CN102876632A - Human NK/T cell line - Google Patents

Abstract

The invention discloses a human NK/T cell line, which is named as human NK/T cell line ZQNK-29 and is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan university, and the preservation number is CCTCC C201278. The human NK/T cell line can realize the long-time transfer of culture, the mass amplification is realized, in addition, higher anti-tumor activity is realized, and a novel experiment model is provided for researchers to deeply developing nature killing cell anti-tumor acting mechanism and clinic adoption immunotherapy.

Description

People NK/T clone

Technical field

The invention belongs to a kind of clone, relate in particular to a strain people NK/T clone ZQNK-29.

Background technology

Before 30 years, the existence of (natural killer cell, NK) that scientist just finds that natural killer cell is arranged in the white corpuscle, it is the important immunocyte of body, because the killing activity of NK cell limits without MHC, does not rely on antibody, therefore be called Nk Cell Activity.The NK cell not only with antitumor, anti-virus infection is relevant with immunomodulatory, and participates in some cases the generation of allergy and autoimmune disorder.From then on, scientists is just studied the concrete function of natural killer cell, has set up the theory that natural killer cell activates other white corpuscles (such as T lymphocyte and scavenger cell) and replys for various infection activate immunities.

The NK cell is considered to the best weapon of body kill cancer cell and infectious cause of disease always, the researchist attempts improving by different approach the antitumor action of NK, but there are certain difficulty technically in the purifying of NK cell and external a large amount of amplification, therefore there are many people to attempt to set up NK clone and carry out tumor biotherapy with allogeneic NK cell, external scientists has been set up Immortalized NK clone: HANK1 in succession, KHYG-1, NK-92, NKL, NK-Y, PLT-2, MOTN, IMC-1, SNK-6 and SNT-8-1 and YT, NK3.3 etc. adopt again the IL-2 Gene transfer techniques to set up IL-2/NK-92, the transgenosis such as IL-2/NKL and IL-2/NK3.3 NK clone.Especially the foundation of NK-92 has promoted its applied research in adoptive immunotherapy, and for furtheing investigate feature, the function of NK cell, further discloses its definite effect in immunity system, and favourable research material is provided.

Chinese patent 200910077749.5 discloses a kind of natural killer cell, and this natural killer cell is that deposit number is people's natural killer cell NKG cell of CGMCC NO.2901.NKG cell of the present invention is strong to the lethality of tumour cell, and survival ability is strong in vivo, steady quality, and antitumor reaction is strong.

Chinese patent 03152968.2 discloses a kind of natural killer cell strain of Interleukin-15 genetic modification, and this cell strain phenotype is the CD56 strong positive, and CD16 and CD3 are all negative, its form for disperse, half adherent growth state.Its preparation method: be that the pcDNA3 carrier for expression of eukaryon is inserted in the cDNA coding region of IL-15, make up secretor type recombinant eukaryon expression vector pcDNA3/IL-15; Utilize liposome transfection NK-92 cell; Adding Geneticin (G418) in screening culture medium screens; The limited dilution cloning cell, the activity with IL-15 in the dependent cells strain mensuration clone cell culture supernatant of IL-15 selects the positive cell clone of expressing IL-15; This is cloned in the complete α of the IL-15 of 20～100 activity units-MEM substratum increases, obtain having the engineering cell strain of clinical value.

Chinese patent application 201010507100.5 discloses the natural killer cell strain of a kind of Alpha-IFN genetic modification of people, that human interferon-alpha gene transfection is entered natural killer cell strain NKL cell, the cell strain of the stably excreting that is built into, expression human interferon-alpha.This cell strain detects through RT-PCR and ELISA that identify can be at intracellular expression, and synthetic IFN-α is secreted in the outer culture supernatant of born of the same parents.This cell strain is the killing hepatoma cell effectively, this anti-tumour effect and NKL-IFN α cell IFN-γ, Granzyme, TNF-α are relevant with the protein expression the level rise with the Perforin gene, and the NKL cell strain after modifying stimulates more responsive to external world.

Although the NK cell of above-mentioned patent disclosure has been obtained certain progress at anti-tumor aspect, but because there are certain difficulty technically in purifying and external a large amount of amplification of NK cell, and people's natural killer cell NKG cell that a domestic rarely seen strain deposit number is CGMCC NO.2901, there are no the report of people NK/T Establishment of Cell Line.Therefore, set up and provide the NK/T that more is appropriate to compatriots clone, antineoplastic basis and the clinical application research of natural killer cell had its prior meaning.

Summary of the invention

The invention provides a strain and can cultivate for a long time the people NK/T clone that can increase in a large number at subculture in vitro separately.

One strain people NK/T clone is named to be to be deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan University on 06 12nd, 2012 by people NK/T clone ZQNK-29, and deposit number is CCTCC NO:C201278.

But inventor NK/T clone Long Term Passages, a large amount of amplifications, and have stronger anti-tumor activity are for the investigator carries out natural killer cell in human function of tumor mechanism in a deep going way and clinical adoptive immunotherapy provides new experimental model.

Description of drawings

Fig. 1 is viable cell form (10 *) under the ZQNK-29 clone light microscopic of the present invention, and cell is the lymphocyte of bright circle, and it is vigorous to grow.

Cellular form (40 *) after Fig. 2 ZQNK-29 clone of the present invention H.E dyeing, cell is rounded, and the caryoplasm ratio is little, and kernel is more obvious, and nuclear membrane is clear, and nuclear fission is as common.

Fig. 3 is the cellular form under the ZQNK-29 clone transmission electron microscope of the present invention, and nucleus is greatly the kidney forming core, and cell surface has microvillus.

Fig. 4 is the cellular form under the ZQNK-29 clone scanning electron microscope of the present invention, and cell surface has many projections.

Fig. 5 is ZQNK-29 cell line cell periodicity analysis results of the present invention.

Fig. 6 is ZQNK-29 cell line cell growth curve chart of the present invention.

Fig. 7 is ZQNK-29 clone karyotype distribution plan of the present invention, and 46 of modal numbers show as 2 times of somatocyte, but the part abnormal karyotype is arranged.

Fig. 8 is ZQNK-29 cell of the present invention, and at the inhibition curve of 5 kinds of tumour cells of In Vitro Anti, (a) effect target ratio is 25: 1, and (b) effect target ratio is 50: 1.

Fig. 9 is ZQNK-29 cell and people's lung cancer A549 cell inoculation experiment of nude mouse figure, and (a) ZQNK-29 cell inoculation nude mice by subcutaneous had no obvious lump on the 13rd day; (b) ZQNK-29 cell inoculation nude mice by subcutaneous had no obvious lump on the 25th day; (c) the 25th day lump of people's lung cancer A549 cell inoculation nude mice by subcutaneous; (d) the 45th day lump of people's lung cancer A549 cell inoculation nude mice by subcutaneous; (e) people's lung cancer A549 adds 7 days visible lumps of ZQNK-29 cell; (f) people's lung cancer A549 adds the lump disappearance after 15 days of ZQNK-29 cell.

Embodiment

(1) foundation of ZQNK-29 clone

1.1 the source of sample:

Take from a peripheral blood 10ml who suffers from nose blood vessel center NK/T cell lymphoma male patient, anticoagulant heparin.And through the patient agree, family members know the inside story.

1.2 the separation of peripheral blood mononuclear cell:

Aseptic extraction peripheral blood in patients 10ml, wherein 2ml takes ordinary method, carried out T cellular immunization Subsets with flow cytometer, its result is: CD25=60%, CD19=1%, CD4=13%, CD8=3%, CD56=81%, CD3=16%, CD44=71%.Remaining 8ml peripheral blood carries out density gradient centrifugation with mixing behind aseptic Hank ' the s solution dilution of equivalent with lymphocyte separation medium, and 2000 rev/mins, 20 minutes centrifugal, room temperature 20 degree.The mononuclearcell of interlayer face in the absorption is washed 2 times through Hank ' s liquid, and 1500 rev/mins, 10 minutes, room temperature 20 degree.Add fresh completely RPMI-1640, in cell suspension 5m1 separating device 30ml plastic culture bottle, at 37 ℃, 5%CO ₂Cultivate in the incubator.

1.3 the separation and purification of cell

After the cell cultures 96 hours, the visible bright and eugonic lymphocyte of Microscopic observation, with negative immunomagnetic beads (the Dynal Biotech ASA of CD56, Oslo, Norway) carry out NK cellular segregation purifying, with the cell behind the purifying, add fresh complete RPMI-1640 mixing, go down to posterity and put in the 30ml plastic culture bottle, cultivate with same culture condition again.

1.4EBV cell transfecting

After the cell cultures 72 hours, take out eugonic lymphocyte, passage is put in 24 orifice plates, add 2 of EBV in every porocyte and carry out cell transfecting.At 37 ℃, 5%CO ₂Continue to cultivate in the incubator, take out 24 well culture plates after 2 days and under inverted microscope, observe, most of hole inner cell well-grown, the visible cell growth is vigorous, and cellular form is dispersed in bright circle, the lymphocyte that differs in size and is grown to the master.After one week, at Microscopic observation, as seen wherein there is the inner cell growth of several holes vigorous especially, cell overlap growth phenomenon is arranged, the intensive growth of lymphocyte that periphery has many bright circles, differs in size, the grape cluster sample that is condensed into that has is grown.Carefully choose out the vigorous especially hole of several Growth of Cells, the hole inner cell is blown and beaten gently, passage put in the 30ml Plastic Bottle cultivate, cell growth condition is good subsequently, and growth velocity is fast, can go down to posterity once in 2-3 days, this cell continous-stable growth was recovered behind liquid nitrogen cryopreservation more than 2 years, and cell state is good, survival rate reaches more than 95%, with this cell called after ZQNK-29 clone.This cell lies on 06 12nd, 2012 and is deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan University, and deposit number is CCTCC NO:C201278.

The used completely RPMI-1640 of ZQNK-29 cell cultures:

RPMI-1640 (South America Hyclon product) includes 10% foetal calf serum (South America Hyclon product), PHA-P HA (Guangzhou Medicine Industry Inst) 20ug/ml, rIL-II (Beijing Sihuan Biopharmaceutical Co., Ltd.) 800U/ml and adds an amount of two anti-(South America Hyclon product).

The negative immunological magnetic bead sorting method of CD56:

Negative immunomagnetic beads (Dynal Biotech ASA, Oslo, the Norway) sorting method of CD56 cell obtains the NK cell of purifying, and concrete operation step is as follows:

1) get 100ul magnetic bead suspension, add 100ulHank ' s liquid, mixing is put on the magnet stand and was placed 1 minute, removes supernatant, adds 100ul Hank ' s liquid again, and mixing is stand-by;

2) collect cell after centrifugal, make about 1 * 10 ⁸/ 100ul MNC adds the heat-inactivated foetal calf serum of 40 ul;

3) add 40ul CD56 antibody, mixing was hatched 20 minutes for 2-8 ℃;

4) add 2ml Hank ' s liquid and clean cell, the inclination test tube is the mixing mixture for several times, and 4 ℃, 300g centrifugal 8 minutes, removes supernatant liquor;

5) add 900ulHank ' s liquid, mixing cell again adds the magnetic bead of 100ul prewashing, hatches 10 minutes companion's inclination and rotation gently for 4 ℃;

6) cell of magnetic bead combination is arranged with TIP head piping and druming gently 5 times again mixing, adding 500ul Hank ' s liquid increases cumulative volume again, and mixing has the cell of magnetic bead combination again, places magnet stand upper 2 minute, shift out supernatant and put in another pipe magnetic bead, be added with the magnetic bead of 50ul prewashing in the new pipe;

7) hatched 10 minutes for 4 ℃, magnet stand upper 2 minute put in inclination gently and rotation, shifts out supernatant liquor, minute installs in 24 well culture plates, and every hole adds the 2ml nutrient solution.

The method of EBV preparation:

EBV liquid is from B958 cell strain (cell biological institute in Shanghai provides).Recover first during extraction and cultivate the B958 cell, when cell is in exponential phase of growth, collect whole cell suspensions, multigelation 3 times transfers in the centrifuge tube, 3000 rev/mins, centrifugal 15 minutes, with supernatant liquor with 0.22 μ m membrane filtration after packing ,-80 ℃ save backup.

(2) observation of morphocytology

2.1 the morphological observation under the light microscopic

2.1.1 the viable cell form of cultivating

Get the cultured cells that goes down to posterity, (Japanese Olympus IMT-2 inverted microscope) observes the viable cell growing state under opticmicroscope.Fig. 1 be the 50th generation cell Optical Morphology picture, visible cell growth is vigorous, background is clear, impurity is rare, take bright medium sized lymphocyte as main, and the unicellular growth of the one-tenth that has, the cell mass sample growth that the mutual aggegation of the cell that has becomes to differ in size can be gone down to posterity in 2-3 days.

2.1.2H.E the cellular form after the dyeing

Collect the cell of logarithmic phase, through 2000 rev/mins, 15 minutes centrifugal, remove supernatant liquor, carry out the paraffin coating with sedimentation cell, H.E dyeing and om observation after the section, as shown in Figure 2, the culturing cell in the same size, nucleus is large, kernel is more obvious, chromatin is dispersed in part cohesion, and nuclear membrane is clear, rule, part is seen multinuclear, and sees that more nuclear fission looks like to be respectively multipolar division picture (Y type).

2.2 the morphological observation under the Electronic Speculum

2.2.1 the observation of cellular form under the transmission electron microscope

Collect the cell of logarithmic phase, Hank ' s liquid is washed two times, and last is all over 1500 rev/mins, and centrifugal 7 minutes, abandoning supernatant added 2.5% glutaraldehyde, sample in 2.5% glutaraldehyde solution 4 ℃ fixedly spend the night, then follow these steps to process sample:

(1) outwells stationary liquid, use 0.1M, the phosphoric acid buffer rinsing sample of pH7.0 three times, each 15min;

(2) with 1% osmic acid solution fixed sample 1-2h;

(3) outwell stationary liquid, use 0.1M, the phosphoric acid buffer rinsing sample of pH7.0 three times, each 15min;

(4) ethanolic soln with gradient concentration (comprise 50%, 70%, 80%, 90% and 95% 5 kind of concentration) carries out processed to sample, and every kind of concentration is processed 15min, uses 100% Ethanol Treatment once, each 20min again; Last excessively to pure acetone processing 20min;

(5) process sample 1h with the mixed solution (V/V=1/1) of embedding medium and acetone;

(6) process sample 3h with the mixed solution (V/V=3/1) of embedding medium and acetone;

(7) pure embedding medium processing sample spends the night;

To get up through the sample embedding of osmotic treated, 70 ℃ of heated overnight namely obtain embedded sample.Sample is cut into slices in the Reichert ultramicrotome, obtains the section of 70-90nm, and this section is through lead citrate solution and the uranyl acetate 50% alcohol saturated solution 15min that respectively dyes, and can observe under the JEM-1230 type transmission electron microscope of Japanese JEOL company.

Cellular form observations under the transmission electron microscope:

As shown in Figure 3, the ultrastructure morphological specificity: cell is rounded, median size is examined greatlyr, and the caryoplasm ratio is little, nuclear is rounded or oval mostly, intact nuclear membrane, the nuclear membrane that has have depression to be the kidney forming core, or a plurality of depression, the nuclear membrane that has visible projection or a plurality of projections different in size, one or more kernels are clearly visible; The even distribution that nuclear chromatin has, the gathering that has is dense, skewness, the close nuclear membrane that has; Nuclear fission is more common, and cell surface has more projection, abundant microvilli.

As shown in Figure 4, each organoid is clear in the cell, more rough surfaced endoplasmic reticulum, plastosome, secretory granules are arranged, caryoplasm and Intermediate filament are also abundanter, endochylema has a large amount of free ribosomes, microfilament, secretory vacuole and dense granule have the nuclear chromatin limit to gather phenomenon in Golgi body, the born of the same parents, and the object line plastochondria enlargement that has, density reduce, the gap is widened.

2.2.2 cellular form is observed under the scanning electron microscope

Collect the cell of logarithmic phase, Hank ' s liquid is washed two times, and last is all over 1500 rev/mins, and centrifugal 7 minutes, abandoning supernatant added 2.5% glutaraldehyde, sample in 2.5% glutaraldehyde solution 4 ℃ fixedly spend the night, then follow these steps to process sample:

(1) outwells stationary liquid, use 0.1M, the phosphoric acid buffer rinsing sample of pH7.0 three times, each 15 minutes;

Osmic acid solution fixed sample 1-2h with 1%;

(2) outwell stationary liquid, use 0.1M, the phosphoric acid buffer rinsing sample of pH7.0 three times, each 15 minutes;

Ethanolic soln with gradient concentration (comprise 50%, 70%, 80%, 90% and 95% 5 kind of concentration) carries out processed to sample, and every kind of concentration is processed 15min, uses 100% Ethanol Treatment twice, each 20 minutes again;

(3) process sample 30min with the mixed solution (V/V=1/1) of ethanol and isoamyl acetate, processed sample 1-2 hour with pure isoamyl acetate again;

(4) critical point drying;

(5) plated film is observed.

The sample of handling well is observed in the XL30 type environmental scanning electronic microscope of Dutch Philips company.

As shown in Figure 5, cell is rounded, and cell surface has the spherical shape projection that differs in size, and the cell surface that has has clump shape or intensive short and small microvillus projection, and the cell condensation that has is grown together, and nuclear fission is common.

(3) cell times body and cell cycle experiment

The cell of taking the logarithm vegetative period, in the 10ml centrifuge tube, 1000 rev/mins, centrifugal 5min is centrifugal with cell harvesting, and supernatant discarded makes 1 * 10 ⁵/ ml cell suspension uses Hank ' s liquid to clean again one time, supernatant discarded, with the cell of centrifugation on turner, the limit rotation, the limit slowly drips 75% cold alcohol 2ml and fixes, it is to be measured to put 4 ℃ of refrigerator overnight.

Get cell 100ul after fixing in another streaming loading pipe, and add an amount of PBS liquid mixing, 1000rpm/5min is centrifugal, removes alcohol.Clean one time with PBS liquid again, supernatant discarded, prepare sample with CycleTESTPLUS DNA Reagent Kit (U.S. B.D company product): add gently mixing room temperature placement 10min of 250ul A liquid (pancreatin damping fluid), add again gently mixing of 200ul B liquid (pancreatin and RNA enzyme inhibitors), room temperature was placed 10 minutes, add at last 200ul precooling C liquid (iodate the third tincture) dyeing, lucifuge refrigeration 10 minutes.The test of FCM loading.Experiment is carried out interpretation of result through 3 repetitions.

The result: ZQNK-29 belongs to diploid cell.66.6% cell be in the G1 phase (mitosis prophase), 6.46% cell is in the G2 phase (mitosis anaphase), 26.94% cell is in the S phase (DNA synthesis phase).

(4) cell growth curve and the mensuration of cell colony doubling time

The passage cell of taking the logarithm vegetative period is collected in the 10ml centrifuge tube 1000 rev/mins, centrifugal 5 minutes, abandon supernatant liquor, use Hank ' s liquid to wash once 1000 rev/mins, centrifugal 5 minutes, abandon supernatant liquor, add RPMI-1640, include 10% foetal calf serum, rIL-II (Beijing Sihuan Biopharmaceutical Co., Ltd.) 800 υ/ml, and an amount of two anti-(South America Hyclon product).

Quantitatively expect blue living cells count counting with platform, total cellular score is 1.1 * 10 ⁷, add to 22ml with nutrient solution, make 5.0 * 10 ⁵The cell suspension of/ml is got the 1ml cell suspension in the 30ml culturing bottle, adds the 4ml fresh medium again, is divided into and fills 22 culturing bottles, places 37 ℃, 5%CO ₂Incubator in cultivate.Cultivate after 24 hours, get 2 bottles of cells every day, expect blue living cells count counting with platform, until till the cell count decline.

Cell colony doubling time (DT) calculation formula: DT (h)=t * lg2/ (LgNt-lgNo) t represents the incubation time that cell is in logarithmic phase, and No and Nt represent respectively the cell count of the initial sum termination of logarithmic phase.

The result of cell colony doubling time:

Table 2-3-1:ZQNK-29 Growth of Cells situation

T=4 * 24 hour (the 2nd day to the 6th day be in logarithmic phase);

Nt＝6.75×10 ⁶，lgNt＝6.8293；No＝1.04×10 ⁶，lgNo＝6.0170；

The cell colony doubling time is DT (h)=4 * 24 * 0.3010 ÷ (6.8293-6.0170)=35.58 hour.

The cell enlargement curve as shown in Figure 6.

(5) cell phenotype analysis

5.1 cell surface CD differentiation antigen is analyzed

The passage cell of taking the logarithm vegetative period, be collected in the centrifuge tube of 10ml, expect blue viable count with platform, cell is collected in streaming loading Guan Zhongfen 2 pipe of 5ml after PBS liquid is washed three times, a tube cell is with an amount of PBS liquid suspendible, for detection of the surface of cell membrane leukocyte differentiation antigen, another tube cell adds Triton100100ul, and room temperature leaves standstill that to add an amount of PBS liquid after 15 minutes resuspended, for detection of leukocyte differentiation antigen in the cell.Other gets the streaming loading pipe of 5ml, adds respectively the corresponding CD antibody of 20ul in every pipe, adds respectively 100ul (1 * 10 again ⁴) cell suspension, the lucifuge room temperature was placed 30 minutes.Add again 400ulPBS liquid, through flow cytometer (FACS Calibur U.S. company BD) sample detection, carry out interpretation of result with the cellquest analysis software.

Positive findings: HLA-DR (+); CycHLA-DR (+); CD3 (+); CycCD3 (+); CD10 (+).

Negative findings: CD56; CD56RO; CD16; CD45; CD45RO; CD34; CD33; CD38CD117; CD2; CD4; CD8; CD19; CD20; CD22; CD7; CD44; CD13; GPATDT.

5.2 Immunohistochemistry detects

5.2.1 cell block handling procedure

(1) with the cell cultures sample, with low speed autobalancing centrifuge (LD-Z5-2 Beijing) 3000 rev/mins, left the heart 10 minutes with per minute 3000 in centrifugal 5 minutes;

(2) getting the precipitation sample wraps up with the tealeaves filter leaf;

(3) get 10% neutral buffered formalin, fix 5 minutes (the quick organization ultrasonic processing instrument of seaquake health in the employing, water temperature arranges 70 ℃);

(4) acetone treatment is 4 minutes, 2 times;

(5) enter 60 ℃ of paraffin, waxdip 30 minutes;

(6) adopt the embedding of Leca JUNG embedding machine;

(7) adopt the section of Leca 2135 paraffin slicing machines, thickness 4 μ;

(8) 60 ℃ of baking boxs, roasting sheet 1 hour;

(9) adopt the dyeing of Leca ST5020 dyeing machinery;

(10) adopt Leca CV5030 mounting machine mounting;

5.2.2 immunohistochemical analysis

For determining the immunophenotype of this cell, application immunohistochemical methods (method) is listed as the cell block of making and puts mark, is respectively CD3, CD4, CD5, CD8, CD20, CD79a, CD16, CD56, GranB, CD45Ro, TIA-1, CD34, CD45/LCA, Perforin.

Concrete steps are:

(1) cell block is through routine dewaxing and aquation.

(2) 3%H ₂O ₂Deionized water soaked 10 minutes, with the blocking-up endogenous peroxydase.

(3) antigen retrieval is with pressure kettle spray vapour half a minute, and damping fluid is 0.01M PH6.0 citrate buffer.

(4) drip primary antibodie, hatched under the room temperature 60 minutes, the flushing of PBS damping fluid, 2 minutes * 3 times.

(5) drip Polymer Hdper, hatched under the room temperature 20 minutes, the flushing of PBS damping fluid, 2 minutes * 3 times.

(6) drip Polyperoxidase-anti-mowse/rabbit ZgG, hatched under the room temperature 30 minutes, the flushing of PBS damping fluid, 2 minutes * 3 times.

(7) dilution preparation DAB solution developed the color Microscopic observation 5～10 minutes.

(8) flushing with clean water, Hematorylin is redyed, conventional dehydration, transparent, mounting.

Used detection reagent PV-9000 Polymer Detection System is available from China fir biotech company in Beijing.Replace an antibody as negative control with PBS.

ImmunohistochemistryResults Results: CD3, CD10, CD20, TIA-1 are all positive; CD4, CD5, CD8, CD16, CD56, GranB, CD45RO, CD45, Perforin are all negative.

(6) cell chromosome experiment

The passage cell of 1) taking the logarithm vegetative period, in the cultivation of going down to posterity the day before yesterday of test, with 10ml RPMI-1640 (including rIL-2800U/ml) suspension culture at 37 ℃ of CO ₂Cultivated 24 hours in the incubator.

2) to add colchicine (Fluka import packing) to ultimate density be 0.8 μ g/ml the same day in test, 5 hours harvested cells of effect are (according to the difference of colchicine concentration in incubator, length that can the Accommodation time, that is: colchicine concentration is high, time effect is short, colchicine concentration is low, and time effect is long).

3) colchicine arrives action time, cell is blown and beaten gently move in the 10ml centrifuge tube, and 1000 rev/mins, centrifugal 5 minutes.

4) abandon supernatant liquor, be added in 37 ℃ of water tanks the 0.075M KCL hypotonic medium 8ml (visual cell what are measured and decide) of pre-temperature, in 37 ℃ of water tanks hypotonic 30 minutes.

5) the hypotonic time arrives, and the stationary liquid 1ml that immediately adding prepares in advance blows and beats gently mixing and plays the effect that pre-fixes, and 1000 rev/mins, centrifugal 5 minutes.(the preparation of stationary liquid: methyl alcohol: Glacial acetic acid=fresh preparation in 3: 1).

6) abandon supernatant liquor, add stationary liquid, blow and beat gently mixing, left standstill 15 minutes, centrifugal 5 minutes of centrifugal 1000rpm repeats 2 times so again.(fixing altogether 3 times).

7) film-making: careful abandoning supernatant, visual cell's amount stays the stationary liquid about 0.5ml, the high-order sheet that drips firmly dispels cell, and cross back and forth on the spirit lamp several lower after, keep flat nature airing (it is clean without oil that slide is wanted, and puts freezing being in store for).

8) repeatedly wash the sample slice, thin piece with front washing lotion before the dyeing, certainly do or dry up (front washing lotion preparation: Glacial acetic acid: methyl alcohol=3: 1).

9) coat skim with Giemsa stoste, leave standstill about 8 minutes (deciding with room temperature height), add with the PBS drop again and be paved with upper 10 minute of slice, thin piece that is added with staining fluid.

10) rinse well with a distilled water high position.

11) washing lotion (40% methyl alcohol) is dripped the son of developing a film repeatedly after, rinses well with distilled water again, waits to do or dry up.(the preparation of rear washing lotion: methyl alcohol 40ml+ distilled water 60ml).

12) counting is scattered and complete at the high power Microscopic observation, and confirms that karyomit(e) does not have the mitotic figure that runs off, and counts under oily mirror, amounts to several 100 mitotic figures, analyzes.

13) result: the human male, modal number is 46, shows as 2 times of somatocyte, and the part abnormal karyotype is arranged, karyotype distributes as shown in Figure 7.

(7) cells in vitro is to the lethal effect research of 5 strain tumour cells

ZQNK-29 cell and 5 strain tumour cells, through the cultivation of going down to posterity, after Growth of Cells was stable, the cell in the vegetative period of taking the logarithm through centrifugal collecting cell, used Hank ' s liquid to wash once again, counting, newly-built cell is quantitatively to 1 * 10 ⁵The cell suspension of/ml is for subsequent use, and 5 strain tumour cells are that 25: 1 and 50: 1 two concentration are diluted by effect target ratio.Get effector cell 100 μ l, about 1 * 10 by group ⁴Cells/well, target cell 100 μ l are sub-packed in 96 orifice plates, total amount 200 μ L, effector cell and target cell are all established control group.Establish 5 multiple holes for every group.Place 37 ℃ of CO ₂Cultivate in the incubator, get respectively rear 1,3,5,7,9 day cell of experiment, add CCK-8, culture plate is knocked gently to help mixing in the 10uL/ hole after adding.37 ℃ hatch 3 hours after, detect absorbancy (OD value) at the enzyme-linked immunoassay instrument, adopt dual wavelength to measure, detect wavelength 450nm, reference wavelength 630nm.Through repeated experiments, average.Detect newly-built cell to the lethal effect situation of 5 strain tumour cells, and each group of in time photomicrography is in the cellular form situation of different time points, experimental result as shown in Figure 8, (a) effect target ratio is 25: 1 visible obviously anti-tumor activities, and (b) effect target ratio is 50: 1 visible obviously anti-tumor activities.

Inhibitory rate of cell growth (IR) is calculated by following formula: cell proliferation inhibition rate (IR)=(the average OD value of the average OD value-treatment group of the negative control group)/average OD value of negative control group * 100%

(8) the cell nude mice becomes experiment in knurl and the anti-human lung cancer A549 nude mouse

Collect the passage cell of logarithmic phase, use Hank ' s liquid to wash once, make 4 * 10 ⁷The cell suspension of/ml.Be used for observing nude mice and become the knurl situation.People's lung cancer A549 cell of taking the logarithm vegetative period makes 4 * 10 ⁷The cell suspension of/ml is used for observing the anti-human lung cancer effect of ZQNK-29 cell situation.Experiment is divided into 3 groups of nude mices to be carried out, and is respectively simple ZQNK-29 groups of cells: get ZQNK-29 cell suspension 0.25ml (1 * 10 ⁷Cell) is inoculated in nude mice by subcutaneous; Simple people's lung cancer A549 group: get A549 cell suspension 0.25ml, (1 * 10 ⁷Cell) is inoculated in nude mice by subcutaneous; The ZQNK-29 cell adds A549 mixing group: get ZQNK-29 cell suspension and each 0.25ml of A549 cell suspension, co-inoculation is at nude mice by subcutaneous, and every nude mice all inoculates 2 points, and repeated experiments, experimental result as shown in Figure 9:

1). simple ZQNK-29 groups of cells: get 1 * 10 ⁷0.25ml the ZQNK-29 cell suspension, be inoculated in nude mice by subcutaneous, 2 points of every nude inoculation through the observation more than 2 months, have no lump growth.Experiment is negative findings (Fig. 9 a, 9b) through triplicate.

2). simple people's lung cancer A549 cell group: get 1 * 10 ⁷0.25ml people's lung cancer A549 cell suspension, be inoculated in nude mice by subcutaneous, 2 points of every nude inoculation, through the observation more than 1 month, visible lump growth, and increase in time, lump increases (Fig. 9 c, 9d).

3). people ZQNK-29 cell adds people's lung cancer A549 cell mixing group: get ZQNK-29 cell suspension and each 0.25ml of A549 cell suspension, co-inoculation is at nude mice by subcutaneous, every nude mice all inoculates 2 points, through the experimental observation more than 1 month, by the lump that begins to grow, disappear gradually after 15 days (Fig. 9 e, 9f).

Claims (1)

1. a strain people NK/T clone is characterized in that, names to be people NK/T clone ZQNK-29, and deposit number is CCTCC NO:C201278.

Images