CN102168061B - Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof - Google Patents
Human lung cell BEAS-2B/CYP2A13, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a human recombination gene lung epithelial cell, in particular to a human lung cell BEAS-2B/CYP2A13 and application thereof. The human lung cell BEAS-2B/CYP2A13 is characterized in that: the cell strain is collected in China Center for Type Culture Collection, and the collection number is CCTCC No: C2010127. In the method, the BEAS-2B cell for stably expressing CYP2A13 is constructed by utilizing a slow virus system for the first time at home and abroad, which is the key technology in the invention. Compared with other virus vector systems, the technology has the advantages of high transfection efficiency, stable expression, easiness in screening and the like, and has the unique advantage on transfection of immortalized cells.
Description
Technical field
The present invention relates to people's recombination pulmonary epithelial cells, particularly a kind of human pneumonocyte BEAS-2B/CYP2A13 and application thereof.
Background technology
Selecting a desirable model is the essential condition of carrying out scientific effort, cell is undoubtedly research model more satisfactory in scientific research, that also be in daily use.Single cell can reduce the error between experiment on the one hand, guarantees controllability and the repeatability of result of study; The test cell line cycle is short on the other hand, cost is low, requirement for experiment condition is not high.Therefore enjoy the favor of vast researcher.Along with deepening continuously of research, the mankind have entered the post-genomic study epoch, and its core content is exactly the function of research gene.Express or do not express because specific protein in most cells system is low, therefore needing the cell model that builds high-expression target proteins badly.
The method of general employing adenovirus builds the cell of transient expression target protein at present, but often effect is unsatisfactory.Mainly the cell due to transient expression, the unstable expression of target protein, and the difference between different experiments is sometimes very large.In addition, the efficient of use adenovirus construction stably express cell is low, success ratio is little, often wastes time and energy.Therefore, need badly and use reliable and stable carrier system to screen by stable transfection and mono-clonal, to obtain to stablize the specific cells of high-expression target proteins.
In view of above-mentioned factor, the present invention utilizes novel slow virus system constructing stably express human-cytochrome P450 2A13(CYP2A13) pulmonary branches tracheal epithelial cell (BEAS-2B), referred to as the BEAS-2B/CYP2A13 cell, to providing desirable cell model for studying medicament metabolism activation and tumor in respiratory system and molecular mechanism.
Summary of the invention
Goal of the invention
The present invention adopts novel slow virus system to build cell model, and a kind of human pneumonocyte BEAS-2B/CYP2A13 and application thereof are provided specifically.
A kind of human pneumonocyte BEAS-2B/CYP2A13 is characterized in that, this cell strain is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C2010127; The preservation time is on December 7th, 2010.The preservation address is Wuhan, China Wuhan University.
The preparation method of a kind of human pneumonocyte BEAS-2B/CYP2A13 is characterized in that
Concrete preparation process is as follows:
A. pLJM1-GFP-CYP2A13 plasmid construction and checking
The method that connects by enzymes double zyme cutting and enzyme builds the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, namely to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out simultaneously enzyme and cuts, reclaim and carry out respectively enzyme even after enzyme is cut product, then transform DH5 α competent cell, screen by penbritin, the picking positive colony extracts recombinant plasmid, uses each gene specific primer to carry out the PCR checking to recombinant plasmid;
B. lentiviral vectors is packed and infection BEAS-2B cell
Pass the 293T cell to the 60mm culture dish, carry out transfection when cell density reaches the 80-90% degrees of fusion; Preparation liposome transfection mixture: DMEM serum-free, unparalleled anti-, the mixture of the every ware of transfection contains 6 μ g pLJM1-GFP-CYPs plasmids, 3 μ g pVSVG, 4.5 μ g delta 8.91 and 15 μ LSofast transfection reagents, mixture is added under DMEM liquid level in culture dish, blow and beat mixing 3-4 time, this cell is put to 37 ℃ of CO
2Incubator is cultivated, and changes fresh perfect medium after 6-8h; After transfection 24h, determine transfection efficiency by observing 293T cell egfp expression situation; Collect viral supernatant, this is and contains
CYP2A13The complete slow virus supernatant of gene; Use this complete slow virus supernatant to filter postoperative infection BEAS-2B cell, add simultaneously polybrene and the HEPES of 10 μ g/mL; After infecting 24h, superinfection once.Build the BEAS-2B cell BEAS-2B/CYP2A13 of stably express CYP2A13;
C. the checking of BEAS-2B/CYP2A13 cell
Collect respectively each BEAS-2B/CYP2A13 total protein of cell in a rear week of slow virus infection success and two weeks, use the specific antibody of CYP2A13, adopt the protein expression in western blotting method checking BEAS-2B/CYP2A13 cell;
D. selected by flow cytometry apoptosis BEAS-2B/CYP2A13 cell strain
For guaranteeing the purity of transfectional cell, use flow cytometer to carry out the sorting of GFP positive cell to the BEAS-2B/CYP2A13 cell strain; The normal cell of untransfected is without fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, by sorting, the cell sorting that fluorescence intensity is stronger out, to guarantee that cell is as 100% transfectional cell;
E. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With the BEAS-2B/CYP2A13 cell of the GFP positive, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days, carry out amplification culture to forming monoclonal cell, until satisfying experiment and preserve, cell requires; The monoclonal cell that filters out is verified, chosen the high and stable cell of CYP2A13 protein expression as the use of subsequent experimental and preservation.
The application of a kind of human pneumonocyte BEAS-2B/CYP2A13, it is characterized in that this cell is for the efficient susceptibility of the characteristic substrate A FB1 reaction of CYP2A13, can apply to the function of any research CYP2A13 and cause at Environmental Chemical Pollutants on the effect and mechanism of tumor in respiratory system, also can be used for screening active ingredients and the research and development of related drugs.
Beneficial effect
1. the present invention adopts novel slow virus system to build cell model.This system has following advantage: high-efficiency transfection, and plasmid transfection efficient can reach more than 95%, and fragment is difficult for losing; The viral system of packing can infect the various kinds of cell type, comprises undifferentiated cell and the cell (as primary cell, hemocyte, stem cell etc.) that is difficult to transfection; Packaging gene is incorporated on genomic dna, and expression level is high, and aberration rate is low, not the normal function of interference cell; Express stable, can heredity.
2, the present invention is take Cytochrome P450 2A13(CYP2A13) be goal gene,, utilize the BEAS-2B cell (BEAS-2B/CYP2A13) of slow virus system constructing stably express CYP2A13 and succeed as carrier cell with Immortalized people normal lung cell (BEAS-2B).By gene, albumen and functional study, result shows that this cell has and expresses the characteristics such as stablize, be quick on the draw, and is the ideal model of studying medicament metabolism activation and tumor in respiratory system and molecular mechanism, is worthy of popularization.
3, taken the lead in utilizing the slow virus system constructing BEAS-2B cell of stably express CYP2A13 at home and abroad, this is the most important gordian technique of the present invention.This technology is compared other virus carrier system, have transfection efficiency high, express advantages such as stablizing, be easy to screening, have the advantage of uniqueness for transfection Immortalized primary cell.
4, utilize this technology, can build primary cell or the cell strain of any stably express goal gene, for the functional study of gene and the Mechanism Study of disease provide important carrier, in case promote, will produce significant social benefit and considerable economic benefit.
Description of drawings
Fig. 1 pLJM1-GFP-CYP2A13 recombinant plasmid PCR checking.3 cloning vectors of random selection utilize the CYP2A13 primer to carry out the PCR checking, found that, bright band has all appearred in 3 plasmids in the position of expection size (1500bp), point out the plasmid construction success.In figure, M is DNA Marker.
Fig. 2 infects GFP protein expression situation in rear different time cell.Respectively before plasmid transfection, 4 days and 1 week after transfection, use the transfection efficiency of the intensity evaluation cell of fluorescent microscope by observing fluorescin GFP.Shown in figure, no matter be BEAS-2B/CYP2A13 cell or unloaded cell (BEAS-2B/Vector), obviously be better than after transfection 4 days after transfection 1 week, non-transfected cells is without fluorescence.
The protein expression checking of Fig. 3 BEAS-2B/CYP2A13 cell.Adopt immunoblotting to detect the expression of CYP2A13 in cell, and with Tubulin as internal reference, 1 and 2 cells that are respectively 1 week and 2 weeks after transfection wherein; The positive contrast of P is the CYP2A13 microsomal protein of vivoexpression; V is unloaded cell BEAS-2B/Vector; N is the BEAS-2B cell of untransfected.
The flow cytometer detected result of each transfectional cell of Fig. 4.The BEAS-2B cell that its left figure is untransfected, its fluorescence intensity is very weak, and right figure is that the BEAS-2B/CYP2A13 cell obviously moves to right.Take the fluorescence intensity of BEAS-2B cell as the line of delimitation, with the cell sorting on right side, line of delimitation out, to obtain the purpose cell of 100% transfection efficiency.
The gene of Fig. 5 BEAS-2B/CYP2A13 monoclonal cell and protein expression checking.Wherein scheming A is the protein expression checking of CYP2A13, and 1-7 is respectively the monoclonal cell albumen that each cell strain is selected; Figure B is the genetic expression checking of No. 6 and No. 7 monoclonal cells; Figure C is the albumen checking of No. 6 and No. 7 monoclonal cells.GADPH is internal reference, and V is unloaded cell, and N is non-transfected cells; The positive contrast of P, M is DNA Marker.
The toxic effect of Fig. 6 AFB1 to the BEAS-2B/CYP2A13 cell.Wherein scheme A and be the activity change that different concns AFB1 processes cell after 24 hours, figure B utilizes the AFB1 of 100nM to process the activity change of cell after different time, and Vector represents unloaded cell, is the control cells of BEAS-2B/CYP2A13 cell.
Embodiment
1. pLJM1-GFP-CYP2A13 plasmid construction and checking
The method that connects by enzymes double zyme cutting and enzyme builds the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, namely to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out simultaneously enzyme and cuts, reclaim and carry out respectively enzyme even after enzyme is cut product, then transform DH5 α competent cell, screen by penbritin, the picking positive colony extracts recombinant plasmid, uses each gene specific primer to carry out the PCR checking to recombinant plasmid.
2. lentiviral vectors is packed and infection BEAS-2B cell
Pass the 293T cell to the 60mm culture dish, carry out transfection when cell density reaches the 80-90% degrees of fusion.Preparation liposome transfection mixture: DMEM (serum-free, unparalleled anti-), the mixture of the every ware of transfection contains 6 μ g pLJM1-GFP-CYPs plasmids, 3 μ g pVSVG, 4.5 μ g delta 8.91 and 15 μ l Sofast transfection reagents, mixture is added under DMEM liquid level in culture dish, blow and beat mixing 3-4 time, this cell is put to 37 ℃ of CO
2Incubator is cultivated, and changes fresh perfect medium after 6-8h.After transfection 24h, determine transfection efficiency by observing 293T cell green fluorescent protein (GFP) expression.Collect viral supernatant, this is and contains
CYP2A13The complete slow virus supernatant of gene.Use this complete slow virus supernatant to filter postoperative infection BEAS-2B cell, add simultaneously polybrene and the HEPES of 10 μ g/mL.After infecting 24h, superinfection once.Build the BEAS-2B cell (BEAS-2B/CYP2A13) of stably express CYP2A13.
3. the checking of BEAS-2B/CYP2A13 cell
Collect respectively each BEAS-2B/CYP2A13 total protein of cell in a rear week of slow virus infection success and two weeks, use the specific antibody of CYP2A13, adopt the protein expression in western blotting method checking BEAS-2B/CYP2A13 cell.
4. selected by flow cytometry apoptosis BEAS-2B/CYP2A13 cell strain
For guaranteeing the purity of transfectional cell, use flow cytometer to carry out the sorting of GFP positive cell to the BEAS-2B/CYP2A13 cell strain.The normal cell of untransfected is without fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, by sorting, the cell sorting that fluorescence intensity is stronger out, to guarantee that cell is as 100% transfectional cell.
5. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With the BEAS-2B/CYP2A13 cell of the GFP positive, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days, carry out amplification culture to forming monoclonal cell, until satisfying experiment and preserve, cell requires.The monoclonal cell that filters out is verified, chosen the high and stable cell of CYP2A13 protein expression as the use of subsequent experimental and preservation.This cell strain is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C2010127.The preservation time is on December 7th, 2010.The preservation address is Wuhan, China Wuhan University.
Result: the structure of human pneumonocyte BEAS-2B/CYP2A13
By recombinant plasmid being used the special primer of CYP2A13 carry out the PCR checking, as seen each PCR product fragment is consistent with gene length, confirms each plasmid construction success (Fig. 1) of pLJM1-GFP-CYPs; Utilize this plasmid cells infected 4 days and 1 week, find by fluorescence microscope, along with the prolongation of incubation time, the green fluorescence intensity of cell strengthens gradually, non-transfected cells is without fluorescence, and prompting cell transfecting success also has higher efficiency of infection (Fig. 2); The specific antibody of employing CYP2A13 carries out the immunoblotting checking to the CYP2A13 albumen of transfectional cell, found that all high expression level CYP2A13 cells of the cell selected, unloaded cell and non-transfected cells are not expressed CYP2A13, the stably express success (Fig. 3) of results suggest cell; In order to obtain monoclonal BEAS-2B/CYP2A13 cell, by flow cytometer, cell is carried out sorting, take non-transfected cells as contrast, with the cell sorting of the fluorescence intensity positive out (Fig. 4); Subsequently above-mentioned cell is carried out the mono-clonal screening, and use respectively immunoblotting and RT-PCR method that CYP2A13 albumen and the mRNA of monoclonal cell verified, found that all cells all expressed CYP2A13 albumen (Fig. 5 A) preferably, the monoclonal cell of selecting is all stablized high expression level CYP2A13 mRNA(Fig. 5 B) and CYP2A13 albumen (Fig. 5 C), the monoclonal BEAS-2B/CYP2A13 of results suggest successfully constructs.
1. the dose-dependently operational research of cell to the AFB1 effect
The BEAS-2B/CYP2A13 cell that utilizes embodiment 1 to obtain, take unloaded cell BEAS-2B/Vector as contrast, give respectively cell 0,10,100,1000 and 10, the AFB1 of 000 nM, processed 24 hours, then adopt mtt assay to detect each dosage and process lower cytoactive, estimate the toxic effect of cell various dose AFB1 under processing, the structure dose-effect relationship.
2. the time-dependent manner operational research of cell to the AFB1 effect
The BEAS-2B/CYP2A13 cell that utilizes embodiment 1 to obtain, take unloaded cell BEAS-2B/Vector as contrast, use 100 nM AFB1 to process respectively cell 24,48 and 72 hours, then adopt mtt assay to detect each time point cytoactive under processing, estimate the toxic effect of cell under AFB1 processing different time, meta-effect relation during structure.
Result: the operational research of human pneumonocyte BEAS-2B/CYP2A13
By Fig. 6 A as seen, for unloaded cell BEAS-2B/Vector, the BEAS-2B/CYP2A13 cell is to ten minutes sensitivities of AFB1, and from minimum concentration, its cytoactive is all significantly lower than BEAS-2B/Vector cell (P＜0.01), half-inhibition concentration (IC
50) be 350nM, and the BEAS-2B/Vector cell still keeps the cytoactive more than 95% when the AFB1 of 10000nM.As shown in Fig. 5 B, along with the prolongation of time, the activity of BEAS-2B/CYP2A13 cell descends gradually, and the BEAS-2B/Vector cells is without obvious change; At each time point, the cytoactive of BEAS-2B/CYP2A13 is all significantly lower than BEAS-2B/Vector cell (P＜0.01).Result proves, this BEAS-2B/CYP2A13 cell is sensitive to the reaction of AFB1, is desirable cell model, applicable to functional study and drug screening and the activity research of CYP2A13.
Claims (3)
1. a human pneumonocyte BEAS-2B/CYP2A13, is characterized in that, this cell strain is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C2010127.
2. the preparation method of a human pneumonocyte BEAS-2B/CYP2A13, is characterized in that
Concrete preparation process is as follows:
A. pLJM1-GFP-CYP2A13 plasmid construction and checking
The method that connects by enzymes double zyme cutting and enzyme builds the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, namely to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out simultaneously enzyme and cuts, reclaim and carry out respectively enzyme even after enzyme is cut product, then transform DH5 α competent cell, screen by penbritin, the picking positive colony extracts recombinant plasmid, uses each gene specific primer to carry out the PCR checking to recombinant plasmid;
B. lentiviral vectors is packed and infection BEAS-2B cell
Pass the 293T cell to the 60mm culture dish, carry out transfection when cell density reaches the 80-90% degrees of fusion; Preparation liposome transfection mixture: DMEM serum-free, unparalleled anti-, the mixture of the every ware of transfection contains 6 μ g pLJM1-GFP-CYPs plasmids, 3 μ g pVSVG, 4.5 μ g delta 8.91 and 15ul Sofast transfection reagent, mixture is added under DMEM liquid level in culture dish, blow and beat mixing 3-4 time, this cell is put to 37 ℃ of CO
2Incubator is cultivated, and changes fresh perfect medium after 6-8h; After transfection 24h, determine transfection efficiency by observing 293T cell egfp expression situation; Collect viral supernatant, this is and contains
CYP2A13The complete slow virus supernatant of gene; Use this complete slow virus supernatant to filter postoperative infection BEAS-2B cell, add simultaneously polybrene and the HEPES of 10 μ g/mL; After infecting 24h, superinfection once; Build the BEAS-2B cell BEAS-2B/CYP2A13 of stably express CYP2A13;
C. the checking of BEAS-2B/CYP2A13 cell
Collect respectively each BEAS-2B/CYP2A13 total protein of cell in a rear week of slow virus infection success and two weeks, use the specific antibody of CYP2A13, adopt the protein expression in western blotting method checking BEAS-2B/CYP2A13 cell;
D. selected by flow cytometry apoptosis BEAS-2B/CYP2A13 cell strain
For guaranteeing the purity of transfectional cell, use flow cytometer to carry out the sorting of GFP positive cell to the BEAS-2B/CYP2A13 cell strain; The normal cell of untransfected is without fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, by sorting, the cell sorting that fluorescence intensity is stronger out, to guarantee that cell is as 100% transfectional cell;
E. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With the BEAS-2B/CYP2A13 cell of the GFP positive, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days, carry out amplification culture to forming monoclonal cell, until satisfying experiment and preserve, cell requires; The monoclonal cell that filters out is verified, chosen the high and stable cell of CYP2A13 protein expression as the use of subsequent experimental and preservation.
3.
According to claim 1The application of a kind of human pneumonocyte BEAS-2B/CYP2A13 is characterized in that utilizing the characteristic substrate aflatoxin B1 of CYP2A13 that cell is tested; Use 0-10, the AFB1 of 000 nM processes cell 24 h, adopts mtt assay to estimate dose-effect relationship; Use 100 nM AFB1 to process cell 24-72 hour, adopt mtt assay evaluation time-effect relation; By comparing with unloaded cell BEAS-2B/Vector, estimate the BEAS-2B/CYP2A13 cell to the susceptibility of AFB1.
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| CN1616674A (en) * | 2003-11-12 | 2005-05-18 | 中国人民解放军军事医学院放射医学研究所 | Method for detecting lung cancer relative CYP2A13 resistance gene and its resistance gene |
| JP2005229967A (en) * | 2004-02-23 | 2005-09-02 | Osaka Industrial Promotion Organization | Recombinant human cell and, method for evaluating at least eithor one of induction of human drug metabolizing enzyme and drug metabolism by using the recombinant human cell |
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| CN1616674A (en) * | 2003-11-12 | 2005-05-18 | 中国人民解放军军事医学院放射医学研究所 | Method for detecting lung cancer relative CYP2A13 resistance gene and its resistance gene |
| JP2005229967A (en) * | 2004-02-23 | 2005-09-02 | Osaka Industrial Promotion Organization | Recombinant human cell and, method for evaluating at least eithor one of induction of human drug metabolizing enzyme and drug metabolism by using the recombinant human cell |
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| 何晓阳.第208位异亮氨酸对人细胞色素P4502A6尼古丁代谢活性的影响.《中国药理学与毒理学杂志》.2009,第23卷(第6期),464-471. * |
| 王守林等.特征性化学物对CYP2A13代谢活化黄曲霉毒素B1的影响.《江苏预防医学》.2006,第17卷(第4期),51-53. * |
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