CN103805564B - Prepare the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast - Google Patents
Prepare the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast Download PDFInfo
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- CN103805564B CN103805564B CN201210444240.1A CN201210444240A CN103805564B CN 103805564 B CN103805564 B CN 103805564B CN 201210444240 A CN201210444240 A CN 201210444240A CN 103805564 B CN103805564 B CN 103805564B
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Abstract
The present invention relates to and prepare the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast, its key step comprises: (1), blood sample are placed in 37 DEG C of constant incubators through phytohemagglutinin process and leave standstill 45-90 minute; (2) mixed-culture medium 37 DEG C, be placed in by the blood sample medium size lymphocyte after phytohaemagglutinin process containing colchicine, ATP, 5% foetal calf serum, CalyculinA and CDK1/CyclinB cultivates 3-12 hour; (3), after cell co-cultivation, 0.075mol is used? the hypotonic 10-20 minute of KCl; (4), add stationary liquid pre-fix 1 time in the hypotonic medium containing Hypotonic treatment cell, then add stationary liquid and fix 2 times, each fixing 10-15 minute, is finally suspended from cell in stationary liquid.The present invention, compared with the precocious aggegation method of chromosome preparation of routine, substantially reduces the time, only needs 3-12 hour.
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) (PrematureCondensedChromosome, PCC) the most classical concept refers to and will be in the cell of division stage (M phase) and be in the cytogamy in cell cycle in other stages, and the chromatin of other phase cells is packaged into karyomit(e) ahead of time.Because the DNA replication dna state of G1, S, G2 is different, the chromosomal of precocious aggegation comes in every shape, and the karyomit(e) as the G1 phase with M phase cytogamy is single line shape, and the S phase is Powdered, and G2 phase karyomit(e) is two-wire.
Precocious aggegation karyomit(e) PCC, as the method for evaluation of radiation dose, receives and pays close attention to widely and approve.Precocious aggegation karyomit(e) has very strong advantage than routine chromosome method: 1. can the division of inducing quiescence phase cell, greatly can 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 drawn is initial injury, is conducive to observing partial irradiation or " bystander cell " quantity of radiant energy in intracellular direct or indirect deposition, adds applicable dose assessment scope, improve 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 providing dosage needs 4-5d, can not provide dose conversion in time to large quantities of wounded.Therefore using PCC for estimating by the radiation dose according to personnel, in the radiation accident of heavy dose, the karyomit(e) of enough Gong analyses can be provided.4. PCC also has other advantage, such as there is strict quantitative relationship with radioactive dose, and the dosage effect calibration curve to set up with isolated experiment and the dose effect curve there was no significant difference (GotohE that obtains of integral experiment, TannoY, TakakuraK.Simplebiodosimetrymethodforuseincasesofhigh-do seradiationexposurethatscoresthechromosomenumberofGiemsa-staineddrug-inducedprematurelycondensedchromosomes (PCC) .IntJRadiatBiol.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. limits its application as biological dosemeter.Recently, some have the discovery and application that promote cell fission working substance, the cell-fusion techniques of instead of in conventional P CC, the requirement of preparation PCC to technology is greatly reduced, shorten analysis time, as okadaic acid (okadaicacid) and calyculin A (calyculinA).Okadaic acid can make peripheral blood lymphocyte, in G1, G2 and M phase, PCC occur, but the lymphocyte cultivation stage in induction PCC process all needs at least 48 hours.CalyculinA can induce the cell of arbitrary phase that PCC occurs, substantially increase the number (PrasannaPG that can be used for analysis of cells, EscaladaND, BlakelyWF.Inductionofprematurechromosomecondensationbyap hosphataseinhibitorandaproteinkinaseinunstimulatedhumanp eripheralbloodlymphocytes:asimpleandrapidtechniquetostud ychromosomeaberrationsusingspecificwhole-chromosomeDNAhy brid.MutatRes, 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 the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast, to solve the problem that prior art prepares the precocious aggegation karyomit(e) of human peripheral lymphocyte length consuming time.
Object of the present invention can by realizing by the following technical solutions:
Prepare the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast, comprise the steps:
(1), phytohemagglutinin process (PHA): gather 3-5ml blood sample, add phytohemagglutinin to final concentration 80-120 μ g/ml in blood sample, mixing is placed in 37 DEG C of constant incubators and leaves standstill 45-90 minute;
(2), mixed culture: by centrifugal for the blood sample after phytohaemagglutinin process, get the lymphocyte of middle level boundary, be placed in the mixed-culture medium containing 250 μ g/ml colchicine, 10mmol/LATP, 5% foetal calf serum, 500nmol/LCalyculinA and 0.2mg/mlCDK1/CyclinB, mixing is placed on quiescent culture 3-12 hour in 37 DEG C of constant incubators;
(3), cell Hypotonic treatment: after cell co-cultivation, centrifugally abandon supernatant, use the hypotonic 10-20 minute of 0.075molKC13-5ml;
(4), cell is fixed: in the hypotonic medium containing Hypotonic treatment cell, add 0.5-1ml stationary liquid pre-fix, centrifugally immediately after mixing abandon supernatant, add 4-6ml stationary liquid again and fix 2 times, each fixing centrifugally after 10-15 minute abandon supernatant, finally cell is suspended from 0.5-1ml stationary liquid; Wherein to be methyl alcohol and Glacial acetic acid be stationary liquid by volume prepares at 3: 1.
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, its advantage is that preparation PCC is consuming time short, wherein after human peripheral lymphocyte phytohaemagglutinin (PHA) process, mixed cultivation process only needs 3-12 hour, the lymphocyte incubation time (about 48 hours) prepared in PCC process than prior art obviously shortens, thus provides fast route 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 in detail by the following examples and provided by the inventionly prepare the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast.Biotechnology involved in embodiment below does not describe part especially and all adopts cytogenetics routine techniques, as the convenient technical process provided in " Molecular Cloning: A Laboratory 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, mixing is placed on containing 5%CO
237 DEG C of constant incubators in leave standstill 1 hour;
(2), blood sample leaves standstill after 1 hour in above-mentioned 37 DEG C 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 containing 3ml250 μ g/ml colchicine (sigma company), 10mmol/LATP (sigma company), 5% foetal calf serum, 500nmolCalyculinA (sigma company) and 0.2mg/ml, Invitrogen company) in mixed-culture medium, mixing is placed in 37 DEG C of constant incubators and leaves standstill 3 hours;
(3), lymphocyte through mixed culture centrifugal 10 minutes of 1000rpm after 3 hours, the 0.075molKCl hypotonic medium adding 5ml is containing 5%CO
237 DEG C of conditions under hypotonic 20 minutes;
(4), pre-fix to containing adding 0.5ml stationary liquid in the lymphocytic hypotonic medium of Hypotonic treatment, after mixing, 1000rpm abandons supernatant in centrifugal 10 minutes immediately, add 5ml stationary liquid again and fix 2 times, after fixing 15 minutes, 1000rpm abandons supernatant in centrifugal 10 minutes at every turn, is finally suspended from by cell in 1ml stationary liquid; Wherein to be methyl alcohol and Glacial acetic acid be stationary liquid by volume prepares at 3: 1.
At 95 DEG C of temperature, in hot steam, dry film-making, with 5% Ji's nurse Sa dyeing after dry air, 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, mixing is placed on containing 5%CO
237 DEG C of constant incubators in leave standstill 1 hour;
(2) blood sample leaves standstill after 1 hour in above-mentioned 37 DEG C of constant incubators, through 400rpm after centrifugal 3 minutes, get 0.8ml middle level boundary lymphocyte, be placed in containing 3ml250 μ g/ml colchicine, 10mmol/LATP, 5% foetal calf serum, 500nmolCalyculinA and 0.2mg/mlCDK1/CyclinB mixed-culture medium, mixing is placed in 37 DEG C of constant incubators and leaves standstill 6 hours;
(3) lymphocyte is through mixed culture centrifugal 10 minutes of 1000rpm after 6 hours, and the 0.075molKCl hypotonic medium adding 5ml is containing 5%CO
237 DEG C of conditions under hypotonic 20 minutes;
(4), pre-fix to containing adding 0.8ml stationary liquid in the lymphocytic hypotonic medium of Hypotonic treatment, after mixing, 1000rpm abandons supernatant in centrifugal 10 minutes immediately, add 5ml stationary liquid again and fix 2 times, after fixing 10 minutes, 1000rpm abandons supernatant in centrifugal 10 minutes at every turn, is finally suspended from by cell in 0.5ml stationary liquid; Wherein to be methyl alcohol and Glacial acetic acid be stationary liquid by volume prepares at 3: 1.
At 95 DEG C of temperature, in hot steam, dry film-making, with 5% Ji's nurse Sa dyeing after dry air, 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, add PHA (Guangzhou Medicine Industry Inst) to final concentration 100 μ g/ml in blood sample, mixing is placed on containing 5%CO
237 DEG C of constant incubators in leave standstill 90 minutes;
(2) blood sample leaves standstill after 90 minutes in above-mentioned 37 DEG C of constant incubators, centrifugal 3 minutes of 300rpm, get 0.5ml middle level boundary lymphocyte, be placed in containing 3ml250 μ g/ml colchicine, 10mmol/LATP, 5% foetal calf serum, 500nmolCalyculinA and 0.2mg/mlCDK1/CyclinB mixed-culture medium, mixing is placed in 37 DEG C of constant incubators and leaves standstill 12 hours;
(3) lymphocyte is through mixed culture centrifugal 10 minutes of 800rpm after 12 hours, and the 0.075molKCl hypotonic medium adding 4ml is containing 5%CO
237 DEG C of conditions under hypotonic 20 minutes;
(4), pre-fix to containing adding 1ml stationary liquid in the lymphocytic hypotonic medium of Hypotonic treatment, after mixing, 800rpm abandons supernatant in centrifugal 10 minutes immediately, add 5ml stationary liquid again and fix 2 times, after fixing 10 minutes, 800rpm abandons supernatant in centrifugal 10 minutes at every turn, is finally suspended from by cell in 0.5ml stationary liquid; Wherein to be methyl alcohol and Glacial acetic acid be stationary liquid by volume prepares at 3: 1.
At 95 DEG C of temperature, in hot steam, dry film-making, with 5% Ji's nurse Sa dyeing after dry air, 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 the chromosomal method of the precocious aggegation of human peripheral lymphocyte fast, its feature comprises the steps:
(1), phytohemagglutinin process: gather 3-5ml blood sample, add phytohemagglutinin to final concentration 80-120 μ g/ml in blood sample, mixing is placed in 37 DEG C of constant incubators and leaves standstill 45-90 minute;
(2), mixed culture: by centrifugal for the blood sample after phytohaemagglutinin process, get the lymphocyte of middle level boundary, be placed in the mixed-culture medium containing 250 μ g/ml colchicine, 10mmol/LATP, 5% foetal calf serum, 500nmol/LCalyculinA and 0.2mg/mlCDKl/CyclinB, mixing is placed on quiescent culture 3-12 hour in 37 DEG C of constant incubators;
(3), cell Hypotonic treatment: after cell co-cultivation, centrifugally abandon supernatant, use the hypotonic 10-20 minute of 0.075molKCl3-5ml;
(4), cell is fixed: in the hypotonic medium containing Hypotonic treatment cell, add 0.5-1ml stationary liquid pre-fix, centrifugally immediately after mixing abandon supernatant, add 4-6ml stationary liquid again and fix 2 times, each fixing centrifugally after 10-15 minute abandon supernatant, finally cell is suspended from 0.5-1ml stationary liquid; Wherein to be methyl alcohol and Glacial acetic acid be stationary liquid by volume prepares at 3: 1.
2. method according to claim 1, is characterized in that, step (2) is described to be centrifugally specially: the centrifugal 3-5 minute of 300-400rpm.
3. method according to claim 1, is characterized in that, step (3) is described to be centrifugally specially: the centrifugal 8-10 minute of 800-1000rpm.
4. method according to claim 1, is characterized in that, step (4) is described to be centrifugally specially: the centrifugal 8-10 minute of 800-1000rpm.
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| 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 |
| 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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| 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 |
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| Calyculin A诱导早熟染色体凝聚的电离辐射剂量效应曲线;陆雪 等;《辐射研究与辐射工艺学报》;20101231;第28卷(第6期);363-367页 * |
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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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