CN102424817B - Cell strain from human lung adenocarcinoma and preparation method thereof - Google Patents
Cell strain from human lung adenocarcinoma and preparation method thereof Download PDFInfo
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- CN102424817B CN102424817B CN 201110461087 CN201110461087A CN102424817B CN 102424817 B CN102424817 B CN 102424817B CN 201110461087 CN201110461087 CN 201110461087 CN 201110461087 A CN201110461087 A CN 201110461087A CN 102424817 B CN102424817 B CN 102424817B
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Abstract
The invention discloses a cell strain from human lung adenocarcinoma. The cell strain is named human lung cancer cell strain 0907, with collection number being CCTCC NO:C201031. Meanwhile, the invention also discloses a preparation method of the cell strain.
Description
Technical field
The present invention relates to a kind of human lung carcinoma cell line and preparation method thereof, especially a kind of cell strain that derives from human lung adenocarcinoma and preparation method thereof.
Background technology
Tumour is one of principal disease of serious harm human health, the research report that the World Health Organization delivers shows, whole world cancer condition will be day by day serious, therefore, the molecular mechanism of further investigation tumorigenesis and transfer, seek more efficiently oncotherapy scheme, become the advanced subject of current medical research.Along with deepening continuously of oncobiology research, the early diagnosis of tumour and prevention level are enhanced, but the height after oncotherapy shifts and high recurrence is still difficult problem that needs to be resolved hurrily in clinical therapy of tumor.In recent years, proposition and the progress thereof of " tumor stem cell " theory go to be familiar with tumour from a brand-new angle.This theory thinks, have the unique stem-like cell subgroup with self and differentiation function of a part in tumour and it be called tumor stem cell, this cell subset just tumour cells of origin and keep the growth of tumour.Therefore, tumor cell line is the important materials of the Biomedical Problems such as research cell carcinogenesis mechanism, metastases, tumor radiotherapy and chemosensitivity molecular basis, sets up and identifies that different tumor cell lines is a job highly significant.
Summary of the invention
The purpose of this invention is to provide a kind of cell strain that derives from human lung adenocarcinoma, described cell strain has typical cellular biology of tumor characteristic; A kind of preparation method of described cell strain also is provided simultaneously.
For achieving the above object, the technical scheme that the present invention takes is: a kind of cell strain that derives from human lung adenocarcinoma, called after human lung carcinoma cell line 0907, preserving number are CCTCC NO:C201031.
Described cell strain is observed under inversion optics phase microscope, and the visible cell volume is large, and core is large, and endochylema is abundant, and iuntercellular connects closely, and between plate, attaching is grown.
The external doubling time of described cell strain is 60.325h.
The protein expression of described cell strain is characterized as the vegf expression positive.0907 immunohistochemical staining shows that its metastasis tumor tissues to the source has similar protein expression, and its outstanding expression characteristic is the VEGF positive expression.This is a vascular endothelial growth factor, and its positive expression has obvious promoter action to tumor vascular formation and tumor growth, and 0907 can be used as a research with the strong instrument cell of VEGF as the targeted drug research of target.
Human lung carcinoma cell line 0907 is that a strain southern china human lung adenocarcinoma cell is, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, the treatments such as radiotherapy and targeted therapy before undergoing surgery.The described cell strain that derives from human lung adenocarcinoma, to excise thing from the lung cancer patient pulmonary masses that histological type is undifferentiated carcinoma to cultivate gained, be accredited as the lung undifferentiated carcinoma through pathology, be the Polygons epithelial cell, have typical cellular biology of tumor characteristic, it can be at external continuous Long Term Passages, external preparation 1 year, pass generation more than 100, cell still growing multiplication is active.
A kind of preparation method who derives from the cell strain of human lung adenocarcinoma comprises the following steps:
(1) tissue block mechanical dissociation: the human lung adenocarcinoma of excision is dissociated, clean and remove impurity, separate the Parenchyma of Lung Carcinoma tissue, after removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue;
(2) collagenase digesting: the remaining tissue that step (1) is obtained is cut into tissue, cleans, and then adds collagenase to tissue, hatches, and filters, and then collects tumour cell, carries out inoculation culture, obtains survivaling cell;
(3) survivaling cell that step (2) is obtained carries out purifying, removes the impurity such as fibrocyte, and the lung carcinoma cell cultured continuously after purifying also goes down to posterity greater than 12 months, namely gets cell strain.
As preparation method's of the present invention preferred implementation, described preparation method comprises the following steps:
(1) tissue block mechanical dissociation: the human lung adenocarcinoma of excision is dissociated, clean impurity such as removing mucus and red corpuscle, separate the Parenchyma of Lung Carcinoma tissue, after removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue;
(2) collagenase digesting: the remaining tissue that step (1) is obtained is cut into 1mm
3Tissue, the cleansing tissue sheet allows tissue precipitation, removes supernatant liquor, residue is organized again frittered, then the cleansing tissue fragment adds collagenase to tissue, hatches under 37 ℃ 4-18 hour; Filter, then collect tumour cell, suspension cell in containing the DMEM in high glucose substratum of 20% foetal calf serum carries out inoculation culture, obtains survivaling cell;
(3) survivaling cell that step (2) is obtained carries out purifying, removes the impurity such as fibrocyte, and the cultured continuously and going down to posterity greater than 12 months in containing the DMEM in high glucose substratum of 20% foetal calf serum of the lung carcinoma cell after purifying namely gets cell strain.
As preparation method's of the present invention preferred implementation, described step (1) is: the human lung adenocarcinoma of excision is dissociated into 0.5cm
3The two anti-solution soaking of green grass or young crops-Streptomycin sulphate with 100% move to Biohazard Safety Equipment, with the PBS damping fluid of sterilization repeatedly cleansing tissue remove the impurity such as mucus and red corpuscle, be soaked in again and move under dissecting microscope in substratum, separate the Parenchyma of Lung Carcinoma tissue with two No. 10 syringe needles, and after removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue.
As preparation method's of the present invention preferred implementation, step (2) is: use aseptic scalper and scissors that the remaining tissue that step (1) obtains is cut into 1mm
3Tissue, with PBS buffer solution for cleaning tissue, allow tissue precipitate, remove supernatant liquor, with aseptic scalper and scissors, residue is organized again and frittered, with PBS cleansing tissue fragment for several times, then add collagenase (50-200 unit/ml, be dissolved in PBS), hatched under 37 ℃ 4-18 hour, by aseptic Stainless Steel Cloth or nylon net filter cell suspension, then collect tumour cell, suspension cell in containing the DMEM in high glucose substratum of 20% foetal calf serum carries out inoculation culture, obtains survivaling cell.More preferably, in step (2), also add the CaCl that 3mM is arranged when adding collagenase
2Add CaCl
2Can improve collagenase to the dissociation efficiency of tissue.
As preparation method's of the present invention preferred implementation, the culture temperature of the lung carcinoma cell in step (3) after purifying is 37 ℃, and atmosphere surrounding is 5%CO
2/ 95% air, humidity are saturated humidity, change a nutrient solution, go down to posterity once in every 5 days in every 3 days.
Description of drawings
Fig. 1 is the viable cell picture that cell strain of the present invention is inverted the lower growth of optics phase microscope (400 times of amplifications);
Fig. 2 is the aspect graph that cell strain of the present invention is planted in NOD-SCID mouse scapular region subcutaneous transplantation tumor tissue.
Embodiment
For the purpose, technical solutions and advantages of the present invention better are described, the invention will be further described below in conjunction with the drawings and specific embodiments.
Embodiment 1
A kind of cell strain that derives from human lung adenocarcinoma of the present invention, according to following preparation method's gained:
(1) tissue block mechanical dissociation: the cancerous lung tissue of excision is dissociated to rapidly 0.5cm with the little scissors of sterilizing
3Size, move to Biohazard Safety Equipment with the two anti-solution soaking of 100% green grass or young crops-Streptomycin sulphate, sterilization PBS damping fluid cleansing tissue is repeatedly removed the impurity such as mucus and red corpuscle, be soaked in again and move under dissecting microscope in substratum, with the careful Parenchyma of Lung Carcinoma tissue that separates of two No. 10 syringe needles, after removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue as far as possible;
(2) collagenase digesting: remove the stroma in lung cancer specimen under dissecting microscope after, use aseptic scalper and scissors that the remaining tissue of step (1) gained is cut into 1mm
3Tissue is with PBS buffer solution for cleaning tissue.Allow tissue precipitation, remove supernatant liquor, with aseptic scalper and scissors, residue is organized again and frittered, with PBS cleansing tissue fragment several, then add collagenase (50-200 unit/ml is dissolved in PBS), 37 ℃ hatch 4-18 hour after.Can add 3mM CaCl in the latex collagenase
2, to improve collagenase to the dissociation efficiency of tissue.By aseptic Stainless Steel Cloth or nylon net filter cell suspension, to separate cell dispersion, fragment of tissue and larger fragment; Can add fresh collagenase further to dissociate to larger fragment of tissue.After digestion fully, collect tumour cell, suspension cell in containing the DMEM in high glucose substratum of 20% foetal calf serum carries out inoculation culture, obtains survivaling cell;
(3) step (2) gained survivaling cell is carried out purifying, remove the impurity cells such as fibrocyte, the lung carcinoma cell after purifying is cultivated in containing the DMEM in high glucose substratum of 20% foetal calf serum, and culture temperature is 37 ℃, and atmosphere surrounding is 5%CO
2/ 95% air, humidity are saturated humidity, change a nutrient solution in every 3 days, go down to posterity once in every 5 days, and cultured continuously also goes down to posterity over 12 months, gets cell strain 0907.
0907 is that a strain southern china human lung adenocarcinoma cell is, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, the treatments such as radiotherapy and targeted therapy before undergoing surgery.
The cell strain that obtains in described step (3) is sent to the center preservation of Chinese Typical Representative culture collection on April 1st, 2010, and deposit number is CCTCC NO:C201031, called after human lung carcinoma cell line 0907.
Embodiment 2
The detection of human lung carcinoma cell line 0907 of the present invention is identified
1, the G6PD isozyme detects source of species (by Chinese Typical Representative prepared product preservation Spot detection).
Human lung carcinoma cell line 0907 of the present invention is that a strain southern china human lung adenocarcinoma cell is, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, the treatments such as radiotherapy and targeted therapy before undergoing surgery.
2, morphologic observation
The gross morphology of main observation of cell, as general form, caryoplasm ratio, chromatin and kernel size, what etc., and the ordered state of cytoskeletal filament microtubule etc.
2.1 inversion observation by light microscope
Main under the normal growth state gross morphology of observation of cell, as general form, caryoplasm ratio, adhesion characteristics etc.
Be inverted and observe human lung carcinoma cell line 0907 of the present invention under the optics phase microscope, the visible cell volume is large, and core is large, and endochylema is abundant, and iuntercellular connects closely, and attaches growth between plate.As shown in Figure 1.
2.2 immunohistochemical methods detects cell marking albumen
Adopt the S-P method to carry out immunohistochemical staining, immunohistochemical staining S-P test kit is available from Fuzhou Maixin biotechnology Development Co., Ltd.Antibody used is: CK7, TTF-1, vimentin, EGFR, VEGF and NSE.
Concrete steps are:
(1) 24 hours in advance creep plates of cell strain to be detected on the sterilization slide glass, after 24 hours, rinse 2 times with the PBS damping fluid, and cold acetone is fixed 15 minutes, is soaked in PBS standby;
(2) get required slide and add under 1 or 50 μ l peroxidase blocking solution (reagent A) room temperatures and hatched 10 minutes, with the activity of blocking-up endogenous peroxydase; PBS rinses three times, each 3 minutes;
(3) remove PBS liquid, every section adds 1 or the normal nonimmune animal serum of 50 μ l (reagent B), hatches under room temperature 10 minutes;
(4) except serum deprivation, every section adds the first antibody of 1 or 50 μ l, hatches under room temperature 60 minutes;
(5) PBS rinses three times, each 3-5 minute; Remove PBS liquid, every section adds one or the 50 biotin labeled second antibody of μ l (reagent C), hatches under room temperature 10 minutes; PBS rinses three times, each 3 minutes;
(6) remove PBS liquid, every section adds 1 or 50 μ l streptomycete antibiotin peroxidase solution (reagent D), hatches under room temperature 10 minutes; PBS rinses three times, each 3 minutes;
(7) remove PBS liquid, every section adds 2 or the 100 freshly prepared DAB of μ l or AEC solution, and microscopically was observed 3-10 minute;
(8) tap water rinses, and Hematorylin is redyed, and PBS or tap water rinse and return indigo plant;
(9) DAB colour developing, section is dry through gradient alcohol dehydration, and dimethylbenzene is transparent, the neutral gum sealing.
Immunohistochemical methods detected result determination methods: press the painted scoring of cell: brown 3 minutes; Brown color 2 minutes; Faint yellow 1 minute; Non-coloring 0 minute.Count positive cell number under same object lens, mark by the quantity of positive cell: visual field intrinsic color cell＞70% is 4 minutes; 51%-75% is 3 minutes; 11%-50% is 2 minutes; 1%-10% is 1 minute; Feminine gender is 0 minute.Two scores multiply each other, and full 3 are divided into "+"; 4 are divided into " ++ "; More than 5 minutes be " +++"." +～+++" positive expression.
The immunohistochemical staining demonstration of human lung carcinoma cell line 0907 of the present invention, its metastasis tumor tissues to the source has similar protein expression, and its outstanding expression characteristic is the VEGF positive expression.This is a vascular endothelial growth factor, and its positive expression has obvious promoter action to tumor vascular formation and tumor growth, and 0907 can be used as a research with the strong instrument cell of VEGF as the targeted drug research of target.
3, growth and proliferation of cell
Detect cell growth curve, cell division index, doubling time, cell cycle time.
3.1MTT method is measured growth and proliferation of cell and to the susceptibility of medicine
(1) get 96 porocytes and prepare plate, add 0.1ml in every hole and contain 2 * 10
4～10 * 10
4The preparation liquid of target cell (DMEM that contains 10% calf serum prepares liquid), preparation is 2-3 hour in the saturation vapour carbonic acid gas of 37 ℃ of 5% CO2 prepares case, allows cell attachment;
(2) prepare 0.1～100 times of successive configuration medicine of liquid with DMEM, every hole adds medicine and the cell to be checked of 0.1ml dilution, 3 repeating holes of each extent of dilution.9 of control wells: 3 positive control holes, every hole add the DMEM that 0.1ml contains 1000 times of medicines and prepare liquid and cell, 3 negative control holes, every hole adds the 0.1ml DMEM that does not contain medicine and prepares liquid and cell, 3 blank holes, every hole adds 0.1ml DMEM and prepares liquid, does not add cell.At 37 ℃ of 5% CO
2The saturation vapour carbonic acid gas prepare and prepared in case 24～48 hours or predetermined time;
(3) suck preparation liquid, with PBS washing once (as being suspension cell, should before sucking supernatant liquor centrifugal preparation plate);
(4) every hole adds 0.1ml PBS and 10 μ l MTT dye liquors, at 37 ℃ of 5% CO
2The saturation vapour carbonic acid gas prepare and prepare 4～6 hours in case;
(5) every hole adds 0.1ml acidifying Virahol, and also the available 10mmol/L HCl that contains 10% SDS replaces the acidifying Virahol, and the mixing that vibrates on vibrator allows reduzate fully dissolve.Put on enzyme connection detector and measure optical density(OD) (OD) value, detect wavelength 570nm, reference wavelength 630nm.To the mapping of diluted sample degree, standard of comparison curve and testing sample curve can be tried to achieve the content of cytokine in testing sample with the OD value.
3.2 the cell flow cytometer detects cell cycle and apoptosis
Flow cytometer is that (Beckman-Ku Erte) company produces U.S. BECKMAN-COULTER.Machine models: ELITE, optical maser wavelength 488nm, power 15MW.Get 10
6Fixing cell washes twice with PBS, goes to add after supernatant 300 μ l DNA dye liquors (to include propidium iodide 100 μ g/ml and RNase 20 units/ml), room temperature 30min.Upper machine: adjust instrument with fluorescent microsphere (U.S. BECKMAN-COULTER company) after laser tube preheating 30min, make the HCV value of the signal that each amplifier receives＜2%, collect 12000 cells, the DNA cycle analysis calculates the percentage ratio of each phase of cell at last with the MULTYCYCLE software of U.S. PHEONIX company.In addition, apoptosis is to carry software processes with instrument, directly draws the apoptosis rate of cell.
The external doubling time of human lung carcinoma cell line 09070907 of the present invention is 60.325h.
5, allosome animal inoculation pvaccination
Inoculating cell suspension in the allosome animal body is observed into the knurl ability.Adopt hypodermic method, pinched gently skin with left hand thumb and forefinger during injection, the right hand is held syringe syringe needle is thrust, and injects at mouse back or forelimb oxter after fixing.
10
6It is subcutaneous that individual human lung carcinoma cell of the present invention 0907 is planted in the NOD-SCID mouse scapular region in 54 ages in week, the transplanted tumor of diameter 2mm all appears in the 3rd week plantation position, the posterior tuberosity body increased to 1.2cm in 2 months, put to death animal after anesthesia, the transplanted tumor tissue dyes form as shown in Figure 2 through embedded section HE, and cell volume is large, and core is large, kernel is obvious, and is similar to the tissue of patient in source.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Claims (1)
1. a cell strain that derives from human lung adenocarcinoma, is characterized in that, called after human lung carcinoma cell line 0907, preserving number are CCTCC NO:C201031.
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| CN105695412B (en) * | 2016-02-16 | 2020-06-16 | 广州医科大学附属第一医院 | Human lung adenocarcinoma cell line HA109 and establishment method thereof |
| CN108913662B (en) * | 2018-08-09 | 2019-03-29 | 爱克精医(北京)生物医药科技有限公司 | A kind of method of the functional high-throughput medication screening of lung cancer |
| CN116355851B (en) * | 2023-03-13 | 2023-09-08 | 广州医科大学附属第一医院（广州呼吸中心） | Primary cell strain derived from human non-small cell lung cancer, and preparation method and application thereof |
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