CN102168061A - 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 an ideal model is the essential condition of carrying out scientific effort, cell is undoubtedly research model more satisfactory in the scientific research, that also be in daily use.Single cell can reduce the error between experiment on the one hand, guarantees the 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 back genome research epoch, and its core content is exactly the function of research gene.Express or do not express because specific protein is low in most of clone, therefore need the cell model that makes up high-expression target proteins badly.
The general at present cell that adopts the method structure transient expression target protein of adenovirus, but often effect is unsatisfactory.Mainly be cell owing to transient expression, the unstable expression of target protein, and the difference between different experiments is very big sometimes.In addition, the efficient of usefulness adenovirus construction stably express cell is low, setting ratio is little, often wastes time and energy.Therefore, need badly and use reliable and stable carrier system to screen, to obtain to stablize the specific cells of high-expression target proteins by stable transfection and mono-clonal.
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), abbreviate the BEAS-2B/CYP2A13 cell as, in the hope of providing the ideal cell model for studying drug 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 make up 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 culture collection center, and deposit number is CCTCC NO:C2010127; The preservation time is on December 7th, 2010.The preservation address is Chinese Wuhan 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 makes up the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, promptly to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out enzyme simultaneously and cuts, reclaim and carry out enzyme respectively even after enzyme is cut product, transform DH5 α competent cell then, 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 6ug pLJM1-GFP-CYPs plasmid, 3ug pVSVG, 4.5ug delta 8.91 and 15ul Sofast transfection reagent, mixture is added under the DMEM liquid level in the 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 behind the 6-8h; Behind the 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 polybrene and the HEPES of 10ug/mL simultaneously; Superinfection once after infecting 24h.Make up the BEAS-2B cell BEAS-2B/CYP2A13 of stably express CYP2A13;
C. the checking of BEAS-2B/CYP2A13 cell
Collect each BEAS-2B/CYP2A13 total protein of cell in one week of slow virus infection success back and two weeks respectively, use the specific antibody of CYP2A13, adopt the protein expression in the 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 that the BEAS-2B/CYP2A13 cell strain has been carried out the sorting of GFP positive cell; The normal cell of untransfected does not have fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, and by sorting, the cell sorting that fluorescence intensity is stronger comes out, and is 100% transfectional cell to guarantee cell;
E. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With GFP positive B EAS-2B/CYP2A13 cell, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days as a result, carry out amplification culture to forming monoclonal cell, up to cell satisfy experiment and preserve require till; The monoclonal cell that filters out is verified, chosen the usefulness of the high and stable cell of CYP2A13 protein expression as subsequent experimental and preservation.
The application of a kind of human pneumonocyte BEAS-2B/CYP2A13, it is characterized in that the efficient susceptibility of this cell for the characteristic substrate A FB1 reaction of CYP2A13, can apply to the function of any research CYP2A13 and cause at the environmental chemistry thing on the effect and mechanism of tumor in respiratory system, also can be used for the screening active ingredients and the research and development of related drugs.
Beneficial effect
1. the present invention adopts novel slow virus system to make up cell model.This system has following advantage: efficient transfection, and plasmid transfection efficient can reach more than 95%, and fragment is endlessly lost; The viral system of packing can infect the various kinds of cell type, comprises the undifferentiated cell and is difficult to cells transfected (as primary cell, hemocyte, stem cell etc.); Packaging gene is incorporated on the genomic dna, the expression level height, and aberration rate is low, not the normal function of interference cell; Express stable, can heredity.
2, the present invention is with 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 people's normal lung cell (BEAS-2B) of immortalization.By gene, albumen and functional study, the result shows that this cell has and expresses characteristics such as stablize, be quick on the draw, and is the ideal model of research drug 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, has the transfection efficiency height, expresses advantages such as stablizing, be easy to screening, has special advantages for the primary cell of transfection immortalization.
4, utilize this technology, can make up the primary cell or the cell strain of any stably express goal gene, for the functional study of gene and
The Mechanism Study of disease provides important carrier, in case promote, will produce remarkable social benefit and considerable economic.
Description of drawings
Fig. 1 pLJM1-GFP-CYP2A13 recombinant plasmid PCR checking.Select 3 cloning vectors at random, utilize the CYP2A13 primer to carry out the PCR checking, found that, bright band, the success of prompting plasmid construction have all appearred in 3 plasmids in the position of expection size (1500bp).M is DNA Maker among the figure.
Fig. 2 infects the proteic expression of GFP in the different time cell of back.Respectively before plasmid transfection, 4 days and 1 week after the transfection, the transfection efficiency of the intensity evaluation cell of utilization fluorescent microscope by observing fluorescin GFP.Shown in the figure, no matter be BEAS-2B/CYP2A13 cell or unloaded cell (BEAS-2B/Vector), obviously be better than commentaries on classics after the transfection 1 week 4 days then, non-transfected cells does not then have fluorescence.
The protein expression checking of Fig. 3 BEAS-2B/CYP2A13 cell.Adopt immunoblotting to detect the expression of CYP2A13 in the cell, and with Tubulin as confidential reference items, 1 and 2 cells that are respectively 1 week and 2 weeks after the 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.Wherein left side figure is the BEAS-2B cell of untransfected, and its fluorescence intensity is very weak, and right figure is that the BEAS-2B/CYP2A13 cell obviously moves to right.Fluorescence intensity with the BEAS-2B cell is the line of delimitation, and the cell sorting on right side, line of delimitation is come out, and has obtained the purpose cell of 100% transfection efficiency.
The gene of Fig. 5 BEAS-2B/CYP2A13 monoclonal cell and protein expression checking.Wherein scheme A and be the protein expression checking of CYP2A13,1-7 is respectively rumbling cell protein of list that each cell strain selects; 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 confidential reference items, and V is unloaded cell, and N is a non-transfected cells; The positive contrast of P, M is DNA Marker.
Fig. 6 AFB1 is to the toxic effect of BEAS-2B/CYP2A13 cell.Wherein scheme A and be that different concns AFB1 handles that cell activity changes after 24 hours, figure B is that cell activity changes after utilizing the AFB1 of 100nM to handle 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 makes up the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, promptly to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out enzyme simultaneously and cuts, reclaim and carry out enzyme respectively even after enzyme is cut product, transform DH5 α competent cell then, 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 6ug pLJM1-GFP-CYPs plasmid, 3ug pVSVG, 4.5ug delta 8.91 and 15ul Sofast transfection reagent, mixture is added under the DMEM liquid level in the 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 behind the 6-8h.Behind the 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 polybrene and the HEPES of 10ug/mL simultaneously.Superinfection once after infecting 24h.Make up the BEAS-2B cell (BEAS-2B/CYP2A13) of stably express CYP2A13.
3. the checking of BEAS-2B/CYP2A13 cell
Collect each BEAS-2B/CYP2A13 total protein of cell in one week of slow virus infection success back and two weeks respectively, use the specific antibody of CYP2A13, adopt the protein expression in the 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 that the BEAS-2B/CYP2A13 cell strain has been carried out the sorting of GFP positive cell.The normal cell of untransfected does not have fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, and by sorting, the cell sorting that fluorescence intensity is stronger comes out, and is 100% transfectional cell to guarantee cell.
5. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With GFP positive B EAS-2B/CYP2A13 cell, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days as a result, carry out amplification culture to forming monoclonal cell, up to cell satisfy experiment and preserve require till.The monoclonal cell that filters out is verified, chosen the usefulness of the high and stable cell of CYP2A13 protein expression as subsequent experimental and preservation.This cell strain is preserved in Chinese typical culture collection center, and deposit number is CCTCC NO:C2010127.The preservation time is on December 7th, 2010.The preservation address is Chinese Wuhan 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 that by fluorescence microscope along with the prolongation of incubation time, the green fluorescence intensity of cell strengthens gradually, non-transfected cells does not then have 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 nucleus non-transfected cells is not then expressed CYP2A13, the stably express success (Fig. 3) of results suggest cell; In order to obtain monoclonal BEAS-2B/CYP2A13 cell, carry out sorting by the flow cytometer pair cell, with the non-transfected cells contrast, the fluorescence intensity positive cells is sorted out (Fig. 4); Subsequently above-mentioned cell is carried out the mono-clonal screening, and use immunoblotting and RT-PCR method that the CYP2A13 albumen and the mRNA of monoclonal cell verified respectively, found that all cells all expressed CYP2A13 albumen (Fig. 5 A) preferably, the monoclonal cell of being selected 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. cell is to the dose-dependently operational research of AFB1 effect
The BEAS-2B/CYP2A13 cell that utilizes embodiment 1 to obtain, with unloaded cell BEAS-2B/Vector is contrast, give cell 0,10,100,1000 and 10 respectively, the AFB1 of 000 nM, handled 24 hours, adopt mtt assay to detect each dosage then and handle cytoactive down, estimate the toxic effect of cell various dose AFB1 under handling, the structure dose-effect relationship.
2. cell is to the time-dependent manner operational research of AFB1 effect
The BEAS-2B/CYP2A13 cell that utilizes embodiment 1 to obtain, with unloaded cell BEAS-2B/Vector is contrast, use 100 nM AFB1 to handle cell respectively 24,48 and 72 hours, adopt mtt assay to detect each time point cytoactive under handling then, estimate the toxic effect of cell under AFB1 processing different time, structure time-effect relation.
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 fens sensitivities of AFB1, and from minimum concentration, its cytoactive all significantly is 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.Shown in Fig. 5 B, along with the prolongation of time, the BEAS-2B/CYP2A13 cell activity descends gradually, and the BEAS-2B/Vector cell does not then have obvious change; At each time point, the cytoactive of BEAS-2B/CYP2A13 all significantly is lower than BEAS-2B/Vector cell (P＜0.01).The result proves that this BEAS-2B/CYP2A13 cell is a sensitive to the reaction of AFB1, is the ideal 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 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 makes up the slow virus plasmid pLJM1-GFP-CYPs that contains purpose CYP gene, promptly to the existing pFastBac in this laboratory
TMThe special-purpose unloaded plasmid pLJM1-GFP of 1-CYP2A13 plasmid and slow virus carries out enzyme simultaneously and cuts, reclaim and carry out enzyme respectively even after enzyme is cut product, transform DH5 α competent cell then, 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 6ug pLJM1-GFP-CYPs plasmid, 3ug pVSVG, 4.5ug delta 8.91 and 15ul Sofast transfection reagent, mixture is added under the DMEM liquid level in the 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 behind the 6-8h; Behind the 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 polybrene and the HEPES of 10ug/mL simultaneously; Superinfection once after infecting 24h; Make up the BEAS-2B cell BEAS-2B/CYP2A13 of stably express CYP2A13;
C. the checking of BEAS-2B/CYP2A13 cell
Collect each BEAS-2B/CYP2A13 total protein of cell in one week of slow virus infection success back and two weeks respectively, use the specific antibody of CYP2A13, adopt the protein expression in the 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 that the BEAS-2B/CYP2A13 cell strain has been carried out the sorting of GFP positive cell; The normal cell of untransfected does not have fluorescence, and the fluorescence intensity of transfectional cell obviously strengthens, and by sorting, the cell sorting that fluorescence intensity is stronger comes out, and is 100% transfectional cell to guarantee cell;
E. the BEAS-2B/CYP2A13 monoclonal cell selecting and verifying
With GFP positive B EAS-2B/CYP2A13 cell, be inoculated into 96 porocyte culture plates with the concentration of 1 cell in every hole, after 7-10 days as a result, carry out amplification culture to forming monoclonal cell, up to cell satisfy experiment and preserve require till; The monoclonal cell that filters out is verified, chosen the usefulness of the high and stable cell of CYP2A13 protein expression as subsequent experimental and preservation.
3. the application of a human pneumonocyte BEAS-2B/CYP2A13 is characterized in that utilizing the characteristic substrate aflatoxin B1 pair cell of CYP2A13 to test; Use 0-10, the AFB1 of 000 nM handles cell 24 h, adopts mtt assay to estimate dose-effect relationship; Use 100 nM to handle cell 24-72 hour, adopt mtt assay evaluation time-effect relation; By comparing, estimate the susceptibility of BEAS-2B/CYP2A13 cell to AFB1 with unloaded cell BEAS-2B/Vector.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106591370A (en) * | 2015-10-19 | 2017-04-26 | 南京华贞生物医药科技有限公司 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
| CN107119074A (en) * | 2017-04-01 | 2017-09-01 | 广州华真医药科技有限公司 | It is a kind of to treat autoimmunity disease and the viral vector and its construction method of diabetes and application |
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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 |
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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 |
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| CN106591370A (en) * | 2015-10-19 | 2017-04-26 | 南京华贞生物医药科技有限公司 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
| CN107119074A (en) * | 2017-04-01 | 2017-09-01 | 广州华真医药科技有限公司 | It is a kind of to treat autoimmunity disease and the viral vector and its construction method of diabetes and application |
| CN111500636A (en) * | 2017-04-01 | 2020-08-07 | 广州华真医药科技有限公司 | Virus vector for treating autoimmune related diseases and construction method and application thereof |
| CN111500636B (en) * | 2017-04-01 | 2024-04-26 | 广州华真医药科技有限公司 | Virus vector for treating autoimmune related diseases and construction method and application thereof |
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