CN103805564A - Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes - Google Patents
Method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes Download PDFInfo
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- CN103805564A CN103805564A CN201210444240.1A CN201210444240A CN103805564A CN 103805564 A CN103805564 A CN 103805564A CN 201210444240 A CN201210444240 A CN 201210444240A CN 103805564 A CN103805564 A CN 103805564A
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Abstract
The invention relates to a method for rapidly preparing prematurely condensed chromosomes of human peripheral blood lymphocytes. The method mainly comprises the following steps: 1, processing a blood sample by phytohemagglutinin, allowing the processed blood sample to stand in a constant temperature incubator with the temperature of 37DEG C for 45-90min; 2, culturing lymphocytes in the phytohemagglutinin processed blood sample in a mixed culturing liquid containing colchicine, ATP, 5% fetal calf serum, CalyculinA and CDK1/CyclinB at 37DEG C for 3-12h; 3, carrying out hypoosmotic treatment of the cultured lymphocytes by using 0.075mol of KCl for 10-20min; and 4, adding an immobile liquid into a hypoosmotic liquid containing the hypoosmotic lymphocytes to realize first-time immobilization, adding the immobile liquid to realize second-time immobilization, and suspending the obtained lymphocytes in the immobile liquid, wherein each of the first-time immobilization and the second-time immobilization is carried out for 10-15min. The preparation method provided by the invention takes a substantially shorter time of 3-12h than routine preparation methods of the prematurely condensed chromosomes.
Description
Technical field
The invention belongs to cytogenetics field, be specifically related to the chromosomal preparation method of the precocious aggegation of human peripheral lymphocyte.
Background technology
Precocious aggegation karyomit(e) (Premature Condensed Chromosome, PCC) the most classical concept refers to that the chromatin of other phase cells is packaged into karyomit(e) ahead of time by the cell in division stage (M phase) and the cytogamy in cell cycle in other stages.Due to the DNA replication dna state difference of G1, S, G2, the chromosomal of precocious aggegation comes in every shape, as with the karyomit(e) of G1 phase of M phase cytogamy be single line shape, the S phase is Powdered, G2 phase karyomit(e) is two-wire.
Precocious aggegation karyomit(e) PCC, as the method for evaluation of radiation dose, has been subject to paying close attention to widely and approving.Precocious aggegation karyomit(e) has very strong advantage than routine chromosome method: 1. can induce the division of cell stationary phase, can greatly increase the quantity of analysis of cells, avoid the deficiency of only division cells being carried out to selectivity analysis in conventional chromosome aberration analysis; 2. the chromosome damage drawing is original damage, is conducive to observe partial irradiation or " bystander cell " quantity of radiant energy in intracellular direct or indirect deposition, has increased applicable dosage scope of assessment, has improved the susceptibility of method and the accuracy of result; 3. PCC easy and simple to handle, save time, and routine chromosome analysis needs rich experience and a twist of the wrist, and the time that provides dosage need 4-5d, can not provide in time dosage estimation to large quantities of wounded.Therefore use PCC to be used for estimating to be subject to the radiation dose according to personnel, in heavy dose of radiation accident, can provide the karyomit(e) of enough Gong analysis.4. PCC also has other advantage, such as having strict quantitative relationship with radioactive dose, and dose effect curve there was no significant difference (the Gotoh E obtaining with dosage effect calibration curve and the integral experiment of isolated experiment foundation, Tanno Y, Takakura K.Simple biodosimetry method for use in cases ofhigh-dose radiation exposure that scores the chromosome number ofGiemsa-stained drug-induced prematurely condensed chromosomes (PCC) .Int J Radiat Biol.2005, 81 (1): 33-40.).
But prepared by early stage cell fusion method PCC cell is time-consuming, technical requirements is high, PCC yield is low, unstable etc. has limited its application as biological dosemeter.Recently, some have the discovery and the application that promote cell fission working substance, replace the cell-fusion techniques in past conventional P CC, making to prepare PCC greatly reduces the requirement of technology, shorten analysis time, as okadaic acid (okadaicacid) and calyculin A (calyculin A).Okadaic acid can make peripheral blood lymphocyte, at G1, G2 and M phase, PCC occur, and still, the lymphocyte cultivation stage in induction PCC process all needs at least 48 hours.Calyculin A can induce the cell of phase when arbitrary that PCC occurs, greatly improve number (the Prasanna PG that can be used for analysis of cells, Escalada ND, Blakely WF.Induction of premature chromosome condensation by a phosphataseinhibitor and a protein kinase in unstimulated human peripheralblood lymphocytes:a simple and rapid technique to study chromosomeaberrations using specific whole-chromosome DNA hybrid.Mutat Res, 2000, 466 (2): 131-41.), but the lymphocyte cultivation stage time in induction PCC process still needs about 48 hours.
Summary of the invention
The object of the invention is to set up and prepare fast the chromosomal method of the precocious aggegation of human peripheral lymphocyte, prepare the problem of the precocious aggegation karyomit(e) of human peripheral lymphocyte length consuming time to solve prior art.
Object of the present invention can be by realizing by the following technical solutions:
Prepare fast the chromosomal method of the precocious aggegation of human peripheral lymphocyte, comprise the steps:
(1), phytohemagglutinin processing (PHA): gather 3-5ml blood sample, add phytohemagglutinin to final concentration 80-120 μ g/ml in blood sample, mix and be placed on standing 45-90 minute in 37 ℃ of constant incubators;
(2), mixed culture: by centrifugal blood sample after treatment phytohaemagglutinin, get the lymphocyte of middle level boundary, be placed in the mixed-culture medium that contains 250 μ g/ml colchicine, 10mmol/L ATP, 5% foetal calf serum, 500nmol/L CalyculinA and 0.2mg/ml CDK1/CyclinB, mix and be placed on the standing 3-12 hour of cultivation in 37 ℃ of constant incubators;
(3), the hypotonic processing of cell: after cell co-cultivation, the centrifugal supernatant of abandoning, uses the hypotonic 10-20 minute of 0.075molKC13-5ml;
(4), cell is fixed: in the hypotonic medium that contains hypotonic processing cell, add 0.5-1ml stationary liquid to pre-fix, the centrifugal supernatant of abandoning immediately after mixing, add again 4-6ml stationary liquid to fix 2 times, fix the centrifugal supernatant of abandoning after 10-15 minute at every turn, finally cell is suspended from 0.5-1ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume.
Wherein, the centrifugal 3-5 minute of the described centrifugal employing 300-400rpm of step (2); The centrifugal 8-10 minute of the described centrifugal employing 800-1000rpm of step (3); The centrifugal 8-10 minute of the described centrifugal employing 800-1000rpm of step (4).
What the present invention set up prepares the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast, it is consuming time short that its advantage is to prepare PCC, wherein human peripheral lymphocyte phytohaemagglutinin (PHA) is processed rear mixed cultivation process and is only needed 3-12 hour, the lymphocyte incubation time (about 48 hours) of preparing in PCC process than prior art obviously shortens, thereby provides quick approach for the preparation of PCC.
Accompanying drawing explanation
Fig. 1 be lymphocyte through mixed culture after 3 hours the precocious aggegation karyomit(e) M phase divide phasor.
Fig. 2 be lymphocyte through mixed culture after 6 hours the precocious aggegation karyomit(e) M phase divide phasor.
Fig. 3 be lymphocyte through mixed culture after 12 hours the precocious aggegation karyomit(e) M phase divide phasor.
Embodiment
Describe by the following examples the chromosomal method of the precocious aggegation of human peripheral lymphocyte of preparing fast provided by the invention in detail.In embodiment, the related not special part of describing of biotechnology all adopts cytogenetics routine techniques below, as the routine techniques method providing in " molecular cloning experiment guide ".
Embodiment 1:
(1), gather Healthy Volunteers blood sample 3ml, in blood sample, add PHA (Guangzhou Medicine Industry Inst) to final concentration 80 μ g/ml, mix and be placed on containing 5%CO
237 ℃ of constant incubators in leave standstill 1 hour;
(2), blood sample leaves standstill after 1 hour in above-mentioned 37 ℃ of constant incubators, through 300rpm centrifugal 3 minutes, get 0.5ml middle level boundary blood lymphocyte, be placed in the CDK1/CyclinB (Cyclin-dependent kinase 1/cyclin B that contains 3ml 250 μ g/ml colchicine (sigma company), 10mmol/L ATP (sigma company), 5% foetal calf serum, 500nmolCalyculinA (sigma company) and 0.2mg/ml, Invitrogen company) in mixed-culture medium, mix to be placed in 37 ℃ of constant incubators and leave standstill 3 hours;
(3), lymphocyte is through mixed culture centrifugal 10 minutes of 1000rpm after 3 hours, the 0.075mol KCl hypotonic medium that adds 5ml is containing 5%CO
237 ℃ of conditions under hypotonic 20 minutes;
(4), add 0.5ml stationary liquid to pre-fix to containing in the lymphocytic hypotonic medium of hypotonic processing, after mixing, 1000rpm abandons supernatant for centrifugal 10 minutes immediately, add again 5ml stationary liquid to fix 2 times, after fixing 15 minutes, 1000rpm abandons supernatant for centrifugal 10 minutes at every turn, finally cell is suspended from 1ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume.
At 95 ℃ of temperature, in hot steam, dry film-making, after dry air with 5% Ji's nurse Sa dyeing, microscopic examination PCC exponential sum form, Fig. 1 be lymphocyte through mixed culture after 3 hours the precocious aggegation karyomit(e) M phase divide phasor.
Embodiment 2:
(1), gather Healthy Volunteers blood sample 3ml, in blood sample, add PHA (Guangzhou Medicine Industry Inst) to final concentration 100 μ g/ml, mix and be placed on containing 5%CO
237 ℃ of constant incubators in leave standstill 1 hour;
(2) blood sample leaves standstill after 1 hour in above-mentioned 37 ℃ of constant incubators, through 400rpm after centrifugal 3 minutes, get 0.8ml middle level boundary lymphocyte, be placed in and contain 3ml 250 μ g/ml colchicine, 10mmol/L ATP, 5% foetal calf serum, 500nmol CalyculinA and 0.2mg/mlCDK1/CyclinB mixed-culture medium, mix and be placed on 37 ℃ of constant incubators interior standing 6 hours;
(3) lymphocyte is through mixed culture centrifugal 10 minutes of 1000rpm after 6 hours, and the 0.075mol KCl hypotonic medium that adds 5ml is containing 5%CO
237 ℃ of conditions under hypotonic 20 minutes;
(4), add 0.8ml stationary liquid to pre-fix to containing in the lymphocytic hypotonic medium of hypotonic processing, after mixing, 1000rpm abandons supernatant for centrifugal 10 minutes immediately, add again 5ml stationary liquid to fix 2 times, after fixing 10 minutes, 1000rpm abandons supernatant for centrifugal 10 minutes at every turn, finally cell is suspended from 0.5ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume.
At 95 ℃ of temperature, in hot steam, dry film-making, after dry air with 5% Ji's nurse Sa dyeing, microscopic examination PCC exponential sum form, Fig. 2 be lymphocyte through mixed culture after 6 hours the precocious aggegation karyomit(e) M phase divide phasor.
Embodiment 3:
(1) gather Healthy Volunteers blood sample 5ml, in blood sample, add PHA (Guangzhou Medicine Industry Inst) to final concentration 100 μ g/ml, mix and be placed on containing 5%CO
237 ℃ of constant incubators in leave standstill 90 minutes;
(2) blood sample leaves standstill after 90 minutes in above-mentioned 37 ℃ of constant incubators, centrifugal 3 minutes of 300rpm, get 0.5ml middle level boundary lymphocyte, be placed in and contain 3ml 250 μ g/ml colchicine, 10mmol/L ATP, 5% foetal calf serum, 500nmol CalyculinA and 0.2mg/mlCDK1/CyclinB mixed-culture medium, mix and be placed on 37 ℃ of constant incubators interior standing 12 hours;
(3) lymphocyte is through mixed culture centrifugal 10 minutes of 800rpm after 12 hours, and the 0.075mol KCl hypotonic medium that adds 4ml is containing 5%CO
237 ℃ of conditions under hypotonic 20 minutes;
(4), add 1ml stationary liquid to pre-fix to containing in the lymphocytic hypotonic medium of hypotonic processing, after mixing, 800rpm abandons supernatant for centrifugal 10 minutes immediately, add again 5ml stationary liquid to fix 2 times, after fixing 10 minutes, 800rpm abandons supernatant for centrifugal 10 minutes at every turn, finally cell is suspended from 0.5ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume.
At 95 ℃ of temperature, in hot steam, dry film-making, after dry air with 5% Ji's nurse Sa dyeing, microscopic examination PCC exponential sum form, Fig. 3 be lymphocyte through mixed culture after 12 hours the precocious aggegation karyomit(e) M phase divide phasor.
Claims (4)
1. prepare fast the chromosomal method of the precocious aggegation of human peripheral lymphocyte, its feature comprises the steps:
(1), phytohemagglutinin processing: gather 3-5ml blood sample, add phytohemagglutinin to final concentration 80-120 μ g/ml in blood sample, mix and be placed on standing 45-90 minute in 37 ℃ of constant incubators;
(2), mixed culture: by centrifugal blood sample after treatment phytohaemagglutinin, get the lymphocyte of middle level boundary, be placed in the mixed-culture medium that contains 250 μ g/ml colchicine, 10mmol/L ATP, 5% foetal calf serum, 500nmol/L CalyculinA and 0.2mg/ml CDK/CyclinB, mix and be placed on the standing 3-12 hour of cultivation in 37 ℃ of constant incubators;
(3), the hypotonic processing of cell: after cell co-cultivation, the centrifugal supernatant of abandoning, uses the hypotonic 10-20 minute of 0.075molKCl3-5ml;
(4), cell is fixed: in the hypotonic medium that contains hypotonic processing cell, add 0.5-1ml stationary liquid to pre-fix, the centrifugal supernatant of abandoning immediately after mixing, add again 4-6ml stationary liquid to fix 2 times, fix the centrifugal supernatant of abandoning after 10-15 minute at every turn, finally cell is suspended from 0.5-1ml stationary liquid; Wherein stationary liquid is that methyl alcohol and Glacial acetic acid are preparation in 3: 1 by volume.
2. method according to claim 1, is characterized in that, described centrifugal being specially of step (2): the centrifugal 3-5 minute of 300-400rpm.
3. method according to claim 1, is characterized in that, described centrifugal being specially of step (3): the centrifugal 8-10 minute of 800-1000rpm.
4. method according to claim 1, is characterized in that, described centrifugal being specially of step (4): the centrifugal 8-10 minute of 800-1000rpm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106970224A (en) * | 2017-03-16 | 2017-07-21 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application |
| CN106980018A (en) * | 2017-03-16 | 2017-07-25 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP17 probe identification circulating tumor cells and its application |
| CN112716505A (en) * | 2020-12-08 | 2021-04-30 | 河北工程大学 | Rapid fatigue detection method |
| CN114563244A (en) * | 2022-02-28 | 2022-05-31 | 河南科技大学第一附属医院 | Method for preparing chromosome karyotype slices of lymphocytes |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106501040A (en) * | 2016-10-24 | 2017-03-15 | 南通大学附属医院 | Human peripheral chromosome synchronizes reagent preparation box |
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| WO2002068603A2 (en) * | 2001-02-28 | 2002-09-06 | The Henry M. Jackson Foundation | Materials and methods for the induction of premature chromosome condensation |
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| WO2002068603A2 (en) * | 2001-02-28 | 2002-09-06 | The Henry M. Jackson Foundation | Materials and methods for the induction of premature chromosome condensation |
Non-Patent Citations (3)
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| P. KEOHAVONG等: "《Molecular Toxicology Protocols》", 31 December 2005, article "Premature Chromosome Condensation in Human Resting Peripheral Blood Lymphocytes for Chromosome Aberration Analysis Using Specific Whole-Chromosome DNA Hybridization Probes", pages: 49-57 * |
| S TSUJI等: "Chemically induced premature chromosome condensation in short-term cultured human peripheral lymphocytes: applications to biodosimetry", 《BIOTECHNIC & HISTOCHEMISTRY》, vol. 82, no. 1, 28 February 2007 (2007-02-28), pages 29 - 34 * |
| 陆雪 等: "Calyculin A诱导早熟染色体凝聚的电离辐射剂量效应曲线", 《辐射研究与辐射工艺学报》, vol. 28, no. 6, 31 December 2010 (2010-12-31), pages 363 - 367 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106970224A (en) * | 2017-03-16 | 2017-07-21 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application |
| CN106980018A (en) * | 2017-03-16 | 2017-07-25 | 武汉康录生物技术股份有限公司 | A kind of kit of application CD45 immunofluorescences joint CEP17 probe identification circulating tumor cells and its application |
| CN106970224B (en) * | 2017-03-16 | 2018-06-26 | 武汉康录生物技术股份有限公司 | A kind of kit and its application using CD45 immunofluorescences joint CEP probe identification circulating tumor cells |
| CN106980018B (en) * | 2017-03-16 | 2018-06-26 | 武汉康录生物技术股份有限公司 | A kind of kit and its application using CD45 immunofluorescences joint CEP17 probe identification circulating tumor cells |
| CN112716505A (en) * | 2020-12-08 | 2021-04-30 | 河北工程大学 | Rapid fatigue detection method |
| CN114563244A (en) * | 2022-02-28 | 2022-05-31 | 河南科技大学第一附属医院 | Method for preparing chromosome karyotype slices of lymphocytes |
| CN114563244B (en) * | 2022-02-28 | 2024-05-24 | 河南科技大学第一附属医院 | Lymphocyte chromosome nuclear type sheet making method |
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