CN105483078A - Isolation and primary culture methods of chicken small intestinal epithelial cells - Google Patents
Isolation and primary culture methods of chicken small intestinal epithelial cells Download PDFInfo
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Abstract
The invention belongs to the technical field of cell isolation and cultivation. In order to solve the problems that the chicken small intestinal epithelial cells are difficult to isolate and culture in vitro at present, the existing isolation process is troublesome and time-consuming, the obtained cells are low in purity and the like, the invention provides isolation and primary culture methods of the chicken small intestinal epithelial cells. The methods comprise the steps of taking chick embryo small intestines, cleaning the small intestines to remove mesenteries, digesting small intestine tissues by protease to obtain the chicken small intestinal epithelial cells, performing adherent difference culture on the cells, removing adherent parenchyma cells, collecting intestinal epithelial cells, inoculating by taking the inoculum density being about 6.5X105/mL for culturing, the chicken small intestinal epithelial cells obtained by culturing is maximum in quantity, low in death rate, and best in growing status. The primary culture is performed by adopting a culture solution in which chicken serum is added, and the growing status of the obtained cells is better. The chicken small intestinal epithelial cells which are good in growing status, normal in shape, high in activity are successfully cultured, the primary and isolation culture methods of the chicken small intestinal epithelial cells are successfully established, and the technical support is provided for follow-up tests.
Description
Technical field
The invention belongs to cellular segregation culture technique field, be specifically related to a kind of separation and primary culture method of chicken intestinal epithelial cell.
Background technology
Intestinal epithelial cells (intestinalepithelialcells, IEC) all plays a very important role to many aspects such as the physiology of enteron aisle and pathology.Enteron aisle is maximum immune organ, and the reaction that it bears immunizing power to the mucous membrane of food antigens is very important.So the research of intestinal epithelial cells contributes to physiology and the pathologic function of exploring enteron aisle.What intestinal epithelial cells can stop potentially harmful substance as one barrier enters the migration with bottom cell, and vitro culture test shows, intestinal epithelial cells affects the change of cell quantity in subenvironment.
The primary culture in vitro of intestinal epithelial cells is more difficult, because they depend on cell and cell or contact growth between cell and matrix, also needs many somatomedins.Shortage or the interacting of extracellular matrix of somatomedin all may cause cell apoptosis or death at short notice.Original cuiture for intestinal epithelial cells have employed many diverse ways, achieve effect in various degree, but apoptosis easily occurs.The immature research that always limit the digestion and absorption function mechanism of intestinal physiology and pathological research, particularly small intestine of intestinal epithelial cells cultural method.Peripheral blood easily obtains from patient, but may there will not be reaction at enteron aisle.The vitro culture of intestinal epithelial cells contributes to the various reactions studying intestinal epithelial cells, and this may play an important role to the research of coeliac disease and small bowel inflammation.
Intestinal epithelial cell has polarity, and its shape is cylindricality, is also functional cell important in enteron aisle, all plays a significant role, and be closely related with enteron aisle endocrine & exocrine function in nutrition digestion, absorption and transhipment etc.Intestinal epithelial cells expresses brush-border hydrolysis enzyme, and alkaline phosphatase and translocator play an important role in absorption of nutrient ingredients process.Therefore, the original cuiture of intestinal epithelial cell has become one of Main Means of the functions such as the mesotrophic absorption and digestion of research enteron aisle, also for its relevant biological function of research provides desirable experimental model.
The original cuiture of intestinal epithelial cells selects best culture environment and nutritional factor to carry out Isolation and culture to intestinal epithelial cells, the influence factor of cell cultures has many, and the best inoculum density of cell is conducive to the number of adherent of cell in culture dish and growing state, if cell density is too little, do not connect between cell, the proliferate speed of cell can be slowly, if and cell density is too large, the space that iuntercellular is not suitable for makes attached cell not reach the density of growth needs, even if cell also very easily comes off after cell growing multiplication, have impact on the growing multiplication of cell.Therefore, the too high or too low adherent growth being all unfavorable for cell of inoculum density of cell, is necessary to find an optimum Initial seeding density, in order that intestinal epithelial cell can play its maximum efficiency.The best inoculum density of different cell is different, finds that the best inoculum density of vitro culture cow mammary gland epithelial cells is l.0 × 10 in the research of Zavizion etc.
4individual/mL.And human intestinal epithelial cells inoculum density is 1.0 × 10 in the test of Liao Xian equality
6~ 6.5 × 10
6/ mL.The isocellular inoculum density of different tests personnel education is also different, as Li Yan etc.
[60]the inoculum density of the chicken intestinal epithelial cell of test is 0.5 × 10
4~ 0.8 × 10
4individual/cm
2, the Initial seeding density of the chicken intestinal epithelial cell that the test of Yang Wen equality uses is 1.0 × 10
5individual/mL.In the culture dish or culturing bottle of different size, the best inoculum density of cell is also different.
Research finds, the Isolation and culture environment of various cell as far as possible close to the physiological environment in its body, as pH value, temperature, gas concentration lwevel, nutritive ingredient and osmotic pressure etc., only in this way just may must successfully turn out required cell.The proliferate of cell needs a series of nutritive substance, and wherein a kind of nutritive substance is exactly the serum collected from blood.Serum plays very crucial role in cell cultures: can provide the necessary hormone of growth and proliferation of cell and somatomedin etc.; Basic nutrition material can be provided, i.e. the required material of the Growth of Cells such as amino acid, lipid material, VITAMIN; Short contact factor can be provided and short stretch the factor, for the adhesion of attached cell in Tissue Culture Dish and stretching, extension, avoiding attached cell to be subject to physical abuse; Can provide associated proteins, associated proteins both can carry important low molecular weight substance, and also Absorbable organic halogens binding substance or change character, play an important role in cellular process; Cultured cells can be avoided to come to harm with toxicant in also containing.Foetal calf serum (FoetalBovineSerum, FBS) is the serum of most widespread use in cell injuring model, but from tire ox, collect the problem of serum except morals, also there is the challenge on Science and Technology.Containing many unknowns, different component in serum, due to this uncertainty, test-results may be different because of the different batches of serum, and this also comprises the possibility that adverse factor pollutes, and may work the mischief to culturing cell.Due to high cost, availability problem and the Main Differences at different iuntercellular nutritional needs, the suitableeest serum that different cell needs may be different.And the size that intestinal epithelial cells cultivates the serum-concentration used is also very important, excessive concentration may promote the growth of other cell, as 10% ~ 20% serum can promote the epithelial division growth of the non-enteric such as inoblast.So, cultivate the concentration of the growing multiplication of intestinal epithelial cells generally no more than 5%, so just can when being unfavorable for that the epithelial raised growth of non-enteric is bred, normal Isolation and culture intestinal epithelial cell.
The patent documentation had not yet to see about chicken intestinal epithelial cell in-vitro separation or cultural method is open, although adopt routine cell culture techniques in existing chicken intestinal epithelial cells cultural method, the object of cultivation can be reached to a certain extent, but there is complex operation, cell is not easily adherent, other heteroproteose cells such as poor growth, inoblast are difficult to the problems such as separation, can not meet the requirement of cell experiment.Have document to adopt the mode of differential velocity adherent or low-speed centrifugal to be separated into fibrocyte composition, but this process is comparatively loaded down with trivial details and consuming time, usually needs 5-7 hour and cell purity is lower.
Application number is 201510137707.1 and is called that the patent of invention of " a kind of chicken intestinal epithelial cells isolation cultivation method " discloses a kind of chicken intestinal epithelial cells isolation cultivation method, said method comprising the steps of: step 1: with proteolytic enzyme, chicken embryo intestinal tissue block is digested, obtain digestion product; Step 2: with cell sieve, digestion product is filtered, collect and be of a size of 100-500 object cell mass; Step 3: by described cell mass centrifugal 5-15min under 800-1500r/min, collecting cell precipitates.Present invention also offers the chicken intestinal epithelial cells utilizing method of the present invention to obtain.Method provided by the present invention can shorten test period greatly, saves reagent, improves the purity of the intestinal epithelial cells be separated.But containing many unknowns, different component in serum, due to this uncertainty, test-results may be different because of the different batches of serum, this also comprises the possibility that adverse factor pollutes, may work the mischief to culturing cell, and then affect separation and the cultivation of chicken intestinal epithelial cell.
Summary of the invention
The problems such as the present invention carries out separation and Culture to solve current chicken intestinal epithelial cell external being difficult to, and the loaded down with trivial details cell purity consuming time, that obtain of existing sepn process is low, provide a kind of separation and primary culture method of chicken intestinal epithelial cell.
The present invention is realized by following technical scheme: a kind of separation of chicken intestinal epithelial cell and primary culture method, take out chicken embryo small intestine, cleaning small intestine removes mesentery, with proteolytic enzyme, small intestine is digested, obtain chicken intestinal epithelial cell, cell is carried out adherent difference and cultivate, remove adherent heteroproteose cell, collect intestinal epithelial cell, concrete steps are:
(1) take out chicken embryo small intestine: get the chicken embryo of hatching 15 days, chicken embryo small intestine is taken out in aseptic technique;
(2) clean small intestine and remove mesentery: the small intestine scavenging solution I of taking-up cleans repeatedly, for subsequent use after using scavenging solution II cleaning after small intestine being shredded into tissue block after limpid to supernatant liquor with scavenging solution II cleaning after removing mesentery;
(3) with proteolytic enzyme, small intestine is digested: adopt thermolysin Digestive system 37 DEG C, 80r/min vibration digestion 120min, piping and druming enzymic digestion liquid 2-3min, the PBS of 37 DEG C dilutes enzymic digestion liquid, draw the suspension after dilution, continue to dilute enzymic digestion liquid suspension cell agglomerate with PBS, draw the suspension of dilution until Digestive system is limpid; By centrifugal for suspension 1000r/min 5min, stay throw out;
(4) cell is carried out adherent difference to cultivate, remove adherent heteroproteose cell: the substratum of suspended centrifugal gained precipitation containing 10% chicken serum suspends piping and druming evenly, is inoculated into by cell suspending liquid in culturing bottle, 40 DEG C, 7%CO
2cultivate 70min in incubator, remove adherent heteroproteose cell;
(5) intestinal epithelial cell is collected: take out culturing bottle, piping and druming culturing bottle 2-3 time is by just adherent intestinal epithelial cell agglomerate Eddy diffusion, by centrifugal for cell suspending liquid 1000r/min 5min, supernatant discarded, precipitation first suspends piping and druming evenly with the DMEM containing 5% chicken serum, with trypan blue diluting cells suspension, for subsequent use after the cell mass number contaminated without indigo plant with blood counting chamber meter cell;
(6) chicken intestinal epithelial cell original cuiture: chicken intestinal epithelial cell is 6.5 × 10 according to inoculum density
5/ mL is inoculated in primary chicken intestinal epithelial cell nutrient solution, 40 DEG C, 7%CO
2cultivate in incubator;
Described scavenging solution I is that 38.8mLPBS+1.2mL is dual anti-, and described scavenging solution II is that 39.8mLPBS+0.2mL is dual anti-.
Described thermolysin Digestive system is: 25mg thermolysin is dissolved in the Hepes damping fluid of 100mL and stirs to dissolving completely, adjusts pH to 7.4, filtration sterilization ,-20 DEG C of preservations; Hepes damping fluid is: 0.5g/LKCl+8.307g/LNaCl+2.38g/LHepes+0.111g/LCaCl2+0.018g/LNaOH, adjust ph to 7.4, filtration sterilization ,-20 DEG C of preservations.
Described primary chicken intestinal epithelial cell nutrient solution is: dual anti-+ 400 μ L Regular Insulin+2mL serum of 40mL liquid low-sugar type DMEM+800 μ L Urogastron+400 μ LL-glutamine+400 μ L heparin sodium+400 μ L; The preparation method of chicken serum is: the chicken of getting fasting 12h carries out Culling heart blood, by the hematology aliquot collected to centrifuge tube; The centrifuge tube containing blood is put in 1h in 37 DEG C of water-baths; After separating out part serum, be placed in 4 DEG C of refrigerator overnight; After serum is separated out naturally, 4 DEG C, 3000r/min, centrifugal 10min, draw serum in centrifuge tube with syringe, discard insolubles; After serum packing, Chu Zang Yu – 80 DEG C of preservations.
Because chicken belongs to birds, and Bos is in Mammals, containing the specific nutritive substance of many birds in chicken serum.Cell difficulty is obtained when therefore selecting bovine serum to carry out the cultivation of chicken intestinal epithelial cell.The chicken serum that market is buied needs through filtration sterilization, and because chicken serum excessive concentration needs through dilution just by the filtering membrane of 0.22 μm, and commercially available chicken serum generally all can through dilution, and its nutritive substance can reduce.So now chicken serum must be adopted in chicken intestinal epithelial cell is cultivated, and filter again with the dilution of low-sugar type basal cell culture medium, then carry out subsequent experimental.
After chicken intestinal epithelial cell digests by the present invention, with inoculum density for 6.5 × 10
5about/mL is inoculated on six well culture plates and cultivates, and the chicken intestinal epithelial cell quantity of cultivating acquisition is maximum, and mortality ratio is low, and growth conditions is best.Adopt the nutrient solution adding chicken serum to carry out the original cuiture of chicken intestinal epithelial cell, the cell growth state of acquisition is better.By the screening to chicken intestinal epithelial cell original cuiture optimum condition and nutritional factor, and Morphological Identification and immunocytochemistry qualification, successfully turn out the chicken intestinal epithelial cell that growth conditions is good, form normal, activity is high, successfully establish the primary isolation cultivation method of chicken intestinal epithelial cell, for follow-up test provides technical support.
Accompanying drawing explanation
Fig. 1 is the cell growth curve figure of different vaccination density; Fig. 2 is the growth column diagram of chicken intestinal epithelial cell at Different growth phases of different vaccination density, note: the first row represents the comparison between same number of days different vaccination density, the comparison between the same inoculum density different number of days of the second line display.Represent between same letter difference not significantly (
p> 0.05); Different capitalization represent difference extremely significantly (
p< 0.01); Fig. 3 is the growing state figure (100 ×) of chicken intestinal epithelial cell different vaccination density, in figure: under A0-A14: the 1 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Under B0-B14: the 2 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Under C0-C14: the 3 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Under D0-D14: the 4 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Under E0-E14: the 5 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Under F0-F14: the 6 group of inoculum density, chicken intestinal epithelial cell is at the growth conditions of 0d, 3d, 7d, 10d, 14d; Fig. 4 is the growth curve of chicken intestinal epithelial cell under different serum free culture system; Fig. 5 be under different serum condition chicken intestinal epithelial cell at the growth column diagram of Different growth phases, note: represent between same serum different number of days same letter difference not significantly (
p> 0.05); Different capitalization and lowercase represent respectively difference extremely significantly (
p< 0.01) and significant difference (
p< 0.05).* represent with * * the different serum significant difference of same number of days (
p< 0.05) and extremely significantly (
p< 0.01), lower same; Fig. 6 adds different serum to chicken intestinal epithelial cell upgrowth situation figure (100 ×), in figure: A-E: chicken intestinal epithelial cell foetal calf serum cultivates 0d, and the growing state of 3d, 7d, 10d, 14d; F-J: chicken intestinal epithelial cell chicken serum cultivates 0d, the growing state of 3d, 7d, 10d, 14d; Fig. 7 is the morphologic observation figure (400 ×) of chicken intestinal epithelial cell; Fig. 8 is the immunohistochemical methods qualification result (400 ×) of chicken intestinal epithelial cell.
Embodiment
A kind of separation of chicken intestinal epithelial cell and primary culture method, take out chicken embryo small intestine, cleaning small intestine removes mesentery, with proteolytic enzyme, small intestine is digested, obtain chicken intestinal epithelial cell, cell is carried out adherent difference and cultivate, remove adherent heteroproteose cell, collect intestinal epithelial cell, concrete steps are:
(1) chicken embryo small intestine is taken out: get the chicken embryo of hatching 15 days, use Iodophor and its shell of 75% wipes of alcohol respectively, dry, be put in the super clean bench of ultraviolet sterilization.First knock air chamber open, then along its edge except shell breaking, remove shell membrane with tweezers, take out chicken embryo, then with tweezers and scissors chicken embryo dissected and take out small intestine, put into the sterilized petri dishes filling the dual anti-scavenging solution of 38mLPBS+2mL.
(2) clean small intestine and remove mesentery: small intestine is cleaned 2-3 time repeatedly, move in small intestine scavenging solution I, remove mesentery gently with tweezers.Then move in small intestine scavenging solution II wash 2-3 time substantially limpid to supernatant, then move into and shred small intestine one-tenth with eye scissors in aseptic 5mL centrifuge tube and be less than 1mm
3tissue block (when shredding small intestine, speed of as far as possible accomplishing is fast, firmly evenly, reduces physical abuse).Use small intestine scavenging solution II cleaning saturating clearly to supernatant liquor, discard scavenging solution.
(3) with proteolytic enzyme, small intestine is digested: the small intestine shredded is put into 15mL centrifuge tube, add the thermolysin Digestive system of 50mg/L, 37 DEG C, 80r/min vibrates at a slow speed and digests 120min, then centrifuge tube is taken out, blow and beat enzymic digestion liquid 2-3min gently, the cell mass that digestion is opened scatter soon as far as possible, with 37 DEG C of preheated PBS, enzymic digestion liquid is diluted, blow and beat gently, draw its suspension, be dispensed in the centrifuge tube of several 15mL, continue to dilute enzymic digestion liquid suspension cell agglomerate with PBS, draw suspension till Digestive system is limpid.Again by the centrifugal 5min of this suspension 1000r/min, stay throw out.
(4) adherent difference is cultivated, and removes adherent heteroproteose cell: the substratum of suspended centrifugal gained precipitation containing 10% serum suspends piping and druming evenly, after piping and druming evenly, is inoculated into by cell suspending liquid in 25mL culturing bottle, then is placed on 40 DEG C, 7%CO
2cultivate 70min in incubator, utilize the different of heteroproteose cell adherent time such as intestinal epithelial cell and inoblast to remove other adherent heteroproteose cells such as inoblast.
(5) intestinal epithelial cell is collected: take out culturing bottle, jiggle several times, blow to each place at the bottom of making at the bottom of glue head dropper featheriness bottle bottle as far as possible again, featheriness makes just adherent intestinal epithelial cell agglomerate Eddy diffusion for 2-3 time, cell suspending liquid is collected in the centrifugal 5min of 1000r/min in 15mL centrifuge tube, supernatant discarded, precipitation first suspends piping and druming evenly with the DMEM containing 5% serum.With trypan blue diluting cells suspension (10 μ L cell suspension+10 μ L0.5% trypan blue), the cell mass number (more than 3 cells) contaminated without indigo plant with blood counting chamber meter cell.
(6) chicken intestinal epithelial cell original cuiture: chicken intestinal epithelial cell is 6.5 × 10 according to inoculum density
5/ mL is inoculated in primary chicken intestinal epithelial cell nutrient solution, 40 DEG C, 7%CO
2cultivate in incubator.
Described scavenging solution I is that 38.8mLPBS+1.2mL is dual anti-, and described scavenging solution II is that 39.8mLPBS+0.2mL is dual anti-.
Experimental example 1: the screening of chicken intestinal epithelial cell optimal cell inoculum density and cultivation
Be divided into six gradients according to the cell count of every milliliter that cell counting draws, as table 1, the data of cell mass diluent good for above-mentioned dilution according to different gradient be inoculated in 6 porocyte culture plates respectively, each gradient repeats 3 times, is put in 40 DEG C, 7%CO
236h is cultivated in incubator, and the growth conditions of observation of cell, calculate adherence rate.Change nutrient solution after 48h, every day observation of cell upgrowth situation and adherent situation, adopt Trypan Blue meter to get viable cell quantity, Continuous Observation 14 days.Using the incubation time of chicken small intestine cells as X-coordinate, viable count makes the growth curve of chicken intestinal epithelial cell as ordinate zou, and observation and comparison also therefrom determines its best inoculum density.
Table 1 cell-seeding-density
| Gradient | 1 | 2 | 3 | 4 | 5 | 6 |
| Cell/mL | 2.0×10 5 | 3.5×10 5 | 5.0×10 5 | 6.5×10 5 | 8.0×10 5 | 9.5×10 5 |
Under six kinds of different cell-seeding-density, chicken intestinal epithelial cell original cuiture 14 days, observation of cell growing state.The growth curve of cell as shown in Figure 1.Can obviously be found out by figure, the 4th group of cell growth state is very good, and cell count is significantly higher than other five groups of cells, obviously can find out latent period, logarithmic phase, stationary phase and apoptosis phase.Within 1-3 days, be wherein latent period, though cell count has propagation change very little, and 3-7 days cells enter logarithmic phase and start continuous division growth, and 7-12 days cell count do not become stationary phase substantially, and after 12 days, the continuous aging death of cell enters the apoptosis phase.1,2 groups do not find that the cell cycle changes, and 3,5,6 groups of changes having a cell cycle, but its stationary phase is very short, have started apoptosis and constantly on a declining curve to the 10th day cell.
By analysis of cells growth curve variation tendency, 0d is the suspended state period that cell has just been inoculated, and substantially enter logarithmic phase at 3d, 7d cell, cytotostatic phase or apoptosis phase is entered from 10d, after 14d, the 4th group of cell just enters the apoptosis phase, curvilinear motion has obvious flex point, choose different vaccination density cells and change the obvious flex point time and contrast its viable count as Fig. 2, and the cell growth state of corresponding time is as Fig. 3.
The inspection of table 2 main effect
| The source of deviation | Sum of square of deviations | df | All square | F | Sig. |
| Calibration model | 1.645×10 6 | 29 | 56714.445 | 1512.236 | 0.000 |
| Cultivated days | 685086.370 | 4 | 171271.593 | 4566.791 | 0.000 |
| Inoculum density | 570312.685 | 5 | 114062.537 | 3041.367 | 0.000 |
| Cultivated days * inoculum density | 389319.852 | 20 | 19465.993 | 519.042 | 0.000 |
| Error | 9000.889 | 240 | 37.504 | ||
| Correct total variation | 1653719.796 | 269 |
Table 2 is known, different vaccination density, cultivated days and reciprocal effects thereof to the growth effect of chicken intestinal epithelial cell all extremely significantly (
p< 0.01), and as shown in Figure 2, the growth rhythm of six groups of cells is all first increase rear minimizing, change all extremely significantly (
p< 0.01).Wherein the 4th group cell count Different growth phases all pole be significantly higher than other group (
p< 0.01), reach the highest at the 10th day cell number, and its cell count is also the highest in all groups; And other group all reaches maximum the 7th day cell count, within the 10th day, cell count reduces all to some extent, wherein the 3rd group of pole be significantly higher than other four groups (
p< 0.01), 5 groups, 6 groups are taken second place, and be finally 1,2 group, Different growth phases cell count is all minimum.
Fig. 3 shows cell attachment and the proliferate situation that different vaccination density cells changes the obvious flex point time, with the 1st, 2, what the cell of the density inoculation of 3 groups observed cell under inverted microscope adherently comparatively disperses (figure A3, B3, C3), the scattered distribution of cell major part, although along with the increase of inoculum density, the number of adherent of cell also significantly increases, but along with the division growth of the increase cell of time weakens gradually, anoikis (figure A7 is just started to the 7th day cell, B7, C7), and mortality thereupon, 1st, zero scattered loose several cells (figure A14 can only be seen for 2 groups to 14 days, B14), 3rd group of cell also all most of blackening death (figure C14), and cell-seeding-density is the 4th group of attached cell observed (figure D3) comparatively first three groups showed increased, within 3rd day, attached cell mostly is the cell that growth is bright in flakes, growth conditions is obviously better compared with other groups, cell can better proliferate in a large number, from the 7th day (figure D7), 10th day (figure D10) observes cell number and reaches maximum, and cell is individual layer is evenly paved with the whole bottom of culture plate, cell senesces death (figure D14) afterwards, the growing state of this group cell is best, and its nutrient solution consumption liquid color turns yellow comparatively when changing rapidly liquid that other groups are obviously, when observation of cell inoculum density is 5, when 6 groups, although find that two groups of cell-seeding-density are maximum, but attached cell does not have to increase because of the increase of density, most cells is all suspended state on the contrary, attached cell only has small part (figure E3, F3), although attached cell has growing multiplication along with the increase of incubation time, but the cell increased is little, suspension cell also has a lot, the growth conditions observing cell (neither scheme E7 very well, F7), to attached cell a large amount of after 10 days with suspension cell is constantly dead (schemes E10, F10), to finally seeing black dead cell in blocks (figure E14, F14).
Experimental example 2: the screening of the best serum of chicken intestinal epithelial cell
Be added in nutrient solution with the foetal calf serum of different concns and chicken serum respectively, two kinds of serum are divided into A group (foetal calf serum) and B group (chicken serum), and serum are divided into 0%, 5%, 10% different concns by the growing state of observation of cell.Before adherent difference, A, B two groups is all that 10% nutrient solution is cultivated enzymic digestion respectively and to be got off cell with serum-concentration, contributes to the epithelial adherent growth of the non-enteric such as inoblast.All with the chicken intestinal epithelial cell that serum-concentration is after the nutrient solution culture purified of 5% after adherent difference, cultivate couple of days and change a not good liquor, change liquid all with the nutrient solution containing 5% serum, be more conducive to intestinal epithelial cell division growth, cell is by about 6.5 × 10
5/ mL is inoculated in 6 well culture plates, often organizes 3 repetitions, is put in 40 DEG C, 7%CO
2cultivate 14 days in incubator, m-cell count growth curve during drafting, and observation of cell growing state, analyze the growth effect of different serum to cell, and concrete cell culture fluid composition is in table 2.
Table 2 chicken embryo intestinal epithelial cell DMEM nutrient solution composition (10mL)
| Serum content | 0% | 5% | 10% |
| Serum | 0 μL | 500 μL | 1 mL |
| Dual anti- | 100 μL | 100 μL | 100 μL |
| Glutamine | 100 μL | 100 μL | 100 μL |
| Heparin sodium | 100 μL | 100 μL | 100 μL |
| Regular Insulin | 100 μL | 100 μL | 100 μL |
| Urogastron | 200 μL | 200 μL | 200 μL |
| Low sugar cell culture medium | 9.40 mL | 8.90 mL | 8.40 mL |
Chicken intestinal epithelial cell uses foetal calf serum and chicken serum original cuiture 14 days respectively, observes the growing state of cell under different serum condition.Cell growth curve as shown in Figure 4.Can obviously be found out by figure, the cell growth state of chicken serum group is better than foetal calf serum group, and two groups of Growth of Cells all experienced by latent period of cell, growth logarithmic phase, stationary phase and apoptosis phase.Two groups of latent period, cell count was substantially identical and growth change is also little all at 1-3 days; The logarithmic phase of cell is similar all at 3-7 days, the continuous division growth of cell, cell count is that logarithm increases, and obviously steep than foetal calf serum cultured cells at logarithmic phase with the chicken intestinal epithelial cell growth curve of chicken serum cultivation, along with the gap of increase by two groups of cell count of time constantly increases; There is notable difference the cytotostatic phase of two groups, chicken serum group is obviously longer than the stationary phase of foetal calf serum group, and its cell count is significantly higher than foetal calf serum group, though it changes at 7-12 days cells, but change is little, and foetal calf serum group was slow downtrending at 7-10 days, but change is very little, basicly stable; Two groups of apoptosis phases are also different, cell aging death rapidly after chicken serum group the 12nd day, and after foetal calf serum group the 10th day, cell is old and feeble apoptosis gradually.
By analyzing the growth curve variation tendency of two groups of different serum free culture system cells, suspended state period when 0d is cell inoculation, 3d is that cell has just entered logarithmic growth period, 7d is stationary phase or the apoptosis phase that cell has just terminated logarithmic growth period, 10d is cell, 14d is apoptosis period, and this five periods are two groups of cellular change obvious period.Change obvious flex point in the past few days contrast its viable count as Fig. 5 period so choose, observe its adherent and growth conditions at corresponding time cell as Fig. 6, both in conjunction with contrast two kinds of serum on the impact of cell.
As shown in Figure 5, the cell of two groups of serum free culture systems change between different number of days all extremely significantly (
p< 0.01), cell count, all along with the increase of cultivated days, is first increase the trend reduced afterwards.Compare discovery between different serum, just during inoculation, two groups of cell-seeding-density are identical, the cell count to the 3rd day chicken serum group be significantly higher than foetal calf serum group (
p< 0.05), and to the 7th, 10,14 day chicken serum group cell count all pole be significantly higher than foetal calf serum group (
p< 0.01), wherein the cell count of the 10th day chicken serum group reaches maximum, and foetal calf serum group reaches maximum the 7th day cell count.
At identical digestive ferment, inoculum density, in serum-concentration situation, cultivates chicken intestinal epithelial cell, and two kinds of dissimilar serum cell growth proliferative conditions as shown in Figure 6.The nutrient solution cultured continuously cell of contrast interpolation two kinds of serum 14 days, the growthhabit situation of continuous viewing duration cell, when just having started to inoculate, a large amount of cell mass is all contained in two groups of cell suspending liquids, also have some individual cells not of uniform size, all limpid bright (figure A, figure F).Within 3rd day, find that the cell of two kinds of serologic group has a large amount of cell attachment (figure B, figure G) although cultivate, and the growth that stretches out, the cell growth state of chicken serum group is better, many growths in flakes.When the 7th day, the chicken intestinal epithelial cell that two kinds of serum free culture systems go out has a large amount of division growths occurs, but chicken serum group cultured cells is all limpid bright, refractivity strong (figure H), and foetal calf serum group culturing process has cell detachment dead, some dead cells (figure C) be mingled with in attached cell, affect the proliferate of cell, so the obvious comparatively foetal calf serum group cultured cells upgrowth situation of chicken serum group cultured cells is good, and proliferative cell also wants many compared with the cell of foetal calf serum group.The 10th day a large amount of cell of foetal calf serum group starts to become circle and comes off (figure D), and the cell of chicken serum group can also continue proliferate, is paved with (figure I) bottom whole culture plate.When the 14th day, two groups of cells all aging death gradually, and the apoptotic cell of foetal calf serum group (figure E) is more more than chicken serum group (figure J).It is all poor compared with the cell of chicken serum group with growth conditions by figure can obviously find out in the cellular form of whole cell cultures stage foetal calf serum group.Therefore later stage adopts chicken serum to cultivate chicken intestinal epithelial cell.
Experimental example 3: the qualification of chicken intestinal epithelial cell
(1) Morphological Identification of cell: after chicken intestinal epithelial cell original cuiture starts, directly observe viable cell by inverted phase contrast microscope, main detection cell attachment growing multiplication situation, comprises cellular form and the size variation of different time.
The cell just got off from the little intestinal digestion of chicken is inoculated into Tissue Culture Plate, examines under a microscope cell as shown in Figure 7, mostly not of uniform size, and shape is circular, and bright, and wherein digesting the major part of getting off is cell mass, at 40 DEG C, and 7%CO
2cultivate within 24h, cell starts adherent, firm attached cell is many in the distribution of island shape, also has the scattered distribution of part then in flat irregular polygon and fusiformis, the adherent in flakes cell of major part is paving stone shape (figure A), and the distribution in swirling, sharpness of border, non-overlapping copies, closely be connected each other and be monolayer alignment, cultivate 48h, paving stone shape cell in blocks starts to grow to external diffusion, cell constantly increases and starts division growth, then logarithmic phase is entered, the continuous merisis propagation of cell, when cultivation was by the 7th day, cell starts slow proliferate, tend towards stability growth gradually, enter stationary phase, after arriving the 10th day, Growth of Cells converges in flakes, the growth of cellular portions overlap, large many cells are oval (figure B), close-packed arrays be pebbles sample distribution and confluent culture plate bottom, stagnating appears in Growth of Cells, Growth of Cells reaches peak value, As time goes on, cell enters decline phase, cell senesces apoptosis, darken, cell quantity starts to reduce and comes off.
(2) the immunohistochemical methods qualification of cytokeratin:
1) cover glass process: the hydrochloric acid soaked overnight of 1%, then rinsed well with tap water, distilled water cleaning subsequently 2-3 time; Then use 95% alcohol immersion 30-60min, distillation washing, puts into the sterilizing of culture dish mesohigh, dry for standby.
2) with before cover glass, soak 5min with 10% poly-lysine and take out, be beneficial to cell paster, 37 DEG C are dried, then put it to one and fill in the ware of substratum, cross and make it become humidity strip, then in the culture plate be put into, on each cultivation plate hole middle berth about 3 pieces, not overlapping.
3) place cell suspending liquid being dripped to respectively cover glass repaves full culture plate, is then put in 40 DEG C, 7%CO
2cultivate in incubator;
4) when chicken intestinal epithelial cell is cultured to cover glass confluent monolayers cell, taken out, PBS cleans 3 times (preheating), each 3-5s, and then the paraformaldehyde of 4% fixes 30min, removes paraformaldehyde, and PBS cleans 3 times, and room temperature is dried;
5) φ=3%H is added
2o
2deionized water (colourless liquid) hatches 5-10min, to remove the activity of endogenous peroxydase.PBS washes 3min × 3 time;
6) drip reagent A (blue liquid is closed with normal rabbit serum working fluid 3ml, 6ml or 15ml), room temperature closes 10-15min, gets rid of surplus liquid (or inclining), does not wash;
7) add the mouse-anti chicken Keratin sulfate monoclonal antibody that 1:150 doubly dilutes, 4 DEG C are spent the night, blank drip equivalent PBS replace primary antibodie, 4 DEG C spend the night after at 37 DEG C of rewarmings, PBS washes 3min × 3 time;
8) reagent B((yellow liquid is dripped, biotinylated goat antimouse IgG bis-anti-working fluid 3ml, 6ml or 15ml), incubated at room temperature 30min, PBS washes 3min × 3 time;
9) drip reagent C (orange liquid, horseradish enzyme labelling strepto-avidin working fluid S-A/HRP3ml, 6ml or 15ml), room temperature or 37 DEG C hatch 10-15min, and PBS washes 3min × 3 time;
10) DAB colour developing: in 1mLDAB substrate buffer solution, drip 1 (about 50 μ L) DAB concentrated solution, mix.Color development at room temperature, controls the time of reacting, is generally room temperature 2-10min under microscope, if do not changed, can continue colour developing; Distilled water wash is with termination reaction;
11) graded ethanol dehydration, 70%(1 × 3min), 80%(1 × 3min), 90%(1 × 3min), 95%(2 × 3min), 100%(2 × 3min);
12) transparent: dimethylbenzene (can not plastics be contacted), 2 × (1-2) min.
13) neutral gum is used to take mounting, last basis of microscopic observation result.
The qualification of the chicken intestinal epithelial cell of original cuiture adopts the Specific marker of monoclonal antibody identification of cell to carry out, and wherein cytokeratin is specific antigenic component in intestinal epithelial cell.Therefore, what this experiment primary antibodie was selected is that the chicken intestinal epithelial cell of a kind of mouse-anti chicken cell Keratin 18 monoclonal antibody to original cuiture carries out immunohistochemical methods qualification, two resist for biotin labeling goat anti-mouse igg, DAB dyes, result is as Fig. 8, and display, dyes brown color and be positive in cytoplasm, and negative control group cytoplasm does not develop the color, illustrate that cultured cells is mainly chicken intestinal epithelial cell.
In experimental example of the present invention, the data obtained adopts statistic software SPSS 17.0 to carry out test of significance, and carries out multiple comparisons by Duncan method.
Compare the impact that different vaccination density grows the vitro culture of chicken intestinal epithelial cell, result shows that the suitableeest inoculum density of chicken intestinal epithelial cell original cuiture is 6.5 × 10
5about/mL, cell-seeding-density is 5.0 × 10
5/ below mL is even lower makes Growth of Cells very slow, and latent period is oversize, can not reach logarithmic phase, or stationary phase very short cell just start mortality, may be that cell can not be survived and be grown in groups because cell density is too low; And cell-seeding-density is 8.0 × 10
5/ mL is unfavorable for Growth of Cells so that upper density is excessive, this may because cell culturing space limited, iuntercellular compete mutually its meta-bolites reach capacity and nutritive substance lack cell all may be caused too early enter the growth-inhibiting phase, cell starts to come off in a large number death
[79].So the too high meeting of inoculum density causes the waste of cell, reduce cell attachment growth rate.Therefore, under same culture conditions, the division growth impact of different vaccination density on chicken intestinal epithelial cell is also different, and suitable inoculum density helps lend some impetus to the propagation of chicken intestinal epithelial cell, and further illustrating inoculum density is one of important factor of growth and proliferation of cell.
The present invention have studied foetal calf serum and chicken serum to the effect of chicken intestinal epithelial cell original cuiture, relatively under two kinds of serum condition, the growth curve chart of cell and aspect graph find, the cell growing survival in the DMEM nutrient solution adding chicken serum is obviously better than adding the chicken intestinal epithelial cell of surviving in the DMEM nutrient solution of foetal calf serum, illustrates that chicken serum more may help lend some impetus to the growing multiplication of chicken intestinal epithelial cell.Also studies have found that, serum can show good proliferate ability for the cell that Species origin is identical or close.The researchs such as willow is blue or green also find that different types of serum is relevant to the sibship of the donor of serum and target cell to the degree of injury of cell, sibship is far away, the damage of serum to cell is larger, homologous serum does not then have obvious effect to cell injury, and the result of this test is consistent with the research of forefathers.So the serum added during culturing cell preferably uses sibship identical or close.
The present invention adopts thermophilic bacteria protein enzyme digestion to be separated by chicken intestinal epithelial cell, enzyme digestion can eliminate intercellular interaction, effect particularly between intestinal epithelial cells and inoblast, also little to the damage of cell, the separation perfecting fine hair crypts unit is the important prerequisite guaranteeing intestinal epithelial cells Isolation and culture.The cell digested is inoculated in the nutritive ingredient (serum containing multiple short chicken intestinal epithelial cell growth by the present invention, glutamine, Regular Insulin, heparin sodium, dual anti-etc.) DMEM low sugar nutrient solution in, and the ratio of nutritive ingredient is the optimum formula in earlier stage filtered out through test, its GLN contributes to the division growth of intestinal epithelial cells and the normal operation of Function of intestinal mucosa, Regular Insulin can stimulate the differentiation of intestinal epithelial cells, and the growing multiplication of Urogastron to intestinal epithelial cells has promoter action, and the growth of heparin sodium to smooth muscle cell has restraining effect, promoter action is played to the growing multiplication of intestinal epithelial cells.The object of purifying intestinal epithelial cells is reached again according to intestinal epithelial cell and the difference of inoblast adherent time.Finally be inoculated in nutrient solution with the suitableeest inoculum density, be put in 40 DEG C, 7%CO
2cell cultures is carried out in incubator.The present invention successfully establishes the primary isolation cultivation method of chicken intestinal epithelial cell.
Claims (3)
1. the separation of a chicken intestinal epithelial cell and primary culture method, take out chicken embryo small intestine, cleaning small intestine removes mesentery, with proteolytic enzyme, small intestine is digested, obtain chicken intestinal epithelial cell, cell is carried out adherent difference and cultivate, remove adherent heteroproteose cell, collect intestinal epithelial cell, it is characterized in that: concrete steps are:
(1) take out chicken embryo small intestine: get the chicken embryo of hatching 15 days, chicken embryo small intestine is taken out in aseptic technique;
(2) clean small intestine and remove mesentery: the small intestine scavenging solution I of taking-up cleans repeatedly, for subsequent use after using scavenging solution II cleaning after small intestine being shredded into tissue block after limpid to supernatant liquor with scavenging solution II cleaning after removing mesentery;
(3) with proteolytic enzyme, small intestine is digested: adopt thermolysin Digestive system 37 DEG C, 80r/min vibration digestion 120min, piping and druming enzymic digestion liquid 2-3min, the PBS of 37 DEG C dilutes enzymic digestion liquid, draw the suspension after dilution, continue to dilute enzymic digestion liquid suspension cell agglomerate with PBS, draw the suspension of dilution until Digestive system is limpid; By centrifugal for suspension 1000r/min 5min, stay throw out;
(4) cell is carried out adherent difference to cultivate, remove adherent heteroproteose cell: the substratum of suspended centrifugal gained precipitation containing 10% chicken serum suspends piping and druming evenly, is inoculated into by cell suspending liquid in culturing bottle, 40 DEG C, 7%CO
2cultivate 70min in incubator, remove adherent heteroproteose cell;
(5) intestinal epithelial cell is collected: take out culturing bottle, piping and druming culturing bottle 2-3 time is by just adherent intestinal epithelial cell agglomerate Eddy diffusion, by centrifugal for cell suspending liquid 1000r/min 5min, supernatant discarded, precipitation first suspends piping and druming evenly with the DMEM containing 5% chicken serum, with trypan blue diluting cells suspension, for subsequent use after the cell mass number contaminated without indigo plant with blood counting chamber meter cell;
(6) chicken intestinal epithelial cell original cuiture: chicken intestinal epithelial cell is 6.5 × 10 according to inoculum density
5/ mL is inoculated in primary chicken intestinal epithelial cell nutrient solution, 40 DEG C, 7%CO
2cultivate in incubator;
Described scavenging solution I is that 38.8mLPBS+1.2mL is dual anti-, and described scavenging solution II is that 39.8mLPBS+0.2mL is dual anti-.
2. the separation of a kind of chicken intestinal epithelial cell according to claim 1 and primary culture method, it is characterized in that: described thermolysin Digestive system is: 25mg thermolysin is dissolved in the Hepes damping fluid of 100mL and stirs to dissolving completely, adjust pH to 7.4, filtration sterilization ,-20 DEG C of preservations; Hepes damping fluid is: 0.5g/LKCl+8.307g/LNaCl+2.38g/LHepes+0.111g/LCaCl2+0.018g/LNaOH, adjust ph to 7.4, filtration sterilization ,-20 DEG C of preservations.
3. the separation of a kind of chicken intestinal epithelial cell according to claim 1 and primary culture method, is characterized in that: described primary chicken intestinal epithelial cell nutrient solution is: dual anti-+ 400 μ L Regular Insulin+2mL serum of 40mL liquid low-sugar type DMEM+800 μ L Urogastron+400 μ LL-glutamine+400 μ L heparin sodium+400 μ L; The preparation method of chicken serum is: the chicken of getting fasting 12h carries out Culling heart blood, by the hematology aliquot collected to centrifuge tube; The centrifuge tube containing blood is put in 1h in 37 DEG C of water-baths; After separating out part serum, be placed in 4 DEG C of refrigerator overnight; After serum is separated out naturally, 4 DEG C, 3000r/min, centrifugal 10min, draw serum in centrifuge tube with syringe, discard insolubles; By ， Yu – 80 DEG C preservation after serum packing.
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| CN104818238A (en) * | 2015-03-26 | 2015-08-05 | 四川农业大学 | Isolated culture method for chicken enterocyte |
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| CN114891722A (en) * | 2022-05-13 | 2022-08-12 | 广东省农业科学院动物卫生研究所 | Avian intestinal epithelial cells and preparation method and application thereof |
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