CN102250840B - Human pancreatic cancer cell line and its application - Google Patents
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Abstract
The invention provides a human pancreatic cancer cell line and its preparation method and application. The human pancreatic cancer cell line is preserved in the China Center for Type Culture Collection and the preservation number is CCTCC C201007. The human pancreatic cancer cell line can be utilized for producing human pancreas cancer in mammals, preparing human pancreas cancer models and screening candidate drugs for treating human pancreas cancer. The human pancreatic cancer cell line has stable characters and is subcultured multiple times. Characters are stably maintained in 20th to 50th generation obtained by subculture in vitro. The human pancreatic cancer cell line has clinical biological characters of human pancreas cancer and provides an experimental material for pancreas cancer research, wherein biological characteristics of the experimental material are close to clinical tumor biological characteristics. Compared with a subcultured parent tumor in a nude mouse, the human pancreatic cancer cell line can be utilized for analysis of correlation of tolerance and susceptibility to drugs in vivo or vitro thus can establish two associated anti-pancreatic cancer drug screening platforms in vitro and in vivo.
Description
Technical field
The invention belongs to clone field, particularly a kind of human pancreatic cancer cell and application thereof.
Background technology
Carcinoma of the pancreas is the disease that a kind of grade malignancy is very high, although relative incidence rate is lower, is generally 9～10/100000, is still the highest disease of Tumor-assaciated mortality ratio in western countries; Also be ascendant trend year by year at China's sickness rate.Carcinoma of the pancreas is difficult for discovery in early days, not yet finds special clinical manifestation and tumor markers at present, and Features is not also true to type; And due to pancreas anatomy and biological property, easily invade surrounding organ and shift, Most patients is in the time making a definite diagnosis, it has been the terminal stage of a disease, and be transferred to the organs such as liver, lung, backbone, kidney or suprarenal gland, can only go and detect or palliative operation, but long-term effect is undesirable, patient dies from hepatic metastases and local recurrence mostly; Can carry out excision radical cure person and only account for 5%～30%; Postoperative recurrence shifts early, incidence is high, and the result for the treatment of of single radiotherapy or chemotherapy is undesirable, poor prognosis.The surgery statistical information demonstration of China, within 5 years, survival rate is only in 5% left and right.
About the biological mechanism of generation, development, transfer and the resistance of carcinoma of the pancreas still not fully aware of at present.Lack at present the experiment material that approaches clinical tumor biological characteristics for carcinoma of the pancreas genesis mechanism and anti-carcinoma of the pancreas drug development.And use clinical tumor to organize directly to set up the success ratio of clone lower, therefore, first set up animal model by using clinical tumor sample, and then the people source tumour cell of setting up by former culture is closer to the Clinical Biological of tumour, the resistance to medicine and susceptibility will have better predictability.
Summary of the invention
The technical problem to be solved in the present invention is exactly that the species diversity existing for existing human pancreatic cancer cell is low, differs larger deficiency with the biological character of carcinoma of the pancreas clinically, and a kind of new human pancreatic cancer cell and uses thereof is provided.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of human pancreatic cancer cell, and it is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C201007.
The present invention also provides the daughter cell of human pancreatic cancer cell as above.
The present invention also provides the purposes of human pancreatic cancer cell as above, for producing carcinoma of the pancreas Mammals.Described Mammals can be various Mammalss, preferably nude mouse.The preferred BALC/c nude mouse of described nude mouse.The low differentiation of the preferred pancreas of described carcinoma of the pancreas or middle differentiation gland cancer.
The present invention also provides a kind of establishment method of above-mentioned human pancreatic cancer cell, comprises the following steps,
1) obtain fresh clinical carcinoma of the pancreas excision sample, be cut into the fritter of 20～50mg, subcutaneous puncture seeded with mammalian;
2) percutaneous puncture-inoculation, after 70～90 days, is put to death tumor animal, takes out tumor tissues, carries out the former culture of cancer cells and the cultivation of going down to posterity.
Wherein, described Mammals, described carcinoma of the pancreas are all described above.
Described fresh clinical carcinoma of the pancreas excision sample is preferably with inoculating after mammaliancellculture liquid or physiological saline rinsing again.Preferably use fresh HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing.
The mode of described inoculation can be subcutaneous puncture inoculation, inoculation in in-situ inoculating or scrotum film.Preferably carry out subcutaneous puncture inoculation for carcinoma of the pancreas.
Described primary culture method can be the primary culture method of conventional mammalian cell.Preferably comprise the following steps: tumor tissues is cut into fritter, insert in culturing bottle, under 37 DEG C of incubator 5%CO2 conditions, cultivate; Next day, culturing bottle is slowly overturn and kept flat, in bottle, add DMEM/F12 nutrient solution (containing 5% foetal calf serum, 10 μ g/ml recombinant human insulins, 6.7ng/ml Sodium Selenite, 5.5 μ g/ml Transferrins,iron complexess, 2 μ g/ml thanomins, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), leave standstill and cultivate;
The described cultural method that goes down to posterity can be the cultural method that goes down to posterity of conventional mammalian cell.Preferably comprise the following steps: inhale and abandon old nutrient solution, in bottle, add 0.05% fresh trypsin solution, after cell detachment, add fresh DMEM/F12 nutrient solution, carefully piping and druming, makes it to depart from bottle wall and form cell suspension; A small amount of cell that becomes circle but do not come off on bottle wall, hangs culturing bottle surface gently with aseptic cell scraper, collects whole cells, centrifugal, is inoculated in respectively new culturing bottle; After five generations of passage to the, nutrient solution is replaced by RPMI RPMI-1640 (containing 10% foetal calf serum, 10 μ g/ml recombinant human insulins, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B).
The present invention also provides a kind of method of the drug candidate that screens treatment carcinoma of the pancreas, comprise the following steps: test compounds is applied to animal model, the test compounds that causes carcinoma of the pancreas symptom to improve or cure after using is exactly the candidate compound for the treatment of carcinoma of the pancreas, and wherein said animal model has the pancreatic tumour that human pancreatic cancer cell as above causes.
Concrete, the method for the drug candidate of screening treatment carcinoma of the pancreas of the present invention comprises the following steps:
(1) described pancreatic cancer cell or its daughter cell are prepared into cell suspension, are inoculated in Mammals subcutaneous, raise, obtain human pancreas cancer animal model;
(2) test compounds is applied to animal model, the test compounds that causes carcinoma of the pancreas symptom to improve or cure after using is exactly the candidate compound for the treatment of carcinoma of the pancreas.
Wherein, the preferred nude mouse of described animal model.The preferred BALB/C nude mouse of described nude mice.Preferably can adopt pallium cell injection to set up animal model.In step of applying, by test compounds by tail vein injection, oral, abdominal injection or be applied to carcinoma of the pancreas tumor animal in modes such as tumor by local medications.Preferably use control experiment, a kind of preferred mode is: also use containing the solvent application of test compounds in carcinoma of the pancreas tumor animal in contrast simultaneously.
In the present invention, above-mentioned optimum condition can arbitrary combination on the basis that meets this area general knowledge, obtains the preferred embodiments of the invention.
The raw material that the present invention is used or reagent except special instruction, all commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows: human pancreatic cancer cell proterties of the present invention is stable, can stablize repeatedly and go down to posterity.For carcinoma of the pancreas research provides the new experiment material closer to clinical tumor biological characteristics.Have and highly become knurl, can successfully prepare carcinoma of the pancreas animal model, made that animal model can be for fundamental research and drug screening.By comparing with nude mouse interior generation parent tumour, can be used to analyze the dependency of external, drug disposition susceptibility and resistance, and then can set up two anti-carcinoma of the pancreas medicine sorting platforms that are associated in external, body.Also can be used for studying the pathogeny of carcinoma of the pancreas transfer, the resistance mechanism of carcinoma of the pancreas, and then can find the feature biomarker of carcinoma of the pancreas transfer and resistance.The desirable clone of human pancreas cancer fundamental research and preclinical phase application.
the preservation of biomaterial
Human pancreatic cancer cell of the present invention, in on March 31st, 2010 be deposited in Chinese Typical Representative culture collection center (CCTCC) (address: China. Wuhan. Wuhan University), culture title behaviour source pancreatic cancer cell PAXC-003, deposit number is CCTCC NO:C201007.
brief description of the drawings
Below in conjunction with brief description of the drawings feature of the present invention and beneficial effect.
The morphological observation (100 ×) of Fig. 1 .PAXC-003 cell.
The chromosome analysis of Fig. 2 .PAXC-003 cell.A.PAXC-003 cell; B. nude mouse karyomit(e) (reference).
Fig. 3 .PAXC-003 Immunohistochemistry dyeing (DAB method): A. cytokeratin Cytokeratin (200 ×); B.CA19-9 (200 ×); C. CEA (200 ×).
Fig. 4 .PAXC-003 cell doubling time curve.
The high intension cell screening of Fig. 5 .Acumen instrument is analyzed the PAXC-003 cell cycle.
The one-tenth knurl of Fig. 6 .PAXC-003 cell.A.PAXC-003 cell is at nude mouse tumor growth curve (gross tumor volume); B. tumor weight when terminal.
Fig. 7. the histopathologic slide of tumour.A. the tumor specimen (100 ×) of obtaining clinically; B. parent's tumour (100 ×) in nude mouse body; C.PAXC-003 cell is in formation in nude mice (100 ×).
embodiment
Further illustrate the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.
The preparation of embodiment 1PAXC-003 cell
Nude mouse: 5 nude mouses, female, body weight 16.0 ± 1.0g, in 5 weeks ages of mouse, raises the environment in SPF.Nude mouse is provided by Shanghai Si Laike laboratory animal Technology Co., Ltd..
Obtain fresh clinical carcinoma of the pancreas Operated Specimens (man from Changhai Hospital, Shanghai City, 62 years old, primary pancreatic carcinoma, pathological diagnosis result is: differentiation gland cancer in (processus uncinatus pancreatis)), immerse immediately in the aseptic HBSS damping fluid of precooling (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs).In Biohazard Safety Equipment, with this fresh aseptic HBSS damping fluid flushing sample, be cut into the fritter of 20～50mg, percutaneous puncture-inoculation nude mouse armpit back is subcutaneous.After animal inoculation pvaccination, state of health is good, and behavior is normal.Inoculate after 40 days, find there is a tumour lesser tubercle inoculation position is subcutaneous by touch, lesser tubercle starts growth obviously in inoculation after 50 days, and when inoculating latter 70 days, gross tumor volume exceedes 300mm
3.
Former culture: subcutaneous puncture inoculation human pancreas cancer is after 70～90 days, lotus knurl nude mouse is put to death with excess carbon dioxide gas anesthesia, aseptic dissection, take out tumor tissues, carry out former culture, method is as follows: use fast 3 times of HBSS damping fluid (containing 500U/ml penicillin G, 500 μ g/ml Vetstreps and 1.25 μ g/ml amphotericin Bs) rinsing tumor tissues, remove reticular tissue and necrotic tissue; Tumor tissues is sheared into about 1mm with aseptic operation blade
3fritter; The tissue block of having sheared is sent into culturing bottle with inoculating needle, and evenly put, interval 0.5cm, builds bottle cap; The culturing bottle that overturns gently, at the bottom of making bottle upwards, in 37 DEG C of incubator 5%CO
2under condition, cultivate; Next day, culturing bottle is slowly overturn and kept flat, in bottle, add 10ml DMEM/F12 nutrient solution (containing 5% foetal calf serum, 10 μ g/ml recombinant human insulins, 6.7ng/ml Sodium Selenite, 5.5 μ g/ml Transferrins,iron complexess, 2 μ g/ml thanomins, 100U/ml penicillin G, 100 μ g/ml Vetstreps and 0.25 μ g/ml amphotericin B), leave standstill and cultivate; Former culture changes liquid once in every 3 days, removes floating tissue block and residual hemocyte.
The cultivation of going down to posterity: the cell growing in by tissue block is covered with behind culturing bottle bottom, carry out passage, concrete steps are as follows: inhale and abandon old nutrient solution, in bottle, add 0.05% trypsin solution that 1ml is fresh, rinse adherent layer gently, suction is abandoned, add again 0.05% trypsin solution that 1ml is fresh, in 37 DEG C of incubators, hatch, observe tenuigenin retraction, intercellular substance increase, after cell detachment, add the DMEM/F12 nutrient solution that 3ml is fresh, carefully piping and druming, makes it to depart from bottle wall and forms cell suspension; A small amount of cell that becomes circle but do not come off on bottle wall, hangs culturing bottle surface gently with aseptic cell scraper, collects whole cells, centrifugal, counts, and is inoculated in respectively new culturing bottle.After 5 generations of passage to the, nutrient solution is progressively replaced by RPMI RPMI-1640 (containing 10% foetal calf serum, 10 μ g/ml recombinant human insulins, 100U/ml penicillin G, 100 μ g/ml Vetstreps), Growth of Cells is good, and form is homogeneous comparatively.More than being passaged to for 50 generations.
In the present invention, the former culture and the cultured cell line that derive from tumor tissues are Epithelial, and cellular form is homogeneous comparatively, contactless inhibition, and primary growth speed is comparatively slow; Along with the increase of passage number, the speed of growth is progressively accelerated; By this clone called after PAXC-003, submit preservation to, deposit number is CCTCC NO:C201007.
Biological characteristics and the application of embodiment 2PAXC-003 cell
The present invention adopts the RPMI RPMI-1640 that contains foetal calf serum and Regular Insulin to cultivate PAXC-003 cell, can external long term growth and stable going down to posterity.More than cell reached for 20 generations, cell proterties is stable gradually, carries out relevant biology, genetics and tissue-derived qualification, until the 50th generation all had identical stable proterties.Through experimental observation and checking, the PAXC-003 cell of growth in vitro has typical Epithelial form, loses contact growth-inhibiting, is malignancy.Genetics research confirms that this cell is heteroploid, and Numerical and structural chromsomal aberrations is serious, meets the genetics characteristics of malignant tumour.This PAXC-003 cell can form tumour in nude mouse body, has tumorigenicity.Clinical pancreatic tumour sample, the nude mouse interior generation parent tumour in this PAXC-003 cell and its source form corresponding relation, can be that research is external, body interior and the dependency of clinical the sensitivity of anti cancer drugs and resistance, and the generation of carcinoma of the pancreas, development, transfer and biomarker provide new test materials.Specific as follows:
A. morphological observation
The culturing bottle of cultivating PAXC-003 cell is placed under inverted microscope, under bright field, takes pictures.The results are shown in Figure 1, visible, PAXC-003 cell has lost contact inhibition, is malignancy, has the feature of overlapping growth, and adherent growth part is flats, taking irregular paving stone sample as main, meets epithelioid cell's feature.
B. chromosomal qualification
The PAXC-003 cell of cultivation was placed in to 4 DEG C after 12 hours, adds colchicine, making its final concentration is 0.4 μ g/ml, then in 37 DEG C of incubators, continues to cultivate 10 hours.Gather the cell of metaphase, be fixed with stationary liquid, then cell suspension is dripped on the microscope slide of precooling, with the dyeing of Giemas staining fluid, under microscope, count chromosome number.The results are shown in Figure 2, visible, after the continuous passage of PAXC-003 cell, karyomit(e) still keeps the chromosomal feature of humanized's tumour cell, show as polyploid, modal number (M) concentrates between 40～46, accounts for 59.4%, has most central authorities and submetacentric chromosome (Fig. 2 A); And the chromosome number 2n=40 of nude mouse, and be kinetochore, top (Fig. 2 B), can distinguish with human chromosomal accordingly.Visible this PAXC-003 cell is heteroploid, and Numerical and structural chromsomal aberrations is serious, meets the genetics characteristics of malignant tumour.
C. tissue-derived qualification
PAXC-003 cell is seeded on cover glass and is cultivated, after cell stretches, fix with 4% formaldehyde, carry out immunohistochemical staining (DAB development process).Result demonstration, cytokeratin (Cytokeratin, Fig. 2 A) and CA19-9 (Fig. 2 B) they are strong positive, carcinomebryonic antigen (CEA, Fig. 2 C) is the weak positive, binding of pathological diagnosis, this cell is pancreatic cancer cell.
D. cytokinetics
PAXC-003 cell is seeded in 96 orifice plates with the density in 2000/ hole, cultivates, respectively at 12 hours, 24 hours, 36 hours, 48 hours, 72 hours and 96 hours fixed cells, and carry out PI dyeing, measure every porocyte number with high intension cell screening instrument Acumen.The demonstration of cytokinetics result of study, the population doubling time of PAXC-003 cell is 33.79 hours (Fig. 4).
E. the cell cycle is detected
PAXC-003 cell is seeded in 96 orifice plates with the density in 2000/ hole, cultivate after 24 hours, fixed cell, and carry out PI dyeing, measure total DNA content (total PI fluorescence intensity of total DNA content of each cell and this cell is proportional) of every porocyte number and each cell with high intension cell screening instrument Acumen; And during according to the different cell cycle, the variation of the total DNA content of cell, calculates the total cellular score of each cell cycle.Result shows, PAX-003CL cell G
1phase is to be 19.01%, G 45.92%, the S phase
2/ M the phase is to be 9.54% 24.52%, the 8N phase.According to cell proliferation exponential formula PI=(G
2/ M+S) ÷ (G
0/ G
1+ S+G
2/ M+8N) × 100%, calculation result: PAXC-003 cell PI=48.66% (Fig. 5).
F. the one-tenth knurl of cell
External large scale culturing and collection PAXC-003 cell, subcutaneous vaccination BALB/C nude mouse (every animal inoculation pvaccination 5.0 × 10
6individual cell, cell suspension and Matrigel mixed with 1: 1, inoculated altogether 5 animals), time investigation the weight of animals and tumor size on every Wendesdays.Inoculate after approximately 10 days, tumour starts to form and grow.Draw tumor growth curve, wherein gross tumor volume=(long × wide × wide) ÷ 2 (seeing Fig. 6 A).Finished experiment at the 26th day and put to death animal, tumor weight is 1.15g ± 0.10g (seeing Fig. 6 B).Visible this PAXC-003 cell can form tumour in nude mouse body, has tumorigenicity.
G. the pathology of tumour qualification
Embodiment 1 obtained to fresh clinical carcinoma of the pancreas Operated Specimens, percutaneous puncture-inoculation in the nude mouse back tumour that subcutaneous 90 days, in the subcutaneous tumour growing and above-mentioned f step, PAXC-003 cell forms in nude mouse subcutaneous vaccination for 10 days afterwards from Changhai Hospital, Shanghai City, carry out specimens paraffin embedding slices and H & E dyeing, the results are shown in Figure 7.Their pathological diagnosis the results are shown in following table 1.Visible clinical samples, nude mouse interior generation parent tumour become the similar of tumour in nude mouse body with PAXC-003 cell, form corresponding relation.
The pathological diagnosis result of the each tumor specimen of table 1.
| Sample title | Pathological diagnosis result |
| Clinical operation sample | Processus uncinatus pancreatis: middle differentiation gland cancer (Fig. 7 A) |
| Nude mouse interior generation parent tumour | Pancreas: middle differentiation gland cancer (Fig. 7 B) |
| PAXC-003 cell becomes tumour in nude mouse body | Pancreas: middle differentiation gland cancer (Fig. 7 C) |
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (4)
1. a human pancreatic cancer cell, is characterized in that, it is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C201007.
2. the daughter cell of human pancreatic cancer cell as claimed in claim 1.
3. the purposes of human pancreatic cancer cell as claimed in claim 1 or 2, is characterized in that, for producing carcinoma of the pancreas Mammals.
4. the purposes of human pancreatic cancer cell as claimed in claim 3, is characterized in that, described Mammals is nude mouse.
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| CN102424815B (en) * | 2011-12-31 | 2013-05-08 | 广州呼吸疾病研究所 | Cell strain from human lung anaplastic cancers and preparation method thereof |
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| CN108977410B (en) * | 2018-08-02 | 2023-04-25 | 郭世伟 | Pancreatic cancer in-situ cancer cell line of Chinese people |
| CN110622922A (en) * | 2019-09-30 | 2019-12-31 | 广西医科大学第一附属医院 | Method for establishing mouse animal model with ascites due to pancreatic cancer |
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