CN102732484A

CN102732484A - Method for establishing human nasopharyngeal carcinoma tumor stem cell line

Info

Publication number: CN102732484A
Application number: CN2012102381901A
Authority: CN
Inventors: 周克元; 李涛; 李彩虹; 冀天星; 黄颖; 林碧华; 张鑫
Original assignee: Guangdong Medical University
Current assignee: Guangdong Medical University
Priority date: 2012-07-10
Filing date: 2012-07-10
Publication date: 2012-10-17
Anticipated expiration: 2032-07-10
Also published as: CN102732484B

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

A kind of human nasopharyngeal carcinoma tumor stem cell line establishment method

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 NPC) is meant the epithelium on cavum nasopharyngeum surface or the epithelial malignancy that nasopharynx crypts epithelium takes place to nasopharyngeal carcinoma.The nasopharyngeal carcinoma morbidity has the region characteristics; China is the highest country of nasopharyngeal carcinoma sickness rate in the world; And the South China; Especially ground such as Guangdong and neighborhood such as Guangxi, Hong Kong is high region of disease, and sickness rate is higher more than 100 times than other most of countries and regions, and the title of " canton tumor " is therefore also arranged.The pathogeny of nasopharyngeal carcinoma is still indeterminate at present, remains further to be furtherd investigate.

Stem cell (stem cell) is a kind of specific function cell that is present in the animal and human, has multiple differentiation capability and self ability.Stem cell biological is learned characteristic and comprised: (1) totipotency or multifunctionality, stem cell have the potential that is divided into the broad variety cell; (2) self ability, stem cell are carried out self and are produced differentiated progenitor cells through the heterogeneity division in case formation all has the self ability throughout one's life at body, keep the stability of body tissue organ; (3) the height multiplication capacity has important effect for keeping the body normal function.

(tumor stem cells TSC), is also referred to as cancer stem cell to tumor stem cell; Stem cell for a kind of specific type; TSC loses the normal regulation to its growth at gene level, and shows characteristics such as infinite multiplication, transfer, heterogeneity because under various tumorigenesis effect of factors.Tumor stem cell has a lot of similar characteristics with stem cell: (1) all has the characteristics of infinite multiplication and differentiation; (2) there is the signal transduction path of similar adjusting self; (3) all have different phenotypes, heterogeneity; (4) all have telomerase activation, can both transfer to various tissue and similar going back to the nest and route of metastasis arranged.Certainly, TSC also has the characteristics that are different from stem cell: the negative feedback mechanism of (1) self signal transduction pathway is destroyed; (2) lack the differentiation and maturation ability; (3) tend to accumulate the mistake of duplicating, and stem cell can prevent the development of misreplication etc.

Start with from the tumor stem cell angle, for the nasopharyngeal carcinoma pathogeny, diagnoses and treatment scheme etc. all can be brought positive pushing effect.And it is at present actually rare for the foundation and the correlation theory research of nasopharyngeal carcinoma tumor stem cell line.

Summary of the invention

To the high problem of present nasopharyngeal carcinoma sickness rate, the present invention provides a kind of method of setting up the human nasopharyngeal carcinoma tumor stem cell line, promotes and promoter action so that the diagnoses and treatment of nasopharyngeal carcinoma is played.

The object of the invention is achieved through following technique means:

A kind of establishment method of human nasopharyngeal carcinoma tumor stem cell line may further comprise the steps:

(1) KB cell of the unicellular state of acquisition;

The KB cell of the unicellular state that (2) step (1) is obtained adds and contains in the serum-free stem cell media of Urogastron and Prostatropin, must single cell suspension;

(3) the single cell suspension concentration in the step (2) is adjusted to 900～1100/ml;

(4) single cell suspension that step (3) is obtained is inoculated in the Tissue Culture Plate, adds the equal-volume stem cell media again and cultivates 72～96h;

(5) collect step (4) gained cell, trysinization to individual cells and the cultivation of going down to posterity.

Nasopharyngeal carcinoma stem cell line establishment method provided by the invention; Easy and simple to handle; The dried cancerous cell line of the nasopharynx that obtains has stronger clonality, has stronger drug tolerance, higher dryness factor expression amount; And nude mice there is stronger tumorigenesis ability, can be used for the research of pathogeny and the diagnoses and treatment aspect of nasopharyngeal carcinoma stem cell.

Description of drawings

The ball rate of formation of Fig. 1 for using different nasopharyngeal carcinoma cell CNE-2, SUNE-1, HONE-1 to obtain in the embodiment of the invention;

Fig. 2 has shown the nasopharyngeal carcinoma cell CNE-2 that uses in the embodiment of the invention and the cellular form of CNE-2s;

Fig. 3 be nasopharyngeal carcinoma cell CNE-2 and CNE-2s in the embodiment of the invention cloning efficiency relatively;

Fig. 4 is that nasopharyngeal carcinoma cell CNE-2 and CNE-2s be to the tolerance of gemcitabine relatively in the embodiment of the invention;

Fig. 5 is the RT-PCR detected result that nasopharyngeal carcinoma cell CNE-2 and CNE-2s express the dryness factor in the embodiment of the invention, and wherein the left side is CNE-2 in the histogram, and the right side is CNE-2s;

Fig. 6 be in the embodiment of the invention nasopharyngeal carcinoma cell CNE-2 and CNE-2s for the comparison of nude mice tumorigenesis ability.

Embodiment

Below will combine accompanying drawing the present invention to be detailed, should understand embodiment described herein only from explaining the object of the invention but not limit it through specific embodiment.

The present invention provides a kind of establishment method of human nasopharyngeal carcinoma tumor stem cell line, may further comprise the steps:

(1) KB cell of the unicellular state of acquisition;

The KB cell who uses in the embodiment of the invention is preferably the cell of logarithmic phase; Logarithmic phase is that phalangeal cell has passed through the of short duration adaptive phase under culture environment; And then quick period of growth, this in period nutritional sufficiency, cell is in the optimum growh state.Choose and the cell of logarithmic phase is all chosen in afterwards detection in identifying in the cultivation of the embodiment of the invention.

More preferably, the nasopharyngeal carcinoma cell that uses in the embodiment of the invention is CNE-2, SUNE-1 or HONE-1, more preferably CNE-2.Wherein CNE-2, SUNE-1 or HONE-1 are PD nasopharyngeal carcinoma cell, i.e. the higher nasopharyngeal carcinoma cell of grade malignancy, 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 of using in the embodiment of the 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 sustenticular cell growth ideally in a lot of serum free culture systems.This substratum can be buied from market.

Preferably, contain Urogastron (EGF) and Prostatropin (bFGF) in the serum-free stem cell media of using in the embodiment of the invention.Urogastron (EGF) is a kind of people or intravital micromolecule polypeptide of other animal of extensively being present in, denier can the intense stimulus cell growth, suppress the appearance of aging gene, delay the epidermic cell aging.Urogastron can activate multiple downstream signal path, produces the various biological effect, and the ras-raf-MEK-erk/MAPK approach is relevant with the activation of propagation, and the P13K-PKC-IKK approach is relevant with the ambulant enhancing of cell.Prostatropin (bFGF) is to contain 155 amino acid whose mitogenetic cationic polypeptides, and molecular weight is 16～18.5 KD.The biological action of bFGF is extremely extensive, and it plays crucial effect in vascularization, promotion wound healing and tissue repair, promotion tissue regeneration and nervous tissue growth and development process.Both concentration is 10～30ng/ml in the serum-free stem cell media of using in the embodiment of the invention, and most preferred concentration is 20ng/ml.

Preferably, the adjustment cell concn is counted through blood counting chamber in the embodiment of the invention step (3), and adjustment back cell concn is preferably 1000/ml.In the cell cultivation process; The too high meeting of inoculum density causes cell to reduce the growing space after growth for some time, and last cells contacting produces and suppresses cytoactive, is unfavorable for that cell reaches optimum regime; And inoculum density is crossed and low can be caused growth cycle long, has prolonged experimental period.

Preferably, the Tissue Culture Plate that uses in the embodiment of the invention step (4) is six orifice plates, and the amount of the single cell suspension that in every hole, adds is 1～2ml/ hole, and preferred concentration is the 1ml/ hole.

Preferably, the pancreas enzyme concentration that uses in the embodiment of the 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. (foetal calf serum Gibco) adds two anti-DMEM/F12 (Gibco) substratum and places 37 ℃, 5%CO to contain 10% FBS with nasopharyngeal carcinoma cell CNE-2, SUNE-1, HONE-1 ₂Cultivate in the incubator;

2. choose nasopharyngeal carcinoma cell CNE-2, SUNE-1, the HONE-1 of logarithmic phase respectively, using the mass and size percentage concentration is that 0.25% trysinization is single cell suspension, adds to contain blood serum medium in right amount and stop the trysinization effect; The centrifugal 5min of 1000rpm; Abandon supernatant, add the serum-free stem cell media that contains 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 20ul and 20ul trypan blue get mix after, get cell counting count board towards the pond, and count the TCS of four big grids, cell concn is: cell count/ml=(four a big TCS/4) 2 * 10 ⁴, behind the counting cell concn is adjusted into 1000/ml;

4. it is dull and stereotyped to get low adhesion six holes, and single cell suspension with the inoculation of 1ml/ hole, is added 1ml serum-free stem cell media, 37 ℃, 5% CO again in every hole ₂Cultivated for 2 weeks in the incubator, form cell quantity at microscopically counting ball;

5. collect the ball formation cell that step 4 obtains, the centrifugal 5min of 2000rpm adds 2ml phosphoric acid buffer PBS rotating wash bowl and forms cell once, adds 1ml 0.25% pancreatin, obtains floss, uses this moment the rifle head to blow and beat and is suspension, puts into 37 ℃, 5% CO ₂Continue to hatch 5min in the cell culture incubator, take out and continue piping and druming several times, when microscopically is viewed as the individual cells suspension; Stop digestion with containing blood serum medium, the centrifugal 5min of 2000rpm abandons supernatant; The stem cell media suspendible cell that adds 1ml, and counting, method of counting is with step 3.

6. the cell concn that step 5 is obtained is adjusted to 1000/ml, is inoculated in low-adhesion six orifice plates in 37 ℃ with the 2ml/ hole, 5% CO ₂Cultivated 14 days in the incubator, ordinary optical microscope each hole ball of counting down forms cell quantity.The result is as shown in Figure 1, and the ball that obtains forms cell and is labeled as CNE-2s, SUNE-1s, HONE-1s respectively, CNE2 and CNE-2s cellular form (all the other two kinds of comparison diagrams do not show) as shown in Figure 2.

Result: as shown in Figure 1; Contain the most a high proportion of ball among the nasopharyngeal carcinoma cell CNE-2 and form cell; Be followed successively by HONE-1 and SUNE-1 afterwards, so will from nasopharyngeal carcinoma cell CNE-2, HONE-1 and SUNE-1, sub-elect the nasopharyngeal carcinoma ball in the embodiment of the invention and form cell CNE-2s, and be contrast with CNE-2; CNE-2s is carried out the detection of tumor stem cell each item index, and experimental procedure and accordingly result are seen embodiment two to embodiment five.

Embodiment two nasopharyngeal carcinoma balls form cell clone formation ability and detect

1. prepare 1.2% and 0.6% agar-agar soln, autoclaving naturally cools to about 45 ℃.

2. the DMEM/F12 substratum that will contain PBS (0.01M) and two anti-(working concentration of penicillium mould is 100U/ml, and the working concentration of Streptomycin sulphate is 0.1mg/ml) is put into 37 ℃ of water baths, and temperature is bathed to 37 ℃;

3. nasopharyngeal carcinoma ball formation in the vegetative period cell nasopharyngeal carcinoma of taking the logarithm respectively ball forms cell CNE-2s, HONE-1s and SUNE-1s; And the nasopharyngeal carcinoma cell CNE-2, HONE-1 and the SUNE-1 that are used as reference; Respectively with 0.25% tryptic digestion and piping and druming gently; Make it to become unicellular, do cell counting, with DMEM/F12 substratum adjustment cell density to 1 * 10 that contain 20% foetal calf serum ⁶Cell/mL.Making the gradient doubling dilution then is 10 ⁴/ mL;

4. after in the 1:1 ratio 1.2% agarose and 2 * DMEM substratum (foetal calf serum that contains 2 * microbiotic and 20%) being mixed, inject in the six hole flat boards with 2mL/ hole mixed solution, cooled and solidified is put 37 ℃, 5% CO ₂Subsequent use in the incubator.

In the 1:1 ratio with 0.6% agarose and 2 * DMEM substratum mixing 4.2mL in sterile test tube, in pipe, add respectively again obtain in the 0.3mL step 3 10 ⁴/ mL cell suspension, abundant mixing, respectively with suspension to form two agar layer in the six hole flat boards that obtain in about 1000/hole implantation step 4, treat that top-layer agar solidifies after, insert 37 ℃ of 5%CO ₂Cultivated 21 days in the incubator;

6. be placed on plate under the inverted microscope observation of cell clone number.Be calculated as follows cloning efficiency, experimental result is got the empirical average value three times.

Cloning efficiency=(clone forms number/inoculating cell number) * 100%

The result: compare with CNE-2, the CNE-2s that embodiment one obtains has stronger clonality, and is as shown in Figure 3.

Embodiment three nasopharyngeal carcinoma balls form the resistance of cell to the antitumor drug gemcitabine

1. prepare 100 μ mol/mL Jixitabin solutions:

The gemcitabine molecular weight is 299.66, and promptly therefore 29.966 mg/mL=0.1mol/L takes by weighing the 0.2997g gemcitabine and be dissolved in the 10ml DMEM/F12 substratum, filtration sterilization, and 4 ℃ of preservations are subsequent use;

2. collect logarithmic phase nasopharyngeal carcinoma ball respectively and form cell CNE-2s, HONE-1s and SUNE-1s; And the nasopharyngeal carcinoma cell CNE-2, HONE-1 and the SUNE-1 that are used as reference; And digestion is single cell suspension; The pair cell counting, counting step is adjusted into 3 * 10 with step 3 among the embodiment one with cell concn ⁴/ mL;

3. MTT detects the susceptibility of two types of cells to gemcitabine:

(1) take the logarithm vegetative period nasopharyngeal carcinoma ball forms 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 ℃, 5% CO ₂, hatch 24～72h;

(2) add 20 μ l MTT, continue to hatch 4h, the centrifugal 5min of 1000rpm abandons supernatant, adds 150 μ l DMSO, shaking table mixing 10min, and ELIASA is measured absorbancy, with the inspection cell survival rate;

The result: compare with CNE-2, the CNE-2s that embodiment one obtains has stronger gemcitabine drug tolerance, and is as shown in Figure 4.

Embodiment four RT-PCR methods detect the dryness factor expression

1. according to the form below 1 carries out design of primers

Table 1

Figure 2012102381901100002DEST_PATH_IMAGE001

2. total RNA extraction (TRIZOL, Invitrogen)

(1) processing of attached cell CNE-2, HONE-1 and SUNE-1: wash pending cell once with ice-cold PBS, directly add Trizol and carry out cracking, the Trizol volume is pressed 10cm ²/ mL ratio adds;

Suspended culture cell CNE-2s, HONE-1s and SUNE-1s handle: collecting cell ball suspension, and centrifugal removal cell culture fluid, and then with ice-cold PBS cleaning once, directly add the Trizol cracking;

(2) add Trizol after, room temperature is placed 5min, makes its abundant cracking;

(3) 4 ℃ of centrifugal 15min of 12000rpm abandon deposition, and this step is in order to remove some undissolved material such as epicytes, high-molecular-weight DNA, polysaccharide etc.;

(4) adds chloroform by 200 μ l chloroform/ml Trizol, vibrator vibration mixing 15s, the room temperature placement 3min 15s that vibrates again, 4 ℃ of centrifugal 15min of 12000rpm;

(5) draw the upper strata water, to another centrifuge tube, press 0.5ml Virahol/ml Trizol and add the Virahol mixing, room temperature is placed 5～10min, 4 ℃ of centrifugal 10min of 12000rpm, and visible white size appearance deposition is RNA at the pipe end, abandons supernatant;

(6) add 75% ethanol by 1ml 75% ethanol/ml Trizol, suspend several times with the piping and druming of rifle head and precipitate 4 ℃ of centrifugal 5min of 7500rpm;

(7) repeating step (6) secondary is inhaled and is removed supernatant, in super clean bench, blows 15min, and to remove ethanol, adding 200ul does not then have RNA enzyme water dissolution RNA.

3. (Cat.No. 70022, Lot.No.130188371) for Oligotex mRNA Mini Kit (12), QIAGEN in the mRNA extraction

(1) add the OBB that is consistent with total RNA, Oligotex Suspension and Rnase-free Water with rifle head piping and druming mixing, are hatched 3min for 70 ℃ and are destroyed the RNA dimer then;

Hatch 10min for (2) 20 ℃～30 ℃, the Oligo dT30 of Oligotex particle is combined with the poly A tract of mRNA is complementary, the centrifugal 2min of 18000rpm, with deposition Oligotex:mRNA mixture, the careful suction removed supernatant;

(3) get the resuspended Oligotex:mRNA mixture of 400 μ l or 600l OW2 damping fluid; Move on in the small spin column (small-sized centrifuge tube) or large spin column (large centrifugal pipe) that contains the 1.5ml centrifuge tube the centrifugal 1min of 18000rpm then;

(4) the spin column (centrifuge tube) with step (3) is installed in another 1.5ml Rnase-free centrifuge tube, adds 400ul or 600ul OW2 damping fluid, and the centrifugal 1min of 18000rpm abandons the liquid that passes through;

(5) the spin column (centrifuge tube) with step (4) is installed in another 1.5ml Rnase-free centrifuge tube, adds 70 ℃ of OEB damping fluids of 20～100ul, blows and beats 3～4 times, and the centrifugal 1min of 18000rpm promptly gets mRNA.

4. RT-PCR (QIAGENOnestepRT-PCRKit, Cat.No.70022, Lot. No .13 0188371) at first utilizes QIAGEN Onestep RT-PCR Kit and Q-Solution method rt and expands purpose mRNA.

The 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

TV 50 μ l

Reaction conditions is:

50℃：2min；

94℃：2min；

95 ℃: 30S, annealing temperature (corresponding to Tm value in the table 1): 30S, 72 ℃: 2min, 30 circulations

5. agarose gel electrophoresis is surveyed the amplified fragments abundance

(1) preparation electrophoretic buffer 5 * TBE (1L)

Tris alkali 54g

Boric acid 27.5g

EDTANa2?4.6g

Above-mentioned substance is dissolved in the 1L ultrapure water;

(2) preparation contains kind buffer that goes up of optical dye

6 * upward appearance buffer 700ul

10000×SYBR?GreenⅠ 1ul

DMSO 99ul

Behind the above-mentioned substance mixing, add 3ul said mixture mixing with the 5ulDNA sample after, last appearance;

(3) be 0.5 * promptly to add the 900ul ultrapure water with 100ul 5 * TBE with 5 * TBE dilution, transferring pH value behind the mixing is 8.0;

(4) preparation 1% sepharose

A) taking by weighing the adding of 0.4g agarose contains in the volumetric flask of 40mL 0.5 * TBE;

B) put into microwave oven, moderate heat heating 3min;

C) take out, room temperature is cooled to about 60 ℃;

D) agarose solution is poured in the electrophoresis support that is inserted with comb;

E) treat gel cohesion after, carefully extract comb;

F) will have the electrophoresis support of gel to put into the electrophoresis chamber that electrophoretic buffer is housed, electrophoretic buffer should not have gel;

G) will contain 6 of SYBR Green I * go up appearance buffer and amplified production with the 1:5 mixing after, in the adding aperture;

H) 80V, electrophoresis 35min;

I) uv analyzer assay products abundance.

The result: compare with CNE-2, the CNE-2s that the embodiment of the invention one obtains has the expression of the dryness factor of the higher level of mRNA level, and is as shown in Figure 5.

Tumorigenesis capability analysis in the embodiment five BALB/C nude mouses

1. cell cultures

Need the cell total: injection number of cells gradient is 10 ⁷～10 ²Cell, totally 6 gradient groups, every group of needs are injected 5 * 100 μ l, and the every kind of TCS that needs is 5.6 * 10 ⁷Cell.CNE-2, HONE-1 and SUNE-1 use 75 cm ²Culturing bottle is cultivated, and after foster expiring, every bottle of cell count is about 2 * 10 ⁷Cell, promptly every kind of cell need be supported full 4 bottles; CNE-2s, HONE-1s and SUNE-1s use the culture dish of 100 mm to cultivate, and after foster expiring, every dish cell count is about 1.5 * 10 ⁷Cell, promptly every kind of cell need be supported full 6 dish.

2. cell packing

(1) packing of CNE-2s, HONE-1s and SUNE-1s

(a) digestion

CNE-2s, HONE-1s and SUNE-1s cell are transferred to 15 ml centrifuge tubes from the low culture dish that adheres to, and centrifugal 5 min of 1,000 r/min abandon supernatant; Add an amount of (1.5 ~ 2 ml/ dish) 0.05% trypsinase-EDTA, merge 2 or 3 tube cells, piping and druming is put 37 ° of C incubators for several times; Hatch 3 min, piping and druming is put 37 ° of C incubators for several times, hatches 3 min; Centrifugal 5 min of 1,000 r/min abandon supernatant.

(b) counting

Step is seen embodiment one step 3.

(c) packing

To contain centrifugal 5 min of centrifuge tube 1,000 r/min of cell suspension, and abandon supernatant, and add the blank substratum of calculated amount, mixing gets 1 * 10 ⁸The cell suspension of/ml is reaffirmed viable cell concentrations as stated above.The cell suspension branch is installed in the centrifuge tube of 62 ml, every 100 μ l gets 1 * 10 ⁷The cell suspension of cell gradient, label; To wherein one add the blank substratum of 900 μ l, mixing divides to install in the centrifuge tube of 62 ml, every 100 μ l, 1 * 10 ⁶The cell suspension of cell gradient, label, by last repetitive operation 4 times, the cell suspension of 6 gradients of preparation, 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) digestion

Abandon the nutrient solution in the most CNE-2 culturing bottle, wash culturing bottle 2 times, add an amount of (1 ~ 1.5 ml/25cm with an amount of PBS ²) 0.05% trypsinase-EDTA, put 37 ° of C incubators, hatch 12 min; Wherein piping and druming for several times, observation of cell digestion situation is during to most of cell detachment; Add an amount of (1 ~ 1.5 ml/25cm2) perfect medium, stop digestion, piping and druming repeatedly; Make it become single cell suspension, be transferred to 15 ml centrifuge tubes.

(b) the counting operation step is the same.

(c) the packaging operation step is the same.

3. inoculation cancer cells

(1) with 30 BALB/c nude mices (BALB/c nude mice BALB-nu) is divided into 6 groups at random, and 5 every group, every group of BALB-nu divides and support in 2 cages, adopts and cuts the ear tag note, numbers as shown in table 3:

Table 3 nude mice numbering

Numbering

	1	2	3	4	5
						The ear of being cut	Right 1	Right 2	A left side 1	A left side 2	Do not have

(2) cell suspension 100 μ l in the numbered 2 ml centrifuge tubes are extracted in injection, the nearly back leg both sides, back of the corresponding grouping BALB-nu of subcutaneous injection (sc), and the left side is CNE-2, HONE-1 and SUNE-1, the right side is CNE-2s, HONE-1s and SUNE-1s.

4. become the knurl subsequent experimental

Record tumor growth situation is in feeding animals, and near the hypodermic changing conditions observation injection point is careful the formation of tumor tissues; The major diameter of body weight and the tumour of record BALB/c-nu (major axis, a) and minor axis (minor axis b), calculates gross tumor volume, and the comparative result of CNE-2 and CNE-2s is shown in Fig. 6 A;

When the major diameter of inoculated tumour on one's body the nude mice reaches 15 mm, use the cervical vertebra dislocation method to put to death and take pictures to nude mice, the comparative result of CNE-2 and CNE-2s is shown in Fig. 6 B;

Use eye scissors and spur tweezer blunt separation tumor tissues; It is separated totally, operational analysis balance weighing tumor tissues weight, record data with the animal epithelium; The comparative result of CNE-2 and CNE-2s is shown in Fig. 6 C; With tumor tissue section and dyeing, microscopically is observed take pictures (operation steps slightly), and the comparative result of CNE-2 and CNE-2s is shown in Fig. 6 D.

Result: compare with CNE-2; The CNE-2s that the embodiment of the invention one obtains has tumorigenesis ability in the higher body for nude mice; As shown in Figure 6; The tumour size comparable situation that the nude mice that has shown four groups among Fig. 6 A obtains behind injection CNE-2 and CNE-2s, visible from figure, the growing tumors in the nude mouse respectively organized of injection CNE-2s is obviously respectively organized nude mice greater than what inject CNE-2; The photo of Fig. 6 B is the long nude mice photo that tumour is arranged behind injection CNE-2 and the CNE-2s, can find out that from picture two groups of nude mice right side tumours are obviously greater than the left side; Fig. 6 C is the weight of the interior growing tumors of nude mouse behind injection CNE-2 and the CNE-2s, and is visible from scheming, and the tumor weight that injection CNE-2s obtains is significantly higher than injects the tumor weight that obtains behind the CNE-2; Fig. 6 D is the photo of the interior growing tumors of nude mouse behind injection CNE-2 and the CNE-2s.

Claims

1. the establishment method of a human nasopharyngeal carcinoma tumor stem cell line may further comprise the steps:

(1) KB cell of the unicellular state of acquisition;

2. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1 is characterized in that, the KB cell who uses in the said step (1) is the logarithmic phase KB cell.

3. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1 is characterized in that, the KB cell who uses in the said step (1) is CNE-2, SUNE-1 or HONE-1.

4. like the establishment method of claim 1 or 3 described human nasopharyngeal carcinoma tumor stem cell lines, it is characterized in that the KB cell who uses in the said step (1) is CNE-2.

5. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1 is characterized in that, said serum-free stem cell media is DMEM/F12.

6. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1; It is characterized in that, saidly contain the serum-free stem cell media mesocuticle growth factor of Urogastron and Prostatropin and the concentration of Prostatropin is 10～30 ng/ml.

7. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1 is characterized in that, the said Tissue Culture Plate in the said step (4) is six orifice plates.

8. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 7 is characterized in that, the amount of the single cell suspension that in said six orifice plates, adds is 1～2ml/ hole.

9. the establishment method of human nasopharyngeal carcinoma tumor stem cell line as claimed in claim 1 is characterized in that, the mass and size percentage concentration of the said pancreatin in the said step (5) is 0.25%.