CN102732484B - Method for establishing human nasopharyngeal carcinoma tumor stem cell line - Google Patents
Method for establishing human nasopharyngeal carcinoma tumor stem cell line Download PDFInfo
- Publication number
- CN102732484B CN102732484B CN201210238190.1A CN201210238190A CN102732484B CN 102732484 B CN102732484 B CN 102732484B CN 201210238190 A CN201210238190 A CN 201210238190A CN 102732484 B CN102732484 B CN 102732484B
- Authority
- CN
- China
- Prior art keywords
- cell
- stem cell
- nasopharyngeal carcinoma
- cne
- single cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for establishing a human nasopharyngeal carcinoma tumor stem cell line. The method comprises the following steps: (1) obtaining human nasopharyngeal carcinoma cells in the single cell state; (2) adding the human nasopharyngeal carcinoma cells in the single cell state, obtained in the step (1), to a serum-free stem cell culture medium containing epidermal growth factors and basic fibroblast growth factors to obtain single cell suspension; (3) adjusting the concentration of the single cell suspension in the step (2) to 900-1100 single cells/ml; (4) inoculating 1ml of the single cell suspension obtained in the step (3) into single pores on a cell culture six-pore plate and then adding the equivoluminal stem cell culture medium for culture for 72-96 hours; and (5) collecting the cells obtained in the step (4), carrying out trypsinization till the single cell and carrying out subculture. The method provided by the invention is simple and convenient to operate; and the obtained nasopharyngeal carcinoma stem cell line has obvious stem cell characteristics.
Description
Technical field
The present invention relates to biology and medical field, particularly, the present invention relates to a kind of human nasopharyngeal carcinoma tumor stem cell line establishment method.
Background technology
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) refers to the epithelium on cavum nasopharyngeum surface or the epithelial malignancy of nasopharynx crypts epithelium generation.Nasopharyngeal carcinoma morbidity has region feature, China is the country that nasopharyngeal carcinoma sickness rate is the highest in the world, and South China, especially Guangdong and such as the ground such as Guangxi, Hong Kong, neighborhood thereof are high regions of disease, sickness rate is higher more than 100 times than other most countries area, therefore also has the title of " canton tumor ".The pathogeny of current nasopharyngeal carcinoma is still not clear, and needs to be furtherd investigate further.
Stem cell (stem cell) is a kind of specific function cell being present in animal and human, has multiple differentiation capability and self-renewal capacity.Stem cell biology characteristic comprises: (1) totipotency or multifunctionality, and stem cell has the potential being divided into broad variety cell; (2) self-renewal capacity, stem cell, once be formed, all has self-renewal capacity throughout one's life at body, carries out self and produces differentiated progenitor cells, maintain the stability of body tissue organ by heterogeneity division; (3) high proliferative capacity, has important effect for maintenance body normal function.
Tumor stem cell (tumor stem cells, TSC), also referred to as cancer stem cell, for a kind of stem cell of specific type, TSC is because under the effect of various tumorigenesis factor, lose the normal regulation to its growth at gene level, and show the features such as infinite multiplication, transfer, heterogeneity.Tumor stem cell and stem cell have much similar feature: (1) all has the feature of infinite multiplication and differentiation; (2) there is the signal transduction path of similar adjustment self; (3) all there is different phenotype, heterogeneous; (4) all there is telomerase activation, various different tissue can be transferred to and have similar going back to the nest and route of metastasis.Certainly, TSC also has the feature being different from stem cell: the negative feedback mechanism of (1) self signal transduction pathway is destroyed; (2) differentiation and maturation ability is lacked; (3) tend to accumulate the mistake copied, and stem cell can prevent from the development etc. of misreplication.
Start with from tumor stem cell angle, for nasopharyngeal carcinoma pathogeny, diagnoses and treatment scheme etc. all can bring positive pushing effect.And at present for the foundation of nasopharyngeal carcinoma tumor stem cell line and correlation theory research actually rare.
Summary of the invention
For the problem that current nasopharyngeal carcinoma sickness rate is high, the invention provides a kind of method setting up human nasopharyngeal carcinoma tumor stem cell line, to play promotion and promoter action to the diagnoses and treatment of nasopharyngeal carcinoma.
Object of the present invention is achieved by following technique means:
An establishment method for human nasopharyngeal carcinoma tumor stem cell line, comprises the following steps:
(1) KB cell of unicellular is obtained;
(2) KB cell of unicellular step (1) obtained adds in the serum-free stem cell media containing Urogastron and Prostatropin, obtains single cell suspension;
(3) the single cell suspension concentration in step (2) is adjusted to 900 ~ 1100/ml;
(4) single cell suspension that step (3) obtains is inoculated in Tissue Culture Plate, then adds equal-volume stem cell media and carry out cultivation 72 ~ 96h;
(5) collect step (4) gained cell, trysinization is to individual cells and carry out Secondary Culture.
Nasopharyngeal carcinoma stem cell line establishment method provided by the invention, easy and simple to handle, the dry cancerous cell line of the nasopharynx obtained has stronger clonality, there is stronger drug tolerance, higher dryness factor expression amount, and have stronger tumorigenesis ability to nude mice, can be used for the pathogeny of nasopharyngeal carcinoma stem cell and the research of diagnoses and treatment aspect.
Accompanying drawing explanation
Fig. 1 is the ball rate of formation using different nasopharyngeal carcinoma cell CNE-2, SUNE-1, HONE-1 to obtain in the embodiment of the present invention;
Fig. 2 shows the cellular form of nasopharyngeal carcinoma cell CNE-2 and the CNE-2s used in the embodiment of the present invention;
Fig. 3 is that the cloning efficiency of nasopharyngeal carcinoma cell CNE-2 and CNE-2s in the embodiment of the present invention compares;
Fig. 4 is that in the embodiment of the present invention, the tolerance of nasopharyngeal carcinoma cell CNE-2 and CNE-2s to gemcitabine compares;
Fig. 5 is the RT-PCR detected result that in the embodiment of the present invention, nasopharyngeal carcinoma cell CNE-2 and CNE-2s expresses the dryness factor, and wherein in histogram, left side is CNE-2, and right side is CNE-2s;
Fig. 6 be in the embodiment of the present invention nasopharyngeal carcinoma cell CNE-2 and CNE-2s for the comparison of nude mice tumorigenesis ability.
Embodiment
By specific embodiment, the present invention is described in detail below with reference to accompanying drawing, embodiment described herein should be understood only for explaining object of the present invention but not being limited.
The invention provides a kind of establishment method of human nasopharyngeal carcinoma tumor stem cell line, comprise the following steps:
(1) KB cell of unicellular is obtained;
(2) KB cell of unicellular step (1) obtained adds in the serum-free stem cell media containing Urogastron and Prostatropin, obtains single cell suspension;
(3) the single cell suspension concentration in step (2) is adjusted to 900 ~ 1100/ml;
(4) single cell suspension that step (3) obtains is inoculated in Tissue Culture Plate, then adds equal-volume stem cell media and carry out cultivation 72 ~ 96h;
(5) collect step (4) gained cell, trysinization is to individual cells and carry out Secondary Culture.
The KB cell used in the embodiment of the present invention is preferably the cell of logarithmic phase, logarithmic phase is that phalangeal cell have passed through the of short duration adaptive phase under culture environment, and then the period of fast growth, this in period nutritional sufficiency, cell is in optimum growh state.Choose in the cultivation of the embodiment of the present invention and in Testing and appraisal afterwards, all choose the cell of logarithmic phase.
More preferably, the nasopharyngeal carcinoma cell used in the embodiment of the present invention is CNE-2, SUNE-1 or HONE-1, is more preferably CNE-2.Wherein CNE-2, SUNE-1 or HONE-1 are PD nasopharyngeal carcinoma cell, the nasopharyngeal carcinoma cell that namely grade malignancy is higher, and the deterioration degree of CNE-2 is the highest in these three kinds of nasopharyngeal carcinoma cells.
Preferably, the serum-free stem cell media used in embodiment of the present invention tumor stem cell screening process is DMEM/F12(Dulbecco's Modified Eagle's Medium/ Nutrient Mixture F12).This DMEM/F12 substratum is mixed by the volume ratio of 1:1 by DMEM and F12, and this substratum can ideally sustenticular cell growth in a lot of serum free culture system.This substratum can be buied from market.
Preferably, containing Urogastron (EGF) and Prostatropin (bFGF) in the serum-free stem cell media used in the embodiment of the present invention.Urogastron (EGF) is a kind of micromolecule polypeptide be extensively present in people or other animal body, denier can intense stimulus Growth of Cells, suppress the appearance of aging gene, delay epidermic cell aging.Urogastron can activate multiple downstream signal path, and produce various biological effect, ras-raf-MEK-erk/MAPK approach is relevant with the activation of propagation, and P13K-PKC-IKK approach is relevant with the enhancing of cell mobility.Prostatropin (bFGF) is that molecular weight is 16 ~ 18.5 KD containing 155 amino acid whose mitogenetic cationic polypeptides.The biological action of bFGF is extremely extensive, it in vascularization, promote wound healing and tissue repair, promote to play a very important role in tissue regeneration and nervous tissue growth and development process.In the serum-free stem cell media used in the embodiment of the present invention, both concentration is 10 ~ 30ng/ml, and most preferred concentration is 20ng/ml.
Preferably, in embodiment of the present invention step (3), adjustment cell concn is counted by blood counting chamber, and after adjustment, cell concn is preferably 1000/ml.In cell cultivation process, after the too high meeting of inoculum density causes Growth of Cells for some time, growing space is reduced, and it is active that last cells contacting produces T suppression cell, is unfavorable for that cell reaches optimum regime, and the too low meeting of inoculum density causes growth cycle long, extend experimental period.
Preferably, the Tissue Culture Plate used in embodiment of the present invention step (4) is six orifice plates, and the amount of the single cell suspension added in every hole is 1 ~ 2ml/ hole, and preferred concentration is 1ml/ hole.
Preferably, the pancreas enzyme concentration used in embodiment of the present invention step (5) is 0.2 ~ 0.3%, and the most preferable concentrations of pancreatin is 0.25%.
embodiment one nasopharyngeal carcinoma ball forms the acquisition of cell
1. nasopharyngeal carcinoma cell CNE-2, SUNE-1, HONE-1 are placed in 37 DEG C, 5%CO to add dual anti-DMEM/F12 (Gibco) substratum containing 10% FBS (foetal calf serum, Gibco)
2cultivate in incubator;
2. choose nasopharyngeal carcinoma cell CNE-2, SUNE-1, HONE-1 of logarithmic phase respectively, be single cell suspension with the trysinization that quality concentration of volume percent is 0.25%, add appropriate containing blood serum medium termination trysinization effect, the centrifugal 5min of 1000rpm, abandon supernatant liquor, add the serum-free stem cell media containing Urogastron EGF and Prostatropin bFGF of 1ml, mixing, this substratum is DMEM/F12, and wherein the final concentration of EGF and bFGF is 20ng/ml;
3. cell counting: get after 20ul and 20ul trypan blue gets mixing, get cell counting count board and rush pond, and count the total cellular score of four block plaid, cell concn is: large total cellular score/4 of cell count/ml=(tetra-) 2 × 10
4, after counting, cell concn is adjusted to 1000/ml;
4. get low adhesion six hole dull and stereotyped, by single cell suspension with the inoculation of 1ml/ hole, then in every hole, add 1ml serum-free stem cell media, 37 DEG C, 5% CO
2cultivate 2 weeks in incubator, count ball under the microscope and form cell quantity;
5. collect the ball formation cell that step 4 obtains, the centrifugal 5min of 2000rpm, add 2ml phosphoric acid buffer PBS rotating wash bowl and form cell once, add 1ml 0.25% pancreatin, obtain floss, now blowing and beating with rifle head is suspension, puts into 37 DEG C, 5% CO
2continue in cell culture incubator to hatch 5min, take out and continue piping and druming several times, when basis of microscopic observation is individual cells suspension, stop digestion with containing blood serum medium, the centrifugal 5min of 2000rpm, abandons supernatant liquor, add the stem cell media suspendible cell of 1ml, and count, method of counting is with step 3.
6. the cell concn that step 5 obtains is adjusted to 1000/ml, is inoculated in low-adhesion six orifice plate in 37 DEG C with 2ml/ hole, 5% CO
2cultivate 14 days in incubator, count each hole ball under ordinary optical microscope and form cell quantity.As shown in Figure 1, the ball obtained forms cell and is labeled as CNE-2s, SUNE-1s, HONE-1s respectively result, and CNE2 and CNE-2s cellular form is (all the other two kinds of comparison diagrams do not show) as shown in Figure 2.
Result: as shown in Figure 1, cell is formed containing the most a high proportion of ball in nasopharyngeal carcinoma cell CNE-2, be followed successively by HONE-1 and SUNE-1 afterwards, therefore will sub-elect nasopharyngeal carcinoma ball in the embodiment of the present invention and form cell CNE-2s from nasopharyngeal carcinoma cell CNE-2, HONE-1 and SUNE-1, and be contrast with CNE-2, CNE-2s is carried out to the detection of tumor stem cell indices, experimental procedure and accordingly result are shown in embodiment two to embodiment five.
embodiment two nasopharyngeal carcinoma ball forms Cell clonality and detects
1. prepare 1.2% and 0.6% agar-agar soln, autoclaving, naturally cools to about 45 DEG C.
2. by containing PBS(0.01M) and the DMEM/F12 substratum of dual anti-(working concentration of penicillin is 100U/ml, and the working concentration of Streptomycin sulphate is 0.1mg/ml) put into 37 DEG C of water baths, temperature bath is to 37 DEG C;
3. nasopharyngeal carcinoma ball formation in vegetative period cell nasopharyngeal carcinoma of taking the logarithm respectively ball forms cell CNE-2s, HONE-1s and SUNE-1s, and be used as nasopharyngeal carcinoma cell CNE-2, HONE-1 and SUNE-1 of reference, also blow and beat gently with 0.25% tryptic digestion respectively, make it to become unicellular, do cell counting, with DMEM/F12 substratum adjustment cell density to 1 × 10 containing 20% foetal calf serum
6cell/mL.Then making gradient doubling dilution is 10
4/ mL;
4. in 1:1 ratio make 1.2% agarose and 2 × DMEM substratum (containing 2 × microbiotic and 20% foetal calf serum) mixing after, inject in six hole flat boards with 2mL/ hole mixed solution, cooled and solidified, puts 37 DEG C, 5% CO
2for subsequent use in incubator.
5. in 1:1 ratio, the agarose of 0.6% and 2 × DMEM substratum are mixed 4.2mL in sterile test tube, then in pipe, add obtain in 0.3mL step 3 10 respectively
4/ mL cell suspension, fully mixes, respectively by suspension to form two agar layer in the six hole flat boards obtained in about 1000/hole implantation step 4, after top-layer agar solidifies, insert 37 DEG C of 5%CO
2cultivate 21 days in incubator;
6. under plate being placed on inverted microscope, observation of cell clone number.Be calculated as follows cloning efficiency, experimental result gets three laboratory mean values.
Cloning efficiency=(Clone formation number/inoculating cell number) × 100%
Result: compared with CNE-2, the CNE-2s that embodiment one obtains has stronger clonality, as shown in Figure 3.
embodiment three nasopharyngeal carcinoma ball forms the resistance of cells against neoplastic medicine gemcitabine
1. prepare 100 μm of ol/mL Jixitabin solutions:
Gemcitabine molecular weight is 299.66, i.e. 29.966 mg/mL=0.1mol/L, and therefore take 0.2997g gemcitabine and be dissolved in 10ml DMEM/F12 substratum, filtration sterilization, 4 DEG C save backup;
2. collect logarithmic phase nasopharyngeal carcinoma ball respectively and form cell CNE-2s, HONE-1s and SUNE-1s, and be used as nasopharyngeal carcinoma cell CNE-2, HONE-1 and SUNE-1 of reference, and digestion is single cell suspension, to cell counting, cell concn, with step 3 in embodiment one, is adjusted to 3 × 10 by counting step
4/ mL;
3. MTT detects two class cells to the susceptibility of gemcitabine:
(1) take the logarithm nasopharyngeal carcinoma ball formation in vegetative period cell CNE-2s, HONE-1s and SUNE-1s, and nasopharyngeal carcinoma cell CNE-2, HONE-1 and SUNE-1, each 100ul adds 96 well culture plates, add following concentration gemcitabine 100 μ l:0.1mmol/L, 0.2 mmol/L, 0.39 mmol/L, 0.78 mmol/L, 1.56 mmol/L, 3.13 mmol/L, 6.25 mmol/L, 12.5 mmol/L, 25 mmol/L, 50 mmol/L respectively, 37 DEG C, 5% CO
2, hatch 24 ~ 72h;
(2) add 20 μ l MTT, continue to hatch 4h, the centrifugal 5min of 1000rpm, abandons supernatant liquor, adds 150 μ l DMSO, shaking table mixing 10min, and microplate reader measures absorbancy, to check cell survival rate;
Result: compared with CNE-2, the CNE-2s that embodiment one obtains has stronger gemcitabine drug tolerance, as shown in Figure 4.
embodiment four RT-PCR method detects dryness factor expression
1. according to the form below 1 carries out design of primers
Table 1
2. Total RNAs extraction (TRIZOL, Invitrogen)
(1) process of attached cell CNE-2, HONE-1 and SUNE-1: rinse pending cell once with ice-cold PBS, directly add Trizol and carry out cracking, Trizol volume presses 10cm
2/ mL ratio adds;
Suspended culture cell CNE-2s, HONE-1s and SUNE-1s process: collecting cell ball suspension, centrifugal segregation cell culture fluid, and then clean once with ice-cold PBS, directly add Trizol cracking;
(2), after adding Trizol, room temperature places 5min, makes its abundant cracking;
(3) 4 DEG C of centrifugal 15min of 12000rpm, abandon precipitation, and this step is in order to remove some undissolved materials as epicyte, high-molecular-weight DNA, polysaccharide etc.;
(4) add chloroform by 200 μ l chloroform/ml Trizol, oscillator vibrates mixing 15s, room temperature is placed 3min and to be vibrated 15s, 4 DEG C of centrifugal 15min of 12000rpm again;
(5) draw upper strata aqueous phase, in another centrifuge tube, add Virahol mixing by 0.5ml Virahol/ml Trizol, room temperature places 5 ~ 10min, 4 DEG C of centrifugal 10min of 12000rpm, and at the bottom of pipe, visible white glue sample precipitation and RNA, abandon supernatant;
(6) add 75% ethanol by 1ml 75% ethanol/ml Trizol, blow and beat with rifle head the precipitation that suspends several times, 4 DEG C of centrifugal 5min of 7500rpm;
(7) repeating step (6) secondary, sucks supernatant liquor, in super clean bench, blows 15min, to remove ethanol, then adds 200ul without RNA enzyme water dissolution RNA.
3. mRNA extracts (Oligotex mRNA Mini Kit (12), QIAGEN, Cat.No. 70022, Lot.No.130188371)
(1) add the OBB be consistent with total serum IgE, Oligotex Suspension and Rnase-free Water, then with rifle head piping and druming mixing, hatch 3min for 70 DEG C and destroy RNA dimer;
Hatch 10min for (2) 20 DEG C ~ 30 DEG C, the poly A tract complementation of Oligo dT30 and the mRNA of Oligotex particle is combined, and the centrifugal 2min of 18000rpm, to precipitate Oligotex:mRNA mixture, carefully sucks supernatant liquor;
(3) 400 μ l or the resuspended Oligotex:mRNA mixture of 600l OW2 damping fluid is got, then the small-sized centrifuge tube of small spin column(containing 1.5ml centrifuge tube is moved on to) or large spin column(large centrifugal pipe) in, the centrifugal 1min of 18000rpm;
(4) by the spin column(centrifuge tube of step (3)) be installed in another 1.5ml Rnase-free centrifuge tube, add 400ul or 600ul OW2 damping fluid, the centrifugal 1min of 18000rpm, abandons the liquid passed through;
(5) by the spin column(centrifuge tube of step (4)) be installed in another 1.5ml Rnase-free centrifuge tube, add 20 ~ 100ul, 70 DEG C of OEB damping fluids, blow and beat 3 ~ 4 times, the centrifugal 1min of 18000rpm, obtains mRNA.
4. RT-PCR (QIAGENOnestepRT-PCRKit, Cat.No.70022, Lot. No .13 0188371), first utilizes QIAGEN Onestep RT-PCR Kit and Q-Solution method reverse transcription and expands object mRNA.
PCR reaction system is:
Taq enzyme 2 μ l
2 x Buffer 25μl
Upstream primer 1 μ l
Downstream primer 1 μ l
MRNA solution 1 μ l
RNase Free dH
2O 20μl
Cumulative volume 50 μ l
Reaction conditions is:
50℃：2min；
94℃：2min；
95 DEG C: 30S, annealing temperature (corresponding to Tm value in table 1): 30S, 72 DEG C: 2min, 30 circulations
5. agarose gel electrophoresis surveys amplified fragments abundance
(1) electrophoretic buffer 5 × TBE(1L is prepared)
Tris alkali 54g
Boric acid 27.5g
EDTANa2 4.6g
Above-mentioned substance is dissolved in 1L ultrapure water;
(2) the loading buffer of preparation containing fluorescence dye
6 × loading buffer 700ul
10000×SYBR GreenⅠ 1ul
DMSO 99ul
After above-mentioned substance is mixed, after adding the mixing of 3ul said mixture with 5ulDNA sample, loading;
(3) be 0.5 by 5 × TBE dilution × namely add 900ul ultrapure water with 100ul 5 × TBE, adjust pH value to be 8.0 after mixing;
(4) 1% sepharose is prepared
A) take in volumetric flask that 0.4g agarose adds containing 40mL 0.5 × TBE;
B) microwave oven is put into, moderate heat heating 3min;
C) take out, room temperature is cooled to about 60 DEG C;
D) agarose solution is poured into be inserted with in the electrophoresis support of comb;
E) after gel condensation, carefully comb is extracted;
F) will the electrophoresis support of gel be had to put into the electrophoresis chamber that electrophoretic buffer is housed, electrophoretic buffer should not have gel;
G), after the 6 × loading buffer containing SYBR Green I and amplified production being mixed with 1:5, add in aperture;
H) 80V, electrophoresis 35min;
I) uv analyzer assay products abundance.
Result: compared with CNE-2, the CNE-2s that the embodiment of the present invention one obtains has the expression of the dryness factor of the higher level of mRNA level in-site, as shown in Figure 5.
tumorigenesis capability analysis in embodiment five BALB/C nude mouse
1. cell cultures
Need cell total: injection number of cells gradient is 10
7~ 10
2cell, totally 6 gradient groups, often group needs injection 5 × 100 μ l, and often kind of total cellular score of needs is 5.6 × 10
7cell.CNE-2, HONE-1 and SUNE-1 use 75 cm
2culturing bottle is cultivated, and support completely, every bottle of cell count is about 2 × 10
7cell, namely often kind of cell needs to support full 4 bottles; CNE-2s, HONE-1s and SUNE-1s use the culture dish of 100 mm to cultivate, and support completely, every dish cell count is about 1.5 × 10
7cell, namely often kind of cell needs to support full 6 dish.
2. cell packing
(1) packing of CNE-2s, HONE-1s and SUNE-1s
A () digests
CNE-2s, HONE-1s and SUNE-1s cell is transferred to 15 ml centrifuge tubes from low adhesion culture dish, centrifugal 5 min of 1,000 r/min, abandon supernatant liquor, add appropriate (1.5 ~ 2 ml/ dish) 0.05% trypsinase-EDTA, merge 2 or 3 tube cells, piping and druming for several times, is put 37 ° of C incubators, is hatched 3 min, piping and druming for several times, put 37 ° of C incubators, hatch 3 min, 1, centrifugal 5 min of 000 r/min, abandon supernatant liquor.
B () counts
Step is shown in embodiment one step 3.
(c) packing
By centrifugal 5 min of centrifuge tube 1,000 r/min containing cell suspension, abandon supernatant liquor, add the blank cultures of calculated amount, mixing, obtains 1 × 10
8the cell suspension of/ml, reaffirms viable cell concentrations as stated above.Cell suspension is dispensed in the centrifuge tube of 62 ml, often props up 100 μ l, obtain 1 × 10
7the cell suspension of cell gradient, label; One adds 900 μ l blank cultures wherein, and mixing, is dispensed in the centrifuge tube of 62 ml, often props up 100 μ l, obtain 1 × 10
6the cell suspension of cell gradient, label, by upper repetitive operation 4 times, the cell suspension of preparation 6 gradients, label sees the following form 2:
Table 2 different concns cell label situation
| 10 7 | 10 6 | 10 5 | 10 4 | 10 3 | 10 2 | |
| CNE-2s | S7 | S6 | S5 | S4 | S3 | S2 |
| CNE-2 | E7 | E6 | E5 | E4 | E3 | E2 |
(2) packing of CNE-2, HONE-1 and SUNE-1
A () digests
Abandon the nutrient solution in most CNE-2 culturing bottle, wash culturing bottle 2 times with appropriate PBS, add appropriate (1 ~ 1.5 ml/25cm
2) 0.05% trypsinase-EDTA, put 37 ° of C incubators, hatch 12 min, wherein blow and beat for several times, observation of cell digestion situation, when coming off to most cells, add appropriate (1 ~ 1.5 ml/25cm2) perfect medium, stop digestion, piping and druming repeatedly, become single cell suspension, be transferred to 15 ml centrifuge tubes.
B () counting operation step is the same.
C () packaging operation step is the same.
3. inoculate cancer cells
(1) 30 BALB/c nude mices (BALB/c nude mice, BALB-nu) are divided into 6 groups at random, often organize 5, often organize BALB-nu and divide and support in 2 cages, adopt and cut ear tag note, number as shown in table 3:
Table 3 nude mice is numbered
| Numbering | 1 | 2 | 3 | 4 | 5 |
| By the ear cut | Right 1 | Right 2 | Left 1 | Left 2 | Nothing |
(2) cell suspension 100 μ l in numbered 2 ml centrifuge tubes is extracted in injection, the nearly back leg both sides, back of subcutaneous injection (sc) correspondence grouping BALB-nu, left side is CNE-2, HONE-1 and SUNE-1, and right side is CNE-2s, HONE-1s and SUNE-1s.
4. become knurl subsequent experimental
Record tumor growth situation, while feeding animals, is observed hypodermic changing conditions near injection point, is noticed the formation of tumor tissues; The major diameter of the record body weight of BALB/c-nu and tumour (major axis, a) and minor axis (minor axis, b), calculate gross tumor volume, the comparative result of CNE-2 and CNE-2s is as shown in Figure 6A;
When with it nude mice, the major diameter of inoculated tumour reaches 15 mm, use cervical dislocation put to death and take pictures to nude mice, the comparative result of CNE-2 and CNE-2s as shown in Figure 6B;
Use eye scissors and spur tweezer blunt separation tumor tissues, it is made to be separated totally with animal epithelium, analytical balance is used to weigh tumor tissue weight, record data, the comparative result of CNE-2 and CNE-2s as shown in Figure 6 C, tumor tissue section dyeed, basis of microscopic observation is taken pictures (operation steps slightly), and the comparative result of CNE-2 and CNE-2s is as shown in Figure 6 D.
Result: compared with CNE-2, the CNE-2s that the embodiment of the present invention one obtains has tumorigenesis ability in higher body for nude mice, as shown in Figure 6, the tumor size comparable situation that the nude mice showing four groups in Fig. 6 A obtains after injection CNE-2 and CNE-2s, as seen from the figure, the tumour of injecting each group of nude mice tumor growth of CNE-2s is obviously greater than each group of nude mice of injection CNE-2; The photo of Fig. 6 B is the long nude mice photo having tumour after injection CNE-2 and CNE-2s, and as can be seen from the picture, two groups of nude mice right side tumor are obviously greater than left side; Fig. 6 C is the weight of the tumour of nude mice tumor growth after injection CNE-2 and CNE-2s, from figure, and the tumor weight that the tumor weight that injection CNE-2s obtains obtains after being significantly higher than injection CNE-2; Fig. 6 D is the photo of the tumour of nude mice tumor growth after injection CNE-2 and CNE-2s.
Claims (3)
1. an establishment method for human nasopharyngeal carcinoma tumor stem cell line, comprises the following steps:
(1) KB cell of unicellular is obtained;
(2) KB cell of unicellular step (1) obtained adds in the serum-free stem cell media containing Urogastron and Prostatropin, obtains single cell suspension;
(3) the single cell suspension concentration in step (2) is adjusted to 900 ~ 1100/ml;
(4) single cell suspension that step (3) obtains is inoculated in Tissue Culture Plate, then adds equal-volume stem cell media and carry out cultivation 72 ~ 96h;
(5) collect step (4) gained cell, trysinization is to individual cells and carry out Secondary Culture;
Wherein, the KB cell used in described step (1) is logarithmic phase KB cell;
The KB cell used in described step (1) is SUNE-1 or HONE-1;
Described serum-free stem cell media is DMEM/F12;
The described Urogastron and the serum-free stem cell media Concentrations of Epidermal Growth Factor of Prostatropin and the concentration of Prostatropin of containing is 10 ~ 30 ng/ml;
The quality concentration of volume percent of the described pancreatin in described step (5) is 0.25%.
2. the establishment method of human nasopharyngeal carcinoma tumor stem cell line according to claim 1, is characterized in that: the described Tissue Culture Plate in described step (4) is six orifice plates.
3. the establishment method of human nasopharyngeal carcinoma tumor stem cell line according to claim 2, is characterized in that: the amount of the single cell suspension added in described six orifice plates is 1 ~ 2ml/ hole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210238190.1A CN102732484B (en) | 2012-07-10 | 2012-07-10 | Method for establishing human nasopharyngeal carcinoma tumor stem cell line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210238190.1A CN102732484B (en) | 2012-07-10 | 2012-07-10 | Method for establishing human nasopharyngeal carcinoma tumor stem cell line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102732484A CN102732484A (en) | 2012-10-17 |
| CN102732484B true CN102732484B (en) | 2015-04-22 |
Family
ID=46988787
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210238190.1A Expired - Fee Related CN102732484B (en) | 2012-07-10 | 2012-07-10 | Method for establishing human nasopharyngeal carcinoma tumor stem cell line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102732484B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593328B (en) * | 2014-12-30 | 2017-11-17 | 中国人民解放军第三军医大学第一附属医院 | Application and method of the Matrigel induced tumors cell reversal for tumor stem cell |
| CN105524886A (en) * | 2015-11-05 | 2016-04-27 | 钱朝南 | Human nasopharynx cancer cell line and applications thereof |
| CN105524885A (en) * | 2015-11-05 | 2016-04-27 | 钱朝南 | Human nasopharynx cancer cell line and applications thereof |
| CN107603949B (en) * | 2017-10-31 | 2020-06-26 | 深圳市人民医院 | Stem cell culture medium and application thereof |
| CN108872603B (en) * | 2018-07-23 | 2020-05-08 | 杭州爱唯生命科技有限公司 | Method for identifying liver cancer stem cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7998674B2 (en) * | 2005-09-22 | 2011-08-16 | China Synthetic Rubber Corporation | Gene expression profiling for identification of prognostic subclasses in nasopharyngeal carcinomas |
-
2012
- 2012-07-10 CN CN201210238190.1A patent/CN102732484B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Pluripotency-associated genes in human nasopharyngeal carcinoma CNE-2 cells are reactivated by a unique epigenetic sub-microenvironment;Jun-Xia Cao et al.;《BMC Cancer》;20100225;第10卷(第68期);1-11 * |
| 人鼻咽癌CD133+干细胞的生物学特性及意义;王海丽等;《中国组织工程研究》;20120205;第16卷(第6期);1007-1010 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102732484A (en) | 2012-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102732484B (en) | Method for establishing human nasopharyngeal carcinoma tumor stem cell line | |
| CN102465113B (en) | Human hepatoma carcinoma cell line and application thereof | |
| CN103898056B (en) | Cell culture medium and the application in cultivating people's primary tumor cell thereof | |
| CN104232574A (en) | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte | |
| CN106939300A (en) | Tree shrew immortalized liver cell cell line and its construction method and application | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN101912618A (en) | Preparation method of bone mesenchymal stem cell carrying NK4 gene and application thereof | |
| CN103881971A (en) | Culture medium and culture method for culturing and/or amplifying mesenchymal stem cells | |
| CN104140968B (en) | A kind of radiotherapy targeting sensitizer and application thereof | |
| CN102409024A (en) | Construction of prostate cancer cell external infiltrate model | |
| CN104974977A (en) | Epinephelus lanceolatus kidney tissue cell line and construction method thereof | |
| CN104031884A (en) | Application of protein arginine methyltransferase 7 in cancer cell metastasis | |
| CN104745530A (en) | Human hepatocellular carcinoma cell line, and establishing method and application thereof | |
| CN103966169A (en) | Chinese people tongue squamous epithelial cell cancer cell line CTSC-1 and establishment method thereof | |
| CN105062973A (en) | HPV negative penial squamous cell carcinoma cell line carrying TP53 mutation and application thereof | |
| CN108841786A (en) | The amnion-derived mescenchymal stem cell preprocess method of people and its application | |
| CN103898043A (en) | Cell culture layer and application in culture of human primary cancer cells thereof | |
| CN105219732B (en) | A kind of immortal human liver cancer Blood vessel endothelial cell line and its preparation method and application | |
| CN104531621A (en) | Separation method of liver cancer stem cells | |
| CN104087682A (en) | Method for detecting astragaloside-induced RMMVECs gene expression profiling by utilizing gene chip | |
| CN108949974A (en) | E3 ubiquitin ligase ASB3 is preparing the application in cancer treatment drug | |
| CN108103023A (en) | People's carcinoma of the rectum primary cell and its application | |
| CN104419676A (en) | Construction of down's syndrome cell model and cell bank of down's syndrome cell by employing hTERT gene recombination | |
| CN102911916A (en) | Chinese tongue squamous cancer cell line (CTSC-2) and establishing method thereof | |
| CN106520695A (en) | Additive containing shen-fu polysaccharide and promoting CIK cell proliferation and differentiation, culture medium, culture method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150422 Termination date: 20200710 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |