CN104087554A - Human thymoma cell line and application thereof - Google Patents
Human thymoma cell line and application thereof Download PDFInfo
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- CN104087554A CN104087554A CN201410312866.6A CN201410312866A CN104087554A CN 104087554 A CN104087554 A CN 104087554A CN 201410312866 A CN201410312866 A CN 201410312866A CN 104087554 A CN104087554 A CN 104087554A
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Abstract
The invention discloses a human thymoma cell line and application thereof, belonging to the field of tumor biology. The human thymoma cell line is named Thy0517 and has a collection number of CGMCC (China General Microbiological Culture Collection Center) No.9328. The human thymoma cell line Thy0517 has main advantages of having protein expression similar to primary tumor tissues, abnormal karyotype and characteristics of tumor cells with in-vitro tumor forming capability. After in-vitro continuous passage culture, the characteristics of thymoma cells keep unchanged, and therefore the human thymoma cell line can be used as a cell material for researching and developing thymoma-related drugs. EGFR (Epidermal Growth Factor Receptor) expression of the human thymoma cell line is positive, so that proliferation, angiogenesis, adhesion, invasion and metastasis of the tumor cells can be promoted; the cell line can be used as a powerful cell tool for researching EGFR-targeted drugs.
Description
Technical field
The present invention relates to a kind of people's thymoma is and uses thereof, belongs to oncobiology field, and especially relating to a kind of people's thymoma is Thy0517 and uses thereof.
Background technology
Thymus gland is immune maincenter organ, is T cell development, differentiation and ripe place, and its function is by part lymphocyte differentiating into T lymphocyte.Other organs of thymus gland and human body are the same, and optimum or malignant tumour can occur.Thymoma is the modal tumour of anterior mediastinum, accounts for 30% of adult's anterior mediastinum tumour, is originate from thymus epithelial or show to the tumour (no matter whether Non-cancerous lymphocyte exists) of thymic epithelial cells's differentiation.Thymoma is higher at Chinese sickness rate, is apt to occur in 55-56 year age group.Although thymoma mortality is low, the incidence of thymoma patient's the second cancer increases.In Clinical symptoms, thymoma is often with autoimmune disorders such as myasthenia gravis, PRCA, systemic lupus erythematouses.In thymoma patient, approximately there is the 1/4 patient myasthenia gravis that occurs together.The formation of thymoma causes the change of thymus microenvironment, is likely the reason of myasthenia gravis with thymoma, and the Thymomas with Myasthenia Gravis state of an illness is conventionally serious than simple myasthenia gravis, need to rely on immunosuppressant treatment.The thymoma cause of disease it be unclear that, and studies have reported that Epstein-Barr virus plays certain effect in thymoma morbidity.
In order to study the biological characteristics of tumour, evaluate the adaptability of external model, tumor cell line has become a kind of important research tool.In recent decades, a large amount of human tumor cell lines is separated and for long-term cultivation, and these cells are that the Molecular level study of tumour provides important model.But due to the shortage of the singularity of thymoma tissue, effectively available thymoma sample and set up the technical difficulty of continuity cell cultures, cause scientific research personnel cannot obtain for a long time thymoma system always, greatly limited the research of thymoma level.
Owing to lacking thymoma in vitro study model, especially lack thymoma system, so cause the external Function detection of thymoma can only be limited to the former culture of limited short-term.For these limited short-term primary cultures, researchist has analyzed the microsatellite instability state of thymoma.Some scholars have carried out an evaluation for the targeted therapy strategy of thymoma from molecule mechanism level, immunohistochemistry demonstration EGF-R ELISA (EGFR) is expressed and is increased in thymoma, and in some patients, EGFR targeted therapy is effective.Also there is scholar's treatment for thymoma by EGFR inhibitor, and also have scholar to find that COX-2 (COX-2) or KIT gene may become the target spot of thymoma second-line chemotherapy.These preliminary study show, part targeted drug to thymoma treatment effectively, but owing to lacking the Function detection of external thymoma system, a lot of new targeted therapies can be used for other tumours, but also do not carry out in thymoma.Therefore, in order in vitro the molecular signal conduction path of thymoma to be studied better, need to set up can prolonged and repeated use thymoma system.
Summary of the invention
The object of this invention is to provide a kind of people's thymoma that a kind of Long Term Passages is in vitro cultivated, stable in properties is.
Second object of the present invention is to provide the purposes that a kind of people's thymoma is preparation thymoma diagnostic reagent.
The 3rd object of the present invention is to provide the purposes that a kind of people's thymoma is preparation thymoma animal model.
It is for developing the purposes that suppresses thymoma medicine as drug target that the 4th object of the present invention is to provide a kind of people's thymoma.
It is the purposes for the in vitro study of thymoma that the 5th object of the present invention is to provide a kind of people's thymoma.
Technical scheme of the present invention is summarized as follows:
People's thymoma system, called after Thy0517, is CGMCC No.9328 at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
A kind of people's thymoma is the purposes that Thy0517 prepares thymoma diagnostic reagent.
A kind of people's thymoma is the purposes that Thy0517 prepares thymoma animal model.
A kind of people's thymoma is Thy0517 as drug target for developing the purposes that suppresses thymoma medicine.
A kind of people's thymoma is the purposes of Thy0517 for the in vitro study of thymoma.
A kind of people's thymoma of the present invention is that Thy0517 main advantage is:
(1) clone of the present invention has similar protein expression to primary tumo(u)r tissue, also has Chromosomal Abnormal Karyotype simultaneously, has the tumour cell characteristic of external one-tenth knurl ability.
(2) clone of the present invention in vitro continuous passage cultivate and keep thymoma tumour cell characteristic constant, be suitable as the cell material of research and development thymoma related drugs.
(3) clone EGFR of the present invention expresses positive, EGFR positive expression can promote propagation, vasculogenesis, adhesion, invasion and attack and the transfer of tumour cell, and this clone can be used as the strong instrument cell of the targeted drug research of a research using EGFR as target.
A kind of people's thymoma of the present invention is, called after Thy0517, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on June 25th, 2014, its deposit number is CGMCC No.9328, and the classification of suggestion is by name: thymoma system.And survive.Preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Brief description of the drawings
Fig. 1 is that a kind of people's thymoma of the present invention is that Thy0517 (CGMCC No.9328) is inverted the cell image (200 times) of growing under opticmicroscope.
Fig. 2 is that a kind of people's thymoma of the present invention is the cell growth curve of Thy0517 (CGMCC No.9328).
Fig. 3 is that a kind of people's thymoma of the present invention is chromosomal a kind of striograph of Thy0517 (CGMCC No.9328).
Fig. 4 is that a kind of people's thymoma of the present invention is the aspect graph (HE dyeing, 200 times) that Thy0517 (CGMCC No.9328) is planted in BALB/C immune deficiency nude mice armpit subcutaneous transplantation tumor tissue.
Embodiment
Describe technical solution of the present invention in detail by the drawings and specific embodiments below, following examples only do not limit practical range of the present invention with this in order to technical scheme of the present invention to be described.
The foundation of embodiment 1 people's thymoma system
Inventor's thymoma is that Thy0517 is the clone of a strain north of China people AB type thymoma, derives from 50 years old male sex's myasthenia gravis with thymoma patient's thymoma tissue.Patient does not accept the treatments such as any chemotherapy, radiotherapy before undergoing surgery.People's thymoma is Thy0517, is to cultivate gained from histological type is the thymoma thoracic adenoma cutting tissue thing of AB type thymoma.
Concrete steps are:
(1) tissue block mechanical separation: the thymoma tissue of excision is separated to rapidly to 0.5 cubic centimetre of size with sterile scissors and scalpel, with containing 100U/ml penicillin, the PBS damping fluid of 100ug/ml Streptomycin sulphate soaks low temperature and moves to Biohazard Safety Equipment, again with containing 100U/ml penicillin, the PBS damping fluid cleansing tissue repeatedly of 100ug/ml Streptomycin sulphate, be soaked in again the penicillin containing 100U/ml, 100ug/ml Streptomycin sulphate, in the DMEM in high glucose substratum of 10% foetal calf serum, softly separate by sterile scissors, remove coating and reticular tissue as far as possible, obtain thymoma parenchymal tissue,
(2) tissue block inoculation: use sterile scissors that the tissue of step (1) gained is cut into the tissue block of 1 cubic millimeter, by the PBS buffer solution for cleaning tissue block containing 100U/ml penicillin, 100ug/ml Streptomycin sulphate.Allow tissue block precipitate, remove supernatant liquor, suspended tissue's piece in the DMEM in high glucose substratum containing 100U/ml penicillin, 100ug/ml Streptomycin sulphate, 10% foetal calf serum, with the soft gripping tumor tissues of aseptic ophthalmology tweezers, sends in 25 square centimeters of Tissue Culture Flasks, tissue block is evenly spread out at the bottom of bottle with elbow straw, every fritter interval 5mm left and right, spreads altogether into 20, has spread after tissue block, put 37 DEG C, 5%CO
2in incubator, cultivate and attach to tissue block for 24 hours, slowly in culturing bottle, add the DMEM in high glucose substratum continuation cultivation of 5ml containing 100U/ml penicillin, 100ug/ml Streptomycin sulphate, 10% foetal calf serum again, obtain tissue block and swim out of cell, continue to cultivate, until cytogamy degree reaches more than 80%;
(3) cell of swimming out of of step (2) gained is carried out to purifying, remove the impurity cells such as inoblast, thymoma after purifying is cultivated in the DMEM in high glucose substratum containing 100U/ml penicillin, 100ug/ml Streptomycin sulphate, 10% foetal calf serum, culture temperature is 37 DEG C, and atmosphere surrounding is 5%CO
2, 95% air, humidity is saturated humidity, within every 2 days, changes a nutrient solution, within every 4 days, goes down to posterity once, cultured continuously also goes down to posterity and exceedes 12 months, obtains clone, called after Thy0517.Clone is deposited in to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and its deposit number is CGMCCNo.9328.
Embodiment 2 people's thymomas are the detection qualification of Thy0517
1, morphologic observation
The gross morphology of main observation of cell, as general form, caryoplasm ratio, adhesion characteristics etc.
Be inverted under optics phase microscope observer's thymoma and be Thy0517 as shown in Figure 1, visible cell is polygon, and core is large, and endochylema is abundant, and iuntercellular connects closely, adherent growth.
2, Immuncytochemical detection cell marking albumen
Adopt SP method to carry out immunocytochemical stain, immunocytochemical stain SP test kit and antibody are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, and antibody used is: CK7, CK8/18, CK19, CK-pan, CD24, BCL-2, P63, Vimentin, EMA, EGFR and CD99.
Concrete steps are undertaken by test kit specification sheets: examine under a microscope coloration result.
Immunohistochemical methods detected result determination methods: +++, >70% positive cell; ++, 30%-70% positive cell; +, <30% positive cell;-, negative reaction.
Inventor's thymoma is that the immunocytochemical stain of Thy0517 shows, people's thymoma is that Thy0517 has similar protein expression to the thymoma tissue in source, its outstanding expression characteristic is EGFR positive expression, this is an EGF-R ELISA, its positive expression can promote propagation, vasculogenesis, adhesion, invasion and attack and the transfer of tumour cell, and Thy0517 can be used as the strong instrument cell of the targeted drug research of a research using EGFR as target.
3, cell counting is drawn cell growth curve
(1) clone to be detected is with after 0.25% tryptic digestion, adjusting cell density with the DMEM in high glucose substratum containing 100U/ml penicillin, 100ug/ml Streptomycin sulphate, 10% foetal calf serum is 8000/ml, be inoculated in 36 holes in 2 24 orifice plates, every hole adds 1ml cell suspension;
(2), from inoculation time, the substratum discarding in 3 holes every 24 hours, adds 0.25% tryptic digestion, suspendible cell is also counted the mean value of the cell density in 3 holes, and every hole counting 3 times, averages, cell in all the other holes continues to cultivate, until the 12nd day; Within every 2 days, change from start to finish a subculture.
(3) draw cell growth curve: taking incubation time (number of days) as X-coordinate, cell number is ordinate zou, marks each corresponding points and is linked to be line, just can obtain cell growth curve.
Inventor's thymoma be Thy0517 growth curve as shown in Figure 2, its logarithmic phase is 6 days.
The cell colony doubling time that is Thy0517 according to cell growth curve calculating inventor thymoma is 37h.
4, nucleus type analysis
Detect caryogram feature, karyomit(e) quantity etc.
(1) cell in logarithmic phase is added to colchicine, make colchicine final concentration 0.1ug/ml, continue to cultivate 3h;
(2) 10ml tip centrifuge tube will be proceeded to after cell dissociation, 1000rpm, centrifugal 10min, removes supernatant, adds the KCl10ml of 0.075mol/L, hatch after 30min for 37 DEG C, in centrifuge tube, add fresh stationary liquid (Glacial acetic acid (v): methyl alcohol (v)=3:1) 1ml again, mix 1000rpm, centrifugal 10min, removes supernatant;
(3) slowly add fresh stationary liquid 10ml, blow gently evenly, room temperature is fixed 20min, centrifugal, removes supernatant, more fixing; 1000rpm, centrifugal 10min, abandons supernatant;
(4) add fresh stationary liquid 250ul, blow gently the even suspension that obtains, at the height apart from slide glass 15cm, drip suspension, air is fully dry, fresh Giemsa dyeing, and tap water rinses, dry rear dimethylbenzene transparent, neutral gum sealing, oily Microscopic observation chromosome morphology counting.
Inventor's thymoma is that chromosome structure and the number of Thy0517 all shows extremely, be heteroploid or polyploid, counting and 100 cell fission phases of analysis, chromosome number is distributed between 27-138 bar, modal number is 65-70 bar, and trunk is hypo-triploid.As shown in Figure 3.
5, allosome animal inoculation pvaccination
To inoculating cell suspension in allosome animal body, observe into knurl ability.
(1) to the sterilization forelimb armpit skin of BALB/C nude mice (being purchased from Test Animal Centre, Academy of Military Medical Sciences, P.L.A) that is numbered 54 week ages of 1,2,3,4,5, injection people thymoma is Thy0517 suspension, every 1,000,000 people's thymomas, applying light after injection, does not make to flow out;
(2) the 14th days, all there is the white projection of a diameter 2mm in injection areas in 5 nude mices, the 30th day, visible tumour is lobulated growth, and tumour major diameter is 15mm-18mm, puts to death nude mice after anesthesia, taking-up be numbered No. 2 transplanted tumor tissue through embedded section HE dyeing form as shown in Figure 4, the large engrain of visible heteroplastic nucleus, kernel is obvious, has the feature of tumour cell.
Preparation filters out the antibody with inhibition and killer T hy0517 cell, the main component using this antibody as anti-thymoma medicine.
Inventor's thymoma is the purposes of Thy0517, also includes but not limited to following purposes:
People's thymoma is the purposes that Thy0517 prepares thymoma diagnostic reagent.
People's thymoma is the purposes of Thy0517 for the in vitro study of thymoma.
People's thymoma is Thy0517 continuous Long Term Passages in vitro, cultivates a year and a half in vitro, goes down to posterity generation more than 150, and cell still growing multiplication is active.
Claims (5)
1. people's thymoma system, is characterized in that called after Thy0517 is CGMCC No.9328 at the preserving number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a kind of people's thymoma claimed in claim 1 is the purposes of preparation thymoma diagnostic reagent.
3. a kind of people's thymoma claimed in claim 1 is the purposes of preparation thymoma animal model.
4. a kind of people's thymoma claimed in claim 1 is for developing the purposes that suppresses thymoma medicine as drug target.
5. a kind of people's thymoma claimed in claim 1 is the purposes for the in vitro study of thymoma.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944755A (en) * | 2020-09-01 | 2020-11-17 | 天津医科大学总医院 | Method for enhancing antigen presenting capability of dendritic cells |
| CN112831472A (en) * | 2021-02-07 | 2021-05-25 | 北京和合医学诊断技术股份有限公司 | Primary human thymoma cell, culture method and application thereof, and serum-free culture medium |
| CN113907049A (en) * | 2021-12-13 | 2022-01-11 | 天津医科大学总医院空港医院 | Method for establishing immune-enhanced EAMG mouse model |
| CN115299405A (en) * | 2022-08-09 | 2022-11-08 | 新疆医科大学第一附属医院 | Method for making nude mouse transplantation tumor model |
-
2014
- 2014-07-02 CN CN201410312866.6A patent/CN104087554B/en not_active Expired - Fee Related
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944755A (en) * | 2020-09-01 | 2020-11-17 | 天津医科大学总医院 | Method for enhancing antigen presenting capability of dendritic cells |
| CN111944755B (en) * | 2020-09-01 | 2022-11-01 | 天津医科大学总医院 | Method for enhancing antigen presenting capability of dendritic cells |
| CN112831472A (en) * | 2021-02-07 | 2021-05-25 | 北京和合医学诊断技术股份有限公司 | Primary human thymoma cell, culture method and application thereof, and serum-free culture medium |
| CN113907049A (en) * | 2021-12-13 | 2022-01-11 | 天津医科大学总医院空港医院 | Method for establishing immune-enhanced EAMG mouse model |
| CN115299405A (en) * | 2022-08-09 | 2022-11-08 | 新疆医科大学第一附属医院 | Method for making nude mouse transplantation tumor model |
| CN115299405B (en) * | 2022-08-09 | 2024-01-30 | 新疆医科大学第一附属医院 | Method for manufacturing nude mouse transplanted tumor model |
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