CN102424817A - 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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- CN102424817A CN102424817A CN2011104610879A CN201110461087A CN102424817A CN 102424817 A CN102424817 A CN 102424817A CN 2011104610879 A CN2011104610879 A CN 2011104610879A CN 201110461087 A CN201110461087 A CN 201110461087A CN 102424817 A CN102424817 A CN 102424817A
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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 RR that the World Health Organization delivers shows; Whole world cancer condition will be serious day by day, therefore, and the molecular mechanism that the further investigation tumour takes place, develops and shift; 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 the oncotherapy shifts and high recurrence is still difficult problem that needs to be resolved hurrily in the clinical therapy of tumor.In recent years, the 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 of a part in the tumour and it be called tumor stem cell with self and differentiation function, this cell subset just tumour cells of origin and keep growth of tumor.Therefore, tumor cell line is the important materials of 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, said cell strain has typical cellular biology of tumor characteristic; A kind of preparation method of said cell strain also is provided simultaneously.
For realizing above-mentioned purpose, 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.
Said cell strain is being inverted observation under the optics phase microscope, and the visible cell volume is big, and nuclear is big, and endochylema is abundant, and iuntercellular connects tight, and attaches growth between the plate.
The external doubling time of said cell strain is 60.325h.
The protein expression of said cell strain is characterized as the vegf expression positive.0907 immunohistochemical staining shows that its MET tumor tissues with the source has similar protein expression, and its outstanding expression characteristic is the VEGF positive expression.This is a VEGF, and its positive expression has obvious facilitation 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 a strain southern china Lu-csf-1, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, treatments such as radiotherapy and targeted therapy before undergoing surgery.The said cell strain that derives from human lung adenocarcinoma is from histological type is the lung cancer patient lung lump excision thing of undifferentiated carcinoma, to cultivate gained, is accredited as the lung undifferentiated carcinoma through pathology; Be the Polygons epithelial cell; Have typical cellular biology of tumor characteristic, it can go down to posterity external continuously for a long time, 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 may further comprise the steps:
(1) tissue block mechanical dissociation: the human lung adenocarcinoma tissue of excision is dissociated, the cleaning and removing removal of impurity, separation lung cancer parenchymal tissue, remove normal alveolar and bronchial tissue and stroma after, obtain remaining tissue;
(2) collagenase digesting: the remaining tissue that step (1) is obtained is cut into tissue, cleans, and adds collagenase to tissue then, hatches, and filters, and collects tumour cell then, carries out inoculation culture, obtains survivaling cell;
(3) survivaling cell that step (2) is obtained carries out purifying, removes impurity such as fibrocyte, and the lung carcinoma cell cultured continuously behind the purifying also goes down to posterity greater than 12 months, promptly gets cell strain.
As preparing method's according to the invention preferred implementation, said preparation method may further comprise the steps:
(1) tissue block mechanical dissociation: the human lung adenocarcinoma tissue of excision is dissociated, and cleaning and removing is removed impurity such as mucus and red corpuscle, separation lung cancer parenchymal tissue, remove normal alveolar and bronchial tissue and stroma after, obtain remaining tissue;
(2) collagenase digesting: the remaining tissue that step (1) is obtained is cut into 1mm
3Tissue, the cleansing tissue sheet lets tissue deposition, removes supernatant, organizes residue again and fritters, the cleansing tissue fragment adds collagenase to tissue then, under 37 ℃, hatches 4-18 hour; Filter, collect tumour cell then, suspension cell in containing the high sugared DMEM 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 impurity such as fibrocyte, and the cultured continuously and going down to posterity greater than 12 months in containing the high sugared DMEM substratum of 20% foetal calf serum of the lung carcinoma cell behind the purifying promptly gets cell strain.
As preparing method's according to the invention preferred implementation, said step (1) is: the human lung adenocarcinoma tissue 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 disinfectant PBS damping fluid repeatedly cleansing tissue remove impurity such as mucus and red corpuscle, be soaked in again and move in the substratum under the dissecting microscope, with two No. 10 syringe needles separation lung cancer parenchymal tissues; And after removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue.
As preparing method's according to the invention preferred implementation, step (2) is: use aseptic scalper and scissors to be cut into 1mm to the remaining tissue that step (1) obtains
3Tissue, with PBS buffer solution for cleaning tissue, let tissue deposition; Remove supernatant, organize residue again with aseptic scalper and scissors and fritter, for several times with PBS cleansing tissue fragment; Add collagenase (50-200 unit/ml is dissolved among the PBS) then, under 37 ℃, hatched 4-18 hour; Through aseptic Stainless Steel Cloth or nylon net filter cell suspension, collect tumour cell then, suspension cell in containing the high sugared DMEM substratum of 20% foetal calf serum; Carry out inoculation culture, obtain survivaling cell.More preferably, in the step (2), also add the CaCl that 3mM is arranged when adding collagenase
2Add CaCl
2Can improve the dissociation efficiency of collagenase to tissue.
As preparing method's according to the invention preferred implementation, the culture temperature of the lung carcinoma cell in the step (3) behind the 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 per 5 days in per 3 days.
Description of drawings
Fig. 1 is that cell strain according to the invention is inverted optics phase microscope (400 times of amplifications) the viable cell picture of growth down;
Fig. 2 is planted in the aspect graph of NOD-SCID mouse omoplate portion subcutaneous transplantation tumor tissue for cell strain according to the invention.
Embodiment
The present invention is further described the object of the invention, technical scheme and advantage for better explaining below in conjunction with accompanying drawing and specific embodiment.
Embodiment 1
A kind of cell strain that derives from human lung adenocarcinoma of the present invention, according to following preparing method's gained:
(1) tissue block mechanical dissociation: with the cancerous lung tissue of excision with the little scissors of the sterilizing 0.5cm that dissociates rapidly
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 impurity such as mucus and red corpuscle, is soaked in to move in the substratum under the dissecting microscope, with the careful lung cancer parenchymal tissue that separates of two No. 10 syringe needles again; After removing normal alveolar and bronchial tissue and stroma, obtain remaining tissue as far as possible;
(2) collagenase digesting: after under dissecting microscope, removing the stroma in the lung cancer specimen, use aseptic scalper and scissors to be cut into 1mm to the remaining tissue of step (1) gained
3Tissue is with PBS buffer solution for cleaning tissue.Let tissue deposition, remove supernatant, organize residue again with aseptic scalper and scissors and fritter,, add collagenase (50-200 unit/ml is dissolved among the PBS) then with PBS cleansing tissue fragment several, 37 ℃ hatch 4-18 hour after.In the latex collagenase, can add 3mM CaCl
2, to improve the dissociation efficiency of collagenase to tissue.Through aseptic Stainless Steel Cloth or nylon net filter cell suspension, to separate cell dispersion, fragment of tissue and bigger fragment; Can add fresh collagenase to bigger fragment of tissue further dissociates.After the digestion fully, collect tumour cell, suspension cell in containing the high sugared DMEM substratum of 20% foetal calf serum carries out inoculation culture, obtains survivaling cell;
(3) step (2) gained survivaling cell is carried out purifying, remove impurity cells such as fibrocyte, the lung carcinoma cell behind the purifying is cultivated in containing the high sugared DMEM 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 per 3 days, go down to posterity once in per 5 days, and cultured continuously also went down to posterity above 12 months, got cell strain 0907.
0907 is a strain southern china Lu-csf-1, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, treatments such as radiotherapy and targeted therapy before undergoing surgery.
The cell strain that obtains in the said step (3) is sent to Chinese typical culture collection center preservation 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 according to the invention is identified
1, the G6PD isozyme detects source of species (being detected by China typical prepared product preservation center).
Human lung carcinoma cell line 0907 according to the invention is a strain southern china Lu-csf-1, derives from 60 years old male lung cancer patient's upper left lung cancer focus.Patient does not accept any chemotherapy, 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, like general form, caryoplasm ratio, adhesion characteristics etc.
Be inverted the optics phase microscope and observe human lung carcinoma cell line 0907 according to the invention down, the visible cell volume is big, and nuclear is big, and endochylema is abundant, and iuntercellular connects closely, and attaches growth between the 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.Used antibody 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 are on the sterilization slide glass, and after 24 hours, with PBS damping fluid flushing 2 times, fixing 15 minutes of cold acetone is soaked among the PBS subsequent use;
(2) get required slide and add under 1 or 50 μ l px blocking solution (reagent A) room temperatures and hatched 10 minutes, with block endogenous property Peroxidase activity; PBS flushing three times, each 3 minutes;
(3) remove PBS liquid, every section adds 1 or the normal non-immune serum of 50 μ l (reagent B), hatches under the room temperature 10 minutes;
(4) remove serum deprivation, every section adds the first antibody of 1 or 50 μ l, hatches under the room temperature 60 minutes;
(5) the PBS flushing is three times, each 3-5 minute; Remove PBS liquid, every section adds one or the biotin labeled SAs of 50 μ l (reagent C), hatches under the room temperature 10 minutes; PBS flushing 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 the room temperature 10 minutes; PBS flushing three times, each 3 minutes;
(7) remove PBS liquid, every section adds 2 or freshly prepared DAB of 100 μ l or AEC solution, and microscopically was observed 3-10 minute;
(8) tap water flushing, Hematorylin is redyed, and indigo plant is returned in PBS or tap water flushing;
(9) DAB colour developing, section is dry through gradient alcohol dehydration, and YLENE is transparent, the neutral gum sealing.
Immunohistochemical methods detected result determination methods: press the painted scoring of cell: brown 3 minutes; Pale brown look 2 minutes; Faint yellow 1 minute; Non-coloring 0 minute.Same object lens are the counting positive cell number down, and 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 of human lung carcinoma cell line 0907 according to the invention shows that its MET tumor tissues with the source has similar protein expression, and its outstanding expression characteristic is the VEGF positive expression.This is a VEGF, and its positive expression has obvious facilitation 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) gets 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) prepared in the case preparation 2-3 hour at the saturation vapour carbonic acid gas of 37 ℃ of 5% CO2, let cell attachment;
(2) prepare 0.1～100 times of successive configuration of liquid medicine with DMEM, every hole adds the 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 not, and the 0.1ml DMEM of drug 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 the case 24～48 hours or preset time;
(3) inhale and to remove to prepare liquid, with PBS washing once (as being suspension cell, should before supernatant is removed in suction 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 the case;
(5) every hole adds 0.1ml acidifying Virahol, and the also available 10mmol/L HCl that contains 10% SDS replaces the acidifying Virahol, and the mixing that on vibrator, vibrates lets reduzate fully dissolve.Put on the enzyme couplet 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 the 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
6The fixed cell washes twice with PBS, removes to add behind the supernatant 300 μ l DNA dye liquors and (includes propidium iodide 100 μ g/ml and RNase 20 units/ml), room temperature 30min.Last machine: adjust instrument with fluorescent microsphere (U.S. BECKMAN-COULTER company) behind the laser tube preheating 30min; Make HCV value＜2% of the signal of each magnifying glass reception; 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 according to the 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 skin gently with left hand thumb and forefinger during injection, the right hand is held syringe syringe needle is thrust, and inject at mouse back or forelimb oxter fixing back.
10
6It is subcutaneous that individual human lung carcinoma cell according to the invention 0907 is planted in the NOD-SCID mouse omoplate portion in 54 ages in week, and the 3rd week was planted the transplanted tumor that the about 2mm of diameter all appears in the position, and the posterior tuberosity body increased to 1.2cm in 2 months; Animal is put to death in the anesthesia back; The transplanted tumor tissue is as shown in Figure 2 through embedded section HE dyeing form, and cell volume is big, and nuclear is big; Kernel is obvious, and is similar with the tissue of patient in source.
Last institute should be noted that; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.
Claims (5)
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.
2. the cell strain that derives from human lung adenocarcinoma as claimed in claim 1 is characterized in that, said cell strain is being inverted observation under the optics phase microscope, and the visible cell volume is big, and nuclear is big, and endochylema is abundant, and iuntercellular connects tight, and attaches growth between the plate.
3. the cell strain that derives from human lung adenocarcinoma as claimed in claim 1 is characterized in that, the external doubling time of said cell strain is 60.325h.
4. the cell strain that derives from human lung adenocarcinoma as claimed in claim 1 is characterized in that the protein expression of said cell strain is characterized as the vegf expression positive.
5. a preparation method who derives from the cell strain of human lung adenocarcinoma is characterized in that, may further comprise the steps:
(1) tissue block mechanical dissociation: the human lung adenocarcinoma tissue of excision is dissociated, the cleaning and removing removal of impurity, separation lung cancer parenchymal tissue, remove normal alveolar and bronchial tissue and stroma after, obtain remaining tissue;
(2) collagenase digesting: the remaining tissue that step (1) is obtained is cut into tissue, cleans, and adds collagenase to tissue then, hatches, and filters, and collects tumour cell then, carries out inoculation culture, obtains survivaling cell;
(3) survivaling cell that step (2) is obtained carries out purifying, removes impurity such as fibrocyte, and the lung carcinoma cell cultured continuously behind the purifying also goes down to posterity greater than 12 months, promptly gets cell strain.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695412A (en) * | 2016-02-16 | 2016-06-22 | 广州医科大学附属第医院 | Human lung adenocarcinoma cell line HA109 and building method thereof |
| CN108913662A (en) * | 2018-08-09 | 2018-11-30 | 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 |
| CN116355851A (en) * | 2023-03-13 | 2023-06-30 | 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2006374A2 (en) * | 2004-09-15 | 2008-12-24 | Apogenix GmbH | Method for the purification and amplification of tumoral stem cells |
| CN101955911A (en) * | 2009-07-14 | 2011-01-26 | 上海市胸科医院 | Chinese lung adenocarcinoma cell line with high metastases potentiality of bone, lever and adrenal gland |
-
2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2006374A2 (en) * | 2004-09-15 | 2008-12-24 | Apogenix GmbH | Method for the purification and amplification of tumoral stem cells |
| CN101955911A (en) * | 2009-07-14 | 2011-01-26 | 上海市胸科医院 | Chinese lung adenocarcinoma cell line with high metastases potentiality of bone, lever and adrenal gland |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695412A (en) * | 2016-02-16 | 2016-06-22 | 广州医科大学附属第医院 | Human lung adenocarcinoma cell line HA109 and building method thereof |
| 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 |
| CN108913662A (en) * | 2018-08-09 | 2018-11-30 | 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 |
| 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 |
| CN116355851A (en) * | 2023-03-13 | 2023-06-30 | 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 |
| 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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| CN102424817B (en) | 2013-11-06 |
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