CN105462928A - Chinese oral melanoma cell line COMM-1 and establishment method thereof - Google Patents

Abstract

The invention discloses a Chinese oral melanoma cell line COMM-1. The Chinese oral melanoma cell line COMM-1 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC:C201586, and the preservation date is June 8th, 2015. The Chinese oral melanoma cell line COMM-1 has the advantages that the Chinese oral melanoma cell line COMM-1 is established, enrichment of tumor stem cells is achieved through the cell line, and a material basis is provided for researching Chinese oral melanoma characters and treatment drug thereof.

Description

A kind of Chinese oral cavity K-1735 COMM-1 and establishment method thereof

Technical field

The invention belongs to cell engineering field, be specifically related to strain Chinese oral cavity K-1735 COMM-1 and an establishment method thereof.

Background technology

The 1.1 melanomatous morbidity in oral cavity and prognosis

Malignant melanoma comes from neural lophocyte, is one of malignant tumour that aggressive is the strongest.Primary oral cavity melanoma is a kind of orphan disease, accounts for malignant tumor of mouth 0.5% and oral cavity biopsy 0.01%.This disease was mainly between 41-60 year, and M-F is 2:1, and patient has no manifest symptom at the ill initial stage, and most of oral cavity melanoma has entered late period when making a definite diagnosis, and visible tumor mass is greater than 4mm.Oral cavity melanoma has a strong impact on patient's sense of taste, and prognosis is poor, and within 5 years, survival rate is only 15-20%.

The foundation of 1.2 clones

Setting up tumor cell line is that research tumour causes the important link in pass.Clone is widely used in each research field of biology.It refers to that directly separation and Culture goes out cell, for follow-up study provides a kind of method of stable cell lines from the tissue of organism.Clone studies the growth of specific cells, metabolism, modulating properties, cell and cell-cell interaction, the effective research material such as the interaction of cell and matrix.The first strain continuous cell line of the mankind is the cervical cancer cell systems (Hela) set up by GeorgeOttoGey laboratory nineteen fifty-one, the foundation of this strain clone is the milestone of human body cell vitro culture, Hela cell advances the process of many research fields such as cell, gene, virus, vaccine, has also deepened the understanding of people to tumour: the reason and process, the screening cancer therapy drug etc. that find the various characteristics of tumour, research tumor development.

Except studying the developing of the characteristic of cell itself and cancer, be also applied to the research field of heredity, immunity, differentiation, growth.In addition, many enterprises can also utilize cell to carry out producing the various factor, albumen etc. both at home and abroad.The widespread use of cell has promoted the flourish of cytobiology.

Original cuiture mainly contains mechanical process and enzyme digestion.Mechanical process mainly utilizes physical shear, is directly shredded by tissue, then these is organized the method for fritter adherent culture.Enzyme digestion is the suitable enzymatic lysis tissue of application thus isolates unicellular, directly the method for suspension or adherent culture.Enzyme for digesting mainly contains: trypsinase, collagenase, pronase, Unidasa etc.

External the building of cancer cells is that difficulty is comparatively large, and one is the pollution that will effectively prevent and remove mycoplasma, mould and bacterium etc.; Two is will ensure that cells survival environment is as serum, substratum, the quality of somatomedin etc.; Three is effectively control fibrocellular growth; Four is to control the going down to posterity and conservation of front 10 generation cells well.Although Moore etc. the seventies establishes HT-144Melanoma in succession, the K-1735s such as RPMI-7951, but its biological characteristics of different melanoma of originating is different, and these clones survival time is long in vitro, its biological characteristics has larger difference compared with solid tumor in body, be difficult to objectively respond the situation of solid tumor in body, therefore, set up the K-1735 of vitro culture, based on energy, research and clinical treatment provide abundant experiment material, for its pathogenesis of discussion, find new treatment means significant.

1.3 the research of melanoma stem cell

In recent years along with the continuous progress to tumor research, tumor stem cell (cancerstemcell is proposed for some recurrent tumors with to radioactivity, chemoresistance, CSC) concept, this theory thinks that tumour cell has heterogeneity, in same tumour, the cell of most cells differentiated has lost Growth and Differentiation potential, only has sub-fraction cell to have the ability of infinite multiplication, differentiation and body outer clone.

Distinguish the basic characteristics of CSC mainly to comprise: one be by tumour transplatation to immunodeficient mouse time, only have little a part of cell to be tumor stem cell in tumour, less cancer cells just can make mouse tumorigenesis.Two is CSC and non-CSC method enrichment of cell by the cell surface marker streaming of uniqueness.Three is that CSC is had self-renewal capacity and can be confirmed by the continuous passage in many generations and transplantation experiments.

Malignant melanoma parental cell suspension culture rear section cell in the unique serum free medium adding somatomedin forms the tumour cell ball of suspension growth, and the melanoma cell surface mark be confirmed in experiment at present is CD20, CD271, JARID1B, ABCB5, ABCG2 etc.CD20 is the surface marker of the malignant melanoma stem cell that the 1st is found, CD20 ⁺melanoma cell subgroup has versatility and Multidirectional Differentiation ability, has external self-renewal capacity and tumorigenicity, can become neural progenitor cell by external evoked.CD271 is low affinity trk C, is also neural spinocellular surface marker, and CD271 is considered to relevant to the peripheroneural Infiltration and metastasis of melanoma cell, and CD271 accounts for greatly the 2.5%-41% of melanoma subgroup.JARID1B is a histone demethylase, has and promotes tumour continuous proliferation, self and versatility.JARID1B low expression or do not express in the normal tissue, and at the strong melanotic nevus tissue abnormalities high expression level of regenerative power, but JARID1B positive cell only accounts for 5%-10%.ATP is in conjunction with boxlike albumen (ATP-bindingcassettetransporter, ABC) be ancient and huge family, it is a class ATP driving pump, ABC family protein hydrolysising ATP generate energy is in order to transport various material, ABCG2 and ABCB5 is considered at the cells with melanoma dryness.

Summary of the invention

The invention provides a kind of Chinese oral cavity K-1735 COMM-1 and establishment method thereof.

First, the invention provides a kind of Chinese oral cavity K-1735 COMM-1, described Chinese oral cavity K-1735 COMM-1 is preserved in China typical culture collection center (CCTCC), its preserving number is CCTCC:C201586, its preservation date is on June 8th, 2015, and preservation address is Wuhan University.

The invention provides the establishment method of a kind of Chinese oral cavity K-1735 COMM-1 further, comprise following concrete steps:

A) tissue explants adherent method is adopted to carry out the original cuiture of oral cavity melanoma cell;

B) carry out Secondary Culture when cell density reaches about 80%, the ratio of going down to posterity is 1:3;

C) the stable cell that goes down to posterity is detected the situation of mrna expression through RT-PCR.

Further, described step c) in, the mRNA K-1735 of detection, neural ridge derived cell tumour antigen mRNA.

Further, described step c) in, the mRNA of detection is Melan-A, TYR, MITF.

The establishment method of described Chinese oral cavity K-1735 COMM-1, step concrete grammar a) is:

1) 1%I Collagen Type VI bag is trained 37 DEG C of static 30min of incubator by culture dish, and cold 1 × PBS washes ware twice, stand-by;

2) configure nutrient solution, to be put in 37 DEG C of thermostat water baths incubation 10 minutes;

3) in super clean bench, take out melanoma tissue, first remove surface fat and epithelium, 75% alcohol disinfecting is no more than 10 seconds, soaks in 1 × PBS, is put in nutrient solution;

4) tissue is cut into fine grained chippings, evenly divides and plant in culture dish, add appropriate nutrient solution, make to organize half to be dipped in nutrient solution;

5) be placed in 37 DEG C, cultivate in the incubator of 5%CO2;

6) appropriate nutrient solution within second day, is supplemented to submergence tissue block slightly;

7) visual cell's growing state suitably changes liquid.

The establishment method of described Chinese oral cavity K-1735 COMM-1, step b) concrete grammar be:

1) abandon nutrient solution, wash once with 1 × PBS, add tryptic digestion;

2) add serum and stop digestion;

3) blow and beat cell, be placed in centrifuge tube, add appropriate nutrient solution piping and druming;

4) centrifugal 1000rpm, 5 minutes;

5) abandon supernatant, add the piping and druming of appropriate nutrient solution and fall apart;

6) cell suspension is added in new culture dish, obtain described Chinese oral cavity K-1735 COMM-1.

Content of the present invention also comprises the purposes of described Chinese oral cavity K-1735 COMM-1 in the melanomatous medicine of research treatment human oral.

Content of the present invention also comprises the purposes of described Chinese oral cavity K-1735 COMM-1 in the melanomatous medicine of preparation human oral.

Content of the present invention also comprises the purposes of described Chinese oral cavity K-1735 COMM-1 in the melanomatous medicine of screening human oral.

Beneficial effect of the present invention is: the present invention establishes Chinese oral cavity K-1735 COMM-1, and achieves the enrichment of tumor stem cell by this clone, for the research melanomatous characteristic in people Chinese oral cavity and medicine thereof provide basic substance.

In order to understand better and implement, describe the present invention in detail below in conjunction with accompanying drawing.

Accompanying drawing explanation

Fig. 1, COMM-1 organize HE to dye

The cellular form of Fig. 2, light Microscopic observation primary cell and COMM-1 continuous cell line.

The ultrastructure figure of COMM-1 cell is observed under Fig. 3, transmission electron microscope.

Fig. 4, sepharose detect the expression of the mRNA of MAGE1, S100B, MITF of COMM-1.

Fig. 5, COMM-1 cell chromosome caryogram microgram (a) and karyomit(e) number statistical graph (b).

The growth curve of Fig. 6, COMM-1 cell

The Clone formation of Fig. 7, COMM-1 cell

The enrichment of tumor stem cell in Fig. 8, melanoma cell COMM-1

Fig. 9, the gene test of tumor stem cell dryness

A kind of Chinese oral cavity K-1735 COMM-1 in the present invention is preserved in China typical culture collection center (CCTCC), and its preserving number is CCTCC:C201586, and its preservation date is on June 8th, 2015, and preservation address is Wuhan University.

Embodiment

The foundation of [embodiment 1] oral cavity K-1735 COMM-1 and CHARACTERISTICS IDENTIFICATION

Material illustrates:

1 clinical samples

This is tested oral cavity tissue used and takes from Zhongshan University's attached brilliance stomatological hospital excision sample.

2 main solution and formula

2.1 cell cultures

1) 10 × PBSBuffer:NaCl80g, KCl2g, Na2HPO411.55g, KH2PO42g, adds the ultrapure water of about 800ml, the abundant stirring and dissolving of magnetic stirring apparatus, and adjust ph to 7.4, is settled to 1L, after autoclave sterilization, and room temperature preservation.

2) foetal calf serum (FBS): purchased from Hyclone company, 4 DEG C of preservations, and substratum is made into suitable proportion.

3) 10 × Trypsin-EDTA (TE): take 1.25gtrypsin, 1.00gEDTA is dissolved in the 1 × PBS of 250ml, filtration sterilization ,-20 DEG C of preservations.Proper concn peptic cell is diluted to 1 × PBSbuffer before using.

4) F12 (DF) substratum: be dissolved in the ultrapure water of 1L, regulate pH value to 7.2, utilize peristaltic pump and 0.22 μm of membrane filtration degerming.4 DEG C of preservations.2 × DF component doubles, and the water yield is constant.DF (-) expression does not add Ampicillin and Kanamycin.

5) mouse tail NTx: during original cuiture, is diluted to the concentration of 1% with 1 × PBS, the ware of each six centimetres adds 1.5mL and cover at the bottom of ware, 37 DEG C of static bags were by 30 minutes.

6) soft agar: softagar adds ultrapure water, autoclave sterilization is made into 0.66% and 1.33%.

7) colchicine: 40 μ g/mL mother liquors, final utilization concentration is 0.2 μ g/mL.

8) melanoma original cuiture liquid: DF+10%FBS.

2.2 nucleic acid electrophoresis

1) 50 × TAE electrophoresis Buffer (pH8.5): add 242gTris in the vial of 1L, 18.6gNa2EDTA2H2O, then adds the secondary water of about 800ml, abundant stirring and dissolving, then the acetic acid adding 57.1ml, fully stir, add secondary water and be settled to 1L, room temperature preservation.Dilute with 1:50 during use.

2) ethidium bromide (EB): storage liquid concentration is 10mg/ml, working concentration is 0.5 μ g/ml.

3) 1% Normal Agarose Gel: take 1g agarose (agarose), add 100ml1 × TAEbuffer, heated and boiled is dissolved completely to agarose, be chilled to about 50 DEG C, add 5 μ lEB storage liquid, make final concentration be 0.5 μ g/ml, shake up rear Casting of gels dull and stereotyped.

3 methods and result:

3.1 human oral cancer melanochrome organize HE to dye

1) 10% formalin or 4%PFA fixing port cavity tissue spend the night;

2) automatic dehydration instrument dewaters: 70% ethanol 2 hours, 80% ethanol 2 hours, 95% ethanol 30 minutes, 95% ethanol 30 minutes, dehydrated alcohol 30 minutes, dehydrated alcohol 2 hours, dehydrated alcohol+TO (1:1) 30 minutes, TO30 minute, TO2 hour, TO+ paraffin (1:1) 30 minutes, paraffin 3 hours, paraffin 2 hours.Totally 12 barrels, 16 hours;

3) embedding machine embeds with paraffin, slicing machine is cut into slices;

4) 60 DEG C, tissue is de-cured, dimethylbenzene 5 minutes, dehydrated alcohol 1 minute, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, distillation washing 4 minutes;

5) Hematorylin liquid dyeing 1-2 minute, tap water 10 minutes, water-soluble eosin stains 2-3 minute;

6) dewater, transparent and mounting: section is added 70% ethanol 30 seconds, 80% ethanol 30 seconds, 90% ethanol 1 minute, dehydrated alcohol 1 minute, dimethylbenzene 5 minutes, finally uses resinene mounting.

Interpretation of result: as shown in Figure 1, COMM-1 organizes HE stained visible black color element knurl, and tumour cell is variform, and as circle, polygon and fusiformis, several cell is mixed to be deposited; Core is not of uniform size, and chromatin enriches, the large and engrain of kernel, and mitosis figures is easily shown in, melanin granule can exist with in tumour cell and interstitial, and part cell is full of melanin granule, is comparatively serious change.

The foundation of 3.2 human oral K-1735 COMM-1 and morphological observation

3.2.1 cell primary is cultivated and Secondary Culture

Tissue explants adherent method is adopted to cultivate the little block organization shredded, within 5th day, cell climbs out of from tissue block, the visible more fibroblastic growth of process of growth, this research adopts gradient digestion and scrapes division removes inoblast, after repeatedly going down to posterity, treat that inoblast ratio declines gradually, melanoma cell propagation is accelerated, become purer clone, called after COMM-1 cell.So far, COMM-1 cell has gone down to posterity more than 50 times, and cell state is stablized.

Interpretation of result: as shown in Figure 2, under light microscopic, visible COMM-1 cell is variform, and from polygon, fusiformis to circle, cell size differs, and most cell caryoplasm ratio is large, and kernel is many and clear, and mitotic division is many.It is agglomerate layer-by-layer growth that cell covers with culture dish, forms clone not of uniform size.Adherent property is poor, 5 days confluent culture wares, and the ratio of going down to posterity is 1:3.

3.2.2COMM-1 cell ultrastructure

1) inoculating cell is in 6cm culture dish, and cell covers with rear 1xTE and digests cell dispersion;

2) FBS stops digestion, adds perfect medium, the centrifugal 5min of 900r/min;

3) cell mass is fixed through 2.5% glutaraldehyde osmic acid, embedding, ultrathin section(ing), and dyeing, at electric Microscopic observation.

Interpretation of result: as shown in Figure 3, transmission electron microscope observing COMM-1 cellular form visible cell has abundant first antenna projection, and cell golgi body is to exocytosis vesicle, and this is vigorous relevant with cellular metabolism.Can be observed golgi body, rrna, endoplasmic reticulum and autophagosome in cell, nucleus is atypia, and have monokaryon, double-core, kernel is not of uniform size, out-of-shape, and be distributed with the melanin granule that film holds around golgi body, plastosome is numerous.Cell tracking is not tight, loose distribution.

3.2.3COMM-1 the qualification of the significant molecule of cell

3.2.3.1COMM-1 the extraction of cell RNA

1) to take the logarithm cell in vegetative period, abandon nutrient solution, in culture dish, add 1mlTrizol, complete to lysis with pipettor piping and druming, liquid is drawn to 1.5ml without in enzyme centrifuge tube;

2) room temperature is placed and within 5 minutes, is made nucleic acid-protein mixture be separated completely, 4 DEG C, and centrifugal 10 minutes of 12000xg, gets supernatant;

3) often use 1mlTrizol to add chloroform 0.2ml, violent jolting 15s, room temperature leaves standstill 3min and makes it layering, 4 DEG C, centrifugal 15 minutes of 12000xg;

4) the upper strata aqueous phase of≤50% is carefully transferred to another 1.5ml without in the clean centrifuge tube of enzyme;

5) often use 1mlTrizol to add 0.5ml Virahol, mix gently, room temperature leaves standstill 10 minutes, 4 DEG C, centrifugal 10 minutes of 12000xg;

6) abandon supernatant, add without enzyme 75% ethanol 1ml along wall, mix gently;

7) 4 DEG C, centrifugal 5 minutes of 7500xg, abandons supernatant, is inverted by 1.5ml centrifuge tube, to exhaust remaining ethanol, dry with the micropipet of 100 μ l, precipitation be dissolved in 20 ~ 40 μ l without in RNase water;

8) get 1 μ l, measure A260nm and A280nm with ultraviolet spectrophotometer (Nanodrop), RNA concentration (μ g/ml)=A260nm × 40 × extension rate; Purity represents with A260/A280 ratio, and reach 1.8-2.0 and can be used for experiment ,-80 DEG C save backup.

3.2.3.2RT-PCR reverse transcription reaction (by TOYOBOReverTraAce-α-test kit specification sheets operation)

1) be sequentially added in PCR reaction tubes:

Total serum IgE 1 μ l (600ng)

10pmol/LOigo(dT)5μl

RNaseFreeH2O6μl

2) ice bath 1min after 65 DEG C of effect 5min, puts and adds on ice:

3) arrange PCR instrument, 42 DEG C, 60min, 99 DEG C, 5min, 4 DEG C, hold, put-20 DEG C and save backup.

3.2.3.3PCR (by the operation of TOYOBOKOD-Plus-test kit specification sheets)

1) be sequentially added in PCR pipe:

2) add successively in PCR pipe

Different primers is according to settings such as various annealing temperature, cycle number, extension times.

Interpretation of result: adopt sepharose to detect the mrna expression situation of MAGE1, S100B, MITF in COMM-1, result as shown in Figure 4.RT-PCR detects COMM-1 and amplifies MAGE1 object fragment 418bp, S100B object fragment 203bp.

3.2.4COMM-1 DNA fingerprint qualification

STR (shorttandemrepeat, STR), also known as microsatellite DNA (microsatelliteDNA), is the DNA polymorphism locus that a class extensively exists in human genome.It forms core sequence by 2-6 base pair, arranges in tandem sequence repeats.Str locus site length is generally between 100-300bp, because DNA fragmentation length between individuality or DNA sequence dna difference form height polymorphism, in gene delivery process, follow the heredity of Mendelian's codominance mode, may be used for proving whether this research cultured cells comes institute by its patient.Get corrective surgery resection organization and cultivate the COMM-1 cell in 14 generations and deliver to Zhongshan University verification centre of forensic medicine extraction DNA, carry out Analysis and Identification, result

As shown in table 1.

Table 1COMM-1 tissue and cell STR genotyping result

COMM-1 organizes

COMM-1 cell

D19S433	13,14.2	13,14.2
			D5S818	11,12	11,12
D21S11	29,32.2	29,32.2 6 -->
			D18S51	13	13
D6S1043	13	13
			D3S1358	17	17
D13S317	8,11	8,11
			D7S820	8,11	8,11
D16S539	9	9
			CSF1P0	10,11	10,11
Penta D	9	9
			Amel	X	X
Vwa	16	16
			D8S1179	10,15	10,15
TPOX	8,11	8,11
			Penta E	12,21	12,21
TH01	7,10	7,10
			D12S391	21	21
D2S1338	19,24	19,24
			FGA	18,21	18,21

Interpretation of result: analyze 20 sites respectively to COMM-1 tissue block and cell, comprise an individual character chromosomal foci and 19 non-sex chromosome sites, all STR are all completely the same, can judge that this tissue and cell are same source.

3.2.5 chromosome karyotype analysis

1) be in logarithmic phase COMM-1 and change to nutrient solution containing colchicine (0.2 μ g/mL), cultivate 2 hours;

2) discard supernatant, 1 × PBS washes ware once, discards 1 × PBS, trypsin digestion and cell;

3), after FBS stops digestion, centrifugal 5 minutes of 1000rpm, removes supernatant;

4) the KCL solution (0.075M) adding preheating 37 DEG C processes 30 minutes (different cell stage is different);

5) add 1mL stationary liquid (methyl alcohol: acetic acid=3:1, now with the current), dispel and beat, centrifugal 5 minutes of 1000rpm, discard supernatant, add Fresh fixative, dispel, effect 20-30 minute, centrifugal 5 minutes of 1000rpm, abandons supernatant, adds 0.5mL stationary liquid, make cell suspension again;

6) drip cell suspension 40cm place above slide glass slide (steeping in 30% ethanol, 4 DEG C of refrigerator precoolings), and dispel immediately, spirit lamp is dried;

7) Gimsa stoste: 1 × PBS=1:9 dyes 20 minutes, and tap water rinses.Result as shown in Figure 6.

Interpretation of result: as shown in Fig. 5 a, 5b, karyomit(e) display is abnormal, and its number all has distribution at 41 to 84, and from 40 to 84 setting 10 intervals, result display 46 to 48 karyomit(e)s are maximum, are similar to diploid.Chromosomal dysploidy is the instable performance of tumorgenesis, except chromosome number differs, also there are the abnormal conditions such as deletion, inversion, repetition and transposition in chromosome structure, these can cause some restructuring of gene, lose the gene being unfavorable for tumor development.

The biological characteristics of 3.3COMM-1 cell

3.3.1 cell counting detects cell growth curve and the doubling time of COMM-1 cell

1) the COMM-1 cell of taking the logarithm vegetative period, is inoculated in 24 orifice plates with the cell concn in 10000/ml/ hole;

2) every 24h counting once, carries out cell counting after 1% trysinization, continuous counter 6 days;

3) with every day cell density average for ordinate zou, incubation time is X-coordinate, draw cell growth curve.Result as shown in Figure 7.

4) by the following formulae discovery doubling time.

TD＝tlog2/(logNt-logN0)

(TD: doubling time; T: incubation time: Nt: cultivate the cell count after t hour; N0: postvaccinal cell count)

Interpretation of result: this experiment have detected the growth curve of COMM-1 cell as shown in Figure 7, under the culture condition of DF+10%FBS, first four days propagation slowly, the 5th day starts fast breeding to cell, and the doubling time of COMM-1 cell is 104.43 hours.

3.3.2COMM-1 the detection of clonality

1) cell of logarithmic phase is digested, counting, resuspended;

2) get 300,500 cells in six orifice plates, add nutrient solution DF+10%FBS;

3) liquid feeding after a week;

5), after 10 days, by violet staining, take pictures, counting clone number;

Interpretation of result: as shown in Figure 7, the cultural method that experiment adopts plate clone to be formed, 300 and 500 cells are planted respectively in six orifice plates, the clone counting diameter afterwards and be greater than 200 microns for 10 days, A375 is owing to being the more clone of passage number, and cell strain is more stable, and clonality is strong, 300 and 500 cells the cloning efficiency of six orifice plates be respectively 59% and 43.27%, COMM-1 be then 8% and 8.06%.

The enrichment of tumor stem cell and CHARACTERISTICS IDENTIFICATION in 3.4COMM-1

3.4.1 serum free suspension cultivates the tumor stem cell in enrichment COMM-1 clone

1) attached cell of logarithmic phase is digested, counting;

2) re-suspended cell inoculating according to the ratio of 3.5 × 105/6cm ware, adds 4mlDF+8F nutrient solution, Continuous Observation;

3) treat that sacculus cell grows to the 7th day, piping and druming sacculus, 40 micron screen are filtered and are collected sacculus, and 400rpm, 10min are centrifugal, abandon waste liquid;

4) add TE and be placed in 37 DEG C of water-bath digestion 5min, Ti termination reactions;

5) the centrifugal receipts cell of 400g, 10min;

6) count, inoculating cell is in culture dish.

Interpretation of result: as shown in Figure 8, in adherent cell, add serum-free cell culture medium DF+8F, originally Growth of Cells is slack-off, and within second day, nearly 5-10% cell is no longer adherent, forms similar spherical suspending cell; Within 4th day, some cell visible forms fine and close cell cluster, and form becomes circle; Within 6th day, cell is constantly bred, and formed the clone that diameter is greater than 200 microns, size is more even, cell is finer and close, edge is comparatively neat, and refractivity is comparatively strong, but along with incubation time elongated, sacculus cell still keeps adhered state, carry out sacculus to go down to posterity when cell is no longer bred, Continuous Observation finds that s-generation sacculus cell is suspension growth, loses adherent proterties, sacculus form is finer and close than the first-generation, and form is more stable.

3.4.2COMM-1 sacculus stem cell markers detects

1) treat that sacculus cell grows to the 7th day, piping and druming sacculus, 40 micron screen are filtered and are collected sacculus, and 400rpm, 10min are centrifugal, abandon waste liquid;

2) add TE and be placed in 37 DEG C of water-bath digestion 5min, Ti termination reactions;

3) the centrifugal receipts cell of 400g, 10min

4) extract RNA and carry out RT-PCR detection;

Interpretation of result: the mRNA that COMM-1 sacculus cell and attached cell carry out stem cell markers Oct4, ABCB5, CD20, CD271, KDM5B, ABCG2 is detected by the method for RT-PCR, as shown in Figure 9, the mrna expression level of COMM-1 sacculus stem cell gene KDM5B, ABCB5, Oct4 is higher than attached cell, adherent COMM-1 cell by the special certain density of serum-free medium process is described, the cell side group with melanoma stem cell properties can be enriched to.

Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, its framework form can be flexible and changeable, can subseries product.Just make some simple deduction or replace, all should be considered as belonging to the scope of patent protection that the present invention is determined by submitted to claims.

Claims (7)

1. a Chinese oral cavity K-1735 COMM-1, described Chinese oral cavity K-1735 COMM-1 is preserved in China typical culture collection center, and its preserving number is CCTCC:C201586.

2. the establishment method of Chinese oral cavity K-1735 COMM-1 according to claim 1, comprises following concrete steps:

A) tissue explants adherent method is adopted to carry out the original cuiture of oral cavity melanoma cell;

B) carry out Secondary Culture when cell density reaches about 80%, the ratio of going down to posterity is 1:3;

C) the stable cell that goes down to posterity is detected the situation of mrna expression through RT-PCR.

3. method according to claim 2, is characterized in that, described step c) in, the mRNA K-1735 of detection, neural ridge derived cell tumour antigen mRNA.

4. method according to claim 2, is characterized in that, described step c) in, the mRNA of detection is MAGE1, S100B, MITF.

5. the purposes of Chinese oral cavity K-1735 COMM-1 according to claim 1 in the melanomatous medicine of research treatment human oral.

6. the purposes of Chinese oral cavity K-1735 COMM-1 according to claim 1 in the melanomatous medicine of preparation human oral.

7. the purposes of Chinese oral cavity K-1735 COMM-1 according to claim 1 in the melanomatous medicine of screening human oral.