CN107102127B - Monokaryon sample marrow source inhibits the detection method of cell in a kind of human peripheral blood - Google Patents
Monokaryon sample marrow source inhibits the detection method of cell in a kind of human peripheral blood Download PDFInfo
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Abstract
Monokaryon sample marrow source inhibits the detection method of cell in a kind of human peripheral blood of the invention, uses antibody are as follows: (a) antibody A PC anti-human CD33；(b) antibody PE anti-human CD15；(c) antibody PE-CF594 anti-human HLA-DR；(d) antibody A PC-H7 anti-human CD14；(e) antibody PE-Cy7 anti-human CD45；(f) antibody FITC anti-mouse CD11b.Prepare streaming sample solution；Use the ratio of mo-MDSCs in flow cytomery streaming sample solution.For detecting mo-MDSCs, the range of specific screening numerical value and the detailed process of screening are given.Detection is simple and convenient to operate, the review time is short, it is only necessary to micro peripheral blood sample label, it is low-cost, and it is easy to widely available.To the peripheral blood blood sample of 151 subjects, detected respectively with method of the invention.The mo-MDSCs proportional numerical value detected shows apparent difference in different groups, and the ratio in patient with breast cancer is significantly higher than Healthy People and Benign Breast tumor patient.Testing result has statistical significance by computer, P < 0.05 for statistical analysis using 6 software of Graphpad Prism, and science is credible.
Description
Technical field
The present invention relates to the detection methods that monokaryon sample marrow source in a kind of human peripheral blood inhibits cell.
Background technique
The inhibitory cells (MDSCs) in medullary system source are the heterogeneous cells of a group derived from bone marrow, according to its form and
The difference of surface marker can be divided into two subgroups: granulocyte sample marrow source inhibits cell (PMN-MDSCs) and monokaryon sample marrow source
Inhibit cell (mo-MDSCs).Wherein, mo-MDSCs has stronger immunosuppression capability1, existing mo-MDSCs in body
It will affect the progress and deterioration of the diseases such as breast cancer.Based on this, screening and detection for mo-MDSCs are very necessary.
Since MDSCs cell mass lacks the identification tag of specificity, existing screening and the method detected are very
Mix.ALMAND B et al. thinks Lin by kinds cancer sample−HLA-DR−Marker be can be used as to screen inspection
Survey MDSCs cell mass2, the work of DIAZ-MONTERO CM et al. then thinks that Lin can be passed through−/lowHLA-DR−CD33+CD11b+
Mark combination carries out selective mechanisms3, also by CD15+The research work etc. of MDSCs cell mass is detected from granulocyte group4。
But the detection method of existing MDSCs and mo-MDSCs relies on operator more and has experience abundant and theoretical background basis,
The detection method of the specific science of mo-MDSCs is not provided.(bibliography:
Summary of the invention
In order to solve the problems, such as that prior art exists, the present invention provides monokaryon sample marrow sources in a kind of human peripheral blood to inhibit
The detection method of cell.
Monokaryon sample marrow source inhibits the step of detection method of cell and condition in a kind of human peripheral blood provided by the invention
It is as follows:
A, antibody used
(101) antibody A PC anti-human CD33, abbreviation CD33；
(102) antibody PE anti-human CD15, abbreviation CD15；
(103) antibody PE-CF594 anti-human HLA-DR, abbreviation HLA-DR；
(104) antibody A PC-H7 anti-human CD14, abbreviation CD14；
(105) antibody PE-Cy7 anti-human CD45, abbreviation CD45；
(106) antibody FITC anti-mouse CD11b, abbreviation CD11b；
B, the preparation of solution used
(201) NaCl 8 g, KCl 0.2 g, Na the preparation of PBS solution: are weighed2HPO41.44 g and KH2PO4 0.24
G is dissolved in 1 L ultrapure water, high pressure sterilization, obtains PBS solution, and 4 DEG C of refrigerators save；
(202) preparation of buffer: taking newborn bovine serum 1mL, is added to and obtains the slow of 10 mL in the PBS solution of 9 mL
Fliud flushing；The new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid: NH is weighed4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultrapure waters
In, solution is adjusted into pH to 7.2, ultrapure water is continuously added until total volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators are protected
It deposits；
(204) preparation of fixer: 10 mL formalins are measured, the PBS solution prepared to 1 L step (201) is added
In, add 20 g of glucose, NaNO3 0.2 g, is completely dissolved, and obtains fixer, and room temperature is spare；
(205) preparation of antibody mixed solution: it is added 4 μ L's in 200 μ L buffers of step (202) preparation
HLA-DR antibody, the CD15 antibody of 6 μ L, the CD45 antibody of 6 μ L, the CD33 antibody of 6 μ L, the CD14 antibody of 8 μ L and 1 μ L
CD11b antibody, mix, obtain antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
C, the preparation of streaming sample solution
(301) it takes 10 μ L of subject's peripheral blood blood sample to be added in 1 mL erythrocyte cracked liquid, is cracked on ice
30 min, every 10 min concussion is primary, obtains peripheral blood suspension；
(302) the peripheral blood suspension centrifuge for obtaining step (301), 1800 rpm are centrifuged 3 min under 4 DEG C of low temperature,
Liquid is discarded supernatant, adds 200 μ L buffers into remaining precipitating, is allowed to hang simultaneously using micropipettor piping and druming precipitating
It mixes；
(303) centrifuge is used, what 1800 rpm were centrifuged 3 min steps (302) under 4 DEG C of low temperature obtains object, discards supernatant
Liquid adds 20 μ L antibody mixed solutions into remaining precipitating, is allowed to hang and be mixed using micropipettor piping and druming precipitating
It is even, it is then placed in 30 min on ice, pays attention to being protected from light；
(304) it obtains that 200 μ L buffers are added in object in step (303), mixes, centrifuge is then used, under 4 DEG C of low temperature
1800 rpm are centrifuged 3 min, discard supernatant liquid；
(305) PBS solution that 200 μ L are added in object is obtained in step (304), is mixed it using micropipettor,
Obtain streaming sample solution；
D, the detection of mo-MDSCs
The streaming sample solution obtained with (305) of step 3, the ratio of mo-MDSCs in detection stream formula sample solution, inspection
Steps are as follows for survey:
(401) according to the common cognition standard of the granularity of cell and size, streaming sample is removed using flow cytometer circle
Adhesion cells and dead cell in solution obtain the work individual cells group i.e. cell mass I in blood；
(402) flow cytometer is used, the case where according to antibody CD45 developed by molecule, circle takes PE-Cy7 in cell mass I glimmering
Luminous intensity is greater than 2 × 102Cell obtain cell mass II, this is the leukocyte population in blood；
(403) antibody PE- is taken from cell mass II centre circle according to the expression of antibody HLA-DR using flow cytometer
CF594 fluorescence intensity is greater than 2 × 102Cell obtain cell mass III；
(404) flow cytometer is used, according to the expression of antibody CD33 and antibody CD11b, from cell mass III
Circle takes APC and FITC fluorescence intensity to be all larger than 2 × 102Cell mass, obtain cell mass IV, this be MDSCs cell mass；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to the expression of antibody CD14 and antibody CD15
Situation takes PE fluorescence intensity less than 2 × 10 from cell mass IV centre circle2And antibody A PC-H7 fluorescence intensity is greater than 3 × 102Cell
Group is mo-MDSCs cell mass；
(406) by the mo-MDSCs cell mass quantity filtered out divided by the number of cell mass II obtained in step (402)
Amount, obtains the proportional numerical value of mo-MDSCs in peripheral white blood cells.
The mo- that monokaryon sample marrow source inhibits the detection method of cell to detect in a kind of human peripheral blood provided by the invention
MDSCs proportional numerical value shows apparent difference in different groups, and the ratio in patient with breast cancer is significantly higher than health
People and Benign Breast tumor patient.
The utility model has the advantages that monokaryon sample marrow source inhibits the detection method of cell, institute in a kind of human peripheral blood provided by the invention
Antibody (a) antibody A PC anti-human CD33, abbreviation CD33；(b) antibody PE anti-human CD15, referred to as
CD15；(c) antibody PE-CF594 anti-human HLA-DR, abbreviation HLA-DR；(d) antibody A PC-H7 anti-human
CD14, abbreviation CD14；(e) antibody PE-Cy7 anti-human CD45, abbreviation CD45；(f) antibody FITC anti-mouse
CD11b；Prepare streaming sample solution；Use the ratio of mo-MDSCs in flow cytomery streaming sample solution.For inspection
It surveys mo-MDSCs and gives the range of specific screening numerical value and the detailed process of screening.When detection is simple and convenient to operate, checks
Between it is short, it is only necessary to micro peripheral blood sample label, it is low-cost, be easy to widely available.The result of detection has carried out Statistics Division
Reason: to the peripheral blood blood sample of 151 subjects, inhibited with monokaryon sample marrow source in a kind of human peripheral blood provided by the invention
The detection method of cell detects, and the information that the detecting step (406) detects is used Graphpad by computer
6 software of Prism is for statistical analysis, and P < 0.05 has statistical significance.Illustrate that methodological science of the invention is credible.
Figure of description
Fig. 1 be method of the invention using flow cytometer circle except in streaming sample solution adhesion cells and dead cell obtain
To cell mass I schematic diagram.
Fig. 2 is that method of the invention using flow cytometer obtains the schematic diagram of cell mass II.
Fig. 3 is that method of the invention using the cell of flow cytometer obtains the schematic diagram of cell mass III.
Fig. 4 is that method of the invention using flow cytometer obtains the schematic diagram of cell mass IV.
Fig. 5 is the schematic diagram for the mo-MDSCs cell mass that method of the invention is obtained using flow cytometer.
Fig. 6 is the ratio that the mo-MDSCs that method of the invention detects shows apparent difference in different groups
Figure.
Specific embodiment
Monokaryon sample marrow source inhibits the step of detection method of cell and condition as follows in a kind of human peripheral blood of embodiment 1:
1, antibody used
(101) antibody A PC anti-human CD33, abbreviation CD33；BD Biosciences company, the U.S.；
(102) antibody PE anti-human CD15, abbreviation CD15；BD Biosciences company, the U.S.；
(103) antibody PE-CF594 anti-human HLA-DR, abbreviation HLA-DR；U.S. BD Biosciences is public
Department；
(104) antibody A PC-H7 anti-human CD14, abbreviation CD14；BD Biosciences company, the U.S.；
(105) antibody PE-Cy7 anti-human CD45, abbreviation CD45；BD Biosciences company, the U.S.；
(106) antibody FITC anti-mouse CD11b, abbreviation CD11b；Tianjin Sungene Biotechnology Co., Ltd..
2, the preparation of solution used
(201) NaCl 8 g, KCl 0.2 g, Na the preparation of PBS solution: are weighed2HPO41.44 g and KH2PO4 0.24
G is dissolved in 1 L ultrapure water, high pressure sterilization, obtains PBS solution, and 4 DEG C of refrigerators save；
(202) preparation of buffer: taking newborn bovine serum 1mL, is added to and obtains the slow of 10 mL in the PBS solution of 9 mL
Fliud flushing；The new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid: NH is weighed4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultrapure waters
In, solution is adjusted into pH to 7.2, ultrapure water is continuously added until total volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators are protected
It deposits；
(204) preparation of fixer: 10 mL formalins are measured, the PBS solution prepared to 1 L step (201) is added
In, add 20 g of glucose, NaNO3 0.2 g, is completely dissolved, and obtains fixer, and room temperature is spare；
(205) preparation of antibody mixed solution: it is added 4 μ L's in 200 μ L buffers of step (202) preparation
HLA-DR antibody, the CD15 antibody of 6 μ L, the CD45 antibody of 6 μ L, the CD33 antibody of 6 μ L, the CD14 antibody of 8 μ L and 1 μ L
CD11b antibody, mix, obtain antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
3, the preparation of streaming sample solution
(301) it takes 10 μ L of subject's peripheral blood blood sample to be added in 1 mL erythrocyte cracked liquid, is cracked on ice
30 min, every 10 min concussion is primary, obtains peripheral blood suspension；
(302) the peripheral blood suspension centrifuge for obtaining step (301), 1800 rpm are centrifuged 3 min under 4 DEG C of low temperature,
Liquid is discarded supernatant, adds 200 μ L buffers into remaining precipitating, is allowed to hang simultaneously using micropipettor piping and druming precipitating
It mixes；
(303) centrifuge is used, what 1800 rpm were centrifuged 3 min steps (302) under 4 DEG C of low temperature obtains object, discards supernatant
Liquid adds 20 μ L antibody mixed solutions into remaining precipitating, is allowed to hang and be mixed using micropipettor piping and druming precipitating
It is even, it is then placed in 30 min on ice, pays attention to being protected from light；
(304) it obtains that 200 μ L buffers are added in object in step (303), mixes, centrifuge is then used, under 4 DEG C of low temperature
1800 rpm are centrifuged 3 min, discard supernatant liquid；
(305) PBS solution that 200 μ L are added in object is obtained in step (304), is mixed it using micropipettor,
Obtain streaming sample solution；
4, the detection of mo-MDSCs
The streaming sample solution obtained with (305) of step 3, the ratio of mo-MDSCs in detection stream formula sample solution, inspection
Steps are as follows for survey:
(401) according to the common cognition standard of the granularity of cell and size, streaming sample is removed using flow cytometer circle
Adhesion cells and dead cell in solution, the i.e. cell mass I(of work individual cells group obtained in blood are shown in Fig. 1)；
(402) flow cytometer is used, the case where according to antibody CD45 developed by molecule, circle takes PE-Cy7 in cell mass I glimmering
Luminous intensity is greater than 2 × 102Cell obtain cell mass II, this is the leukocyte population (see figure 2) in blood；
(403) antibody PE- is taken from cell mass II centre circle according to the expression of antibody HLA-DR using flow cytometer
CF594 fluorescence intensity is greater than 2 × 102Cell obtain cell mass III(and see Fig. 3)；
(404) flow cytometer is used, according to the expression of antibody CD33 and antibody CD11b, from cell mass III
Circle takes APC and FITC fluorescence intensity to be all larger than 2 × 102Cell mass, obtain cell mass IV, this for MDSCs cell mass (see figure
4)；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to the expression of antibody CD14 and antibody CD15
Situation takes PE fluorescence intensity less than 2 × 10 from cell mass IV centre circle2And antibody A PC-H7 fluorescence intensity is greater than 3 × 102Cell
Group is mo-MDSCs cell mass (see figure 5)；
(406) by the mo-MDSCs cell mass quantity filtered out divided by the number of cell mass II obtained in step (402)
Amount, obtains the proportional numerical value of mo-MDSCs in peripheral white blood cells.
Thus the mo-MDSCs proportional numerical value that method detects shows apparent difference in different groups, in breast cancer
Ratio in patient is significantly higher than Healthy People and Benign Breast tumor patient (see Fig. 6).
The present embodiment, to the peripheral blood blood sample of 151 subjects, respectively with a kind of human peripheral provided by the invention
Monokaryon sample marrow source inhibits the detection method detection of cell in blood.The result of detection has carried out statistical disposition: the detection is walked
Suddenly the information of (406) detection, for statistical analysis using 6 software of Graphpad Prism by computer, P < 0.05, tool
Standby statistical significance.Illustrate that methodological science of the invention is credible.
Claims (1)
1. monokaryon sample marrow source inhibits the detection method of cell in a kind of human peripheral blood, feature, step and condition are as follows:
A, antibody used
(101) antibody A PC anti-human CD33, abbreviation CD33；
(102) antibody PE anti-human CD15, abbreviation CD15；
(103) antibody PE-CF594 anti-human HLA-DR, abbreviation HLA-DR；
(104) antibody A PC-H7 anti-human CD14, abbreviation CD14；
(105) antibody PE-Cy7 anti-human CD45, abbreviation CD45；
(106) antibody FITC anti-mouse CD11b, abbreviation CD11b；
B, the preparation of solution used
(201) NaCl 8 g, KCl 0.2 g, Na the preparation of PBS solution: are weighed2HPO41.44 g and KH2PO40.24 g is molten
In 1 L ultrapure water, high pressure sterilization obtains PBS solution, and 4 DEG C of refrigerators save；
(202) preparation of buffer: taking newborn bovine serum 1mL, is added in the PBS solution of 9 mL and obtains the buffering of 10 mL
Liquid；The new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid: NH is weighed4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultrapure waters, will
Solution adjusts pH to 7.2, continuously adds ultrapure water until total volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators save；
(204) preparation of fixer: measuring 10 mL formalins and be added in the PBS solution prepared to 1 L step (201),
Add 20 g of glucose, NaNO3 0.2 g, is completely dissolved, and obtains fixer, and room temperature is spare；
(205) HLA-DR of 4 μ L the preparation of antibody mixed solution: is added in 200 μ L buffers of step (202) preparation
Antibody, the CD15 antibody of 6 μ L, the CD45 antibody of 6 μ L, the CD33 antibody of 6 μ L, the CD11b of the CD14 antibody of 8 μ L and 1 μ L
Antibody mixes, obtains antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
C, the preparation of streaming sample solution
(301) 10 μ L of subject's peripheral blood blood sample is taken to be added in 1 mL erythrocyte cracked liquid, in cracking 30 on ice
Min, every 10 min concussion is primary, obtains peripheral blood suspension；
(302) the peripheral blood suspension centrifuge for obtaining step (301), 1800 rpm are centrifuged 3 min under 4 DEG C of low temperature, discard
Supernatant adds 200 μ L buffers into remaining precipitating, is allowed to hang and be mixed using micropipettor piping and druming precipitating；
(303) centrifuge is used, what 1800 rpm were centrifuged 3 min steps (302) under 4 DEG C of low temperature obtains object, liquid is discarded supernatant, then
20 μ L antibody mixed solutions are added into remaining precipitating, is allowed to hang and mix using micropipettor piping and druming precipitating, then
It is placed in 30 min on ice, pays attention to being protected from light；
(304) it obtains that 200 μ L buffers are added in object in step (303), mixes, then use centrifuge, 1800 under 4 DEG C of low temperature
Rpm is centrifuged 3 min, discards supernatant liquid；
(305) PBS solution that 200 μ L are added in object is obtained in step (304), it is mixed using micropipettor, is obtained
Streaming sample solution；
D, the detection of mo-MDSCs
The streaming sample solution obtained with (305) of step 3, the ratio of mo-MDSCs in detection stream formula sample solution, detection step
It is rapid as follows:
(401) according to the common cognition standard of the granularity of cell and size, streaming sample solution is removed using flow cytometer circle
In adhesion cells and dead cell, obtain the work individual cells group i.e. cell mass I in blood；
(402) flow cytometer is used, the case where according to antibody CD45 developed by molecule, circle takes PE-Cy7 fluorescence in cell mass I strong
Degree is greater than 2 × 102Cell obtain cell mass II, this is the leukocyte population in blood；
(403) antibody PE-CF594 is taken from cell mass II centre circle according to the expression of antibody HLA-DR using flow cytometer
Fluorescence intensity is greater than 2 × 102Cell obtain cell mass III；
(404) it is taken according to the expression of antibody CD33 and antibody CD11b from cell mass III centre circle using flow cytometer
APC and FITC fluorescence intensity is all larger than 2 × 102Cell mass, obtain cell mass IV, this be MDSCs cell mass；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to the expression of antibody CD14 and antibody CD15,
Take PE fluorescence intensity less than 2 × 10 from cell mass IV centre circle2And antibody A PC-H7 fluorescence intensity is greater than 3 × 102Cell mass, be
Mo-MDSCs cell mass；
(406) the mo-MDSCs cell mass quantity filtered out is obtained divided by the quantity of cell mass II obtained in step (402)
Out in peripheral white blood cells mo-MDSCs proportional numerical value.
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| CN103180739A (en) * | 2010-09-21 | 2013-06-26 | 伊玛提克斯生物技术有限公司 | Use of myeloid cell biomarkers for the diagnosis of cancer |
| CN103260650A (en) * | 2010-10-20 | 2013-08-21 | 西奈山医学院 | Methods and compositions for treating tumors by using myeloid derived suppressor cells |
| CN105378066A (en) * | 2013-05-20 | 2016-03-02 | 细胞治疗有限公司 | Multicolor flow cytometry method for identifying population of cells, in particular mesenchymal stem cells |
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| CN103180739A (en) * | 2010-09-21 | 2013-06-26 | 伊玛提克斯生物技术有限公司 | Use of myeloid cell biomarkers for the diagnosis of cancer |
| CN103260650A (en) * | 2010-10-20 | 2013-08-21 | 西奈山医学院 | Methods and compositions for treating tumors by using myeloid derived suppressor cells |
| CN105378066A (en) * | 2013-05-20 | 2016-03-02 | 细胞治疗有限公司 | Multicolor flow cytometry method for identifying population of cells, in particular mesenchymal stem cells |
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| 非小细胞肺癌患者外周血CD11b+CD33+CD15+CD14-及CD11b+CD33+CD15-CD14+髓系细胞的比例变化及临床意义;罗斌等;《现代免疫学》;20170131;第37卷(第1期);正文第3.1节 |
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