CN102586188B - Cell line from human lung large cell cancer and preparation method thereof - Google Patents
Cell line from human lung large cell cancer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cell line from human lung large cell cancer. The cell line is named as human lung cancer cell line 1125 with the preservation number of CCTCC NO: C201029. Meanwhile, the invention also discloses a preparation method of the cell line.
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 people's lung large cell carcinoma 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, find 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 a difficult problem urgently 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 to tumor stem cell, this cell subset just tumour cells of origin and maintain 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 cell strain that the purpose of this invention is to provide a kind of people's of deriving from lung large cell carcinoma, 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 of the people's of deriving from lung large cell carcinoma, and called after human lung carcinoma cell line 1125, preserving number is CCTCC NO:C201029.
Described cell strain is observed under inverted 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 not tight, and dispersion easy to digest has cell that the suspension growth tendency is arranged.
The external doubling time of described cell strain is 35.775h.
The protein expression of described cell strain is characterized as vimentin and NSE all expresses the positive.
The cell strain 1125 of the people's of deriving from lung large cell carcinoma companion NE differentiation of the present invention is that the contriver cultivates successfully from lung cancer patient pulmonary masses excision thing, through pathology, is accredited as lung large cell carcinoma companion NE differentiation.For epithelial cell, there is typical cellular biology of tumor characteristic.It is continuous Long Term Passages in vitro, cultivates in vitro 1 year, passes generation more than 100, and cell still growing multiplication is active.
After testing, its general biological characteristics shows that this cell is the Polygons giant cells to clone of the present invention, adherent growth.The contact growth-inhibiting disappears.
A kind of preparation method who derives from the cell strain of people's lung large cell carcinoma comprises the following steps:
(1) tissue block mechanical dissociation: people's lung large cell carcinoma tissue 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 to tissue, adds collagenase, hatches, and filters, and then collects tumour cell, carries out inoculation culture, obtains survivaling cell;
(3) survivaling cell step (2) obtained carries out purifying, removes the impurity such as fibrocyte, and the lung carcinoma cell cultured continuously after purifying also goes down to posterity and is greater than 12 months, obtains cell strain.
As preparation method's of the present invention preferred implementation, described preparation method comprises the following steps:
(1) tissue block mechanical dissociation: people's lung large cell carcinoma tissue 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, allow tissue precipitation, removes supernatant liquor, and residue is organized and frittered, the cleansing tissue fragment, then add collagenase to tissue, under 37 ℃, hatches 4-18 hour; Filter, then collect tumour cell, containing suspension cell in the DMEM in high glucose substratum of 20% foetal calf serum, carry out inoculation culture, obtain survivaling cell;
(3) survivaling cell step (2) obtained carries out purifying, removes the impurity such as fibrocyte, and the lung carcinoma cell after purifying, containing cultured continuously in the DMEM in high glucose substratum of 20% foetal calf serum and go down to posterity and be greater than 12 months, obtains cell strain.
As preparation method's of the present invention preferred implementation, described step (1) is: people's lung large cell carcinoma tissue of excision is dissociated into to 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 in substratum and move under dissecting microscope, 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: the remaining tissue that uses aseptic scalper and scissors that step (1) is obtained is cut into 1mm
3tissue, by PBS buffer solution for cleaning tissue, allow tissue precipitate, remove supernatant liquor, with aseptic scalper and scissors, residue is organized and frittered, with PBS cleansing tissue fragment for several times, then add collagenase (50-200 unit/ml, be dissolved in PBS), hatch 4-18 hour under 37 ℃, by aseptic Stainless Steel Cloth or nylon net filter cell suspension, then collect tumour cell, containing suspension cell in the DMEM in high glucose substratum of 20% foetal calf serum, carry out inoculation culture, obtain survivaling cell.More preferably, in step (2), also add the CaCl that 3mM is arranged when adding collagenase
2.Add CaCl
2can improve the dissociation efficiency of collagenase to 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 is saturated humidity, within every 3 days, changes a nutrient solution, within every 5 days, goes down to posterity once.
The accompanying drawing explanation
Fig. 1 is that cell strain of the present invention is inverted (400 times of amplifications) growth viable cell picture under the optics phase microscope;
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, below in conjunction with the drawings and specific embodiments, the invention will be further described.
Embodiment 1
A kind of cell strain that derives from people's lung large cell carcinoma 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 to 0.5cm with the little scissors of sterilizing
3size, move to Biohazard Safety Equipment by 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 in substratum and move under dissecting microscope, 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 to 1mm
3tissue, by PBS buffer solution for cleaning tissue.Allow tissue precipitation, remove supernatant liquor, with aseptic scalper and scissors, residue is organized and frittered, with PBS cleansing tissue fragment several, then add collagenase (50-200 unit/ml, be dissolved in PBS), 37 ℃ hatch 4-18 hour after.Can add 3mM CaCl in the latex collagenase
2, to improve the dissociation efficiency of collagenase to tissue.By aseptic Stainless Steel Cloth or nylon net filter cell suspension, to separate cell dispersion, fragment of tissue and larger fragment; To larger fragment of tissue, can add fresh collagenase further to dissociate.After digestion fully, collect tumour cell, containing suspension cell in the DMEM in high glucose substratum of 20% foetal calf serum, carry out inoculation culture, obtain survivaling cell;
(3) step (2) gained survivaling cell is carried out to purifying, remove the impurity cells such as fibrocyte, the lung carcinoma cell after purifying is being cultivated containing in 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 is saturated humidity, within every 3 days, changes a nutrient solution, within every 5 days, goes down to posterity once, cultured continuously also goes down to posterity over 12 months, obtains cell strain.
Cancerous lung tissue in described step (1), derive from 59 years old female lung cancer patient's lower-left lung cancer focus.Patient does not accept the treatments such as any chemotherapy, radiotherapy and targeted therapy before undergoing surgery, and finds in art that lung tumors is positioned at the lower-left lung, and quality is crisp soft, a large amount of downright bad, and the profit growth is invaded in part, and parietal pleura extensively shifts.
The cell strain obtained in described step (3), send to the center preservation of Chinese Typical Representative culture collection on April 1st, 2010, and deposit number is CCTCC NO:C201029, called after human lung carcinoma cell line 1125.
Embodiment 2
The detection of human lung carcinoma cell line 1125 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 1125 of the present invention is factor analysis lung large cell carcinoma systems of a strain companion neuroendocrine function, derives from 53 years old male lung cancer patient's right upper lung carninomatosis kitchen range.Patient does not accept the treatments such as any chemotherapy, radiotherapy and targeted therapy before undergoing surgery.
2, morphologic observation
The gross morphology of main observation of cell, as big or small as general form, caryoplasm ratio, chromatin and kernel, how many 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.
Observe human lung carcinoma cell line 1125 of the present invention under inversion optics phase microscope, the visible cell volume is large, and core is large, and endochylema is abundant, and iuntercellular connects closely, and between plate, attaching is not tight, and dispersion easy to digest has cell that the suspension growth tendency is arranged.As shown in Figure 1.
2.2 projection electron microscopic observation
(1) take out sample from 2.5% glutaraldehyde stationary liquid, with 0.2mol/L PBS (pH7.4) rinsing 3 times, each 15min;
(2) 1%OsO
4rear fixing, room temperature 1h;
(3) 0.2mol/L PBS (pH7.4) rinsing is 3 times, each 15min;
(4) 50%, 70%, 90% and 100% acetone dewaters step by step, every gradient concentration 15min;
(5) soak into: use acetone: resin=soak 1h at 1: 1, then use acetone: resin=soak at 1: the 2 last virgin resin of 2h soaks and spends the night;
(6) carry out polymerization after the sample embedding, 70 ℃, 24h;
(7) ultrathin section(ing), thickness 80nm;
(8) after lead-uranium dyeing, upper machine is observed.
The Electronic Speculum ultrastructure shows, human lung carcinoma cell line 1125 cells of the present invention have impressive cell surface long wool hair, and iuntercellular fine hair is entwined, and connects closely.Core is large, a little heterochromatin of nuclear membrane marginal distribution, and organoid is more with plastosome and endoplasmic reticulum, and plastosome mostly is zebra line-transect plastochondria.
2.3 immunohistochemical methods detects cell marking albumen
Adopt the S-P method to carry out immunohistochemical staining, immunohistochemical staining S-P test kit is purchased 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 hatch 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), under room temperature, hatches 10 minutes;
(4), except serum deprivation, every section adds the first antibody of 1 or 50 μ l, under room temperature, hatches 60 minutes;
(5) PBS rinses three times, each 3-5 minute; Remove PBS liquid, every section adds one or the biotin labeled second antibody of 50 μ l (reagent C), under room temperature, hatches 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), under room temperature, hatches 10 minutes; PBS rinses three times, each 3 minutes;
(7) remove PBS liquid, every section adds 2 or the freshly prepared DAB of 100 μ l or AEC solution, micro-Microscopic observation 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 through the gradient alcohol dehydration drying, 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, by the quantity of positive cell, mark: 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 1125 of the present invention, 1125 immunohistochemical staining shows that its metastasis tumor tissues to source has similar protein expression, the positive expression that its outstanding expression characteristic is NSE.1125 cancer cells have neuroendocrine function.
1125 also express Vimentin simultaneously, and the metastatic potential of this prompting clone is higher.
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 containing 2 * 10
4～10 * 10
4target cell prepare liquid (containing the DMEM of 10% calf serum, preparing liquid), preparation 2-3 hour in the saturation vapour carbonic acid gas of 37 ℃ of 5%CO2 prepares case, allow 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: ，Mei hole, 3 positive control holes adds 0.1ml and prepares liquid and cell, 3 negative control holes containing the DMEM of 1000 times of medicines, every hole adds the 0.1ml DMEM that does not contain medicine and prepares liquid and cell, ，Mei hole, 3 blank holes adds 0.1ml DMEM and prepares liquid, does not add cell.At 37 ℃ of 5%CO
2the saturation vapour carbonic acid gas prepare in case and prepare 24～48 hours or predetermined time;
(3) suck and prepare liquid, with PBS washing once (as be suspension cell, should before sucking supernatant liquor the centrifugal plate for preparing);
(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 in case and prepare 4～6 hours;
(5) every hole adds 0.1ml acidifying Virahol, and also the available 10mmol/L HCl containing 10%SDS replaces the acidifying Virahol, on vibrator, vibrates and mixes, and allows reduzate fully dissolve.Put on enzyme connection detector and measure optical density(OD) (OD) value, detect wavelength 570nm, reference wavelength 630nm.With the OD value, 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.
3.2 the cell flow cytometer detects cell cycle and apoptosis
Flow cytometer is that U.S. BECKMAN-COULTER (Beckman-Ku Erte) company produces.Machine models: ELITE, optical maser wavelength 488nm, power 15MW.Get 10
6fixing cell, wash twice with PBS, goes to add 300 μ l DNA dye liquors (including propidium iodide 100 μ g/ml and RNase 20 units/ml), room temperature 30min after supernatant.Upper machine: after laser tube preheating 30min, with fluorescent microsphere (U.S. BECKMAN-COULTER company), adjust instrument, the HCV value of the signal that each amplifier is received<2%, collect 12000 cells, the MULTYCYCLE software of U.S. PHEONIX company for the DNA cycle analysis, finally calculate the percentage ratio of each phase of cell.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 1125 of the present invention is 35.775h.
4, nucleus type analysis
Detect the caryogram characteristics, the having or not of karyomit(e) quantity, marker chromosomes, banding pattern etc.
4.1 prepare: colchicine (colchicine), physiological saline is mixed with 10 μ g/ml concentration, 15 pounds of sterilizings in 20 minutes, packing, put-20 ℃; Hypotonic medium: 0.35%KCl; Stationary liquid (Carnoy stationary liquid) methyl alcohol: glacial acetic acid (3: 1), interim preparation; Giemsa working fluid: 1 part of stoste and 10 parts of phosphoric acid buffers, interim preparation.
4.2 colchicine is processed: stop the front 2-4 hour of preparation, add colchicine (drip 2 with No. 5 needle points of 1ml syringe, making final concentration is 0.07 μ g/ml) in preparing liquid.
4.3 karyomit(e) preparation
(1) collecting cell: prepared product is all proceeded in clean centrifuge tube, with the centrifugal 8-10 minute of 1000rpm, abandon supernatant liquor;
(2) hypotonic processing: the hypotonic medium 8ml to adding 37 ℃ of pre-temperature in graduated centrifuge tube, with dropper, mix, put hypotonic 15-25 minute in 37 ℃ of waters bath with thermostatic control;
(3) pre-fix: add the 0.5ml stationary liquid after hypotonic, mix gently the centrifugal 8-10 minute of rear 1000rpm;
(4) one is fixing: abandon supernatant liquor, add the 5ml stationary liquid, mix gently, standing 20 minutes; 1000rpm is centrifugal, abandons supernatant liquor;
(5) two fixing, three fixing: same fixing;
(6) suspension processed: after abandoning supernatant liquor, visual cell's quantity adds appropriate stationary liquid to make cell suspension;
(7) drip sheet: draw cell suspension on the high slide glass that drops in a dried and clean of 10-20cm, featheriness is loose, and gas is dry;
(8) dyeing: the 1:10Giemsa 5-10 minute that dye, unnecessary dye liquor is removed in thin washing, and gas is done;
(9) microscopy: the division phase that find good dispersion under low power lens, dyes moderate, oily Microscopic observation chromosome morphology counting.
5, allosome animal inoculation pvaccination
To inoculating cell suspension in the allosome animal body, observe into the knurl ability.Adopt hypodermic method, during injection, with left hand thumb and forefinger, pinched gently skin, the right hand is held syringe syringe needle is thrust, and after fixing, at mouse back or forelimb oxter, is injected.
10
6individual human lung carcinoma cell line of the present invention 1125 cell seedings are subcutaneous at the NOD-SCID mouse scapular region in 54 week ages, and the transplanted tumor of diameter 2mm all appears in the 3rd week plantation position, after 2 months, the knurl body increases to 1.2cm, put to death animal after anesthesia, the transplanted tumor tissue is through embedded section HE dyeing, and form as shown in Figure 2, cell volume is large, atypia is obvious, and core is large, and nuclear atypia is obvious, kernel is obvious, 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 is explained in detail; 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 (4)
1. a cell strain that derives from people's lung large cell carcinoma, is characterized in that, called after human lung carcinoma cell line 1125, and preserving number is CCTCCNO:C201029.
2. the cell strain that derives from people's lung large cell carcinoma as claimed in claim 1, it is characterized in that, described cell strain is observed under inverted microscope, the visible cell volume is large, and core is large, and endochylema is abundant, iuntercellular connects closely, and between plate, attaching is not tight, dispersion easy to digest, have cell that the suspension growth tendency is arranged.
3. the cell strain that derives from people's lung large cell carcinoma as claimed in claim 1, is characterized in that, the external doubling time of described cell strain is 35.775h.
4. the cell strain that derives from people's lung large cell carcinoma as claimed in claim 1, is characterized in that, the protein expression of described cell strain is characterized as vimentin and NSE all expresses the positive.
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| CN105647869B (en) * | 2016-02-16 | 2020-12-15 | 广州医科大学附属第一医院 | Human lung adenocarcinoma cell strain HA1221 and establishment method thereof |
| CN107312752A (en) * | 2017-07-03 | 2017-11-03 | 浙江省肿瘤医院 | A kind of lung cancer cell line and its application |
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| Establishment and Identification of small lung cancer cell lines having classic and variant features;Desmond N.Carney et al;《CANCER RESEARCH》;19851231;第45卷;2913-2923 * |
| 人肝癌组织中肿瘤干细胞样细胞的分离培养及鉴定;赵璇等;《中国肿瘤生物治疗杂志》;20091031;第16卷(第5期);436-441 * |
| 吴婕等.肺癌细胞系中肿瘤干细胞的分离鉴定与功能分析.《中国组织工程研究与临床康复》.2010,第14卷(第32期),5988-5991. |
| 肺癌细胞系中肿瘤干细胞的分离鉴定与功能分析;吴婕等;《中国组织工程研究与临床康复》;20100806;第14卷(第32期);5988-5991 * |
| 赵璇等.人肝癌组织中肿瘤干细胞样细胞的分离培养及鉴定.《中国肿瘤生物治疗杂志》.2009,第16卷(第5期),436-441. |
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