i.e. gaseous environment contain according to volume percentage 5%O2、10%CO2And 85%N2,
Count daily afterwards in blake bottle liquid after the viable bacteria amount of helicobacter pylori and the morphological change of observation of cell, collecting action
Cell and bacterium.
2. the method that a kind of helicobacter pylori according to claim 1 and gastric epithelial cells are co-cultured, its feature exists
In the step 1）In, the preparation method of Colombia's Solid agar culture for cultivating helicobacter pylori is specially：Claim
Columbia agar basis powder 4.25g is taken in triangular flask, ddH is added2O 85ml, fill in after culture medium fully mixes dissolving
Enter bottle stopper, HTHP moist heat sterilization, it is subject to sterilization after culture medium when being cooled to 50 DEG C -60 DEG C, add the glucose of 5ml 10% note
Liquid is penetrated, the final concentration of glucose is calculated as 0.5% with mass volume ratio, adds 10ml newborn calf serum, makes newly to calve
The final concentration of serum obtains heiicobacter pylori cultivation base using volume basis as 10% after fully mixing.
3. the method that a kind of helicobacter pylori according to claim 2 and gastric epithelial cells are co-cultured, its feature exists
In the step 1）In, the temperature of HTHP moist heat sterilization is 121 DEG C, and the time is 15min.
4. the method that a kind of helicobacter pylori according to claim 3 and gastric epithelial cells are co-cultured, its feature exists
In the step 3）In, containing gastric epithelial cells 5 × 104The preparation method of individual/ml cell suspension is specially：Discard training
Supported it is adherent up to 90% gastric epithelial cells nutrient solution, add after 2ml PBSs 3 times, discard PBS, add
The complete medium that 1ml is added after 2ml 0.25% trypsinized enzyme, digestion 5min terminates digestion, and complete medium contains
Amount proportioning contains according to the DMEM 90% that volume basis is high glycoform, the complete medium of newborn calf serum 10%, i.e. 100ml
90ml DMEM and 10ml newborn calf serum, draw the mixed liquor of the tryptic digestive juice and complete medium, with
After rotating speed 800rpm centrifugations 5min, supernatant discarded, the complete medium piping and druming for drawing 1ml mixes precipitation, separately takes 5ml complete training
Base is supported, the celliferous nutrient solution for having blown and beaten mixing is added dropwise, the concentration of cell is reached 5 × 104Individual/ml, is produced
To containing gastric epithelial cells 5 × 104Individual/ml cell suspension.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109880801A (en) * | 2019-03-27 | 2019-06-14 | 蔺莉 | A kind of method of inducing cell vicious transformation culture model |
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| CN1654472A (en) * | 2003-04-17 | 2005-08-17 | 南方医院 | Recombinant helicobacter pylori outer-membrane protein 25 |
| CN103757088A (en) * | 2014-01-20 | 2014-04-30 | 四川万可泰生物技术有限责任公司 | Quantitative determination and drug allergy determination kits for helicobacter pylori viable bacteria and determination method |
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| 梁昌盛等: ""幽门螺杆菌体外培养条件的研究"", 《临床医学工程》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880801A (en) * | 2019-03-27 | 2019-06-14 | 蔺莉 | A kind of method of inducing cell vicious transformation culture model |
| CN109880801B (en) * | 2019-03-27 | 2022-07-12 | 蔺莉 | Method for inducing cell malignant transformation culture model |
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