CN101126758A - Flow cytometry synchronous detection method for multiple protein expression of tumor cell - Google Patents
Flow cytometry synchronous detection method for multiple protein expression of tumor cell Download PDFInfo
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- CN101126758A CN101126758A CNA2007101320030A CN200710132003A CN101126758A CN 101126758 A CN101126758 A CN 101126758A CN A2007101320030 A CNA2007101320030 A CN A2007101320030A CN 200710132003 A CN200710132003 A CN 200710132003A CN 101126758 A CN101126758 A CN 101126758A
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Abstract
The utility model discloses an expression method of multi cell protein in the tumor tested synchronously by a flow cytometry, comprising the following steps: the fresh tumor organ is made into a single cell suspension solution; the concentration of the singe cell suspension solution is adjusted to +/- 106 cell/100ul; the single cell suspension solution attained by the steps is input into a test tube, a fluorescence symbolized antibody of 5 to 20 mul discriminating the different cells is filled into the test tube according to the mixing ratio of the reagent, mixed evenly and sufficiently, and reacted for 15 to 45 minutes in ambient temperature and light shielded; a protein antibody under test is filled in the test tube and mixed evenly and sufficiently and is reacted for 15 to 45 minutes in the ambient temperature and the light shielded; the upper clean liquid is removed by the centrifugal force, so that the non-combined fluorescence symbolized antibody is removed too; the suspended cell is tested and analyzed on the flow cytometer apparatus. The utility model has advantages of improving the accuracy and precision of examining the objective cell, lowering the examination cost, and reducing the system error caused by separate experiments in multi times.
Description
Technical field
The present invention relates to the tumor cell assay detection range, relate in particular to the expression of utilization Flow cytometry tumour cell albumen.
Background technology
At present, in entity tumor research, the unicellular tumour cell that all is considered as usually by tumor tissue's preparation is measured and is studied, when needs are done patient self tumour cell and normal cell comparison, often need to gather in addition normal tissue cell, this relates to serial problems such as patient's wish, multidisciplinary cooperation, sample storage transportation, often is difficult for perfect; When comparison of tumor cell and Normocellular protein expression level, then in different test tubes, detect respectively, analyze respectively.
In fact in tumor tissues, except that a large amount of tumour cells, also contain normal cells such as haemocyte, vascular endothelial cell.If can be distinguished with suitable labelled antibody, just can in a test tube, finish certain protein expression level of different cells and measure, reach synchronous detection, Synchronization Analysis, mutual purpose relatively.
Flow cytometry is to finish a kind of analytical technology that the pair cell various characteristics carries out qualitative, quantitative by flow cytometer by antigen, antibody response and fluorescent marker method.Its principle is the unicellular measurement zone that passes through one by one that will be suspended in the liquid stream, reaches a series of physical characteristicss and the biochemical characteristic of measuring cell fast, in large quantities with the optical parametric of instrument detecting cell.
Summary of the invention
Goal of the invention: technical matters to be solved by this invention provides a kind of method that adopts expression of cellular proteins in the Flow cytometry tumour, can satisfy the requirement of the protein expression level of synchronous mensuration various kinds of cell (two kinds or above cell).
Technical scheme: for solving the problems of the technologies described above, thinking of the present invention is to use fluorescence labeling to distinguish the method and the method combination of using antibody labeling specific recognition albumen of different cells, to reach the purpose of a certain protein expression on the different cells of synchronous detection.
The concrete steps of the protein expression of various kinds of cell are as follows in the flow cytometry synchronous detection tumour:
1, fresh tumor tissues is prepared into single cell suspension.
2, regulating single cell suspension concentration is ± 10
6Cell/100 μ l.
3, add the single cell suspension that step 2 obtains in a test tube, the matched proportion density by the reagent explanation of fluorescent-labeled antibody adds the fluorescent-labeled antibody 5-20 μ l that can distinguish different cells to be measured respectively, abundant mixing, room temperature lucifuge reaction 15～45 minutes.By adding suitable fluorescence labeling and the cell effect method of distinguishing different cells that develops the color is technology well known in the art, for example uses to have different fluorescently-labeled antibody CD3, CD19 and CD14 distinguishes T cell, B cell and monocyte simultaneously.
4, in above-mentioned test tube, add the fluorescence antibody of testing protein, abundant mixing, room temperature lucifuge reaction 15～45 minutes.The color mark of this antibody will be distinguished mutually with the fluorescence color of labeled cell in the step 3.For different albumen kinds, the adding mode of selecting suitable antibody and antibody is a technology well known in the art, for the expressing protein that is positioned at surface of cell membrane, can directly add antibody response; For being positioned at intracellular expressing protein, the first pair cell of needs is fixed, rupture of membranes, adds antibody response then.The MDR glycoprotein Pgp that for example detects on the cell membrane expresses, and directly adds the fluorescence antibody different with cell fluorescence and reacts; When detecting endonuclear apoptotic proteins Bcl-2, proliferating cell nuclear antigen PCNA, the first pair cell of needs is fixed, rupture of membranes, adds the fluorescence antibody different with cell fluorescence then and reacts.
5, centrifugal removal supernatant is to remove unconjugated fluorescent-labeled antibody.
6, the protein expression of up flow type cell instrument detection behind the suspension cell, analysis various kinds of cell.
Wherein the cell to be measured described in the step 3 is two or more the cell in tumour cell, stroma cell, vascular endothelial cell, lymphocyte infiltration or the haemocyte.Tumour cell can be squamous cell carcinoma, adenocarcinoma cell, undifferentiated cancer cell, sarcoma cell or tumor stem cell.Haemocyte can be lymphocyte, monocyte, granulocyte or candidate stem cell.
Before tumor tissues was prepared into single cell suspension, fresh tumor tissue can put into the physiological saline that contains the 30-60IU/ml anti-coagulants, to reach the purpose of the transportation of preserving moisture.
Beneficial effect: the method for various kinds of cell protein expression compared with prior art has following advantage in the flow cytometry synchronous detection tumour of the present invention:
1, can understand the content of different cells in the tumor tissue.
2, looked tumor tissue's cell in the past be tumour cell and measure and study, distinguish cell by labelled antibody now after, can effectively improve accuracy and degree of accuracy that target cell is measured.
3, can not only solve the problem that is difficult for obtaining with reference to cell, and can in same data plot, carry out Synchronization Analysis with existing normal cell in the tumor tissue as interior reference, enlarged quantity of information.
4, reduce cost of determination, reduce the systematic error that the gradation experiment brings.
Description of drawings
Fig. 1 detects the flow cytometry figure of gland cancer sample (embodiment 1) for adopting the inventive method.
Fig. 2 detects the flow cytometry figure of squama cancer sample (embodiment 2) for adopting the inventive method.
Fig. 3 a does not promptly carry out the flow cytometry figure that cell marking detects thyroid gland lump sample (embodiment 3) for adopting classic method.
Fig. 3 b to 3e detects the flow cytometry figure of thyroid gland lump sample (embodiment 3) for adopting the inventive method.
Embodiment
Embodiment 1: gland cancer is carried out the research of tumor suppressor gene sudden change product P 53 (mutant) expression.
The gland cancer sample is put into the physiological saline that contains the 50IU/ml anti-coagulants immediately and is sent the prepared in laboratory single cell suspension, and transferring single cell suspension concentration is ± 10
6Cell/100 μ l.Single cell suspension is divided into 2 parts, every part 100 μ l.A earlier with fluorescence labeling CD45 mouse-anti people's monoclonal antibody (Immunotech company) 10 μ l body tag haemocytes, fully behind the mixing, the room temperature lucifuge was reacted 30 minutes; Again with kit IntraPREP fixing and rupture of membranes
TMPair cell fix and rupture of membranes after, P53 (mutant) mouse-anti people's monoclonal antibody (Ancell company) 20 μ l labeled cell albumen, fully behind the mixing, room temperature lucifuge reaction 25 minutes; Through centrifugal removal supernatant, up flow type cell instrument FACSCalibur detects, analyzes behind the suspension cell.Another part is done homotype negative control pipe.Fig. 1 shows this sample removal haemocyte (CD45
+) after the P53 (mutant) of adenocarcinoma cell to express positive cell rate be 16.88%.
Embodiment 2: observe a routine squamous cell carcinoma propagation level, carry out the mensuration of proliferating cell nuclear antigen (PCNA).
Squama cancer sample is put into the physiological saline that contains the 60IU/ml anti-coagulants immediately and is sent prepared in laboratory to become single cell suspension, and transferring single cell suspension concentration is ± 10
6Cell/100 μ l.Single cell suspension is divided into 2 parts, every part 100 μ l.A earlier with fluorescence labeling CD45 mouse-anti people's monoclonal antibody (Immunotech company) 10 μ l body tag haemocytes, fully behind the mixing, the room temperature lucifuge was reacted 45 minutes; Again with kit IntraPREP fixing and rupture of membranes
TMPair cell fix and rupture of membranes after, with mouse-anti people monoclonal antibody PCNA (Pharmingen company) 10 μ l labeled cell albumen, fully behind the mixing, room temperature lucifuge reaction 45 minutes; Through centrifugal removal supernatant, up flow type cell instrument FACSCalibur detects, analyzes behind the suspension cell.Another part is done homotype negative control pipe.Fig. 2 shows haemocyte (CD45 in this squama cancer
+) the PCNA positive cell rate be 30.35%, the PCNA positive cell rate of adenocarcinoma cell is 40.75%.
Embodiment 3: the mensuration of a routine thyroid gland lump being carried out MDR glycoprotein Pgp expression.
Sample becomes single cell suspension in prepared in laboratory, and transferring concentration is ± 10
6Cell/100 μ l.Single cell suspension is divided into 2 parts, every part 100 μ l.Portion is used mouse-anti people monoclonal antibody (Immunotech company) CD45, CD14 each 10 μ l difference mark haemocyte and the macrophage that has fluorescence labeling 1 and have fluorescence labeling 2 earlier, and fully behind the mixing, the room temperature lucifuge was reacted 45 minutes; With MDR glycoprotein Pgp monoclonal antibody (Immunotech company) the 10 μ l labeled cell albumen that have fluorescence labeling 3, fully behind the mixing, the room temperature lucifuge was reacted 30 minutes again, and up flow type cell instrument FC500 detects, analyzes.Fig. 3 a shows the thyroid gland lump Pgp expression 4.5% that records when adopting classic method promptly not carry out cell marking; Fig. 3 b records when show adopting the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour of the present invention that tumour cell accounts for 80.4% in the thyroid gland lump, and haemocyte accounts for 15.8%, and macrophage accounts for 1.4%.Fig. 3 c records tumour cell Pgp expression 2.6% in the thyroid gland lump when showing the method that adopts various kinds of cell protein expression in the flow cytometry synchronous detection tumour of the present invention.Fig. 3 d records haemocyte Pgp expression 3.6% in the thyroid gland lump when showing the method that adopts various kinds of cell protein expression in the flow cytometry synchronous detection tumour of the present invention.Fig. 3 e records macrophage Pgp expression 65.5% in the thyroid gland lump when adopting the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour of the present invention.
Claims (4)
1. the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour is characterized in that this method may further comprise the steps:
(1) fresh tumor tissues is prepared into single cell suspension;
(2) regulating single cell suspension concentration is ± 10
6Cell/100 μ l;
(3) in a test tube, add the single cell suspension that step (2) obtains, matched proportion density by the explanation of the reagent of fluorescent-labeled antibody adds the fluorescent-labeled antibody 5-20 μ l that can distinguish different cells to be measured respectively, abundant mixing, room temperature lucifuge reaction 15～45 minutes;
(4) in above-mentioned test tube, add the antibody of testing protein, abundant mixing, room temperature lucifuge reaction 15～45 minutes;
(5) centrifugal removal supernatant is to remove unconjugated fluorescent-labeled antibody;
(6) protein expression of up flow type cell instrument detection behind the suspension cell, analysis various kinds of cell.
2. the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour according to claim 1 is characterized in that the cell to be measured described in the step (3) is two or more the cell in tumour cell, stroma cell, vascular endothelial cell, lymphocyte infiltration or the haemocyte.
3. the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour according to claim 2 is characterized in that described tumour cell is squamous cell carcinoma, adenocarcinoma cell, undifferentiated cancer cell, sarcoma cell or tumor stem cell.
4. the method for various kinds of cell protein expression in the flow cytometry synchronous detection tumour according to claim 2 is characterized in that described haemocyte is lymphocyte, monocyte, granulocyte or candidate stem cell.
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| CN102879319A (en) * | 2012-10-17 | 2013-01-16 | 许旭欣 | Flow cell velocity measuring method for adjusting concentration of responding cells |
| CN102914495A (en) * | 2012-10-10 | 2013-02-06 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for evaluating DNA (Deoxyribose Nucleic Acid) damages of peripheral blood lymphocytes caused by ionizing radiation |
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| CN102914495B (en) * | 2012-10-10 | 2015-04-15 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Method for evaluating DNA (Deoxyribose Nucleic Acid) damages of peripheral blood lymphocytes caused by ionizing radiation |
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| CN105683752A (en) * | 2013-06-21 | 2016-06-15 | 国立大学法人冈山大学 | Method using abnormally-activated-cell detection to test for malignant tumors and abnormally-activated-cell apheresis-therapy apparatus |
| CN104725511B (en) * | 2015-02-16 | 2018-01-12 | 华南农业大学 | A kind of isolated culture method of pig intestinal stem cell |
| CN104725511A (en) * | 2015-02-16 | 2015-06-24 | 华南农业大学 | Separation culture method for pig intestinal stem cells |
| CN105223360A (en) * | 2015-08-28 | 2016-01-06 | 北京大学人民医院 | Differentiate to detect normal plasma cells and Clonal plasmacytic kit and application thereof |
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