Summary of the invention
Technical problem to be solved by the invention is to provide a kind of easy-to-use inducing cell vicious transformation culture modelsMethod.
To solve the above problems, a kind of method of inducing cell vicious transformation culture model of the present invention, including withLower step:
(1) immortal human gastric epithelial GES-1 is put into the DMEM culture solution containing 10% fetal calf serum, in 37 DEG C, 5%CO2Routine culture in incubator, when cell Proliferation to logarithmic phase 1 × 105Cell after being cultivated when a/mL；
(2) the helicobacter pylori J99 of the Cag A positive is put into the brain-heart infusion medium containing 10% newborn calf serum and is trainedIt supports, is cultivated 3 ~ 4 days in 37 DEG C of incubators, bacterial multiplication to 1 × 108Supernatant is removed in centrifugation when the ratio of a/mL, is cultivatedBacterium afterwards；
(3) the bacterium after the culture is diluted to 1 × 10 using DMEM7After a/mL, by the next day kind bacterium and bacterium and cell bodyProduct is added in the cell after the culture than the mode for 100:1 to be induced, while the daily MNNG solution that 5uM is added,Helicobacter pylori (H. pylori) infection model induced after 48 weeks.
The step (1) in the DMEM culture solution containing 10% fetal calf serum refer to 50mL fetal calf serum be added to 450mLIt is prepared in DMEM culture solution.
The step (2) in the brain-heart infusion medium containing 10% newborn calf serum refer to 10mL newly calve bloodReset and add in 90mL brain-heart infusion medium and is prepared.
(2) middle centrifugal condition refers to that revolving speed is 10000 turns/min, time 5min to the step.
Compared with the prior art, the present invention has the following advantages:
1, in order to simulate human body Long Term Contact helicobacter pylori and NOCs, the present invention is thin using normal person's Gastric Mucosal Cells GES-1Born of the same parents are, the helicobacter pylori J99 and MNNG of the CagA positive are induced jointly, can not only observe the items that malignant transformation occurs for cellIndex, and can establish the cell model of helicobacter pylori and the long-term collective effect stomach lining generation malignant transformation of MNNG, it isGood experiment basis is established in subsequent gastric cancer Forming Mechanism research and curing cancer drug development.
2, it is detected through to the inducing cell after present invention modeling, it can be found that being lured jointly in helicobacter pylori with MNNGUnder the conditions of leading, the change of tumor stem cell sample is not only had occurred in Gastric Mucosal Cells, it is more likely that while Epithelial and stromal conversion has occurred(EMT).Helicobacter pylori joint MNNG infection simultaneously can make the relevant marker of stemness in Gastric Mucosal Cells, drug resistance and resistThe marker expression of apoptosis characteristic obviously increases, and makes cell while expressing EMT and CSCs characteristic.
[MTT method] adjustment cell concentration simultaneously be inoculated in 96 well culture plates, 5000 cells/wells, grouping culture 24,48,72 hours, the MTT solution (3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide of 5mg/ml was added in 4h before culture terminatesBromide)；After continuing culture 4 hours, 10%SDS solution is added and terminates reaction, 37 DEG C overnight, and automatic microplate reader measures each hole and existsAbsorbance at 370nm wavelength calculates the proliferation rate of cell.
[cell scratch experiment] cell is planted in six orifice plate culture 24 hours respectively, when cell Proliferation fusion area reaches90%, cell scratch is manufactured perpendicular to orifice plate with 200ul pipette tips, guarantees that each scratch width is consistent as far as possible, replaces free serum cultureCulture plate is put into incubator culture by liquid DMEM, and taking-up in 0,24,36,48 hour is taken pictures, according to the data analysis experiment that collects picturesAs a result.
[Transwell Matrigel] is first diluted 1% Matrigel and serum-free DMEM culture solution by 1:5, in additionRoom is put into 37 DEG C of incubators and is incubated for 4 ~ 5 hours, and DMEM of the 600ul containing 10% fetal calf serum is then added in lower room and cultivatesLiquid；Vitellophag counts 2 × 10 after serum-free medium is added4Cells/ml, upper chamber addition 400ul cell suspending liquid, 37In DEG C incubator overnight；It takes out transwell to be washed twice with PBS, 100% methanol is fixed, 1% violet staining, under microscopeObservation.
PBS is washed one time after [flow cytometry] vitellophag, with -20 degree refrigerator overnight, next day after 70% ice alcohol fixationAfter PBS washes twice, it is protected from light at room temperature with propidium iodide (PI) and RNase and is incubated for 30min, pass through flow cytometry analysis cellPeriod；Cell is resuspended in 100ul and combined and delayed by the cells rinsed with PBS of apoptosis-inducing agent cis-platinum (DDP) before and after the processing 2 timesIn fliud flushing, the Annexin V and PI marked with fluorescein isothiocynate (FITC) is protected from light and is incubated for 30min, pass through flow cytometerDetect apoptosis rate；After vitellophag after PBS washing, cell is resuspended in 100ul combination buffer, anti-CD44- is usedFITC and anti-CD24-PE antibodies dyeing is protected from light room temperature and is incubated for 15min, then washed with PBS, passes through fluidic cellInstrument meter number cell.
[clone's spherical is at experiment] counts 1000 cells, is planted in low adherency using serum-free DMEM/F12 culture solutionIn 6 orifice plates, cell factor B27, basic-FGF 20ng/ml and EGF 20ng/ml are added in culture solution.It is added every three daysSame amount of culture solution, after 7 days under the microscope observation clone spherical at size and number.
[Western blotting detects protein content] collects cell, and RIPA lysate extracts cell after PBS washingTotal protein takes the sample of phase homogenous quantities that PAGE gel is added using BCA kit measurement protein concentration after protein quantificationLoading hole in be separated by electrophoresis, protein band is transferred on PVDF film by electroporation, 5% skimmed milk power close 1 hour afterPrimary antibody, 4 degree of refrigerator overnights are incubated, next day PBS-T washs 3x5min, and it is separately added into luminous secondary antibody and is incubated for one hour, PBS-T washing3x5min sprays luminescent solution, exposes observation protein band expression in darkroom.
[statistical analysis] all data count soft using SPSS 22.0 using mean ± standard deviation (means ± SD)Part is analyzed.
[result]
(1) long-term helicobacter pylori infections joint MNNG induces GES-1 abnormal cell proliferation.
With MTT have detected processing after 24,48,72 hours group of cells proliferation rate, as shown in Figure 1: with control group(control) compare, J99 infection can be such that cell proliferation rate obviously slows down, and cell activity reduces, MNNG effect and MNNG/J99Combined induction accelerates cell Proliferation obviously, increased activity.Cell growth curve shows (such as Fig. 2): compared with the control group,The cell growth of J99 infection is suppressed；The cell of MNNG effect and MNNG/J99 combined induction is raw after proliferation second dayLong speed is accelerated.The doubling time of each group is calculated according to growth curve, is found compared with control group, when J99 infection is doubledBetween slightly shorten, it is not statistically significant, and MNNG effect, MNNG/J99 combined induction cell doubling time be obviously shortened.It finds that the cell of (such as Fig. 3): J99 infection is arrested in the G0/G1 phase by the flow cytomery cell cycle, is proliferated suppressed；The cell of MNNG effect and MNNG/J99 combined induction is arrested in the S phase, and cell Proliferation is active.To CyclinD1 and P53 albumenCarry out Western blotting(WB) detection, as shown in Figure 4: compared with the control group, it is obvious that J99 infects CyclinD1 expressionDecline, and MNNG effect and the CyclinD1 expression of MNNG/J99 combined induction are remarkably reinforced；And under three groups of P53 are expressedDrop, wherein the cell of MNNG/J99 combined induction is the most obvious.P53 both has the work for inhibiting cell Proliferation as tumor suppressor geneWith while also inhibiting the process of reprogramming of somatic cells, which body cell can be made to dedifferente and to be provided with Tumor Stem thinBorn of the same parents' feature.
Selecting cis-platinum (DDP) is inducer of apoptosis, has carried out streaming Annexin V/PI to group of cells before and after inductionDouble labelled staining method, flow cytometer (FCM) detect apoptosis rate, as the result is shown apoptosis-induced 24 hours of (such as Fig. 5): DDPAfterwards, J99 and MNNG/J99 group apoptosis rate is significantly lower than control and MNNG group；In addition, the control group after DDP effectBefore being apparently higher than induction with MNNG group apoptosis rate, and the apoptosis rate of J99 and MNNG/J99 group increases amplitude significantly lower than controlWith MNNG function cells.Protein factor WB relevant to apoptosis detection, as shown in Figure 6: compared with control group, J99 infection andMNNG/J99 combined induction cell Bcl2 expression is remarkably reinforced, and promotes the Bax of apoptosis to express and be decreased obviously.It is related with drug resistanceProtein factor WB detects (such as Fig. 7), and discovery J99 and MNNG/J99 group cell Mdr1, Mrp1 expression is remarkably reinforced.Anti-apoptotic andDrug resistance is also one of characteristic of tumor stem cell, this is also exactly the main reason for chemotherapeutics can not cure tumour.
(2) long-term helicobacter pylori infections joint MNNG induces GES-1 cell to convert to tumor stem cell sample.
The change that group of cells form after induction 1 year is observed by light microscopic, Giemsa dyeing and Electronic Speculum, such as Fig. 8 instituteShow: control cell is in monolayer growth, orderly aligned, and cell is in shuttle shape, and core is located among cytoplasm, core in oval or similar roundBenevolence is may be seen indistinctly, and cytoplasm is careful sparse, and cell size is than more uniform, and boundary is than more visible；Cell size after MNNG effect is notOne, it comes in every shape, disorganized, nucleus is deformed, and is located at cytoplasm on one side, kernel increases, and cytoplasm is uneven, thicker(visible coarse granule in it)；It is in spindle sample that J99 infected cell shape, which is elongated, and disorderly and unsystematic, multi ANN is unevenly distributedEven, core is oval, and kernel increases, and cytoplasm is thicker, it is seen that vacuole increases, and cytoplasm periphery hairiness pricked changes, cell dispersion pointCloth；MNNG/J99 combined induction cell is then not of uniform size, dispersed distribution, cell traction elongation, and leaf is thin between similar fibroblastBorn of the same parents' morphologic change, active proliferation, nuclei dyeing chromaticness concentration are in pyknosis state, and cytoplasm is uneven, and nucleus is squeezed, cytoplasmPeriphery sees that burr sample changes.Clone ball has then been carried out under conditions of low adhesion sheet serum-free medium forms experiment, such as Fig. 9It is shown: compared with control group, J99 infection, J99/MNN combined induction Cell clonality and quantity obviously higher thanControl group and MNNG group.OCT4, C-MYC, SOX2, CD44 have high expression in tumor stem cell, the detection through WB, such as Figure 10Shown: compared with control group, processed three groups of cells expression has different degrees of enhancing, wherein J99 infection, MNNG/The enhancing of J99 combined induction cell is the most obvious.Further through the cell number of Flow cytometry CD44+ and CD24+, such as Figure 11 instituteShow: compared with control group, other three groups processed cell CD44+/CD24+ cell numbers be increased significantly；And MNNG/J99The CD44+/CD24+ cell number of combined induction cell is apparently higher than J99 infection cell again.Still Tumor Stem is not thin by CD44 and CD24Cellular surface marker also expresses the ability of cell migration and DISTANT METASTASES IN.Individually being infected by the above description of test J99 can be withMake GES-1 cell that there is tumor stem cell characteristic changing, is moved when joint MNNG induction is very possible while enhancing cell invasionShifting ability, or Epithelial and stromal sample conversion (EMT) has occurred.
(3) EMT. occurs for long-term helicobacter pylori infections joint MNNG induction GES-1 cell
By the invasive ability of Transwell Germicidal efficacy cell, as shown in figure 12, MNNG effect, MNNG/J99 combined inductionThe quantity that cell passes through cell is significantly more than control group, and statistics has significant difference.Scratch experiment observation cell movesShifting ability, as shown in figure 13,12,24,36,48 hours scratch area percentage, finds compared with control group by comparing,MNNG effect, MNNG/J99 combined induction cell migration ability are remarkably reinforced in different time, but the invasion of J99 infection cell are movedShifting ability increases without apparent.Detect the WB of protein factor relevant to EMT and Wnt/ β-Catenin signal path as the result is shown(as shown in figure 14) compares with control group, and E-cad albumen is bright in MNNG effect, the expression of MNNG/J99 combined induction cellAobvious decline；The Vimentin expression of three kinds of processing mode cells is remarkably reinforced；And it is related to Wnt/ β-Catenin signal pathWnt2 albumen J99 infection, MNNG/J99 combined induction cell express enhancing, MNNG function cells are without significant change.C-The target gene of myc, CD44 as the signal path downstream, wherein the cell expression of tri- kinds of processing modes of C-myc enhances；AndCD44 expresses enhancing in the cell that J99 infects, MNNG/J99 is induced jointly, it is also the marker of tumor stem cell.
(4) cell cycle network is regulated and controled by many signal schemes, decides four kinds of entirely different knots of cell respectivelyOffice, including aging, apoptosis, proliferation, differentiation.Those are considered as the molecular product of proto-oncogene, can promote cell after mutationProliferation, and tumor suppressor gene can then inhibit cell Proliferation.CyclinD1 observed by an experiment belongs to oncogene, activation or high tableUp to can promote cell Proliferation, the generating process of tumour is participated in.P53 albumen belongs to cancer suppressor protein, inhibits cell Proliferation, helpsAging death or apoptosis occur for DNA damaged cell.When cell encounters environmental stimuli or pressure, the proliferating cycle of cell can all haveChanged, be to rest on the G0/G1 phase, still enter the S phase as early as possible, different inducements make cell make second selecting, when impairedWhen resting on the G0/G1 phase, the powerful repair function of gene can help cell ageing dead or apoptosis is without entering the S phase, and p53 is justBe assist the key factor of the process, but the loss of the factor or mutation can make cell that another selection occur, such as help atRipe body cell occurs reprogramming or dedifferentes, and is provided with the characteristic of tumor stem cell.It is well known that having infected the pylorus of CagA+Helicobacter, gastric epithelial cells will appear the change of " hummingbird sample " phenotype: cell traction elongation, dispersed distribution.In the present inventionHelicobacter pylori joint MNNG induction, not only make cell that modal change have occurred, suppression expressed by p53 in WB resultIt makes and rises in value suppressed, it is more likely that dedifferente Gastric Mucosal Cells selection and change to the direction CSCs, canceration may finally be caused.MNNG makes cell be provided with apparent proliferation activity as mature carcinogen, long-term chronic induction.When helicobacter pylori andWhen the common inducing cell of MNNG, one side MNNG promotes cell Proliferation, increases the chance of DISTANT METASTASES IN, on the other hand, carefullyBorn of the same parents are stretched like interstitial cell, arrange disorderly and unsystematic, polarity missing.Malignant tumour originating from epithelial cell, oncocyte are pastIt is past to be a lack of polarity, it is disorderly and unsystematic, inorganization, polarity missing can also make the function of epithelial cell loss adhesive connection.?In neoplastic process, the missing of cell polarity also becomes key factor, can not only cause dedifferenting, being pernicious swollen for cellThe increase of tumor cells of origin can also occur EMT, have transfer ability.So the proliferative disorder of cell, tumour stemness and EMTWith malignant growth have it is close contact, the EMT phenomenon in tumour cell includes the ginseng of many genes and signal pathWith, and these accesses have all played an active part in proliferation, differentiation and the apoptosis of cell.Combining form changes, HP and MNNG of the present inventionCommon induction makes cell that the change of tumor stem cell sample not only have occurred, while having the characteristics that EMT.
3, the present invention is simple and easy to do.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, uses MTTThe variation of 24,48,72 hours group of cells proliferation rates after method detection effect (* indicates that p < 0.05, * * indicate p < 0.01).
Fig. 2 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, uses MTTThe cell proliferating number of continuous detection 6 days, the proliferation activity of cell, the doubling time of group of cells are observed by cell growth curve(* indicates that p < 0.05, * * indicate p < 0.01).
Fig. 3 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, streamingThe characteristic distributions in group of cells period after cell instrument observation processing 24 hours.
Fig. 4 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, processingCell is collected after 24 hours, CyclinD1, P53 protein expression level are observed by WB.
Fig. 5 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, and DDP makeesFor inducer, the comparison of effect front and back group of cells apoptosis rate.
Fig. 6 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, and is witheredThe western blot image for dying relevant Bcl-2, Bax protein factor compares (* indicates that p < 0.05, * * indicate p < 0.01).
Fig. 7 is that long-term helicobacter pylori J99 of the invention infects joint MNNG induction GES-1 abnormal cell proliferation, and resistance toThe western blot image of relevant Mdr1, Mrp1 protein factor of medicine compares (* indicates that p < 0.05, * * indicate p < 0.01).
Fig. 8 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell to convert to tumor stem cell sampleMorphological changes of cell.Light microscopic observation cellular morphology changes (x200)；Observation cellular morphology changes after Giemsa dyeing(x1000)；The change (x10000) of electric microscopic observation cell ultrastructure.
Fig. 9 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell to convert to tumor stem cell sample,The group of cells gathered is planted in low adhesion sheet culture 7 days, observation clone ball formed size and quantity (* indicate p <0.05, * * indicates p < 0.01).
Figure 10 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell to turn to tumor stem cell sampleChange, collect group of cells, OCT4, C-MYC, SOX2 relevant to tumor stem cell, CD44 protein expression water are observed by WBIt is flat.
Figure 11 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell to turn to tumor stem cell sampleChange, ratio shared by CD44+/CD24+ positive cell in flow cytometry group of cells.(* expression p < 0.05, * * expression p <0.01).
Figure 12 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell that EMT, Transwell occursCell experimental result observes the invasive ability of group of cells (* indicates that p < 0.05, * * indicate p < 0.01).
Figure 13 is that the long-term helicobacter pylori infections joint MNNG of the present invention induces GES-1 cell that EMT, scratch experiment knot occursFruit observes the transfer ability of group of cells (* indicates that p < 0.05, * * indicate p < 0.01).
Figure 14 is that the long-term helicobacter pylori infections of the present invention combine MNNG and induce GES-1 cell that EMT occurs, with EMT andRelevant E-cadherin, Vimentin, Wnt2, β-Catenin of Wnt/ β-Catenin signal path, CD44, C-myc albumenThe image of factor western blotting compares (* indicates that p < 0.05, * * indicate p < 0.01).