CN103196817A - Identification method of EB (Epstein-Barr) virus transformation cell apoptosis of excellent ice athletes - Google Patents
Identification method of EB (Epstein-Barr) virus transformation cell apoptosis of excellent ice athletes Download PDFInfo
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- CN103196817A CN103196817A CN2013101216899A CN201310121689A CN103196817A CN 103196817 A CN103196817 A CN 103196817A CN 2013101216899 A CN2013101216899 A CN 2013101216899A CN 201310121689 A CN201310121689 A CN 201310121689A CN 103196817 A CN103196817 A CN 103196817A
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Abstract
The invention discloses an identification method of EB (Epstein-Barr) virus transformation cell apoptosis of excellent ice athletes. The implementation steps of the identification method of the EB virus transformation cell apoptosis of the excellent ice athletes are shown in a specific implement mode part. The identification method provided by the invention provides a researching mode for the EB virus transformation cell apoptosis of the excellent ice athletes, the EB virus transformation cell apoptosis conditions of the excellent ice athletes are subjected to quantitative analysis, according to the identification method, viable apoptotic cells, non-viable apoptotic cells and non-viable non-apoptotic cells can be distinguished quantitatively, and the effect and the accuracy are reliable through the electron microscope inspection and Tunel method inspection.
Description
Technical field
The present invention relates to a kind of flow cytometry that utilizes and identify outstanding winter sports person's epstein-Barr virus (epstein-barr virus, EB) transformant apoptosis authentication method.
Background technology
The cell immortality technical development has had significant progress to today, (Simian vacuolating virus40 SV40) is immortalized cells transforming virus commonly used for Epstein-Barr virus, vacuolating virus of monkey 40.The immortality cell strain that Epstein-Barr virus transforms outstanding winter sports person's peripheral blood bone-marrow-derived lymphocyte foundation allows script have the human body cell in certain life-span to breed down incessantly, and the multiplication capacity of some special cells is strengthened, and apoptosis rate reduces.Outstanding winter sports person's Epstein-Barr virus transformant can permanently be preserved the human genetic resources relevant with outstanding locomitivity, solve the relay-type of experimental study in the past simultaneously and carry out the contradiction that exists in the temporary collection with sample, in order to effectively same research object is carried out lasting continuity research.Epstein-Barr virus transformant apoptosis shows the morphological feature identical with the ordinary cells apoptosis and life-stylize feature, but because its apoptosis rate is very little, many detection methods be difficult to detect or the testing result accuracy rate very low.Apoptosis is identified and is mainly utilized cytomorphology characteristics and biochemical character to carry out.Normally by optical microscope, fluorescent microscope and electron microscope tissue or cell are carried out various dyeing and come the observing apoptosis phenomenon, next is to adopt situ end labeling (Terminal deoxynucleotidyl trasnferase-mediated deoxyuridine triphosphate nick-end labelling assay, TUNEL) and the DNA agarose gel electrophoresis method analyze the apoptosis situation, but these methods all exist testing cost costliness, subjectivity too strong and drawback such as can not quantitatively detect.In recent years, along with the continuous development of flow cytometry, use this technology for detection Apoptosis and used fully.Fluorescein isothiocynate (fluorescein isothiocyanate, FITC) with propidium iodide (Propidium iodide, PI) staining counter, be that Annexin V-FITC/PI double label method can the quantitative test Apoptosis, (specificity PS) can take place with the plain V of connection (Annexin V) and be combined phosphatidylserine in Phosphatidylserine.PI is nucleotide fluorescent dye, can not see through human cell membrane, can only enter damaged cell membrane.During cell generation apoptosis, the phosphatide symmetry of its cell membrane changes and makes PS be exposed to cell membrane outer (being that PS turns up), and apoptotic cell still keeps the integrality of its cell membrane, thereby can with Annexin V (fluorescein-labeled Annexin V, as FITC-Annexin V) combination, and can not bind nucleic acid fluorescent dye PI, though still keeping the non-viable non-apoptotic cell PS of membrane structure does not overturn, but because change has taken place in the permeability of its cell membrane, Annexin V still can enter in the cell and be combined with the PS of cell membrane inside surface, but simultaneously also can be painted by PI; Late period, change took place because PS turns up with membrane permeability in apoptotic cells, by Annexin V and PI mark, also was Annexin V
+/ PI
+Normal cell is complete and the PS flop phenomenon does not take place because of after birth, so can not be shown as jack to jack adapter by Annexin V and PI mark.The two marks of Annexin V-FITC/PI have overcome in conjunction with the Flow cytometry method and have singly dyed the inaccurate shortcoming of testing result, but and the outstanding winter sports person's Epstein-Barr virus of quantitative test transformant apoptosis situation, can quantitatively distinguish viable apoptotic cell, non-viable apoptotic cell and non-viable non-apoptotic cell.
Summary of the invention
The purpose of this invention is to provide the quantitative analysis method of a kind of outstanding winter sports person's Epstein-Barr virus transformant apoptosis; A kind of outstanding winter sports person's Epstein-Barr virus transformant apoptosis authentication method step is provided.
Description of drawings
The outstanding winter sports person's Epstein-Barr virus of Fig. 1 Flow cytometry transformant apoptosis rate
C1: downright bad and mechanical injuries cell
C2: viable apoptotic cell
C3: living cells
C4: non-viable apoptotic cell
Embodiment
(1) preparation single cell suspension is collected the Epstein-Barr virus transformant in the streaming pipe, 1200rpm, centrifugal 8min.
(2) (phosphate buffered solution, PBS) washed cell secondary (the centrifugal 8min of 1200rpm) is collected (1-5) * 10 with phosphate buffer
5Cell.
(3) each sample adds connection damping fluid (Binding Buffer) suspension cell of 100 μ l.
(4) add 5 μ l FITC and 5 μ l PI pairs simultaneously and dye mixing.
(5) with known medicine (as the camptothecine) cell death inducing that makes Epstein-Barr virus transformant apoptosis, as positive control.Dye with 5 μ l FITC and PI respectively before the detection.
(6) room temperature, lucifuge, reaction 5-10min.
(7) the apoptosis situation of observation of cell under 488nm copolymerization Jiao at first.
(8) replenish 400 μ l Binding Buffe, 400 eye mesh screens sieve, and carry out flow cytometry in the 1h and quantitatively detect.
(9) result judges: dyestuff such as PI that apoptotic cell is identified being useful on cytoactive have anti-metachromia, and non-viable non-apoptotic cell then can not.Cell membrane has the DNA of the cell of damage by PI to dye the generation red fluorescence, and the cell that cell membrane remains intact does not then have red fluorescence and produces.Therefore, apoptotic early stage PI not can dye and do not have the red fluorescence signal.Normal living cells similarly.On the scatter diagram of bivariate flow cytometer, left lower quadrant shows living cells, is (FITC-/PI-); Right upper quadrant is non-living cells, and namely non-viable non-apoptotic cell is (FITC+/PI+); And right lower quadrant is apoptotic cell, manifests (FITC+/PI-).
Claims (1)
1. outstanding winter sports person's Epstein-Barr virus transformant apoptosis quantitative analysis method, its principal character are the apoptosis that adopts the two marks of AnnexinV-FITC/PI and detect outstanding winter sports person's Epstein-Barr virus transformant in conjunction with flow cytometry.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107782598A (en) * | 2017-10-16 | 2018-03-09 | 四川省中医药科学院 | A kind of colouring method of apoptotic cell |
| CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798230A (en) * | 1995-11-09 | 1998-08-25 | Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh | Process for the preparation of human monoclonal antibodies and their use |
| CN101624581A (en) * | 2009-06-03 | 2010-01-13 | 中国人民解放军第三军医大学 | Pseudovirus based on Vasohibin gene and preparation method and application thereof |
| CN101892261A (en) * | 2009-05-18 | 2010-11-24 | 中国医学科学院肿瘤研究所 | Recombinant adenovirus vector and its application |
| CN102648197A (en) * | 2009-08-12 | 2012-08-22 | 铂雅制药公司 | Method of promoting apoptosis and inhibiting metastasis |
-
2013
- 2013-04-10 CN CN2013101216899A patent/CN103196817A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5798230A (en) * | 1995-11-09 | 1998-08-25 | Gsf-Forschungszentrum Fur Umwelt Und Gesundheit Gmbh | Process for the preparation of human monoclonal antibodies and their use |
| CN101892261A (en) * | 2009-05-18 | 2010-11-24 | 中国医学科学院肿瘤研究所 | Recombinant adenovirus vector and its application |
| CN101624581A (en) * | 2009-06-03 | 2010-01-13 | 中国人民解放军第三军医大学 | Pseudovirus based on Vasohibin gene and preparation method and application thereof |
| CN102648197A (en) * | 2009-08-12 | 2012-08-22 | 铂雅制药公司 | Method of promoting apoptosis and inhibiting metastasis |
Non-Patent Citations (7)
| Title |
|---|
| ALAN WELLS ET. AL.: "Epstein-barr virus receptor expression is correlated to cell cycle phase", 《JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION》, vol. 2, no. 3, 31 December 1981 (1981-12-31) * |
| 冯永东等: "MOLT-4细胞凋亡不同阶段活性caspase-3在细胞内的空间位相移行", 《癌症》, vol. 23, no. 9, 5 September 2004 (2004-09-05) * |
| 刘畅等: "激光扫描共聚焦显微镜在细胞凋亡研究中的应用", 《医学综述》, vol. 7, no. 10, 15 October 2001 (2001-10-15) * |
| 季宇彬等: "龙葵碱诱导HepG2细胞凋亡的线粒体通路研究", 《中国药学杂志》, vol. 43, no. 4, 22 February 2008 (2008-02-22) * |
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| 钟国华等: "印楝素对SL-1 的细胞凋亡诱导作用", 《昆虫学报》, vol. 51, no. 6, 20 June 2008 (2008-06-20) * |
| 陶怡等: "EB病毒感染对骨髓瘤细胞生物学特性的影响", 《现代免疫学》, vol. 25, no. 6, 20 November 2005 (2005-11-20) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107782598A (en) * | 2017-10-16 | 2018-03-09 | 四川省中医药科学院 | A kind of colouring method of apoptotic cell |
| CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
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Application publication date: 20130710 |