CN105505876A - XL-2 human lung cancer highly metastatic cell line and applications - Google Patents
XL-2 human lung cancer highly metastatic cell line and applications Download PDFInfo
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Abstract
The invention belongs to the field of cytobiology, and discloses a human lung cancer XL-2 highly metastatic cell line, called XL-2sci for short. The human lung cancer XL-2 highly metastatic cell line is characterized by being prepared through the step that conventional lung cancer cells XL-2 cells are subjected to animal in-vivo cyclic screening, and the conventional lung cancer cells XL-2 cells are preserved in the China Center for Type Culture Collection (CCTCC), and have the preservation number CCTCC NO C201282. The human lung cancer XL-2 highly metastatic cell line has the advantages that (1) through the repeated in-vivo screening, a human lung cancer XL-2 highly metastatic cell line model and a human lung cancer XL-2 highly metastatic cell line are successfully established, the metastasis rate is high, the metastasis is stable, the intuition is good, and the targeting is strong; (2) with the human lung cancer XL-2 highly metastatic cell line provided by the invention, an ideal model is provided for the prevention and cure research and anti-metastasis treatment on lung cancer.
Description
Technical field
The present invention relates to cell biology, particularly a kind of people's lung cancer high-transfer cell system and purposes.
Background technology
Extremely complicated process that metastases is a multistage, multi-step, multifactor, polygene interact, from cancer cells from knurl parent depart from metastasis formed to stick with solution through sticking, protease hydrolysis, complex process and the mechanism such as motion.In malignant tumor patient body, the oncocyte entering lymphatic vessel or blood vessel can have a lot, but only has some oncocyte subgroup to be able to dominant growth, and obtain metastatic phenotype, these oncocytes with high metastatic potential just easily shift.Experimentation on animals also proves: transplant T241 sarcoma cell in C
57after BL/6 mouse, within 24 hours, just can look in blood and see oncocyte alive, but occur transfer to the 7th day rear; Radiolabeled tumour cell is injected animal blood vessels, enters circulation of blood after 24 hours, the oncocyte that can survive only 1%, and finally arrive at certain part produce secondary stove less than 0.1%.
At present, although adopt the method for screening successfully to establish high metastasis model by scholar both at home and abroad, such as: Krasagakis etc. once established cancerous cell line from cutaneous nerve through the Metastatic Lymph Nodes of endocrine cell cancer.Lee etc. establish cancerous cell line from prostate cancer endocranium metastasis.Seki etc. establish a clone from people's liver cancer Metastatic Lymph Nodes.But, the rare report successfully building transplanting metastasis model in desirable people's lung cancer body at present.Therefore this area also needs to research and develop people's lung cancer height metastasis model and clone further.
Current through the external artificial subculture of continuous print in addition, a lot of tumour cell can not maintain its most original biological characteristics.Therefore, its dlinial prediction ability of the metastasis model using this tumour cell to set up is lower.
Summary of the invention
The object of the present invention is to provide a strain people lung cancer XL-2 high-transfer cell system.
Another object of the present invention is the purposes providing this people lung cancer XL-2 high-transfer cell system.
In a first aspect of the present invention, provide a kind of people lung cancer XL-2 high-transfer cell system, be called for short XL-2sci, it is with conventional lung carcinoma cell XL-2 cell, obtains through the screening of animal body internal recycle; Described human lung carcinoma cell XL-2 cell is in China typical culture collection center preservation, and preserving number is CCTCCNOC201282, China typical culture collection center address: China. Wuhan. and Wuhan University, preservation date on June 19th, 2012.
In a preferred embodiment of the invention, the karyomit(e) of described people lung cancer XL-2 high-transfer cell system also has another dystopy except having two chromosome translocations occurring in XL-2 cell: der (9) (9pter → p11::1q11 → qter); In addition, a marker karyomit(e) is also had in XL-2 high-transfer cell.
In a preferred embodiment of the invention, the Lung metastases rate 90% of described people lung cancer XL-2 high-transfer cell system.
In a second aspect of the present invention, provide the purposes of described people lung cancer XL-2 high-transfer cell system, for screening Lung cancer metastasis related gene.
In a third aspect of the present invention, a kind of method of screening potential Lung cancer metastasis related gene is provided, described method comprises the steps: the genetic comparison will expressed in described people lung cancer XL-2 high-transfer cell system and human lung carcinoma cell XL-2, select the gene of up-regulated statistically in described people lung cancer XL-2 high-transfer cell system, this gene is potential Lung cancer metastasis related gene.
In another preference, the method also comprises: carry out further test cell line and/or animal experiment at obtained potential Lung cancer metastasis related gene, with select for lung cancer and shifted the gene of definite effect.
In a fourth aspect of the present invention, provide the purposes of described people lung cancer XL-2 high-transfer cell system, for screening medicine for anti transfer of tumor.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1 is XL-2 cell epithelia characteristic and chromosome karyotype analysis schematic diagram.
Fig. 2 is XL-2 cell II type alveolar origin and adenocarcinoma of lung feature schematic diagram.
Fig. 3 first and third, five takes turns the external migration of people lung cancer XL-2 high-transfer cell system, the invasive ability that screening obtains and compares schematic diagram.
Fig. 4 is XL-2 high-transfer cell chromosome karyotype analysis schematic diagram.
Fig. 5 lung cancer XL-2 cell of behaving compares schematic diagram with transfer ability in people lung cancer XL-2 high-transfer cell system body.
Fig. 6 behave between lung cancer XL-2 cell with people lung cancer XL-2 high-transfer cell system attack, move, multiplication capacity compare schematic diagram.
Fig. 7 is that colony formation detects individual cells multiplication capacity schematic diagram between people's lung cancer XL-2 cell and people lung cancer XL-2 high-transfer cell system.
Embodiment
The present inventor, through long-term research and screening repeatedly, finds a kind of lung cancer metastasis rate high and the people lung cancer XL-2 high-transfer cell system that grade malignancy is high, and described people lung cancer XL-2 high-transfer cell system is a kind of model of desirable research metastases.The present invention is completed based on this.There is certain heterogeneity in people's lung cancer high-transfer cell tumour cell, the metastatic potential between them there are differences.
The present invention carries out screening repeatedly, is intended to the people lung cancer XL-2 high-transfer cell system finding metastatic potential high.
Compared with primary tumor cell, the cell after screening may have some special proterties to impart their special transfer abilities.By finding these mechanism, design blocks target spot accordingly, namely can suppress transfer.Further, the tumour cell of high metastatic potential after screening of going down to posterity can have stronger aggressive and transitivity, easily adheres to, high yield lytic enzyme and have the characteristic of various anti-host immune defenses, thus also more easily shifts.
Therefore, the present inventor, from primary tumors cells, through too much generation screening, have found the tumour cell subbreed with special high transfer ability.Specifically, people lung cancer XL-2 high-transfer cell system of the present invention is by the following method, obtain through long-term analysis and screening: by people lung cancer XL-2 Transplanted cells to T cell, B cell, the NOD-SCID mouse of NK cell defect subcutaneous, ocal resection when tumour is grown to enough large, Pulmonary metastasis focuses is obtained in Mice Body, then building by cell subcutaneous vaccination → Subcutaneous tumor excision (screening only for front three-wheel) → Pulmonary metastasis focuses → subcutaneous transplantation → cell is repeatedly that many generation screenings obtain spontaneous high transfer animal model, set up corresponding high-transfer cell system simultaneously.This clone is named as people lung cancer XL-2 high-transfer cell system by the present invention, and described people lung cancer XL-2 high-transfer cell system is called for short XL-2sci, and it is with conventional lung carcinoma cell XL-2 cell, obtains through the screening of animal body internal recycle; Described conventional lung carcinoma cell XL-2 cell is in China typical culture collection center preservation, and preserving number is CCTCCNOC201282.
Set up the important means that human cancer cell is research human tumor in vitro.The vitro culture of tumour cell builds the larger engineering of strain formula difficulty, and it is lower that primary tumor tissue is directly used in the external chance of success being of building.The present invention builds using the subcutaneous transplantation knurl of people's lung carcinoma subcutaneous height metastasis model that fresh 5 repeated screenings in body obtain the knurl source being as vitro culture, the vigor of cancer cells can be strengthened on the one hand, newly-built clone can be made itself to have higher metastatic potential on the other hand.Building in the process being, the present inventor constantly gropes and adjusts culture condition and cultural method, ensures the relatively stable of culture environment simultaneously, successfully establishes people lung cancer XL-2 high-transfer cell system and survives.
Morphological observation shows; Described people lung cancer XL-2 high-transfer cell is typical epithelioid cell, and vitro growth rates is very fast, and the doubling time is 37.10 ± 4.90 hours, and the doubling time of people lung cancer XL-2 cell is 31.92 ± 2.73 hours.
Chromosome analysis confirms: people lung cancer XL-2 high-transfer cell also has another dystopy except having two chromosome translocations occurring in people lung cancer XL-2 cell: der (9) (9pter → p11::1q11 → qter); In addition, a marker karyomit(e) is also had in XL-2 high-transfer cell.
People lung cancer XL-2 high-transfer cell system applies
Present invention also offers people lung cancer XL-2 high-transfer cell system to transplant or the purposes of people lung cancer XL-2 high-transfer cell system, include but not limited to for the mechanism of research tumour (particularly lung cancer) transfer and/or invasion and attack; Screen potential Lung cancer metastasis related gene; Screen the medicine etc. that antitumor (particularly lung cancer) shifts.
Present invention also offers a kind of method of screening potential Lung cancer metastasis related gene, described method is the genetic comparison by the gene of expressing in described people lung cancer XL-2 high-transfer cell system and people lung cancer XL-2 cells, select the gene that XL-2 high-transfer cell ties up to statistically up-regulated, this gene is because potential Lung cancer metastasis related gene.
The expression (or raise or lower) detecting genes involved in cell is technology well known to those skilled in the art, include but not limited to: biochip technology, immunohistochemistry technology etc., such as the related gene expression of the expression conditions of XL-2 high-transfer cell and people lung cancer XL-2 cell can be compared, thus find out the gene with statistical significance differential expression.
The invention has the advantages that:
(1) by screening in body repeatedly, successfully establish people lung cancer XL-2 high-transfer cell system's model and people lung cancer XL-2 high-transfer cell system, its rate of transform is high, transfer is stable, intuitive is good, targeting is strong.
(2) people lung cancer XL-2 high-transfer cell system of the present invention is utilized, for the study on prevention of lung cancer and anti-metastatic therapy provide desirable model.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (NewYorK:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
1 material
1.1 Specimen origin
Patient, man, 45 years old, within 06 year, be diagnosed as right lung gland cancer in Shanghai City Ruijin Hospital, after right upper lung surgical blanking, 1 year right adrenal gland, two kidney, two lungs, liver, posterior peritoneum lymphoglandula extensively shift, simultaneously with a large amount of ascites.Uncontaminated ascites sample 100mL is got for cellular segregation, cultivation when surgical operation.All operations all follows human tissue sample using priciple.
1.2 laboratory animal
The male BALB/c nude mice of SPF level, mouse 6-8w in age, body weight 18-22g, is provided by Shanghai Inst. of Tumor, and production licence number is SCXK (Shanghai) 2012-0001; All mouse are all raised in barrier system, and feed and water are freely taken in, and occupancy permit number is SYXK (Shanghai) 2012-0001.
1.3 plasmid
PWPXL-eGFP-2A-CBGr99 plasmid, pA2 packaging plasmid, pMDG2 envelope plasmid is so kind as to give by national oncogene and genes involved key lab.HB101 competence bacterium is purchased from TAKARA company.
1.4 main antibody
Immunohistochemical experiment antibody used, in table 1.
Antibody used tested by table 1
Immunofluorescence experiment antibody used, in table 2.
Antibody used tested by table 2
2 methods
2.1 cellular segregation are cultivated
60ml the is pollution-free centrifugal 5min of ascites 1500 × g, abandon supernatant, the resuspended precipitation of PBS, 1500 × g centrifugal 5min, PBS again resuspended precipitation are for subsequent use.Gradient centrifugation is adopted to carry out cellular segregation.Percoll stock solution and 1.5MPBS carry out 9:1 and dilute and obtain 100%Percoll suspension, and this 100%Percoll suspension 0.15MPBS carries out diluting the Percoll parting liquid obtaining 60% and 40% respectively.Above-mentioned cell suspension, 60%, the centrifugal 30min of 40%Percoll parting liquid (volume ratio 1:1:1) 3000 × g, white cellular layer PBS between two-layer Percoll parting liquid washs centrifugal, containing the DMEM nutrient solution mixing cell precipitation of 10% foetal calf serum, 100U/mL penicillin and 100mg/mL Streptomycin sulphate, be placed in 37 DEG C, CO
2volume fraction is cultivate in the incubator of 5%.After several days, start there is Growth of Cells at the bottom of culture dish, go down to posterity through trysinization, fibrocyte quantity reduces gradually, and part XL-2 cell continues Secondary Culture and is used for subsequent experimental, and part is stored in liquid nitrogen container.
2.2 morphology tests
The XL-2 cell of taking the logarithm vegetative period, trysinization, is inoculated in 75cm after re-suspended cell
2in culture dish, when Growth of Cells degree of converging reaches about 80%, utilize phase microscope observation of cell form and take pictures.
2.3 chromosome karyotype analysis
3h before collecting cell, adds the colchicine 70 μ l of 20 μ g/ml in culture dish, makes its final concentration be 0.2 μ g/ml; Trypsin solution peptic cell, centrifugal; In centrifuge tube, add the 0.075mol/LKCL8ml of 37 DEG C of preheatings, with dropper repeatedly blow and beat, cell dispersion, put 25-30min in 37 DEG C of water-baths; Add the stationary liquid (methyl alcohol: Glacial acetic acid=3:1) of fresh configuration
[39]1ml-1.5ml pre-fixes, and room temperature leaves standstill 5min, centrifugal; Add stationary liquid 8ml, be placed in 37 DEG C of water-bath 30min, centrifugal, add stationary liquid 6ml and be placed on 2 DEG C of-8 DEG C of preservations and spend the night; Second day centrifugal after rejoin stationary liquid 0.5ml, with suction pipe cell suspension dropped in that precooled slide glass to cross spirit lamp dry; Spend the night in 60 DEG C of baking boxs aging; The aobvious band of dyeing, basis of microscopic observation analysis of accounts.
2.4 immunofluorescence
Cell dissociation counting is plated on slide glass, takes out slide glass in 24h, and PBS cleans, and 4% paraformaldehyde of precooling fixes 30min; PBS cleans 3 times; Add the penetrating 15min of 0.3%TritonX-100 room temperature; After PBS cleans 3 times, 3%BSA room temperature closes 2h; Primary antibodie incubated at room 1h; PBST washs 3 times; Add two anti-room temperature lucifuges and hatch 1h; PBST washs 3 times; DAPI contaminates core room temperature lucifuge and hatches 10min; Confocal laser scanning microscope is carried out after PBST washs 3 times.
2.5Westernblot
Extract protein, BCA method measures protein concentration, SDS-PAGE electrophoresis, wet method transferring film 50min, and 1h closed by room temperature 5% skimmed milk, primary antibodie 4 DEG C of overnight incubation, and PBST washs three times, and two anti-hatch 2h, luminous after PBST washs three times, development, fixing.
2.6 experimentation on animals
2.6.1 induce tumor test
Taking the logarithm XL-2 cell in vegetative period, is 1 × 10 with serum-free DMEM nutrient solution adjustment cell concn
7/ mL, 10 BALB/c-nu/nu mouse are every only in the nearly armpit subcutaneous vaccination 0.2mL cell suspension in back of the body right side, routine observation mouse subcutaneous tumors growing state and tumor formation rate.When subcutaneous tumors major diameter reaches 2cm, cut knurl, 10% neutral formalin is fixed, paraffin section, determines that subcutaneous tumors form and IHC analyze for follow-up H & E dyeing.
2.6.2 the screening of high-transfer cell system
Taking the logarithm XL-2 cell in vegetative period, is 1 × 10 with serum-free DMEM nutrient solution adjustment cell concn
7/ mL, 10 BALB/c-nu/nu mouse are every only in the nearly armpit subcutaneous vaccination 0.2mL cell suspensions in back of the body right side, and 4 weeks rear subcutaneously cuts knurl, to extend the Bearing Mice Life cycle, until mouse enter obviously become thin, moribund condition time, put to death mouse, getting its lung's Nodules, to be inoculated into an other mouse subcutaneous, when this subcutaneous tumors major diameter reaches 2cm, cut knurl, Ti Jingliu surface vascular tissue and reticular tissue, knurl body is cut into 0.1cm × 0.1cm × 0.1cm size, be placed in culture dish, 37 DEG C, 5%CO
2incubator in cultivate, add after two hours containing 10%FBS, 100IU/mL penicillin and 100 μ g/mL Streptomycin sulphates DMEM substratum.Reach until cell and after certain number, to inoculate that mouse is subcutaneous enters new round screening; Repeat three-wheel in this approach.Afterwards owing to shifting the shortening of required time, when fourth round and the 5th take turns screening, cut knurl without the need to subcutaneous, and directly use mouse lung Nodules to set up clone, subcutaneous without the need to again pulmonary nodule being inoculated into, built by subcutaneous become knurl and be.
And transfer in high-transfer cell body, multiplication capacity compares 2.6.3XL-2
20 BALB/c-nu/nu mouse stochastic averagina are divided into two groups, and it is 1 × 10 that XL-2 and high transfer two kinds of cells thereof all adjust cell concn with the DMEM nutrient solution not containing serum
7individual/mL, every mouse inoculation 0.2mL cell suspension is subcutaneous in the nearly armpit in back of the body right side, the growing state of routine observation mouse nutritional status, general activity and transplanted tumor and the rate of transform after subcutaneous vaccination.Until subcutaneous touch tumor mass after, start rule measure Subcutaneous tumor knurl footpath, four days are once, continuously measured 8 times, and draw growth curve.Knurl footpath measured by vernier callipers calculates gross tumor volume, and formula is:
Volume＝1/2×L×W
2
Wherein L is major diameter, and W is minor axis.Occur that when there being some animals in two groups that is emaciation terminates experiment on the 9th week.Take out mouse lungs, normal saline flushing, naked eyes detect lungs transfer case, after drawing sample surface physiological saline, take pictures, then put into the 15mL centrifuge tube containing 10% neutral formalin, fixing, dye for H & E, calculate the rate of transform and the metastatic nodules of each group.
2.7 paraffin sections, H & E dye
Subcutaneous transplantation knurl and lungs are after 10% neutral formalin is fixing, and routine paraffin wax embeds, and 4 μm of thick sections, H & E dyes, om observation.
2.8 immunohistochemical analysis
Roasting sheet is carried out in the section of being made by subcutaneous tumors tissue, the dewaxing of dimethylbenzene gradient and aquation; 3%H2O2 incubated at room 10min; PBS rinses 3 times; Antigen retrieval; PBS cleans 3 times, and 10% lowlenthal serum room temperature closes 30min; Add the primary antibodie of suitable Dilution ratio, 4 DEG C of overnight incubation; Add after PBS cleaning two anti-37 DEG C hatch 1h; PBS cleans 2 times; DAB develops the color, and resinene mounting, in basis of microscopic observation.Result judges: redye nucleus under mirror in blue ,/brown color brown in specificity be positive staining.
2.9 slow virus packagings, mensuration and infection
Packaging plasmid pA2, envelope plasmid pMDG2 and recombinant plasmid pWPXL-eGFP-2A-CBGr99 common transfection 293T cell are carried out slow virus packaging by the cell transfecting method of application Lipofectamine2000 mediation; Carry out slow virus titer determination subsequently; By XL-2 and high-transfer cell bed board thereof, in 24h, in each cell, add polybrene (final concentration is 6 μ g/mL), put into incubator and continue to cultivate.After 30min, in each hole, add 6.0 × 10
6unit virus; Change liquid after 8h to continue to cultivate; Cell GFP expression in each hole of fluorescence microscopy Microscopic observation.
2.10 animalcule noclilucences and fluorescence imaging detect mouse model tumor growth in vivo and transfer case
Mouse Nembutal sodium solution intraperitoneal injection of anesthesia, subsequently abdominal injection D-Luciferin working fluid; The detection of animalcule chemiluminescence imaging is carried out after 15min.Adopt fluorescence imaging mode, application 470nm excitation wavelength, 525nm emission wavelength, the time shutter is 5s, carries out small animal living body imaging fluorescence imaging and detects.
2.11Micro-computedtomography (Micro-CT) scans
Use Micro-CT (Skyscan1076, Belgium) to carry out monitoring XL-2 and high transfer group mouse lung transfer case thereof, take a normal mouse as negative control simultaneously.Micro-CT parameter is as follows: voltage 50kV, electric current 200 μ A, time shutter 130ms, angle gain 0.7o, 360o rotation sweep.Before shooting, mouse first uses 1% Nembutal sodium solution (10ml/kg body weight) intraperitoneal injection of anesthesia, and shooting terminates to carry out three-dimensional reconstruction to lung mechanics afterwards.
2.12Trans-well cell migration and Matrigel
Cell invasion experimental technique: by Matrigel glue by joining on Transwell on indoor film after 1:7 dilution, each cell 40 μ l, hatches 4h for 37 DEG C.Cell dissociation counts, and concentration is adjusted to 1 × 105/0.2ml, is inoculated in each upper room, adds 800 μ l containing the DMEM substratum of 10%FBS, hatch, take out cell after 18h in 37 DEG C of incubators in each lower room, fixing dyeing, and microscope is taken pictures counting.Cell migration assay is substantially identical with Matrigel method, but upper room does not need to add Matrigel glue, and cell concn is 5 × 10
4individual/0.2ml, incubation time is about 12h.
2.13CCK-8 measure
Cell proliferation adopts cellcountingkit-8 (CCK-8) reagent to detect.2000 cells are planted, three wells in the 96 every holes of orifice plate, and set time every day every hole adds 10 μ LCCK-8 and 100 μ LDMEM reagent mixtures, hatches 2h for 37 DEG C, detect 450nm wavelength absorption value, and METHOD FOR CONTINUOUS DETERMINATION draws growth curve in 7 days.
2.14FACS the analyzing and testing cell cycle
Trypsin digestion cell, centrifugal, PBS washes 2 times; Add 70% ethanol mixing of precooling (-20 DEG C) ,-20 DEG C are fixedly spent the night fixing, and second day centrifugal, and PBS washes cell once, adds dyestuff, and 4 DEG C of lucifuges leave standstill 30min; The flow cytomery cell cycle, by ModFitsoftware analyzing and testing result.
2.15 soft-agar clonings and Clone forming Test
Configuration 0.6% and 1% agar-agar soln; Get 7mL20%FBSDMEM and 7mL1%agar, mix, join in each hole of 2 piece of 24 orifice plate, 0.25mL/well, 4 DEG C solidify, for subsequent use; Take the logarithm cell in vegetative period, adjustment concentration is 1.0 × 10
5/ mL, adds 0.15mL two kinds of cell suspensions, 1.85mL20%FBSDMEM, 2mL0.6%agar solution respectively, fully mixes, for subsequent use; Cell suspension and agar mixture are added in each 12 holes of 24 orifice plates, 4 DEG C solidify 10min, put into 37 DEG C of incubators and continue to cultivate; After 3 weeks, take pictures under inverted microscope.
2.16 Clone forming Test
The cell of taking the logarithm vegetative period, adjusting its concentration is 1.0 × 104/mL, by 0.01mL/ hole, joins in each 12 holes in 24 orifice plates respectively, continues to cultivate in 37 DEG C of incubators; After 2 weeks, PBS washs 3 times, and 100% methyl alcohol room temperature fixes 30min; Viola crystallina room temperature dyeing 30min; Tap water cleans, and room temperature is dried, and basis of microscopic observation result is also taken pictures.
2.17 statistical analysis
Quantitative test result is expressed as means ± SD, between two groups, difference adopts student ' st-test to carry out analyzing (two-tailed, P<0.05 is that difference has statistical significance, during P<0.01, difference has remarkable statistical significance), the comparison between qualitative data adopts χ 2test.
3 results
3.1XL-2 cellular form and phenotype
From ascites, we are separated, have cultivated the new lung cancer cell line of a strain (XL-2).The cell of morphological observation showed individual layer, Polygons, similar Epithelial morphology is tightly affixed on (Figure 1A) bottom culture dish.For its epithelial phenotype, we take immunofluorescence and immunohistochemical experiment to detect the expression of epithelium mark pan-cytokeratin in cell and subcutaneous tumors tissue slice respectively, and result is all shown as the positive, as shown in Figure 1B, Fig. 1 C.XL-2 cell tumorigenesis rate is 100% (10 whole subcutaneous one-tenth knurl of mouse), and subcutaneous tumors organizes H & E to dye as shown in figure ip.
3.2XL-2 cell chromosome Karyotyping
We have carried out chromosome karyotype analysis to XL-2 cell.As referring to figure 1e, 50 at Stochastic choice are in the cell of mitosis metaphase, and its chromosome number is distributed between 44 ~ 55 more, have 51 and 53 chromosomal cells and are in the great majority.Der (1) (q11 → q11::qter) and der (14) (14pter → q11.2::1q12 → qter) are the topmost two kinds of chromosome translocations of XL-2 cell, and in its karyogram, we can also see that a large amount of karyomit(e) increases, disappearance simultaneously.Therefore, XL-2 cells show has gone out an important feature of tumour cell: chromosomal unstable and mitotic abnormality.In addition, because mouse tool has plenty of 40 acrocentric chromosomes, unlike this, the results of karyotype of XL-2 cell also show its human origin.
3.3 lung adenocarcinoma cell CHARACTERISTICS IDENTIFICATION
SP-C albumen is produced by II type alveolar epithelial cells (AECII), is considered to a mark of AECII.Therefore, we adopt immunofluorescence and Westernblot to detect the expression of XL-2 cell SP-C albumen.As shown in Figure 2 A, immunofluorescence is shown as the positive; Fig. 2 B shows that XL-2 cell and A549 cell (originating from AECII, positive control) SP-C albumen have similar secretion level; More than proving that XL-2 origin of cell is in AECII, is a lung cancer cell line.Bibliographical information, CEA and Tif-1 is usually used in the diagnosis of adenocarcinoma of lung, and therefore, we adopt ImmunohistochemistryMethods Methods to detect the expression of these two kinds of albumen in subcutaneous tumors tissue caused by XL-2 cell.Result display (Fig. 2 C), during CEA and Tif-1 dyeing, tumour cell shows as corresponding tenuigenin and nucleus positive reaction respectively.Therefore, the biological characteristics of XL-2 cell meets the diagnosis of clinical patients.
The foundation of 3.4XL-2 high-transfer cell system
First and third, five mouse lung rates of transform when taking turns screening and occur that the transfer time is as shown in table 3, the cells in vitro invasion and attack obtained in three-wheel screening, transfer ability are more as shown in Figure 3.Drawn by above result, the 5th when taking turns screening, and the mouse lung rate of transform is the highest and the 5th takes turns cell line in vitro invasion and attack that screening sets up, transfer ability is the strongest.Therefore, utilize XL-2 clone in body, screen us and successfully establish the XL-2 clone that a strain has high transfer ability, be called for short XL-2sci.Carry out chromosome karyotype analysis to XL-2 cell, as shown in Figure 4,50 of Stochastic choice are in the cell of mitosis metaphase, and its chromosome number is distributed between 45 ~ 58 more, have 49 and 51 chromosomal cells and are in the great majority.XL-2 high-transfer cell also has another dystopy except having two chromosome translocations occurring in XL-2 cell: der (9) (9pter → p11::1q11 → qter); In addition, a marker karyomit(e) is also had in XL-2 high-transfer cell.Also similarity is to a certain degree there is in above result while showing to there is heterogeneity between XL-2 and its high-transfer cell.In the body of two kinds of cells, transfer ability as shown in Figure 5, and the transfer of XL-2 height transfer group mouse lung obviously (Fig. 5 A); Pathological section shows same result (Fig. 5 B).Statistical analysis (Fig. 5 C) shows XL-2 height transfer group mouse lung Nodules number obviously more than XL-2 group (P<0.01), and difference has statistical significance.XL-2 height transfer group and the XL-2 group lung rate of transform are respectively 9/10 and 3/10 (P<0.05) (Fig. 5 D).Therefore, XL-2 high-transfer cell system has transfer ability in stronger body than XL-2 cell.
The mouse lung rate of transform and occur transfer time during screening in table 3 first and third, five wheel body
3.5 optical imageries and micro-CT compare transfer ability in XL-2 and high-transfer cell body thereof
In order to compare transfer ability in XL-2 and high-transfer cell body thereof more intuitively, we are according to aforementioned manner subcutaneous vaccination XL-2-GFP-Luc and high transfer two kinds of cells thereof.Occur when there being some animals in two groups becoming thin, dying time (after 9 weeks), we use invivoBLI to monitor its Lung metastases situation.But, because the signal from subcutaneous tumors is too strong so that the observation (Fig. 5 F (b)) of impact to lung's transfer case.Therefore, we use micro-CT instead and carry out lung's transfer case detection.As shown in fig. 5e, the transfer of XL-2 height transfer group mouse lung obviously; Also there is Lung metastases to a certain degree in XL-2 group mouse, but metastasis degree is limited.Micro-CT puts to death mouse, cuts open lung, utilize exvivoBFI to observe two groups of mouse lung transfer case further after observing and terminating.ExvivoBFI and micro-CT observations is consistent, and as shown in Fig. 5 F (a), signal mainly concentrates on XL-2 height transfer group mouse lung tissue, and obviously more weak at XL-2 group signal.Therefore, in XL-2 high-transfer cell body, transfer ability is obviously better than XL-2 cell.
3.6 vitro invasions, transfer ability compare
Test us by Trans-well and analysis is compared to XL-2 and high-transfer cell vitro invasion thereof, transfer ability.As shown in figure (6A), in invasion and attack test, XL-2 high-transfer cell number is obviously more than XL-2 cell; Migration is tested us and be have also been obtained similar result; More than show, XL-2 high-transfer cell has stronger invasion and attack, transfer ability equally than XL-2 cell in vitro.Therefore, screen in body, we successfully obtain the clone that one has high invasion and attack, transfer ability.
2.3.7 multiplication capacity compares
Growth curve and colony formation is adopted to compare XL-2 and high-transfer cell multiplication capacity thereof.XL-2 cell doubling time is 31.92 ± 2.73h and the doubling time of XL-2 high-transfer cell is 37.10 ± 4.90h (P<0.05), and XL-2 high-transfer cell proliferation rates in vitro is considerably slower than XL-2 cell (Fig. 6 B) as can be seen here.Compared two kinds of cell proliferation in vivo abilities by subcutaneous tumors growth curve, we obtain analog result (Fig. 6 C).From Fig. 5 F (b) invivoBLI, we also can see that XL-2 group mouse subcutaneous tumors volume is obviously greater than XL-2 height transfer group mouse subcutaneous tumors volume.About have 35.28% to be in the S phase in Fig. 6 D, cell cycle experiment display XL-2 cell, 8.54% is in the G2/M phase; And the cell proportion being in S phase and G2/M phase in XL-2 high-transfer cell is respectively 28.15% and 18.79%; Statistical analysis display is in two kinds of cell proportion differences of S phase and G2/M phase all has statistical significance.During soft-agar cloning is formed and tests, but the high transfer of XL-2, XL-2 two kinds of cells can form clone clonality obvious difference.The plastidogenetic colony counts of XL-2 and diameter obviously more than be greater than XL-2 high-transfer cell (Fig. 6 E (ab)).Carry out statistical analysis to the colony counts that the two is formed, difference has statistical significance (Fig. 6 E (c)).In colony formation, we obtain same conclusion (Fig. 7).
Claims (6)
1. a people lung cancer XL-2 high-transfer cell system, be called for short XL-2sci, it is characterized in that, be with conventional lung carcinoma cell XL-2 cell, obtains through the screening of animal body internal recycle; Described conventional lung carcinoma cell XL-2 cell is in China typical culture collection center preservation, and preserving number is CCTCCNOC201282.
2. people lung cancer XL-2 high-transfer cell system as claimed in claim 1, it is characterized in that, the karyomit(e) of described people lung cancer XL-2 high-transfer cell system also has another dystopy except having two chromosome translocations occurring in XL-2 cell: der (9) (9pter → p11::1q11 → qter); In addition, a marker karyomit(e) is also had in XL-2 high-transfer cell.
3. people lung cancer XL-2 high-transfer cell system as claimed in claim 1, it is characterized in that, the Lung metastases rate of described people lung cancer XL-2 high-transfer cell system is 90%.
4. one kind is screened the method for potential Lung cancer metastasis related gene, described method comprises the steps: the genetic comparison will expressed in people lung cancer XL-2 high-transfer cell system according to claim 1 and human lung carcinoma cell XL-2, select the gene of up-regulated statistically in described people lung cancer XL-2 high-transfer cell system, this gene is potential Lung cancer metastasis related gene.
5. the method for claim 1, is characterized in that, also comprises: carry out further test cell line and/or animal experiment at obtained potential Lung cancer metastasis related gene, with select for lung cancer and shifted the gene of definite effect.
6. the purposes of described people lung cancer XL-2 high-transfer cell system according to claim 1, is characterized in that, for screening medicine for anti transfer of tumor.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998414A (en) * | 2018-01-17 | 2018-12-14 | 上海中医药大学附属龙华医院 | A kind of human lung cancer circulating tumor cell system and its application |
| CN109837306A (en) * | 2019-03-20 | 2019-06-04 | 江南大学附属医院（无锡市第四人民医院） | Contain the excretion body and its preparation method and application of miRNA-204-5p |
| CN113817684A (en) * | 2021-09-30 | 2021-12-21 | 天津医科大学总医院 | Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof |
| CN114634913A (en) * | 2022-02-27 | 2022-06-17 | 复旦大学 | Method for constructing tumor dura mater metastasis model |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434935A (en) * | 2007-11-14 | 2009-05-20 | 上海市肿瘤研究所 | Construction of human lung cancer highly metastatic cell lines SPC-A-1sci |
| CN101775369A (en) * | 2008-11-04 | 2010-07-14 | 复旦大学附属肿瘤医院 | High-lung-metastasis human breast cancer cell line |
| CN101906400A (en) * | 2009-06-08 | 2010-12-08 | 南京大学 | Spontaneous high-metastasis tendency mice lung cancer cell line and establishment method thereof |
| CN102071168A (en) * | 2009-11-24 | 2011-05-25 | 上海市肿瘤研究所 | High metastasis human breast carcinoma cell subline and establishing method thereof |
-
2014
- 2014-09-26 CN CN201410504969.2A patent/CN105505876A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434935A (en) * | 2007-11-14 | 2009-05-20 | 上海市肿瘤研究所 | Construction of human lung cancer highly metastatic cell lines SPC-A-1sci |
| CN101775369A (en) * | 2008-11-04 | 2010-07-14 | 复旦大学附属肿瘤医院 | High-lung-metastasis human breast cancer cell line |
| CN101906400A (en) * | 2009-06-08 | 2010-12-08 | 南京大学 | Spontaneous high-metastasis tendency mice lung cancer cell line and establishment method thereof |
| CN102071168A (en) * | 2009-11-24 | 2011-05-25 | 上海市肿瘤研究所 | High metastasis human breast carcinoma cell subline and establishing method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 周清华等: "人大细胞肺癌细胞系NL9980和L9981的建立及其生物学特性研究", 《中国肺癌杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998414A (en) * | 2018-01-17 | 2018-12-14 | 上海中医药大学附属龙华医院 | A kind of human lung cancer circulating tumor cell system and its application |
| CN109837306A (en) * | 2019-03-20 | 2019-06-04 | 江南大学附属医院（无锡市第四人民医院） | Contain the excretion body and its preparation method and application of miRNA-204-5p |
| CN113817684A (en) * | 2021-09-30 | 2021-12-21 | 天津医科大学总医院 | Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof |
| CN113817684B (en) * | 2021-09-30 | 2023-09-19 | 天津医科大学总医院 | Chinese lung adenocarcinoma cell line with high lymph node metastasis potential and application thereof |
| CN114634913A (en) * | 2022-02-27 | 2022-06-17 | 复旦大学 | Method for constructing tumor dura mater metastasis model |
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