CN103756968A - Spinal highly-metastatic human lung adenocarcinoma cell strain and construction method and application thereof - Google Patents
Spinal highly-metastatic human lung adenocarcinoma cell strain and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses spinal highly-metastatic human lung adenocarcinoma cell strain A549L6, the cell strain is preserved in China Center for Type Culture Collection (CCTCC) in April 25, 2013 with the accession number of CCTCC No:C201346. The invention also discloses a construction method and application of the spinal highly-metastatic human lung adenocarcinoma cell strain. The spinal highly-metastatic human lung adenocarcinoma cell strain is high in spinal metastatic rate, and can be widely applied to metastatic study models of lung adenocarcinoma, studies on metastatic mechanisms of the lung adenocarcinoma and screening and preparing of anti-metastasis drugs.
Description
Technical field
The present invention relates to oncobiology technical field and microorganism animal cell line technical field, be specifically related to a kind of backbone high-transfer human lung adenocarcinoma cell line and construction process and application.
Background technology
Cancer is the No.1 killer of current threat human health, and according to World Health Organization's incomplete statistics, the patient that cancer is died from the whole world every year reaches more than 700 ten thousand people.Lung cancer is one of cancer that current whole world M & M is the highest.Annual nearly 170000 people of the U.S. die from lung cancer, account for the 30%[1 of whole cancer mortality sum].Along with economic in recent years rapid growth, the sickness rate of China's lung cancer starts to be multiplied, and approximately has every year 400000 people to be made a definite diagnosis and suffers from lung cancer, and sickness rate, up to 61.4/10 ten thousand, becomes the maximum country of patients with lung cancer in the world.Expect 2025, the number of dying from lung cancer is often only by China will approach 1,000,000 people.
The same with other malignant tumours, the transfer of lung cancer is to cause the first cause [2] for the treatment of unsuccessfully with death.Because lung cancer early symptom is not obvious and disease progression speed is very fast, when extremely medical, approximately there is 40% patient to occur distant metastasis [3].Bone is one of the most common metastasis site of lung cancer, and the progressive stage lung cancer of about 30-40% occurs that bone shifts [3].
Backbone is one of the most common bone metastasis site of lung cancer, accounts for the 50%[4 that bone shifts].The complication such as the transfer of lung cancer backbone often causes that ostalgia, hypercalcemia, pathologic fracture, spinal compression are even paralysed, have a strong impact on patient's life quality and lifetime, accelerate patient's dead process, bring heavy burden [5,6] to patient and family thereof and society.Although the complex therapy such as the surgical excision of code requirement, chemicotherapy, Diphosphonate, immunotherapy clinically, the median survival interval that lung cancer backbone shifts was still hovered in about half a year, became medical circle a great problem urgently to be resolved hurrily.
In recent years, biomedical sector has been obtained the development of advancing by leaps and bounds, but to lung cancer backbone shift to occur, development mechanism still understands very less, lacks correlative study.Its major cause is to lack reliable lung cancer backbone and shifts animal model.Therefore, be necessary to set up a kind of meet Clinical symptoms, reproducible, can real-time tracing monitoring, reliable and stable lung cancer backbone shifts animal model.The molecular mechanism that cancer backbone is shifted, clinical diagnosis and treatment will have great promoter action, for further finding new drug targets and developing corresponding medicine, provide research tool, the treatment of shifting to lung cancer and bone is brought to more wide prospect.
A549 cell strain (ATCC CCL-185), within 1972, by the male sex white man's of 58 years old of the employings such as Giard cancerous lung tissue, through vitro culture, set up, be one of the most widely used lung adenocarcinoma cell line [7], the present invention adopts the A549 cell strain in ATCC source to build lung cancer backbone metastasis model as source cell.
The background technology relevant with the present invention comprises below with reference to document.
[1]Dela Cruz CS，Tanoue LT，Matthay RA.Lung cancer：epidemiology,etiology,and prevention.Clin Chest Med.2011Dec；32(4)：605-44.
[2]Pantel K，Alix-Panabières C.Circulating tumour cells in cancer patients：challenges and perspectives.Trends Mol Med.2010Sep；16(9)：398-406.Epub2010Jul29。
[3]McErlean A，Ginsberg MS.Epidemiology of lung cancer.Semin Roentgenol[J].2011Jul；46(3)：l73-7.
[4]Tsuya A，Kurata T，Tamura K，Fukuoka M.Skeletal metastases in non-small cell lung cancer：a retrospective study.Lung Cancer.2007Aug；57(2)：229-32
[5]Coleman RE.Skeletal complications of malignancy.Cancer1997；80：1588-94.
[6] Xiao Jianru. backbone metastatic carcinoma Surgical Strategy is familiar with to [J] again. JISUI ZAZHI AUTHOR AND, 2011,21 (7): 531-532.
[7]Giard DJ，Aaronson SA，Todaro GJ，et al：In vitro cultivation of human tumors：establishment of cell lines derived from a series of solid tumors.J Natl Cancer Inst51：1417-1423，1973
[8]Kang Y，Siegel PM，Shu W，etal.A multigenic program mediating breast cancer metastasis to bone.Cancer Cell.2003Jun；3(6)：537-49.
Summary of the invention
The invention discloses a kind of backbone high-transfer human lung adenocarcinoma cell line and construction process and application.
One of object of the present invention is to provide a kind of backbone high-transfer human lung adenocarcinoma cell line, and this cell strain is deposited on April 25th, 2013 the Chinese Typical Representative culture collection center (being called for short CCTCC) that is positioned at Wuhan, and preserving number is CCTCC No:C201346.
The present invention is usingd human lung adenocarcinoma cell line A549 cell strain (ATCC CCL-185) as source cell, by the method for nude mice Vivo Studies on Screening, builds the human lung adenocarcinoma cell line that a strain has the high transfer abilities of organ such as backbone, brain, called after A549L6.
The detection method that detection backbone high-transfer cell strain A549L6 biological characteristics adopts is with following step B.Nude mice left ventricle injection A549L6 cell, and use IVIS live body detection system and x-ray, the image detection equipment such as micro-CT, MRI to monitor its transfer performance, and use HE pathological section to confirm, adopt the experiment in vitro such as wound healing, transwell to detect its migrate attribute, adopt cell proliferation experiment to detect its multiplication characteristic in vitro.
A549L0 compares with source cell strain, and backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 cell strain of the present invention has following biological characteristics:
(1) through wound healing and transwell, detect A549L6 cell strain, observe 36 hours, the ability of migrating of A549L6 cell is respectively 11.5 times and 22.4 times of source cell A549L0; Experimentation on animals shows that its backbone rate of transform is up to 80% (8/10), compared with the backbone rate of transform (7.7%, 1/13) of source cell A549L0, significantly improves.
(2) the distant metastasis of human rate after A549L6 cell strain nude mice left ventricle injection is 100%, 70% for many organs, to shift simultaneously.The bone rate of transform is 90% (9/10), and the backbone rate of transform is 80% (8/10), and the four limbs rate of transform is 30% (3/10); The brain rate of transform is 60% (6/10), and wherein, the median time that backbone shifts is 50 days (40-106).
(3) backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 cell strain of the present invention is through luciferase mark, can pass through for example IVIS live body Real-Time Monitoring tumour growth, transfer case in vivo of living imaging instrument equipment, and backbone transfer signal within 6.00 ± 0.63 days, be detected early than x-ray test set.
(4) Micro-CT, MRI all can successfully detect A549L6 backbone metastatic lesion.After described cell strain shifts, can detect backbone metastasis through x-ray, micro-CT, MRI, can detect backbone and brain metastasis through HE pathological section.
(5) A549L6 is transferred to after backbone, has to human cancer backbone and shifts the rear similar symptom of spinal compression that occurs.According to the development of the course of disease, it can be divided into successively to five obvious observable nodes: tail mops floor, instep lands gait, the walking step state of sweeping the floor, paralysis, death.It betides respectively 1.43 ± 1.72 days, 3.71 ± 2.06 days, 8.29 ± 2.06 days, 11.57 ± 3.69 days, 14.29 ± 3.59 days after living imaging system discovery backbone metastasis.
(6) A549L6 is transferred to after backbone, its body weight significantly declines, during with left ventricle injection A549L6 cell, tail mops floor, instep lands gait, the walking step state of sweeping the floor, paralysis, dead corresponding body weight are respectively 17.84 ± 0.83g, 26.20 ± 1.85g, 24.96 ± 1.52g, 22.78 ± 1.44g, 21.02 ± 1.72g, 18.76 ± 1.73g.
The nutrient solution of cell strain of the present invention, go down to posterity, frozen, recovery condition is all identical with A549, in-vitro multiplication ability is similar to A549; There is notable difference in cellular form and the A549L0 of A549L6, A549L0 and A549 plesiomorphism, major part is the poor cell clone of light transmission, only contain the cell clone that part light transmission is strong, and A549L6 (under Figure 13) is almost the cell clone that light transmission is strong.
Another aspect of the present invention is to provide the construction process of described backbone high-transfer human Adenocarcinoma of lung cell line A549 L6, comprises the steps:
(A) human lung adenocarcinoma cell line A549 cell is inoculated in to culture plate, carries out PGL4 plasmid transfection, the cell clone that transfection is obtained by G418 screening carries out luciferase assays, obtains the cell clone A549L0 of the luciferase expression positive;
(B) A549 expressing luciferase positive cell clone A549L0 suspension is injected in nude mice left ventricle;
(C) screening backbone transfer cell strain, selects the destruction of bone of backbone metastasis the most by force and the strongest metastatic tumour cell of fluorescence carries out amplification in vitro cultivation; The cell that cultivation is obtained is injected in nude mice left ventricle by step (B) method, then carries out amplification in vitro cultivation by step (C); The screening of repetition three-wheel;
(D) the A549 cell of fluorescent mark positive step (C) being obtained carries out mono-clonal to be selected, and amplification in vitro is cultivated, and obtains backbone high-transfer human lung adenocarcinoma cell cell line A549 L6 of the present invention.
Concrete steps comprise as follows:
Steps A, luciferase (Luciferase) mark human lung adenocarcinoma cell line A549
By human lung adenocarcinoma cell line A549 (ATCC CCL-185) cell according to 1.0 * 10
5/ hole is inoculated in 24 well culture plates, carries out PGL4 plasmid (Promega Biotech Co.Ltd) transfection next day; Transfection step and method are carried out according to Lipofectamin2000 reagent specification sheets; After transfection 24h, change liquid, add 600ug/ml G418 (purchased from Merck company) screening three weeks, obtain positive cell clone; And the clone who obtains is carried out to luciferase (Luciferase) fluorescence activity and measure.
Step B, left ventricle injecting method are set up human lung adenocarcinoma Metastatic nude model
The method [8] of setting up with reference to structure bone metastases of lung cancer models such as Kang, by the A549 cell (called after A549L0) of luciferase (Luciferase) the fluorescent mark positive, is adjusted into 1.0 * 10
5the cell suspension of/100ul, gets 100ul and is injected in nude mice left ventricle, and uses the monitoring adenocarcinoma of lung backbone transfer case such as IVIS living imaging system and x-ray imaging system.
Step C, screening backbone transfer cell strain
Get the destruction of bone of backbone metastasis is the strongest and fluorescence is the strongest metastatic tumour cell through vitro culture, amplification, culture condition is that DMEM adds 10% (V/V) foetal calf serum, 37 ℃, and 5%C0
2/ 95% air, saturated humidity is cultivated; The cell of cultivating gained is inoculated in to the recycle system of nude mice according to step B, according to step C amplification in vitro, with method, repeats three-wheel screening.
Step D, mono-clonal are selected
The A549 cell of the fluorescent mark positive that step C is taken out carries out mono-clonal to be selected, and amplification in vitro, sets up cell strain, called after A549L6, changes nutrient solution every other day, within every three days, goes down to posterity once, conventional frozen, recovery, obtain backbone high-transfer human Adenocarcinoma of lung cell line A549 L6.
Another aspect of the present invention is to provide described backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 and at the backbone of preparing human lung adenocarcinoma, shifts the application of animal model.The preparation process of aforementioned backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 is the present invention and shifts the application in animal model structure at the backbone of human lung adenocarcinoma.The present invention also provide the application of described backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 in bone metastases of lung cancer Mechanism Study, with and the application of the anti-human adenocarcinoma of lung medicine of preparation, with and in screening with assess the application in anti-human adenocarcinoma of lung medicine.
Backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 of the present invention has the advantages that the backbone rate of transform is high, be a kind of more preferably adenocarcinoma of lung bone transfer research model, it will have wide prospect in disclosing the metastasis of adenocarcinoma of lung and the application of anti-metastasis drug screening.
Accompanying drawing explanation
Backbone high-transfer human lung adenocarcinoma cell line A549 L6 of the present invention, is preserved in Chinese Typical Representative culture collection center (being called for short CCTCC, China, Wuhan, Wuhan University) on April 25th, 2013, and preserving number is CCTCC No:C201346.
Fig. 1 represents the luciferase expression of A549L0 and A549L6.Through IVIS living imaging system, detect, the equal stably express fluorescence of A549L0 and A549L6, after multi-turns screen, the luciferase expression value of A549L6 is significantly higher than A549L0.
Fig. 2 represents the live body detected result that lung cancer backbone shifts.Live body detected result shows 100% mouse generation distant metastasis of human, and mostly is many places organ and shifts simultaneously, and 80% mouse generation backbone shifts, and 60% brain occurs shifts.
Fig. 3 represents that x-ray imaging system detects A549L6 the X-ray film image that backbone shifts occurs.
Fig. 4 represents that micro-CT detects A549L6 CT imaging performance and the three-dimensional reconstruction image that backbone shifts occurs.
Fig. 5 represents that the MRI image that backbone shifts occurs A549L6.
Fig. 6 represents A549L6 backbone metastasis histology profile section HE staining pathologic section figure.
Fig. 7 represents A549L6 backbone metastasis histology square section section HE staining pathologic section figure.
Fig. 8 represents that spinal compression symptom schematic diagram shift to occur A549L6 backbone, the Node Events mop floor along with tail occurs PD successively, instep landing gait, the walking step state of sweeping the floor, paralysis, death etc. can be easy to observation.
Fig. 9 represents that A549L6 backbone shifts generation spinal compression symptom schematic diagram, along with tail occurs PD successively, mop floor, instep lands gait, the walking step state of sweeping the floor, paralysis, death etc. can be easy to the Node Events of observing, along with PD, also there is noticeable change in its body weight thereupon.
Figure 10 represents that wound healing tests the cell migration ability that compares A549L6 and A549L0.A left side is schematic diagram, and the right side is statistical graph.
Figure 11 represents that Transwell tests the cell migration ability that compares A549L6 and A549L0.A left side is schematic diagram, and the right side is statistical graph.
Figure 12 represents that A549L6 is similar to A549L0 and A549 multiplication capacity in vitro.
Figure 13 represents there is notable difference in the cellular form of A549L6 and A549L0, A549, and A549L6 is almost the cell clone that light transmission is strong, and A549L0, A549 all only contain the cell clone that part light transmission is strong.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Do not deviating under the spirit and scope of inventive concept, variation and advantage that those skilled in the art can expect are all included in the present invention, and to take appending claims and equivalent thereof be protection domain.Implement process of the present invention, condition, reagent, experimental technique etc., except mentioning specially below explanation, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.Below in conjunction with embodiment and accompanying drawing, describe the present invention.But the following example should not regarded limitation of the scope of the invention as.
Embodiment 1: the structure of backbone high-transfer human Adenocarcinoma of lung cell line A549 L6
By human lung adenocarcinoma cell line A549 cell strain (ATCC CCL-185) cell according to 1.0 * 10
5/ hole is inoculated in 24 well culture plates, carries out P6L4 plasmid (Promega Biotech Co.Ltd) transfection next day; Transfection step and method are carried out according to Lipofectamin2000 reagent specification sheets; After transfection 24h, change liquid, add 600ug/ml G418 (purchased from German merck company) screening three weeks, obtain positive cell clone; And the clone who obtains is carried out to luciferase (Luciferase) fluorescence activity and measure.
Human lung adenocarcinoma cell line A549 cell suspension 0.1ml (is contained to 1x10
5individual cell) be injected in nude mice left ventricle, and use IVIS living imaging system and x-ray imaging system monitoring adenocarcinoma of lung backbone transfer case.Nude mice is purchased from (Shanghai western pul-Bi Kai laboratory animal company limited), and product are nu/nu nude mice, male, in age in 4-6 week, raises in SPF level environment.
The method that cell is injected by left ventricle is inoculated in after nude mice blood circulation, anatomy experiment animal, get the destruction of bone of backbone metastasis is the strongest and fluorescence is the strongest metastatic tumour cell through vitro culture, amplification, nutrient solution is that DMEM (gibico) adds 10% (v/v) foetal calf serum (Hyclone, Utah, USA), 37 ℃, 5%CO
2/ 95% air, saturated humidity is cultivated.
The cell of cultivating gained is inoculated in to the recycle system of nude mice according to above-mentioned steps, according to this step amplification in vitro, with method, repeats three-wheel screening.
The A549 cell of the fluorescent mark positive that above-mentioned steps is taken out carries out mono-clonal to be selected, and amplification in vitro, sets up cell strain, called after A549L6, changes nutrient solution every other day, within every three days, goes down to posterity once, conventional frozen, recovery, obtain backbone high-transfer human Adenocarcinoma of lung cell line A549 L6.
Embodiment 2: the Characteristics Detection of backbone high-transfer human Adenocarcinoma of lung cell line A549 L6 of the present invention
The cell line A549 L6 of the present invention that serial experiment obtains embodiment 1 structure routinely detects:
1, after cell routine trysinization is centrifugal, density is adjusted into 1x10
6/ ml, gets 100ul/ hole and adds black 96 orifice plates, carries out successively doubling dilution, and each gradient is established 2 multiple holes.Every hole adds the luciferase substrate 100ul of 300 μ g/ml, within 10 minutes, be placed on IVIS living imaging system and take (Caliper Life Sciences IVIS Lumina II, model: DS934N-BV-286), time shutter is 5 minutes, shows after testing the equal stably express luciferase of A549L0 and A549L6.As shown in Fig. 1 left side, through too much taking turns after Vivo Studies on Screening, when cell count is 1x10
6time, A549L6 luciferase expression value is approximately 5.512 times of A549L0; When cell count dilution is 5x10
4time A549L0 almost without macroscopic fluorescence, and A549L6 is until lower than 1.25x10
4the macroscopic fluorescence of Shi Caiwu.Fig. 1 right side is depicted as the luciferase expression value curve of A549L6 under each cell density condition.As seen from Figure 1, through IVIS living imaging system, detect, the equal stably express fluorescence of A549L0 and A549L6, after multi-turns screen, the luciferase expression value of A549L6 is significantly higher than A549L0.
2, get 1x10
5cell (0.1ml) is injected in nude mice left ventricle, within latter 25 days, starts to carry out the live body detection of lung cancer backbone transfer in injection.Method is: abdominal injection injected fluorescein enzyme substrates 200ul/ is (D-luciferin only, potassium salt, 15mg/ml, sigma), within 10 minutes, be placed on IVIS living imaging system and take (Caliper Life Sciences IVIS LuminaII, model: DS934N-BV-286), the time shutter is 5 minutes.As shown in Figure 2, the distant metastasis ability of cell line A549 L6 of the present invention significantly improves compared with A549L0 (distant metastasis of human rate is 7.7%), overall Distant metastasis rates is 100%, mostly be bone (the bone rate of transform is 90%) and brain (the brain rate of transform is 60%) shifts simultaneously, and mostly be many places organ and shift simultaneously.Wherein, the backbone rate of transform is 80%, and the four limbs rate of transform is 30%, and the meta number of days that backbone transfer occurs is 50 days (40-106 days).
3, through animalcule x-ray test set (Kodak DXS4000Pro System, model: Micro Focus X-Ray Imaging Source, 35KVP, the parameter that instrument uses is Bringing:4XBringing, f-stop:2.51, Fov:59.4mm, Resolution:220ppi, CCD temperature:-29 ℃, time shutter is 1 minute) monitoring, in IVIS, 6.00 ± 0.63 days after cell line A549 L6 backbone metastasis signal of the present invention detected, can utilize x-ray test set the metastasis of mouse backbone to be detected.As shown in Figure 3, on Fig. 3, be IVIS living imaging figure, the next corresponding x-ray image of Fig. 3.
4, use animalcule CT (Skyscan1076,40Kv, 250uA, Bruker microCT NV, Antwerp, Belgium) and 3.0T MRI (Magnetom Verio, Siemens, Germany) can effectively detect the destruction situation of A549L6 backbone metastasis.As shown in Figure 4, Fig. 4 upper left represents that the centrum of mouse obviously destroyed by tumour, and upper right is normal control; Under Fig. 4, be corresponding three-dimensional reconstruction figure.Fig. 5 right side is respectively coronal-plane, sagittal plane, the transverse section T2 weighted mri imaging of backbone metastasis, and left side is respectively corresponding normal control.
5, through HE pathological section, finding that A549L6 backbone transfer characteristic is similar to the clinical picture that lung cancer backbone shifts, as shown in Figure 6, represent the whole centrum of tumor cell invasion, but do not invade intervertebral disk under Fig. 6, is normal control on Fig. 6.As shown in Figure 7, representing that whole centrum is tumor cell invasion under Fig. 7, is normal control on Fig. 7.
6, by observing the spinal compression symptom of mouse every day, find: along with the development of the course of disease also can occur and clinical similar spinal compression symptom, along with tail occurs PD successively, mop floor, instep lands gait, the walking step state of sweeping the floor, paralysis, death etc. can be easy to the Node Events (shown in Fig. 8) of observing, and betides respectively 1.43 ± 1.72 days, 3.71 ± 2.06 days, 8.29 ± 2.06 days, 11.57 ± 3.69 days, 14.29 ± 3.59 days after living imaging system discovery backbone metastasis.
7, through the monitoring to Mouse Weight, find: A549L6 is transferred to after backbone, its body weight significantly declines, during with left ventricle injection A549L6 cell, tail mops floor, instep lands gait, the walking step state of sweeping the floor, paralysis, dead corresponding body weight are respectively 17.84 ± 0.83g, 26.20 ± 1.85g, 24.96 ± 1.52g, 22.78 ± 1.44g, 21.02 ± 1.72g, 18.76 ± 1.73g.(as shown in Figure 9).
8, through wound healing (by tumor cell inoculation in 12 orifice plates, when tumour cell 90% polymerization, hungry 12 hours, use aseptic pipettor gun head along culture plate center cut, then in the migration situation of different timing node observation of cell), after 27 hours and 36 hours, the ability of migrating of A549L6 cell is respectively 5.5 times and 11.5 times of source cell, as shown in figure 10, wherein, left figure is schematic diagram, and right figure is statistical graph.
9, through transwell, (tumour cell is resuspended in to the DMEM of serum-free, concentration adjustment is 50,000/200ul, gets the upper strata that 200ul is inoculated in the cell that shuttles back and forth, and inserts the DMEM substratum 600ul containing 10% foetal calf serum in cell below.At different time points, use 4% paraformaldehyde to fix, violet staining, and the tumour cell of the statistics migration of taking pictures under the microscope) detect, after 36 hours, the ability of migrating of A549L6 cell is 22.4 times of source cell, as shown in figure 11, and wherein, left figure is schematic diagram, and right figure is statistical graph.
10, the nutrient solution of A549L6, go down to posterity, frozen, recovery condition is all identical with A549, in-vitro multiplication experiment is (by cell with 1x10
4the density in/hole is inoculated in 6 orifice plates, respectively at postvaccinal the 2nd, 4,6,8,10 days by cell dissociation, counting) show that its multiplication capacity is similar to A549L0 and A549, as shown in figure 12, show that A549L6 is similar to A549L0 and A549 multiplication capacity in vitro.There is notable difference in cellular form and the A549L0 of A549L6, Figure 13 shows there is notable difference in the cellular form of A549L6 and A549L0, A549, A549L6 is almost the cell clone that light transmission is strong, and A549L0, A549 all only contain the cell clone that part light transmission is strong.As on Figure 13 with to represent A549L0 in Figure 13 similar to A549 all only containing strong cell clone and the poor cell clone of light transmission of part light transmission, and A549L6 (under Figure 13) is almost the cell clone that light transmission is strong.
Claims (10)
1. a backbone high-transfer human lung adenocarcinoma cell line, is characterized in that, described cell strain is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC No:C201346, and the preservation time is on April 25th, 2013.
2. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, it is characterized in that, described cell strain is with luciferase mark, can pass through the whole body transfer case of IVIS living imaging system live body Real-Time Monitoring tumour, and backbone transfer signal within 6.00 ± 0.63 days, be detected early than animalcule x-ray equipment.
3. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, the bone rate of transform of described cell strain is 90%, and the backbone rate of transform is 80%, and the brain rate of transform is 60%, and the meta number of days that backbone generation backbone shifts is 50 days.
4. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, after described cell strain shifts, can detect backbone metastasis through x-ray, micro-CT, MRI, can detect backbone and brain metastasis through HE pathological section.
5. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, described cell strain detects through wound healing and transwell, and after 36 hours, the ability of migrating of A549L6 cell is respectively 11.5 times and 22.4 times of source cell.
6. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, described cell strain is transferred to after backbone, has to people's lung cancer backbone and shifts the rear similar symptom of spinal compression that occurs; According to the development of the course of disease, it can be divided into successively to five obvious observable nodes: tail mops floor, instep lands gait, the walking step state of sweeping the floor, paralysis, death; It betides respectively 1.43 ± 1.72 days, 3.71 ± 2.06 days, 8.29 ± 2.06 days, 11.57 ± 3.69 days, 14.29 ± 3.59 days after IVIS living imaging system discovery backbone metastasis.
7. backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, it is characterized in that, described cell line A549 L6 is transferred to after backbone, its body weight significantly declines, during with left ventricle injection A549L6 cell, tail mops floor, instep lands gait, the walking step state of sweeping the floor, paralysis, dead corresponding body weight are respectively 17.84 ± 0.83g, 26.20 ± 1.85g, 24.96 ± 1.52g, 22.78 ± 1.44g, 21.02 ± 1.72g, 18.76 ± 1.73g.
8. the preparation method of backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that,
(A) human lung adenocarcinoma cell line A549 cell is inoculated in to culture plate, carries out PGL4 plasmid transfection, the cell clone that transfection is obtained by G418 screening carries out luciferase assays, obtains the cell clone A549L0 of the luciferase expression positive;
(B) A549 positive cell clone A549L0 suspension is injected in nude mice left ventricle;
(C) screening backbone transfer cell strain, selects the destruction of bone of backbone metastasis the most by force and the strongest metastatic tumour cell of fluorescence carries out amplification in vitro cultivation; The cell that cultivation is obtained is injected in nude mice left ventricle by step (B) method, then carries out amplification in vitro cultivation by step (C); The screening of repetition three-wheel;
(D) the positive A549 cell of fluorescent mark step (C) being obtained carries out mono-clonal to be selected, and amplification in vitro is cultivated, and obtains described backbone high-transfer human lung adenocarcinoma cell cell line A549 L6.
9. the application of backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, described cell strain shifts the application in animal model at the backbone of preparing human lung adenocarcinoma.
10. the application of backbone high-transfer human lung adenocarcinoma cell line as claimed in claim 1, is characterized in that, the application of described cell strain in bone metastases of lung cancer mechanism and in the anti-human adenocarcinoma of lung medicine of preparation.
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| CN111187753A (en) * | 2020-01-16 | 2020-05-22 | 杭州市第一人民医院 | High-potential brain metastasis lung cancer cell strain and application and construction method thereof |
| CN114561360A (en) * | 2022-01-25 | 2022-05-31 | 南方医科大学 | Mouse lung cancer brain metastasis cell line LLC-BMT3 and construction method and application thereof |
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| CN111187753A (en) * | 2020-01-16 | 2020-05-22 | 杭州市第一人民医院 | High-potential brain metastasis lung cancer cell strain and application and construction method thereof |
| CN114561360A (en) * | 2022-01-25 | 2022-05-31 | 南方医科大学 | Mouse lung cancer brain metastasis cell line LLC-BMT3 and construction method and application thereof |
| CN114561360B (en) * | 2022-01-25 | 2023-09-08 | 南方医科大学 | Mouse lung cancer brain transfer cell LLC-BMT3 and construction method and application thereof |
| CN117402828A (en) * | 2023-04-13 | 2024-01-16 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Human lung cancer brain metastasis cell line and application thereof |
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