CN102465168A - Method for preparing metaphase specimen of mammalian cell chromosome - Google Patents
Method for preparing metaphase specimen of mammalian cell chromosome Download PDFInfo
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- CN102465168A CN102465168A CN2010105603420A CN201010560342A CN102465168A CN 102465168 A CN102465168 A CN 102465168A CN 2010105603420 A CN2010105603420 A CN 2010105603420A CN 201010560342 A CN201010560342 A CN 201010560342A CN 102465168 A CN102465168 A CN 102465168A
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Abstract
The invention provides a method for preparing a metaphase specimen of mammalian cell chromosome. The method comprises the following step of: dripping mammalian cell suspension onto a glass slide at the temperature of 20-30 DEG C and under the relative humidity of 45-50%. The metaphase chromosome obtained with the method is good in quality, shape and dispersion degree, and is a metaphase chromosome specimen easy for chromosome evaluation.
Description
Technical field
The present invention relates to a kind of preparation method of mammalian cell chromosome specimen, be specifically related to a kind of preparation method of mammalian cell karyomit(e) phase in mid-term sample.
Background technology
In new drug development, healthcare products evaluation and radiation safety research, it is content important in the genetics toxicity assessment that the mammalian cell chromosome aberration is observed.In chromosome aberration was estimated, karyomit(e) phase in mid-term sample preparations was unusual the key link, and karyomit(e) dispersive quality has great importance to the experiment quality, and then the safety estimated of influence.Disperse to be easy between the insufficient Metaphase Chromosome intersection, overlapping, cause when analyzing chromatid and chromosomal fracture, disappearance and exchanging, being difficult to make accurate judgement.How to obtain the good Metaphase Chromosome of form, be the difficult problem that many new drug safety evaluation genetic experiments chamber faces always.
Find in the real work that karyomit(e) disperses to be subject to the external environment influence in the sample preparations process, owing to there is not unified standard, cause each laboratory karyomit(e) phase in mid-term specimen quality different, result's comparability is poor between the different experiments chamber.Temperature and humidity is conspicuous to the unfolded influence that karyomit(e) drips behind the sheet.In dripping the sheet process, karyomit(e) relies on and absorbs extraneous moisture and loose the shop rapidly, so the humidity of external environment is unusual The key factor.Because temperature can exert an influence to the time of drying of dripping sheet, so also have influence on dispersion effect to a certain extent.Because China is vast in territory, weather condition significant difference throughout the year, various places testing laboratory temperature, humidity etc. differ greatly, and only rely on outside physical environment, and the suitable condition when being difficult to reach Chromosome Preparation also just is difficult to guarantee the stable of appraisement system.Therefore, creating a metastable local environment through related measure, is helpful to preparing good sample.Optimal control through to outside atmosphere will significantly reduce artificial factor, and it is reliable and stable to have guaranteed that karyomit(e) prepares mid-term mutually.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of mammalian cell karyomit(e) phase in mid-term sample, Metaphase Chromosome quality, form and dispersity are good to obtain, karyomit(e) mid-term of being easy to carry out evaluated chromosome sample mutually.
Technical scheme of the present invention is following:
A kind of preparation method of mammalian cell karyomit(e) phase in mid-term sample, this preparation method may further comprise the steps: under the relative humidity of 20～30 ℃ temperature and 45～50%, the mammalian cell hanging drop is added on the slide glass.Preferably, under the relative humidity of 25 ℃ temperature and 50%, the mammalian cell hanging drop is added on the slide glass.
In above-mentioned preparation method, preferably, it highly is apart from slide glass 15～25cm that the mammalian cell hanging drop adds, preferred 20cm.Preferably, the mammalian cell hanging drop added-time, slide glass becomes 15 ° of angles with horizontal plane.Preferably, the dripping quantity of mammalian cell suspension-s is every slide glass 10 μ L.
In above-mentioned preparation method, the preparation method of mammalian cell suspension-s can may further comprise the steps:
The mammalian cell that (1) will be cultured to exponential phase of growth is handled with NST-757;
(2) with the volume ratio be methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1 is mammalian cell fixedly.
Particularly, the preparation method of above-mentioned mammalian cell suspension-s may further comprise the steps:
The mammalian cell that (1) will be cultured to exponential phase of growth makes every milliliter with the digestion of 0.25% (g/100ml) trypsinase aqueous solution and comprises 3～5 * 10
4The cell suspending liquid of individual cell, and cultivated 21 hours;
(2) add NST-757 and handled 24 hours, the concentration of NST-757 is that every ml cells suspension-s 15 μ g confirm;
(3) discard substratum, with 0.25% (g/100ml) trypsinase aqueous solution digestion mammalian cell and centrifugal, preferably under 1000rpm centrifugal 10 minutes;
(4) supernatant discarded adds under 37 ℃ of the KCl aqueous solution of 0.075mol/L and left standstill 20 minutes;
(5) adding volume ratio again is methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1, and centrifugal behind the piping and druming cell mixing, preferably under 1000rpm centrifugal 10 minutes;
(6) supernatant discarded, adding volume ratio again is methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1, leave standstill after 20 minutes under 37 ℃ centrifugal.Preferably, step (6) is carried out 3 times repeatedly.
In above-mentioned preparation method, preferably, the mammalian cell hanging drop is added to seasoning behind the slide glass, uses 10% (1 part of Ji's nurse Sa stoste: Ji's nurse Sa dye liquor dyeing 9 portions of damping fluids) again.
The present invention also provides mammalian cell karyomit(e) phase in the mid-term sample according to above-mentioned preparing method's preparation.
Below be detailed description of the present invention:
1. cell is prepared:
The recovery mammalian cell obtains the required cell of capacity, adds an amount of substratum, is cultured to exponential phase of growth.
Trypsinase aqueous solution had digestive transfer culture cell with 0.25% (g/100ml) is prepared into 3～5 * 10
4Cell/mL cell suspension was cultivated 21 hours, added NST-757 (concentration 15 μ g/mL cell suspensions), was cultured to 24 hours and outwelled substratum, harvested cell.
2. cell fixation:
Behind the trypsinase aqueous solution peptic cell with 0.25% (g/100ml), move to the 15mL centrifuge tube, centrifugal 10 minutes of 1000rpm, abandoning supernatant.
The KCl aqueous solution 5mL that adds 0.075mol/L in the centrifuge tube, hypotonic 20 minutes (37 ℃).Every afterwards pipe adds the 0.5mL stationary liquid again, and (volume ratio: methyl alcohol: Glacial acetic acid min. 99.5=3: 1), blow and beat gently with suction pipe, 1000rpm is centrifugal 10 minutes behind the mixing, removes supernatant.
In centrifuge tube, add again stationary liquid (volume ratio: methyl alcohol: 5mL Glacial acetic acid min. 99.5=3: 1), behind the mixing 37 ℃ fixing 20 minutes down, centrifugal, remove supernatant, above-mentioned steps is repeatable operation 2 times again, accomplishes karyomit(e) and fixes.
3. Chromosome Preparation:
Last fixing back is centrifugal, upper strata liquid major part is outwelled only remaining about 0.5mL upper strata stationary liquid.With dropper piping and druming cell precipitation thing, make into even cell suspending liquid.
Ambient temperature range was that 20～30 ℃ and ambient relative humidity scope are 45～55% when sheet was dripped in control.Sample injector drips apart from slide glass 15～25cm place, and cell is dispersed on the slide, dries naturally.Slide glass and horizontal plane angle: 15 °.Sample drop sheet volume: 10 μ L/ slide glasss.
4. chromosome dyeing:
Slide glass places 10% Ji's nurse Sa dye liquor dyeing 15 minutes.Take out behind the slide sample earlier with the tap water flushing, again with distilled water flushing to there not being loose colour, put seasoning in the air.
Chromosome Preparation has very crucial meaning, also is the emphasis of technical scheme of the present invention.The present invention finds after deliberation, and temperature and relative humidity when dripping sheet are most important for the control of time of drying, and the karyomit(e) dispersity that whether can obtain is had very big influence.Specifically, chromosomal dispersion depends on phase karyomit(e) exsiccant speed in mid-term, and rate of drying is too fast, and cell does not reach optimum diameter, forms closely the phase in mid-term, causes between the karyomit(e) folding each other; In contrast,, will cause two kinds of results if rate of drying is too slow, the one, membranolysis, karyomit(e) shifts out, and the 2nd, cytolemma does not break, and karyomit(e) interweaves in cell.Therefore, just very crucial to the optimization of karyomit(e) rate of drying, time of drying is controlled and then controlled to ambient temperature, relative humidity when dripping sheet, the karyomit(e) dispersity that will help obtaining.Simultaneously, drip sheet height, slide glass and horizontal plane angle and a sheet volume and also can disperse to produce certain influence karyomit(e).Therefore, emphasis of the present invention is further optimized other correlation parameters on the basis of screening and optimization external environment temperature and humidity, to reach the purpose of preparation good dispersion karyomit(e) phase in mid-term sample.Technical scheme of the present invention is following with respect to the beneficial effect of prior art:
Ambient temperature range was 20～30 ℃ when at first, sheet was dripped in control.Design temperature is that 20～30 ℃ reason need to be time enough absorbing suff water, the evaporation of fixing agent when satisfying chromosomal spread.The present invention's experiment shows that when temperature was higher than 30 ℃, stationary liquid fast drying, karyomit(e) can fully not spread; Temperature is lower than 20 ℃, and stationary liquid is dry slow, will cause two kinds of results, and the one, membranolysis, karyomit(e) shifts out, and can cause chromosome elimination.The 2nd, cytolemma does not break, and karyomit(e) interweaves in cell, can't observe karyomit(e) phase in mid-term.The ambient relative humidity scope was 45～55% when simultaneously, sheet was dripped in control.When the assessment Metaphase Chromosome disperseed, ambient moisture also was an interfering factors to slide glass time of drying, because the ambient moisture height means identical cell suspension under identical temperature, the fixing agent water cut reduces slowly, and causing needs long time of drying.Be higher than at 55% o'clock in relative humidity, karyomit(e) is of poor quality.For being lower than at 45% o'clock, Metaphase Chromosome disperses area little, and chromosome repeat is many in relative humidity.
Secondly, the present invention's experiment shows that sample injector drips apart from slide glass 20cm place and makes cell be dispersed on slide, helps nature and dries.
The 3rd, slide glass and horizontal plane angle are 15 °.15 ° of angles make cell suspension be tilted to down along slide glass, in karyomit(e) prepares mid-term mutually, are beneficial to cell and disperse to spread.
The 4th, sample drop sheet volume is 10 μ L/ slide glasss.Sample drop sheet volume prolongs its dry jitter time more than 10 μ L, causes chromosome repeat.
The 5th; The preparation method of Mammals karyomit(e) phase in mid-term sample of the present invention can be used for the influence of detection of drugs for mammalian cell; Can be with the medicine and the mammalian cell co-cultivation of different concns; Adopt method of the present invention to prepare karyomit(e) phase in mid-term then, observe and to estimate for the influence of mammalian cell with regard to medicine through carrying out chromosome aberration, and then obtain information about aspects such as drug safeties.
Description of drawings
Below, specify embodiment of the present invention in conjunction with accompanying drawing, wherein:
Fig. 1 shows the karyomit(e) in karyomit(e) phase in the mid-term sample of the preparation method preparation of adopting embodiment 1;
Fig. 2 displays temperature is higher than 30 ℃, relative humidity greater than the karyomit(e) in karyomit(e) phase in the mid-term sample of preparation in 55% o'clock;
Fig. 3 displays temperature be lower than 20 ℃, relative humidity be lower than 45% and karyomit(e) phase in the mid-term sample for preparing during the slide glass horizontal positioned in karyomit(e).
Embodiment
Specify the present invention through specific embodiment below, but scope of the present invention does not receive the restriction of these embodiment.
Embodiment 1
Mammalian cell is an example with CHL cell (the Chinese hamster lung fibroblast is available from Shanghai cell institute).CHL cell culture condition: contain the RPMI1640 nutrient solution of 10～15% (10～15ml serum/100ml RPMI1640 nutrient solution) foetal calf serum, 37 ℃, 5%CO
2Incubator is cultivated.
Recovery CHL cell, the trypsinase aqueous solution had digestive transfer culture cell with 0.25% (g/100ml) is prepared into 3～5 * 10
4Cell/mL cell suspension adds 2mL cell suspension and 8mL fresh culture in each 50mL culturing bottle, under 37 ℃, 5%CO
2Cultivated 2 days in the incubator, change nutrient solution, continue to cultivate.Cultivated 21 hours, and added NST-757 0.2mL (concentration 15 μ g/mL), be cultured to 24 hours and outwell substratum, harvested cell.
Behind 0.25% (g/100ml) trypsinase aqueous solution peptic cell, move to the 15mL centrifuge tube, centrifugal 10 minutes of 1000rpm removes supernatant, adds the KCl aqueous solution 5mL of 0.075mol/L, hypotonic 20 minutes (37 ℃).Every pipe adding stationary liquid (volume ratio: methyl alcohol: 0.5mL Glacial acetic acid min. 99.5=3: 1), pre-fix, centrifugal 10 minutes of 1000rpm removes supernatant; In centrifuge tube, add stationary liquid 5mL, behind the mixing under 37 ℃ of conditions fixing 20 minutes, centrifugal, abandoning supernatant, this step be repeatable operation 2 times again.
After last the fixing, centrifugal, upper strata liquid major part is outwelled only remaining about 0.5mL upper strata liquid.With dropper piping and druming cell precipitation thing, make into even cell suspending liquid.Envrionment temperature is 25 ℃ when regulate dripping sheet, and ambient relative humidity is 50% when dripping sheet, places slide glass, with horizontal plane angle be 15 °, draw cell suspension with sample injector, sample volume is 10 μ L.Sample injector drips apart from slide glass 20cm place, and cell is dispersed on the slide, dries naturally.
Slide glass places 10%, and (1 part of Ji's nurse Sa stoste: 9 portions of damping fluids) dyeing of Ji's nurse Sa dye liquor is 15 minutes.Take out behind the slide glass sample earlier with the tap water flushing, again with distilled water flushing to there not being loose colour.Reading sheet observes.
Embodiment 2
Mammalian cell is example with the human peripheral lymphocyte.CHL cell culture condition: contain the RPMI1640 nutrient solution of 10～20% (10～20ml serum/100mlRPMI1640 nutrient solution) foetal calf serum, 37 ℃, 5%CO
2Incubator is cultivated.
Extract healthy volunteer's venous blood, the anti-freezing isolated lymphocytes adds 1mL lymphocyte suspension and 9mL fresh culture in each 50mL culturing bottle, under 37 ℃, 5%CO
2Cultivated 64 hours in the incubator, add NST-757 0.2mL (concentration 15 μ g/mL), be cultured to 68 hours and outwell substratum, harvested cell.
Cell culture fluid moves to the 15mL centrifuge tube, and centrifugal 10 minutes of 1000rpm removes supernatant, adds the KCl aqueous solution 5mL of 0.075mol/L, hypotonic 20 minutes (37 ℃).Every pipe adding stationary liquid (volume ratio: methyl alcohol: 0.5mL Glacial acetic acid min. 99.5=3: 1), pre-fix, centrifugal 10 minutes of 1000rpm removes supernatant; In centrifuge tube, add stationary liquid 5mL, behind the mixing under 37 ℃ of conditions fixing 20 minutes, centrifugal, abandoning supernatant, this step be repeatable operation 2 times again.
After last the fixing, centrifugal, upper strata liquid major part is outwelled only remaining about 0.5mL upper strata liquid.With dropper piping and druming cell precipitation thing, make into even cell suspending liquid.Envrionment temperature is 20～30 ℃ when regulate dripping sheet, and ambient relative humidity is 45～55% when dripping sheet, places slide glass, with horizontal plane angle be 15 °, draw cell suspension with sample injector, sample volume is 10 μ L.Sample injector drips apart from slide glass 15～25cm place, and cell is dispersed on the slide, dries naturally.
Slide glass places 10%, and (1 part of Ji's nurse Sa stoste: 9 portions of damping fluids) dyeing of Ji's nurse Sa dye liquor is 15 minutes.Take out behind the slide glass sample earlier with the tap water flushing, again with distilled water flushing to there not being loose colour.Reading sheet observes.
Claims (10)
1. the preparation method of mammalian cell karyomit(e) phase in a mid-term sample, this preparation method may further comprise the steps: under the relative humidity of 20～30 ℃ temperature and 45～50%, the mammalian cell hanging drop is added on the slide glass.
2. preparation method according to claim 1 is characterized in that, under the relative humidity of 25 ℃ temperature and 50%, the mammalian cell hanging drop is added on the slide glass.
3. preparation method according to claim 1 and 2 is characterized in that, it highly is apart from slide glass 15～25cm that said mammalian cell hanging drop adds, preferred 20cm.
4. according to each described preparation method in the claim 1 to 3, it is characterized in that the said mammalian cell hanging drop added-time, slide glass becomes 15 ° of angles with horizontal plane.
5. according to each described preparation method in the claim 1 to 4, it is characterized in that the dripping quantity of said mammalian cell suspension-s is every slide glass 10 μ L.
6. according to each described preparation method in the claim 1 to 5, it is characterized in that the preparation method of said mammalian cell suspension-s may further comprise the steps:
The mammalian cell that (1) will be cultured to exponential phase of growth is handled with NST-757;
(2) with the volume ratio be methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1 is mammalian cell fixedly.
7. according to each described preparation method in the claim 1 to 6, it is characterized in that the preparation method of said mammalian cell suspension-s may further comprise the steps:
The mammalian cell that (1) will be cultured to exponential phase of growth makes every milliliter with the digestion of the 0.25%W/V trypsinase aqueous solution and comprises 3～5 * 10
4The cell suspending liquid of individual cell, and cultivated 21 hours;
(2) add NST-757 and handled 24 hours, the concentration of NST-757 is every ml cells suspension-s 15 μ g;
(3) discard substratum, with 0.25%W/V trypsinase aqueous solution digestion mammalian cell and centrifugal, preferably under 1000rpm centrifugal 10 minutes;
(4) supernatant discarded adds under 37 ℃ of the KCl aqueous solution of 0.075mol/L and left standstill 20 minutes;
(5) adding volume ratio again is methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1, and centrifugal behind the piping and druming cell mixing, preferably under 1000rpm centrifugal 10 minutes;
(6) supernatant discarded, adding volume ratio again is methyl alcohol: the stationary liquid of Glacial acetic acid min. 99.5=3: 1, leave standstill after 20 minutes under 37 ℃ centrifugal.
8. preparation method according to claim 7 is characterized in that, said step (6) is carried out 3 times repeatedly.
9. according to each described preparation method in the claim 1 to 8, it is characterized in that said mammalian cell hanging drop is added to seasoning behind the slide glass, dyes with 10%V/V Ji's nurse Sa dye liquor again.
10. mammalian cell karyomit(e) phase in the mid-term sample for preparing according to each described preparation method in the claim 1 to 9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107384912A (en) * | 2017-07-31 | 2017-11-24 | 四川金域医学检验中心有限公司 | The preparation method of G-band chromosome |
| CN108444779A (en) * | 2018-01-30 | 2018-08-24 | 昆明金域医学检验所有限公司 | A kind of chromosome drop piece production method |
| CN109115566A (en) * | 2017-11-10 | 2019-01-01 | 佛山市艾达思精密仪器有限公司 | A method of it goes to prepare cell chromosome karyotyping glass slide with spraying humidity strip |
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| CN1428419A (en) * | 2002-03-01 | 2003-07-09 | 中国石油化工股份有限公司 | Method for preparing fertilized ovum chromosome |
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2010
- 2010-11-18 CN CN2010105603420A patent/CN102465168A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1428419A (en) * | 2002-03-01 | 2003-07-09 | 中国石油化工股份有限公司 | Method for preparing fertilized ovum chromosome |
Non-Patent Citations (2)
| Title |
|---|
| LYNDA J. CAMPBELL: "Cytogenetic and FISH Techniques in Myeloid Malignancies", 《METHODS IN MOLECULAR MEDICINE》 * |
| 张小建等: "牦牛成纤维细胞的体外培养研究", 《湖北农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107384912A (en) * | 2017-07-31 | 2017-11-24 | 四川金域医学检验中心有限公司 | The preparation method of G-band chromosome |
| CN109115566A (en) * | 2017-11-10 | 2019-01-01 | 佛山市艾达思精密仪器有限公司 | A method of it goes to prepare cell chromosome karyotyping glass slide with spraying humidity strip |
| CN108444779A (en) * | 2018-01-30 | 2018-08-24 | 昆明金域医学检验所有限公司 | A kind of chromosome drop piece production method |
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Application publication date: 20120523 |