CN101892197A - Human bronchial epithelial cell - Google Patents
Human bronchial epithelial cell Download PDFInfo
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- CN101892197A CN101892197A CN 201010233630 CN201010233630A CN101892197A CN 101892197 A CN101892197 A CN 101892197A CN 201010233630 CN201010233630 CN 201010233630 CN 201010233630 A CN201010233630 A CN 201010233630A CN 101892197 A CN101892197 A CN 101892197A
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Abstract
The invention discloses a human bronchial epithelial cell BERP35T4-Ta with the collection number of CGMCC NO.3748. The human bronchial epithelial cell BERP35T4-Ta has the advantages of high growth speed, high soft agar cloning formation rate, high resistance to serum differentiation, high incidence of polyploidy of chromosome, and strong migration capacity, is considered to have typical phenotype of a malignant cell and also has higher metastasis potentiality. The cell has important value for studying molecular transformation incidents and cancer cell metastatic mechanisms, and searching tumor markers and ideal cell model systems for experimental treatment of malignant tumors in the process that radiating factors induce cells to generate canceration.
Description
Technical field
The present invention relates to a human bronchial epithelial cell, particularly a kind ofly bring out the human bronchial epithelial that the human bronchial epithelial cell vicious transformation obtains by alpha-radiation and become oncocyte.
Background technology
According to estimates, the alpha-particle that sends when 50% dosage comes from radon and daughter disintegration in the human suffered natural radiation, epidemiological study shows and is exposed to high-caliber radon and daughter can cause the sickness rate of lung cancer to increase, therefore, the research radon in other words the relation of alpha-particle and Human Lung Cancer be the focus of radiation induced carcinogenesis research field.
The main path of research radiation induced carcinogenesis has at present: 1. epidemiology survey, comprise case-control study, cohort study etc., and promptly various dose is subjected to the long-time healthy investigation according to the crowd, determine the relation that contact radioactive rays and radiation dose and cancer take place.2. animal experiment study.This be determine certain factor whether have carinogenicity the experimental study that must do, can be used for the Study on Correlative Mechanisms of canceration simultaneously.3. cell in vitro and molecular biology research.Be in-vitro simulated cell carcinogenesis and observe that cytology behavior in the canceration process, molecules change and the important means and the technological approaches of genesis mechanism to have irreplaceable advantage.
The cell in vitro transformation experiment is meant under the condition of vitro culture, cell is spontaneous or under the effect of handling factor, grow, the property out of control variation of aspects such as propagation and differentiation, and then show a series of vicious transformation phenotypes and final performance tumorigenicity in animal body, be an effectively method of research oncogenic process, the multistage process that target cell develops to tumour cell in the energy analogue body.In addition, under the isolated cells culture condition, carry out canceration and observe, can be at the process and the influence condition that do not have research canceration under the disturbed condition of other factors.Therefore, for cell and the molecule mechanism of illustrating radiation induced carcinogenesis, it is significant that the method for employing cell in vitro vicious transformation is set up radiation induced external canceration model.
The Study on Transformation of cell in vitro starts from 1963, and the research of this respect has after this obtained developing rapidly.The fibroblastic vicious transformation model of SHE, C3H10T1/2, BALB/3T3, induced lung that the alpha-particle extracorporeal irradiation brings out has successively been set up in the Duo Jia laboratory, and above-mentioned cell is all the inoblast of rodent.Since the late nineteen eighties, the cell transformation system has occurred from rodent zooblast to the human cell, from the trend of inoblast to the epithelial cell transformation, the several strain immortal human epithelial cell strains that particularly use the serum-free culture technology to set up are more pressed close to human model for external canceration research provides.Compare with rodent zooblast, the human cell has better gene stability, less spontaneous transformation and the resistance stronger to procarcinogen.80% Zou You of human tumor originates from epithelium, and the metabolism of epithelium, reparation, differentiation etc. are obviously different with inoblast.Therefore, the vicious transformation experimental model that adopts people's epithelial cell to set up more approaches the evolution process of human tumor.
BEP2D is the human bronchial epithelial cell strain of immortalization, be by Harris CC and colleague's transfection human papillomavirus 18 (Human Papillomaviruses 18, HPV18) build in normal people's bronchial epithelial cell (NHBE) back immortalization and be, karyotype is a near diploid, external continuous passage does not take place old and feeble and dead more than 100 times, the inoculation nude mice is raised not observe more than 12 months has into knurl.
Summary of the invention
The purpose of this invention is to provide a human bronchial epithelial cell and application thereof.
Human bronchial epithelial cell provided by the present invention is to bring out the human bronchial epithelial that human bronchial epithelial cell BEP2D vicious transformation obtains by alpha-radiation to become oncocyte system, and title is human bronchial epithelial cell BERP35T4-Ta.
Human bronchial epithelial cell BERP35T4-Ta is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2010 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .3748.
The present invention adopts 1.5Gy's
238The human bronchial epithelial cell BEP2D of Pu αLi Zizhaoshe immortalization reached for 35 generations according to the back cells in vitro and sets up clone BERP35, and the vicious transformation phenotypic characteristic appears in this cell, formed high differentiation squamous cell carcinoma in the inoculation nude mouse.The tumor tissue cell of separation and Culture BERP35 tumour that forms in nude mouse is set up clone BERP35T4 after the external continuous passage.This cell is inoculated nude mice once more, become after the knurl, adopt tissue mass cell culture, from the nude mice tumor tissues, separate, cultivate and set up clone BERP35T4-Ta once more tumor tissue continuous passage three times in nude mouse.Human bronchial epithelial cell BERP35T4-Ta fast growth, soft-agar cloning rate of formation height, the strong resistance that differentiation produces to serum, chromosomal polyploid incidence height, transfer ability is strong, think that this cell has the phenotype of comparatively typical malignant cell, and have higher metastatic potential.
The comparative analysis of clone BERP35T4-Ta and its parental cell line BERP35, BERP35T4 can reflect the three phases of radiation induced carcinogenesis process from in-vitro simulated mode: startup, promotion and malignant progression to a certain extent.BERP35T4-Ta is that the research radiation factor brings out molecule change events in the cell carcinogenesis, cancer metastasis mechanism, seeks the desirable cell model system of tumor marker and malignant tumour experimental therapy, has crucial value.
Description of drawings
Fig. 1 is BERP35T4-Ta cytokeratin immunohistochemical analysis (DAB colour developing, * 100)
Fig. 2 is BEP2D, BERP35T4, BERP35T4-Ta growth curve
Fig. 3 be on the om observation Transwell film from BERP35T4-Ta isolating transitional cell (* 100)
Embodiment
Experimental technique described in the following embodiment if no special instructions, is ordinary method;
Percentage composition described in the following enforcement if no special instructions, is the quality percentage composition.
The foundation of embodiment 1, human bronchial epithelial cell BERP35T4-Ta
1, alpha-radiation brings out the tumorigenicity of immortal human bronchiolar epithelium malignant conversioning cell BERP35T4
Adopt the BALB/c nude mouse, mouse age is 5-6 week, and body weight 11-15g provides (license licensed licenser licence is numbered: the capital is moving is betrothed to No. 017) by Institute of Experimental Animals, Chinese Academy of Medical Sciences.
The 25th generation BEP2D cell: BEP2D is the human bronchial epithelial cell strain of immortalization, be by Harris CC and colleague's transfection human papillomavirus 18 (Human Papillomaviruse 18, HPV18) build in normal people's bronchial epithelial cell (NHBE) back immortalization and be, karyotype is a near diploid, external continuous passage does not take place old and feeble and dead more than 100 times, the inoculation nude mice is raised not observe more than 12 months has into knurl.Professor Harris presents this cell in this laboratory, document Willey JC, Broussoud A, Sleemi A, et al.Immortalization ofnormal human bronchial epithelial cells by human papillomavirus 16 or 18.Cancer Res., 1991,51 (19): 5370-77 discloses this cell, and the applicant has preserved this cell.The laboratory is 75cm with this cell cultures in floorage
2Culturing bottle in, changed a not good liquor (nutrient solution is LHC-8, and available from Biosource company, article No. is P141-500) in three days, treat that cell grows to 80% and merges (approximately week age) and go down to posterity, the ratio of going down to posterity is 1: 3, this cell is called the 2nd generation BEP2D cell.So passed for 25 generations, be the 25th generation BEP2D cell.
The 15th generation BERP35T4 cell: with 1.5Gy's
238Pu αLi Zizhaoshe BEP2D cell is normally cultivated, is gone down to posterity according to the back cell, will after becoming the aseptic separation of tumor tissue of knurl nude mice, cultivate and obtain tumor tissue cell BERP35T4 according to back 35 generation cell inoculation nude mice.This cell continuous passage is the 15th generation BERP35T4 for 15 times.Nutrient solution used in this process is LHC-8.
This nude mice is divided into 2 groups, and 9 every group, male and female hold concurrently half: experimental group is inoculated the 15th generation BERP35T4 cell; Control group is inoculated the 25th generation BEP2D cell.Every the about 2-3 of nude inoculation * 10
6Cell.Through three latent period in week, in 9 nude mices of inoculation BERP35T4 cell, 1 tumour occurs, has 3 tumour to occur subsequently successively, and 9 nude mices of inoculation BEP2D cell none tumour appears.Choose a band knurl nude mice, break spinal cord and put to death, extract the subcutaneous transplantation knurl, be cut into 0.2 * 0.2 * 0.2cm
3Fritter, place that to be inoculated in new nude mice in the trochar subcutaneous, continue to go down to posterity, continuous passage is 3 times altogether, 14 of inoculation nude mices, and transplanted tumor all can survive and grow, the tumour tumor formation rate that goes down to posterity reaches 100% between mouse, become knurl obviously to shorten latent period, be about 7-14 days, be about for 3.5 weeks (table 1) pitch time of going down to posterity.
Go down to posterity between table former pickup kind of 1BERP35T4 nude mice and mouse
2, alpha-radiation brings out the foundation that the immortal human bronchiolar epithelium becomes oncocyte BERP35T4-Ta
Adopt tissue mass cell culture: under aseptic condition, get fresh tumor tissues and carefully divest film and vascular components, avoid regression of tumor tissues center and necrotic area, the better position of picking vigor, with aseptic PBS damping fluid (PH 7.2) flushing three times, move in the little culture dish, drip the wetting tissue block of several LHC-8 substratum (Gibco company), be cut into 2-3mm with eye scissors
3Fritter is displaced to 25cm with the ophthalmology tweezer with fritter
2On at the bottom of the culturing bottle bottle, distance is about 0.5cm between fritter, and culturing bottle is turned gently, and the order bottle end adds LHC-8 substratum 5ml up, and the bottle cap of screwing on is in 37 ℃, 5%CO
2Placed 2 hours in the incubator.Slowly the upset culturing bottle allows substratum cover bottle end tissue block, notes avoiding tissue block to be dashed by substratum.Observation of cell climbs out of situation in several weeks backward, changes the LHC-8 substratum once in per three days.After cell to be climbed out of grows to the 70-80% fusion, with the 0.25% trysinization cultivation of going down to posterity: on cell, add 2ml 0.25% pancreatin (available from Gibco company, article No. is: 25200-056), 37 ℃ digested 5-7 minute, after treating that cell all becomes circle and comes off from the bottle wall, add the 2ml stop buffer and (contain 10% (volumn concentration) serum (available from PAA company, article No. is A15-101) the DMEM substratum (available from Gibco company, article No. is 12800-017), with suction pipe cell suspension is transferred in the centrifuge tube, 1500 rev/mins centrifugal 5 minutes, supernatant is siphoned away, in cell precipitation, add 5ml PBS damping fluid (pH 7.2) again, lightly cell precipitation is dispelled recentrifuge with suction pipe, 1500 rev/mins centrifugal 5 minutes, supernatant is siphoned away, add 2ml LHC-8 substratum cell precipitation is dispelled, evenly assign to three 25cm
2In the culturing bottle, in each culturing bottle, add 5ml LHC-8 substratum again, promptly finish and go down to posterity, finish the tumour cell that goes down to posterity and be BERP35T4-T.(substratum is the LHC-8 substratum, 37 ℃ of culture condition, 5%CO in continuous passage culture in vitro when this cell
2Incubator, the incubation time in per generation are 72 hours) after 20 generations, with this cell with 1 * 10
3The density of/ware is inoculated in and makes it to be unicellular growth in the 60mm culture dish, split into thousands of cells and become when clone when unicellular, select the good mono-clonal of growth conditions, with the rifle of decaptitating point picking mono-clonal and cultivate and set up alpha-radiation and bring out the immortal human bronchiolar epithelium and become oncocyte BERP35T4-Ta.
Human bronchial epithelial cell BERP35T4-Ta is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2010 and (is called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .3748.
3, tumour cell BERP35T4-Ta biological Characteristics Study
3.1BERP35T4-Ta cellular immunization chemical analysis
Cover glass culturing cell BERP34T4-Ta fixes 10 minutes with the methyl alcohol of precooling, and acetone fixed is after 5 minutes, with 0.3% hydrogen peroxide effect 20 minutes to eliminate endogenous peroxydase; Non-specific combination site is sealed in 10% foetal calf serum effect 20 minutes; Add the anti-human keratinous of rabbit and resist (available from Beijing Zhong Shan biotech company) (1: 50), 4 ℃ of overnight incubation more; PBS (pH 7.2) flushing 3 times, the goat anti-rabbit igg (available from Beijing Zhong Shan biotech company) (1: 50) of adding horseradish peroxidase-labeled was hatched 4 hours for 4 ℃; PBS damping fluid (pH 7.2) flushing 3 times, DAB colour developing liquid (0.1%H
2O
2) colour developing 5-10 minute, the result shows, endochylema engrain (Fig. 1) has confirmed the epithelial origin of tumour cell BERP35T4-Ta.
3.2BERP35T4-Ta the mensuration of cell growth curve
Respectively with BERP35T4, BERP35T4-Ta and BEP2D cell with 1 * 10
4The density in/hole is inoculated in (substratum: LHC-8 on the 12 porocyte culture plates; Culture condition: 37 ℃, 5%CO
2), digest 3 porocytes and counting every day after 24 hours, continues 8 days, draws cell growth curve (Fig. 2) according to measurement result, and calculate cell growth index (n), thereby further calculate cell doubling time (PDT).
Calculation formula is as follows:
n＝(lnN
1-lnN
0)/(t-t
0)
PDT(h)＝0.693/n
N is cell counting; T is a minute
Calculating cell doubling time is followed successively by: BEP2D 66 hours, BERP35T430 hour, BERP35T4-Ta18 hour.As seen, the speed of growth of tumour cell BERP35T4-Ta obviously speeds.
3.3 soft-agar cloning experiment
(colony forming efficiency CFE) is the important symbol of cell grade malignancy height to the colony forming efficiency of cell in agar.With PBS damping fluid compound concentration is 3.5% agar, autoclaving 10 minutes.With the LHC-8 substratum with 5 times of 3.5% agar dilution to 0.7% as lower floor's agar, with shop, 3ml/ hole system bottom-layer agar, it is standby to solidify the back in 6 orifice plates.Use again the LHC-8 substratum with one times of 0.7% agar dilution to 0.35% as top-layer agar, be placed in 40 ℃ of water-baths standby.After BERP35T4, BERP35T4-Ta and BEP2D cell digested according to a conventional method and counting, with 0.35% top-layer agar mixing, the 3ml/ hole is laid on lower floor's agar of 0.7%, and the inoculum size of cell is 1 * 10
4/ hole.After treating that top-layer agar solidifies, the surface adds 2ml LHC-8 substratum, 37 ℃, 5%CO
2Cultivate in the incubator, changed liquid once in per three days.Table 2 has been listed the CFE of various types of cells, and as can be seen, the CFE of BERP35T4, BERP35T4-Ta cell is all apparently higher than control cells BEP2D (p＜0.05), and the CFE of BERP35T4-Ta also is higher than BERP35T4.Show that the grade malignancy of BERP35T4-Ta cell improves a lot than BERP35T4.
The soft-agar cloning rate of formation of table 2.BEP2D, BERP35T4 and BERP35T4-Ta cell
| The soft-agar cloning number | CFE(％) | The | |
| BEP2D | |||
| 5±0.816 | 0.05±0.008 | ||
| BERP35T4 | 64±3.266 | 0.64±0.033 ＊ | ＜0.05 |
| BERP35T4-Ta | 126±4.899 | 1.26±0.049 ＊ | ＜0.05 |
" * " represents p＜0.05
3.4 seroresistance detects
Under the induction of differentiation of serum, the squamous differentiation can take place in normal epithelial cell, and malignant conversioning cell and tumour cell then have resistivity.The cell of inoculating 1000 exponentials phase of growth is in 6 orifice plates, and adding 3ml contains the LHC-8 substratum of 10% (volumn concentration) foetal calf serum, cultivates after 4 days, and the squamous differentiation appears in control group BEP2D cell, and growth is slowed down rapidly, and it is flat greatly also dead gradually that cell becomes.And BERP35T4 and BERP35T4-Ta cell cultures form colony (table 3) gradually after 14 days, by in the table as seen, tumour cell BERP35T4-Ta to the serum inductive eventually the end resistibility of breaking up obviously strengthen (p＜0.05).
Table 3.BEP2D, BERP35T4 and BERP35T4-Ta cell are to the resistance of the short differentiation capability of serum
" * " represents p＜0.05
3.5Transwell the mobility of technical measurement cell
Lower floor adds LHC-8 substratum 2.6ml in the Transwell device, and the upper strata adds substratum 1.2ml, places 37 ℃ of incubators to spend the night.Second day, with BEP2D, BERP35T4 and BERP35T4-Ta cell with 3 * 10
4The density in/hole is inoculated on the film of Transwell device, and the parameter of this device is as follows: poly-carbon ester film diameter is 24mm, and thick is 10 μ m, and the aperture is 0.8 μ m, and the density in hole is 1 * 10
5/ cm
3Place 37 ℃, 5%CO
2Hatched in the incubator about 6 hours, and film was taken out carry out conventional Giemsa dyeing.Coloration result shows: the BEP2D cell does not move, and the mobility of BERP35T4-Ta is higher than BERP35T4 cell (as Fig. 3).
Claims (1)
1. human bronchial epithelial cell, its name is called BERP35T4-Ta, and preservation registration number is CGMCC № .3748.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451148A (en) * | 2013-09-22 | 2013-12-18 | 武汉大学 | Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU7547491A (en) * | 1990-03-02 | 1991-09-18 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | An immortalized human bronchial epithelial cell line |
| WO2002074979A2 (en) * | 2001-03-20 | 2002-09-26 | Ortho-Clinical Diagnostics, Inc. | Expression profiles and methods of use |
-
2010
- 2010-07-19 CN CN 201010233630 patent/CN101892197A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU7547491A (en) * | 1990-03-02 | 1991-09-18 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | An immortalized human bronchial epithelial cell line |
| WO2002074979A2 (en) * | 2001-03-20 | 2002-09-26 | Ortho-Clinical Diagnostics, Inc. | Expression profiles and methods of use |
Non-Patent Citations (2)
| Title |
|---|
| 《中国人民解放军军事医学科学院硕士学位论文》 20040903 胡迎春 alpha粒子辐射诱发人支气管上皮恶性转化成瘤细胞的生物学特性研究 第二部分第22页 1 , 2 * |
| 《中国肿瘤生物治疗杂志》 20071031 高福,蔡建明; 崔建国,黄定德,李百龙,杨平,闵锐,倪瑾,孙顶： ~(60)C0gamma射线诱发人支气管上皮细胞恶性转化模型的建立 435－439 1 第14卷, 第5期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451148A (en) * | 2013-09-22 | 2013-12-18 | 武汉大学 | Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof |
| CN103451148B (en) * | 2013-09-22 | 2015-08-19 | 武汉大学 | People's normal bronchial epithelial cell and primary separation and Culture thereof and Secondary Culture method and purposes |
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Open date: 20101124 |