CN107102127A - Monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood - Google Patents
Monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood Download PDFInfo
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Abstract
Monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood of the present invention, is using antibody：(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 detection mo MDSCs, the clearly scope of screening numerical value and the idiographic flow 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 mark, low-cost, it is easy to widely available.To the peripheral blood blood sample of 151 subjects, detected respectively with the method for the present invention.The mo MDSCs proportional numerical values detected show obvious difference in different groups, and the ratio in patient with breast cancer is significantly higher than Healthy People and Benign Breast knurl patient.Testing result carries out statistical analysis, P by computer using the softwares of Graphpad Prism 6<0.05, possess statistical significance, science is credible.
Description
Technical field
The present invention relates to the detection method that monokaryon sample marrow source in a kind of human peripheral blood suppresses cell.
Background technology
The inhibitory cells in medullary system source（MDSCs）The heterogeneous cell 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 suppresses cell（PMN-MDSCs）With monokaryon sample marrow source
Suppress cell（mo-MDSCs）.Wherein, mo-MDSCs has stronger immunosuppression capability1, the interior mo-MDSCs existed of body
Progress and the deterioration of the diseases such as breast cancer can be influenceed.Based on this, screening and detection for mo-MDSCs are very necessary.
Because MDSCs cell masses lack specific identification tag, therefore the existing method screened and detected is very
Mix.ALMAND B et al. think Lin by kinds cancer sample−HLA-DR−Inspection can be screened as mark
Survey MDSCs cell masses2, DIAZ-MONTERO CM et al. work is then thought can be by Lin−/lowHLA-DR−CD33+CD11b+
Mark combination carries out selective mechanisms3, also by CD15+Research work of detection MDSCs cell masses etc. from granulocyte group4。
But, operator is relied on existing MDSCs and mo-MDSCs detection method has rich experience and theoretical background basis more,
The detection method of the clear and definite science of mo-MDSCs is not provided.（Bibliography：
The content of the invention
In order to solve the problem of prior art is present, suppress the invention provides monokaryon sample marrow source in a kind of human peripheral blood
The detection method of cell.
The step of monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood that the present invention is provided and condition
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, solution used preparation
(201) preparation of PBS solution：Weigh NaCl 8 g, KCl 0.2 g, Na2HPO41.44 g and KH2PO40.24 g is molten
In 1 L ultra-pure waters, autoclaving obtains PBS solution, and 4 DEG C of refrigerators are preserved；
(202) preparation of buffer solution：NBCS 1mL is taken, 10 mL buffering is obtained in the PBS solution for being added to 9 mL
Liquid；Described new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid：Weigh NH4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultra-pure waters,
Solution is adjusted into pH to 7.2, ultra-pure water is continuously added up to cumulative volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators are preserved；
(204) preparation of fixer：10 mL formalins are measured to add in the PBS solution prepared to 1 L steps (201),
Add glucose 20 g, NaNO3 0.2 g, is completely dissolved, and is fixed liquid, and room temperature is standby；
(205) preparation of antibody mixed solution：4 μ L HLA-DR is added in 200 μ L buffer solutions prepared by step (202)
Antibody, 6 μ L CD15 antibody, 6 μ L CD45 antibody, 6 μ L CD33 antibody, 8 μ L CD14 antibody and 1 μ L CD11b
Antibody, mixes, obtains antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
C, streaming sample solution preparation
(301) the μ L of subject's peripheral blood blood sample 10 are taken to be added in 1 mL erythrocyte cracked liquids, in cracking 30 on ice
Min, every 10 min concussions once, obtain peripheral blood suspension；
(302) 1800 rpm centrifuge 3 min under the peripheral blood suspension centrifuge for obtaining step (301), 4 DEG C of low temperature, discard
Supernatant, adds 200 μ L buffer solutions into remaining precipitation, is allowed to hang and is mixed using micropipettor piping and druming precipitation；
(303) 1800 rpm under centrifuge, 4 DEG C of low temperature are used to centrifuge the thing that obtains of 3 min steps (302), abandoning supernatant, then
20 μ L antibody mixed solutions are added into remaining precipitation, is allowed to hang and mixes using micropipettor piping and druming precipitation, then
It is placed in 30 min on ice, lucifuge is noted；
(304) 200 μ L buffer solutions are added in step (303) obtains thing, mixed, then with centrifuge, 1800 under 4 DEG C of low temperature
Rpm centrifuges 3 min, abandoning supernatant；
(305) 200 μ L PBS solution is added in step (304) obtains thing, it is mixed using micropipettor, obtained
Streaming sample solution；
D, mo-MDSCs detection
Mo-MDSCs ratio in the streaming sample solution obtained with (305) of step 3, detection stream formula sample solution, detection step
It is rapid as follows：
(401) according to the granularity of cell and the common cognition standard of size, streaming sample solution is removed using flow cytometer circle
In adhesion cells and dead cell, obtain the i.e. cell mass I of work individual cells group in blood；
(402) flow cytometer is used, according to the situation of antibody CD45 developed by molecule, circle takes PE-Cy7 fluorescence in cell mass I strong
Degree is more than 2 × 102Cell obtain cell mass II, this be blood in leukocyte population；
(403) flow cytometer is used, according to antibody HLA-DR expression, antibody PE-CF594 is taken from cell mass II centre circles
Fluorescence intensity is more than 2 × 102Cell obtain cell mass III；
(404) flow cytometer is used, according to antibody CD33 and antibody CD11b expression, is taken from cell mass III centre circles
APC and FITC fluorescence intensities are all higher than 2 × 102Cell mass, obtain cell mass IV, this be MDSCs cell masses；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to antibody CD14 and antibody CD15 expression,
PE fluorescence intensities are taken to be less than 2 × 10 from cell mass IV centre circles2And antibody A PC-H7 fluorescence intensities are more than 3 × 102Cell mass, be
Mo-MDSCs cell masses；
(406) by the cell mass II obtained in the mo-MDSCs cell masses quantity divided by step (402) that filter out quantity, obtain
Go out the proportional numerical value of mo-MDSCs in peripheral white blood cells.
Monokaryon sample marrow source suppresses the mo- that the detection method of cell is detected in a kind of human peripheral blood that the present invention is provided
MDSCs proportional numerical values show obvious difference in different groups, and the ratio in patient with breast cancer is significantly higher than health
People and Benign Breast knurl patient.
Beneficial effect：Monokaryon sample marrow source suppresses the detection method of cell, institute in a kind of human peripheral blood that the present invention is provided
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
Survey mo-MDSCs and give the clearly scope of screening numerical value and the idiographic flow of screening.When detection is simple and convenient to operate, checked
Between it is short, it is only necessary to micro peripheral blood sample mark, it is low-cost, it is easy to widely available.The result of detection has carried out Statistics Division
Reason：To the peripheral blood blood sample of 151 subjects, monokaryon sample marrow source suppresses in a kind of human peripheral blood provided with the present invention
The detection method detection of cell, the information that described detecting step (406) is detected, by computer, uses Graphpad
The softwares of Prism 6 carry out statistical analysis, P<0.05, possess statistical significance.Illustrate that the methodological science of the present invention is credible.
Figure of description
Fig. 1 is that the adhesion cells and dead cell that method of the invention is removed using flow cytometer circle in streaming sample solution are obtained carefully
Born of the same parents' group's I schematic diagrames.
Fig. 2 is that the method for the present invention obtains cell mass II schematic diagram using flow cytometer.
Fig. 3 is that the method for the present invention obtains cell mass III schematic diagram using the cell of flow cytometer.
Fig. 4 is that the method for the present invention obtains cell mass IV schematic diagram using flow cytometer.
Fig. 5 is the schematic diagram for the mo-MDSCs cell masses that method of the invention is obtained using flow cytometer.
Fig. 6 is the ratio that the mo-MDSCs that method of the invention is detected shows obvious difference in different groups
Figure.
Embodiment
The step of monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood of embodiment 1 and condition are as follows：
1st, antibody used
(101) antibody A PC anti-human CD33, abbreviation CD33；BD Biosciences companies of the U.S.；
(102) antibody PE anti-human CD15, abbreviation CD15；BD Biosciences companies of the U.S.；
(103) antibody PE-CF594 anti-human HLA-DR, abbreviation HLA-DR；BD Biosciences companies of the U.S.；
(104) antibody A PC-H7 anti-human CD14, abbreviation CD14；BD Biosciences companies of the U.S.；
(105) antibody PE-Cy7 anti-human CD45, abbreviation CD45；BD Biosciences companies of the U.S.；
(106) antibody FITC anti-mouse CD11b, abbreviation CD11b；Tianjin Sungene Biotechnology Co., Ltd..
2nd, the preparation of solution used
(201) preparation of PBS solution：Weigh NaCl 8 g, KCl 0.2 g, Na2HPO41.44 g and KH2PO40.24 g is molten
In 1 L ultra-pure waters, autoclaving obtains PBS solution, and 4 DEG C of refrigerators are preserved；
(202) preparation of buffer solution：NBCS 1mL is taken, 10 mL buffering is obtained in the PBS solution for being added to 9 mL
Liquid；Described new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid：Weigh NH4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultra-pure waters,
Solution is adjusted into pH to 7.2, ultra-pure water is continuously added up to cumulative volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators are preserved；
(204) preparation of fixer：10 mL formalins are measured to add in the PBS solution prepared to 1 L steps (201),
Add glucose 20 g, NaNO3 0.2 g, is completely dissolved, and is fixed liquid, and room temperature is standby；
(205) preparation of antibody mixed solution：4 μ L HLA-DR is added in 200 μ L buffer solutions prepared by step (202)
Antibody, 6 μ L CD15 antibody, 6 μ L CD45 antibody, 6 μ L CD33 antibody, 8 μ L CD14 antibody and 1 μ L CD11b
Antibody, mixes, obtains antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
3rd, the preparation of streaming sample solution
(301) the μ L of subject's peripheral blood blood sample 10 are taken to be added in 1 mL erythrocyte cracked liquids, in cracking 30 on ice
Min, every 10 min concussions once, obtain peripheral blood suspension；
(302) 1800 rpm centrifuge 3 min under the peripheral blood suspension centrifuge for obtaining step (301), 4 DEG C of low temperature, discard
Supernatant, adds 200 μ L buffer solutions into remaining precipitation, is allowed to hang and is mixed using micropipettor piping and druming precipitation；
(303) 1800 rpm under centrifuge, 4 DEG C of low temperature are used to centrifuge the thing that obtains of 3 min steps (302), abandoning supernatant, then
20 μ L antibody mixed solutions are added into remaining precipitation, is allowed to hang and mixes using micropipettor piping and druming precipitation, then
It is placed in 30 min on ice, lucifuge is noted；
(304) 200 μ L buffer solutions are added in step (303) obtains thing, mixed, then with centrifuge, 1800 under 4 DEG C of low temperature
Rpm centrifuges 3 min, abandoning supernatant；
(305) 200 μ L PBS solution is added in step (304) obtains thing, it is mixed using micropipettor, obtained
Streaming sample solution；
4th, mo-MDSCs detection
Mo-MDSCs ratio in the streaming sample solution obtained with (305) of step 3, detection stream formula sample solution, detection step
It is rapid as follows：
(401) according to the granularity of cell and the common cognition standard of size, streaming sample solution is removed using flow cytometer circle
In adhesion cells and dead cell, obtain the i.e. cell mass I of work individual cells group in blood（See Fig. 1）；
(402) flow cytometer is used, according to the situation of antibody CD45 developed by molecule, circle takes PE-Cy7 fluorescence in cell mass I strong
Degree is more than 2 × 102Cell obtain cell mass II, this be blood in leukocyte population（See Fig. 2）；
(403) flow cytometer is used, according to antibody HLA-DR expression, antibody PE-CF594 is taken from cell mass II centre circles
Fluorescence intensity is more than 2 × 102Cell obtain cell mass III（See Fig. 3）；
(404) flow cytometer is used, according to antibody CD33 and antibody CD11b expression, is taken from cell mass III centre circles
APC and FITC fluorescence intensities are all higher than 2 × 102Cell mass, obtain cell mass IV, this be MDSCs cell masses（See Fig. 4）；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to antibody CD14 and antibody CD15 expression,
PE fluorescence intensities are taken to be less than 2 × 10 from cell mass IV centre circles2And antibody A PC-H7 fluorescence intensities are more than 3 × 102Cell mass, be
Mo-MDSCs cell masses（See Fig. 5）；
(406) by the cell mass II obtained in the mo-MDSCs cell masses quantity divided by step (402) that filter out quantity, obtain
Go out the proportional numerical value of mo-MDSCs in peripheral white blood cells.
Thus the mo-MDSCs proportional numerical values that method is detected show obvious difference in different groups, in breast cancer
Ratio in patient is significantly higher than Healthy People and Benign Breast knurl patient (see Fig. 6).
The present embodiment, to the peripheral blood blood sample of 151 subjects, a kind of human peripheral provided respectively with the present invention
Monokaryon sample marrow source suppresses the detection method detection of cell in blood.The result of detection has carried out statistical disposition：Described detection is walked
Suddenly the information of (406) detection, by computer, statistical analysis, P are carried out using the softwares of Graphpad Prism 6<0.05, tool
Standby statistical significance.Illustrate that the methodological science of the present invention is credible.
Claims (1)
1. monokaryon sample marrow source suppresses the detection method of cell in a kind of human peripheral blood, its 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, solution used preparation
(201) preparation of PBS solution：Weigh NaCl 8 g, KCl 0.2 g, Na2HPO41.44 g and KH2PO40.24 g is molten
In 1 L ultra-pure waters, autoclaving obtains PBS solution, and 4 DEG C of refrigerators are preserved；
(202) preparation of buffer solution：NBCS 1mL is taken, 10 mL buffering is obtained in the PBS solution for being added to 9 mL
Liquid；Described new born bovine is the calf being born in 6 hours；
(203) preparation of erythrocyte cracked liquid：Weigh NH4Cl 7.47 g, Tris 2.06 g be dissolved in 500 mL ultra-pure waters, will
Solution adjusts pH to 7.2, continuously adds ultra-pure water up to cumulative volume is 1 L, obtains erythrocyte cracked liquid, 4 DEG C of refrigerators are preserved；
(204) preparation of fixer：10 mL formalins are measured to add in the PBS solution prepared to 1 L steps (201),
Add glucose 20 g, NaNO3 0.2 g, is completely dissolved, and is fixed liquid, and room temperature is standby；
(205) preparation of antibody mixed solution：4 μ L HLA-DR is added in 200 μ L buffer solutions prepared by step (202)
Antibody, 6 μ L CD15 antibody, 6 μ L CD45 antibody, 6 μ L CD33 antibody, 8 μ L CD14 antibody and 1 μ L CD11b
Antibody, mixes, obtains antibody mixed solution, 4 DEG C of refrigerators are kept in dark place；
C, streaming sample solution preparation
(301) the μ L of subject's peripheral blood blood sample 10 are taken to be added in 1 mL erythrocyte cracked liquids, in cracking 30 on ice
Min, every 10 min concussions once, obtain peripheral blood suspension；
(302) 1800 rpm centrifuge 3 min under the peripheral blood suspension centrifuge for obtaining step (301), 4 DEG C of low temperature, discard
Supernatant, adds 200 μ L buffer solutions into remaining precipitation, is allowed to hang and is mixed using micropipettor piping and druming precipitation；
(303) 1800 rpm under centrifuge, 4 DEG C of low temperature are used to centrifuge the thing that obtains of 3 min steps (302), abandoning supernatant, then
20 μ L antibody mixed solutions are added into remaining precipitation, is allowed to hang and mixes using micropipettor piping and druming precipitation, then
It is placed in 30 min on ice, lucifuge is noted；
(304) 200 μ L buffer solutions are added in step (303) obtains thing, mixed, then with centrifuge, 1800 under 4 DEG C of low temperature
Rpm centrifuges 3 min, abandoning supernatant；
(305) 200 μ L PBS solution is added in step (304) obtains thing, it is mixed using micropipettor, obtained
Streaming sample solution；
D, mo-MDSCs detection
Mo-MDSCs ratio in the streaming sample solution obtained with (305) of step 3, detection stream formula sample solution, detection step
It is rapid as follows：
(401) according to the granularity of cell and the common cognition standard of size, streaming sample solution is removed using flow cytometer circle
In adhesion cells and dead cell, obtain the i.e. cell mass I of work individual cells group in blood；
(402) flow cytometer is used, according to the situation of antibody CD45 developed by molecule, circle takes PE-Cy7 fluorescence in cell mass I strong
Degree is more than 2 × 102Cell obtain cell mass II, this be blood in leukocyte population；
(403) flow cytometer is used, according to antibody HLA-DR expression, antibody PE-CF594 is taken from cell mass II centre circles
Fluorescence intensity is more than 2 × 102Cell obtain cell mass III；
(404) flow cytometer is used, according to antibody CD33 and antibody CD11b expression, is taken from cell mass III centre circles
APC and FITC fluorescence intensities are all higher than 2 × 102Cell mass, obtain cell mass IV, this be MDSCs cell masses；
(405) in detection stream formula sample solution mo-MDSCs ratio, according to antibody CD14 and antibody CD15 expression,
PE fluorescence intensities are taken to be less than 2 × 10 from cell mass IV centre circles2And antibody A PC-H7 fluorescence intensities are more than 3 × 102Cell mass, be
Mo-MDSCs cell masses；
(406) by the cell mass II obtained in the mo-MDSCs cell masses quantity divided by step (402) that filter out quantity, obtain
Go out the proportional numerical value of mo-MDSCs in peripheral white blood cells.
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| CN109765369A (en) * | 2018-11-21 | 2019-05-17 | 深圳市人民医院 | A kind of evaluation diversification breast cancer adjuvant chemotherapy device |
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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 |
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| CN109765369A (en) * | 2018-11-21 | 2019-05-17 | 深圳市人民医院 | A kind of evaluation diversification breast cancer adjuvant chemotherapy device |
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