CN102443623A - Chromosome preparation method, as well as required culture medium and preparation method thereof - Google Patents
Chromosome preparation method, as well as required culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chromosome preparation method, as well as a required culture medium and a preparation method thereof, and is used for solving karyotype analysis problem of chromosome. The culture medium consists of RPMI (Roswell Park Memorial Institute) 1640, heparin sodium, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), L-glutamine, NaHCO3, benzylpenicillin potassium, streptomycin sulphate, bovine serum and phytohemagglutinin (PHA). The detection method comprises the following steps: implanting 0.3 to 0.4ml of human peripheral blood into the culture medium; adding colchicinamide in 2-4 hours before culture is terminated to realize that the cell is terminated in anaphase; culturing the cell after 68 to 72 hours to harvest the cell; performing hypotonicity for 40 minutes, three times of fixation, banding, dyeing and other treatments; and performing chromosome analysis under a microscope to determine whether the peripheral blood supplier has a phenomenon of chromosome abnormality. The culture medium disclosed by the invention has convenience for use, simpleness in operation, low cost and low patient detection fee, and is suitable for genetic diagnosis, infertility and prenatal diagnosis in each level of hospitals.
Description
Technical field
The present invention relates to a kind of effective karyomit(e) molecular agents material in physianthropy genetics, the reproductive medicine field, be specifically related to a kind of method of chromosome preparation and required substratum of chromosomal disorders diagnosis and preparation method thereof.
Background technology
Karyomit(e) is that genetic material is the carrier of gene, in human each cell 46 karyomit(e)s is arranged, 44 euchromosomes wherein, 2 sex chromosome.No matter be euchromosome, or sex chromosome, they are all carrying thousands of genes, so chromosome number or small textural anomaly all will cause the increase or the disappearance of many genes, and produce multiple deformity or unusual syndrome.The caused disease of chromosome number or textural anomaly is called chromosomal disorder, and chromosomal disorder often exists with syndromic form.
Chromosomal disorder is a kind of common inherited disease, is that numerical abnormalities of chromosomes or chromosomal structural abnormality all can cause congenital malformation, mental retardation, miscarriage, infertile, dysplasia etc., the physical and mental health that is seriously endangering people.
Chromosome examination is mainly used in state of an illness diagnosis.Clinical every discovery has that obvious growth heteroplasia, multiple deformity, skin texture are unusual, reproductive system heteroplasia etc., needs clearly whether there is defective or unusual in karyomit(e); Suspect individuality and parents thereof that mongolism is arranged, the mental retardation person who causes by heredity; Agnogenic spontaneous abortion, fetation deformity, embryo or fetus stasi, the neonatal death of birth once repeatedly, such Mr. and Mrs need check karyomit(e); Menstrual onset never, normally pregnancy, hypoplasia of uterus, congenital testis are sent out unusually for a long time, severely subnormals such as azoospermia or sperm development, reproductive development are unusual etc., need inspection karyomit(e) to make a definite diagnosis.
Chromosome examination is fairly simple, and China can check to go to the hospital in general county at present.The method of inspection is to adopt the cell cultures banding technique.Mr. and Mrs two people will check when suspecting that both sides might have problems.The check result male sex should be 46XY, and the women then is 46XX, and result in addition is unusual, morbid state.
At present clinical chromosome examination method commonly used has: fluorescence in situ hybridization technique, polymerase chain reaction technique, primed in situlabelling technology, karyotyping are technological.In view of characteristics such as low, the accuracy height of karyotyping technical costs, for large and medium-sized hospital widely used.
But; Not unified standard of the used substratum of chromosome examination is carried out in domestic each hospital and laboratory; Quality is extremely unstable, and is few and karyomit(e) is elongated inadequately by the karyomit(e) division phase number of its cultivation, is not easy to clinical chromosome examination analysis and scientific research; Often cause to carry out chromosome analysis, influence the diagnosis of disease.
Summary of the invention
For addressing the above problem; The objective of the invention is to overcome above-mentioned deficiency; A kind of steady quality is provided, and numerous and karyomit(e) is elongated by the karyomit(e) of its cultivation division phase number, what be convenient to that clinical chromosome examination analysis and scientific research use is used for substratum of Chromosome Preparation and preparation method thereof.
For achieving the above object, technical scheme of the present invention is: a kind of preparation method who is used for the substratum of Chromosome Preparation may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
RPMI1640:10-11g/L, heparin sodium: 6400-7000IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.06-0.1g/L, Ox blood serum: 100-200ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 50-200mg/L in the initial soln after filtration, stir, and makes substratum.
Preferably, increase following steps:
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
A kind of substratum that is used for Chromosome Preparation, the prescription of said substratum is: RPMI1640:10-11g/L, heparin sodium: 6400-7000IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.06-0.1g/L, Ox blood serum: 100-200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
Preferably, the prescription of said substratum is: RPMI1640:10.2-10.6g/L, heparin sodium: 6400-6500IU, HEPES:2-4g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.07-0.09g/L, Ox blood serum: 150-200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
Preferably, the prescription of said substratum is: RPMI1640:10.4g/L, heparin sodium: 6400IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.6g/L, NaHCO3:2g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.08g/L, Ox blood serum: 200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
A kind of method of chromosome preparation may further comprise the steps:
Step 1, medium preparation prepares substratum according to the said method of claim 1;
Step 2; Cell cultures goes into human peripheral blood 0.3ml-0.4ml kind in the said substratum to cultivate, and is the Colchiceinamidum of 0.1ug/ml stopping cultivating preceding 2.5 hours adding concentration; Make cell stop at metaphase, cultivate the back harvested cell through the 68-72 hour cell;
Step 3, the preparation cell suspension passes through the hypotonic 40min of KCL hypotonic medium of 0.075mol/L again, pre-fix after three times fixing, the preparation cell suspension is used for Chromosome Preparation;
Step 4, film-making promptly prepares chromosome specimen; Beat even and fine born of the same parents' suspension with dropper, draw to drip to the cleaning of soaking through frozen water from the 20-30cm eminence on a small quantity and do not have on the slide of fat, every drips 2-3 and drips; Drip off the back on spirit lamp flame back and forth for several times, make its drying, make chromosome specimen;
Step 5 is carried out chromosome banding analysis or non-apparent band analysis according to the diagnosis needs with above-mentioned chromosome specimen.
Preferably, said cell cultures may further comprise the steps:
The first step, blood sampling, human vein is got blood, anticoagulant heparin;
Second step, plant blood, prepare the karyomit(e) substratum, return to room temperature, the plug that the syringe needle of syringe is passed culturing bottle adds the 0.3-0.4ml whole blood in every flask culture base, shake up immediately, puts into 37 ℃ of constant incubators,
The 3rd step, cell cultures, respectively shake up once every day sooner or later behind the kind blood, stopped cultivating in the 3rd day after the cell cultures, and whole incubation time is 68-72h;
The 4th step, detention metacinesis phase, 2.5h adds Colchiceinamidum before stopping cultivating, and the Colchiceinamidum final concentration is that 0.1ug/ml shakes up, and continues to put into 37 ℃ of constant incubators, continues to be cultured to 68h-72h;
In the 5th step, harvested cell takes out substratum from 37 ℃ of constant incubators, shakes up, and pours culture into conical centrifuge tube, 1000 commentaries on classics/min, and centrifugal 10min after the centrifugal end, inhales and removes supernatant, only keeps about blood preparation 1ml.
Preferably, said preparation cell suspension may further comprise the steps:
The first step, hypotonic, in above-mentioned centrifuge tube, add temperature in advance to 0.075mol/LKCL hypotonic medium liquid 6～8ml of 37 ℃, with the evenly rearmounted 37 ℃ of hypotonic 40min of water-bath of dropper piping and druming;
Second step pre-fixed, according to Glacial acetic acid min. 99.5: methyl alcohol=1: 3 preparation stationary liquid, after the hypotonic end, add stationary liquid 1ml along centrifugal tube wall, behind the piping and druming mixing, 1000 commentaries on classics/min, centrifugal 10min;
The 3rd step, fixing for the first time, inhale and remove supernatant, add the 7ml stationary liquid along centrifugal tube wall, with placing more than 30 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 4th step, fixing for the second time, inhale and remove supernatant, keep 1.5ml.Add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 5th step, fixing for the third time, outwell supernatant, add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
In the 6th step, the preparation of cell suspension and preservation are inhaled and to be removed supernatant, and it is an amount of to add stationary liquid, beat evenly gently, process the suitable cell suspension of concentration.
Adopt the beneficial effect of present technique scheme to be: the substratum quality of production is stable, and is numerous and karyomit(e) is elongated by the karyomit(e) division phase number of its cultivation, is convenient to clinical chromosome examination analysis and scientific research and uses.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.
Embodiment 1,
A kind of preparation method who is used for the substratum of Chromosome Preparation may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
RPMI1640:10g/L, heparin sodium: 7000IU, HEPES:1.19g/L, L-glutaminate: 0.6g/L, NaHCO3:1g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.06g/L, Ox blood serum: 200ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 50mg/L in the initial soln after filtration, stir, and makes substratum.
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
Embodiment 2,
A kind of preparation method who is used for the substratum of Chromosome Preparation may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
RPMI1640:11g/L, heparin sodium: 6400IU, HEPES:4.05g/L, L-glutaminate: 0.5g/L, NaHCO3:2g/L, penicilline g potassium: 0.05g/L, Vetstrep: 0.1g/L, Ox blood serum: 100ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 200mg/L in the initial soln after filtration, stir, and makes substratum.
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
Embodiment 3,
A kind of preparation method who is used for the substratum of Chromosome Preparation may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
RPMI1640:10.5g/L, heparin sodium: 6600IU, HEPES:3g/L, L-glutaminate: 0.55g/L, NaHCO3:1.5g/L, penicilline g potassium: 0.08g/L, Vetstrep: 0.08g/L, Ox blood serum: 150ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 120mg/L in the initial soln after filtration, stir, and makes substratum.
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
Embodiment 4,
A kind of preparation method who is used for the substratum of Chromosome Preparation may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
The prescription of said substratum is: RPMI1640:10.4g/L, heparin sodium: 6400IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.6g/L, NaHCO3:2g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.08g/L, Ox blood serum: 200ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 50-200mg/L in the initial soln after filtration, stir, and makes substratum.
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
Present embodiment is optimum scheme.
Embodiment 5,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10g/L, heparin sodium: 7000IU, HEPES:1.19g/L, L-glutaminate: 0.6g/L, NaHCO3:1g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.06g/L, Ox blood serum: 200ml/L, phytoh(a)emagglutinin PHA:50mg/L, water for injection add to 1L.
Substratum of the present invention is easy to use, and is simple to operate, and cost is low, and patient's check fee is low, is suitable for hospitals at different levels and is used for genetic diagnosis, infertile and antenatal diagnosis.
Embodiment 6,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:11g/L, heparin sodium: 6400IU, HEPES:4.05g/L, L-glutaminate: 0.5g/L, NaHCO3:2g/L, penicilline g potassium: 0.05g/L, Vetstrep: 0.1g/L, Ox blood serum: 100ml/L, phytoh(a)emagglutinin PHA:200mg/L, water for injection add to 1L.
Embodiment 7,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10.5g/L, heparin sodium: 6600IU, HEPES:3g/L, L-glutaminate: 0.55g/L, NaHCO3:1.5g/L, penicilline g potassium: 0.08g/L, Vetstrep: 0.08g/L, Ox blood serum: 150ml/L, phytoh(a)emagglutinin PHA:150mg/L, water for injection add to 1L.
Embodiment 8,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10.2g/L, heparin sodium: 6500IU, HEPES:2g/L, L-glutaminate: 0.6g/L, NaHCO3:1g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.07g/L, Ox blood serum: 200ml/L, phytoh(a)emagglutinin PHA:50mg/L, water for injection add to 1L.
Embodiment 9,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10.6g/L, heparin sodium: 6400IU, HEPES:4g/L, L-glutaminate: 0.5g/L, NaHCO3:2g/L, penicilline g potassium: 0.05g/L, Vetstrep: 0.09g/L, Ox blood serum: 150ml/L, phytoh(a)emagglutinin PHA:200mg/L, water for injection add to 1L.
Embodiment 10,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10.4g/L, heparin sodium: 6450IU, HEPES:3g/L, L-glutaminate: 0.55g/L, NaHCO3:1.5g/L, penicilline g potassium: 0.055g/L, Vetstrep: 0.08g/L, Ox blood serum: 180ml/L, phytoh(a)emagglutinin PHA:180mg/L, water for injection add to 1L.
Embodiment 11,
A kind of culture medium prescription that is used for Chromosome Preparation is: RPMI1640:10.4g/L, heparin sodium: 6400IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.6g/L, NaHCO3:2g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.08g/L, Ox blood serum: 200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
Embodiment 12,
A kind of method of chromosome preparation may further comprise the steps:
Step 1, medium preparation prepares substratum according to the said method of claim 1;
Step 2, cell cultures comprises following five steps:
The first step, blood sampling, human vein is got blood, anticoagulant heparin;
Second step, plant blood, prepare the karyomit(e) substratum, return to room temperature, the plug that the syringe needle of syringe is passed culturing bottle adds the 0.3ml whole blood in every flask culture base, shake up immediately, puts into 37 ℃ of constant incubators,
The 3rd step, cell cultures, respectively shake up once every day sooner or later behind the kind blood, stopped cultivating in the 3rd day after the cell cultures, and whole incubation time is 68h;
The 4th step, detention metacinesis phase, 2.5h adds Colchiceinamidum before stopping cultivating, and the Colchiceinamidum final concentration is that 0.1ug/ml shakes up, and continues to put into 37 ℃ of constant incubators, continues to be cultured to 68h;
In the 5th step, harvested cell takes out substratum from 37 ℃ of constant incubators, shakes up, and pours culture into conical centrifuge tube, 1000 commentaries on classics/min, and centrifugal 10min after the centrifugal end, inhales and removes supernatant, only keeps about blood preparation 1ml.
Step 3, the preparation cell suspension, said preparation cell suspension may further comprise the steps:
The first step, hypotonic, in above-mentioned centrifuge tube, add temperature in advance to 37 ℃ 0.075mol/LKCL hypotonic medium liquid 6ml, with the evenly rearmounted 37 ℃ of hypotonic 40min of water-bath of dropper piping and druming;
Second step pre-fixed, according to Glacial acetic acid min. 99.5: methyl alcohol=1: 3 preparation stationary liquid, after the hypotonic end, add stationary liquid 1ml along centrifugal tube wall, behind the piping and druming mixing, 1000 commentaries on classics/min, centrifugal 10min;
The 3rd step, fixing for the first time, inhale and remove supernatant, add the 7ml stationary liquid along centrifugal tube wall, with placing more than 30 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 4th step, fixing for the second time, inhale and remove supernatant, keep 1.5ml.Add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 5th step, fixing for the third time, outwell supernatant, add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
In the 6th step, the preparation of cell suspension and preservation are inhaled and to be removed supernatant, and it is an amount of to add stationary liquid, beat evenly gently, process the suitable cell suspension of concentration.
Step 4, film-making promptly prepares chromosome specimen; Beat even and fine born of the same parents' suspension with dropper, draw to drip to the cleaning of soaking through frozen water from the 20cm eminence on a small quantity and do not have on the slide of fat, every drips 2; Drip off the back on spirit lamp flame back and forth for several times, make its drying, make chromosome specimen;
Step 5 is carried out chromosome banding analysis or non-apparent band analysis according to the diagnosis needs with above-mentioned chromosome specimen.
Embodiment 13,
A kind of method of chromosome preparation may further comprise the steps:
Step 1, medium preparation prepares substratum according to the said method of claim 1;
Step 2, cell cultures comprises following five steps:
The first step, blood sampling, human vein is got blood, anticoagulant heparin;
Second step, plant blood, prepare the karyomit(e) substratum, return to room temperature, the plug that the syringe needle of syringe is passed culturing bottle adds the 0.4ml whole blood in every flask culture base, shake up immediately, puts into 37 ℃ of constant incubators,
The 3rd step, cell cultures, respectively shake up once every day sooner or later behind the kind blood, stopped cultivating in the 3rd day after the cell cultures, and whole incubation time is 72h;
The 4th step, detention metacinesis phase, 2.5h adds Colchiceinamidum before stopping cultivating, and the Colchiceinamidum final concentration is that 0.1ug/ml shakes up, and continues to put into 37 ℃ of constant incubators, and continuing is cultured to 72h;
In the 5th step, harvested cell takes out substratum from 37 ℃ of constant incubators, shakes up, and pours culture into conical centrifuge tube, 1000 commentaries on classics/min, and centrifugal 10min after the centrifugal end, inhales and removes supernatant, only keeps about blood preparation 1ml.
Step 3, the preparation cell suspension, said preparation cell suspension may further comprise the steps:
The first step, hypotonic, in above-mentioned centrifuge tube, add temperature in advance to 0.075mol/LKCL hypotonic medium liquid 6～8ml of 37 ℃, with the evenly rearmounted 37 ℃ of hypotonic 40min of water-bath of dropper piping and druming;
Second step pre-fixed, according to Glacial acetic acid min. 99.5: methyl alcohol=1: 3 preparation stationary liquid, after the hypotonic end, add stationary liquid 1ml along centrifugal tube wall, behind the piping and druming mixing, 1000 commentaries on classics/min, centrifugal 10min;
The 3rd step, fixing for the first time, inhale and remove supernatant, add the 7ml stationary liquid along centrifugal tube wall, with placing more than 30 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 4th step, fixing for the second time, inhale and remove supernatant, keep 1.5ml.Add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 5th step, fixing for the third time, outwell supernatant, add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
In the 6th step, the preparation of cell suspension and preservation are inhaled and to be removed supernatant, and it is an amount of to add stationary liquid, beat evenly gently, process the suitable cell suspension of concentration.
Step 4, film-making promptly prepares chromosome specimen; Beat even and fine born of the same parents' suspension with dropper, draw to drip to the cleaning of soaking through frozen water from the 30cm eminence on a small quantity and do not have on the slide of fat, every drips 3; Drip off the back on spirit lamp flame back and forth for several times, make its drying, make chromosome specimen;
Step 5 is carried out chromosome banding analysis or non-apparent band analysis according to the diagnosis needs with above-mentioned chromosome specimen.
Embodiment 14,
A kind of method of chromosome preparation may further comprise the steps:
Step 1, medium preparation prepares substratum according to the said method of claim 1;
Step 2, cell cultures comprises following five steps:
The first step, blood sampling, human vein is got blood, anticoagulant heparin;
Second step, plant blood, prepare the karyomit(e) substratum, return to room temperature, the plug that the syringe needle of syringe is passed culturing bottle adds the 0.35ml whole blood in every flask culture base, shake up immediately, puts into 37 ℃ of constant incubators,
The 3rd step, cell cultures, respectively shake up once every day sooner or later behind the kind blood, stopped cultivating in the 3rd day after the cell cultures, and whole incubation time is 70h;
The 4th step, detention metacinesis phase, 2.5h adds Colchiceinamidum before stopping cultivating, and the Colchiceinamidum final concentration is that 0.1ug/ml shakes up, and continues to put into 37 ℃ of constant incubators, continues to be cultured to 70h;
In the 5th step, harvested cell takes out substratum from 37 ℃ of constant incubators, shakes up, and pours culture into conical centrifuge tube, 1000 commentaries on classics/min, and centrifugal 10min after the centrifugal end, inhales and removes supernatant, only keeps about blood preparation 1ml.
Step 3, the preparation cell suspension, said preparation cell suspension may further comprise the steps:
The first step, hypotonic, in above-mentioned centrifuge tube, add temperature in advance to 37 ℃ 0.075mol/LKCL hypotonic medium liquid 7ml, with the evenly rearmounted 37 ℃ of hypotonic 40min of water-bath of dropper piping and druming;
Second step pre-fixed, according to Glacial acetic acid min. 99.5: methyl alcohol=1: 3 preparation stationary liquid, after the hypotonic end, add stationary liquid 1ml along centrifugal tube wall, behind the piping and druming mixing, 1000 commentaries on classics/min, centrifugal 10min;
The 3rd step, fixing for the first time, inhale and remove supernatant, add the 7ml stationary liquid along centrifugal tube wall, with placing more than 30 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 4th step, fixing for the second time, inhale and remove supernatant, keep 1.5ml.Add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 5th step, fixing for the third time, outwell supernatant, add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
In the 6th step, the preparation of cell suspension and preservation are inhaled and to be removed supernatant, and it is an amount of to add stationary liquid, beat evenly gently, process the suitable cell suspension of concentration.
Step 4, film-making promptly prepares chromosome specimen; Beat even and fine born of the same parents' suspension with dropper, draw to drip to the cleaning of soaking through frozen water from the 20-30cm eminence on a small quantity and do not have on the slide of fat, every drips 2-3 and drips; Drip off the back on spirit lamp flame back and forth for several times, make its drying, make chromosome specimen;
Step 5 is carried out chromosome banding analysis or non-apparent band analysis according to the diagnosis needs with above-mentioned chromosome specimen.
Adopt the beneficial effect of present technique scheme to be: the substratum quality of production is stable, and is numerous and karyomit(e) is elongated by the karyomit(e) division phase number of its cultivation, is convenient to clinical chromosome examination analysis and scientific research and uses.
Above-described only is preferred implementation of the present invention, should be pointed out that for the person of ordinary skill of the art, under the prerequisite that does not break away from the invention design, can also make some distortion and improvement, and these all belong to protection scope of the present invention.
Claims (8)
1. a preparation method who is used for the substratum of Chromosome Preparation is characterized in that, may further comprise the steps:
Step 1, the preparation initial soln, said initial soln prescription is following:
RPMI1640:10-11g/L, heparin sodium: 6400-7000IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.06-0.1g/L, Ox blood serum: 100-200ml/L, water for injection add to 1L, stir to settled solution;
Step 2, filtration sterilization is carried out filtration sterilization with filter membrane with above-mentioned initial soln;
Step 3, preparation phytoh(a)emagglutinin PHA solution is prepared 2% phytoh(a)emagglutinin PHA solution with water for injection, and is subsequent use;
Step 4, the substratum preparation adds said phytoh(a)emagglutinin PHA solution: 50-200mg/L in the initial soln after filtration, stir, and makes substratum.
2. the preparation method who is used for the substratum of Chromosome Preparation according to claim 1 is characterized in that, increases following steps:
Step 5: packing
The described solution branch of step 4 is installed in the cillin bottle, seal.
Step 6: labeling
With the described product labeling of step 5, promptly form the karyomit(e) substratum.
3. substratum that is used for Chromosome Preparation; It is characterized in that the prescription of said substratum is: RPMI1640:10-11g/L, heparin sodium: 6400-7000IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.06-0.1g/L, Ox blood serum: 100-200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
4. a kind of substratum that is used for Chromosome Preparation according to claim 3; It is characterized in that the prescription of said substratum is: RPMI1640:10.2-10.6g/L, heparin sodium: 6400-6500IU, HEPES:2-4g/L, L-glutaminate: 0.5-0.6g/L, NaHCO3:1-2g/L, penicilline g potassium: 0.05-0.1g/L, Vetstrep: 0.07-0.09g/L, Ox blood serum: 150-200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
5. a kind of substratum that is used for Chromosome Preparation according to claim 3; It is characterized in that the prescription of said substratum is: RPMI1640:10.4g/L, heparin sodium: 6400IU, HEPES:1.19-4.05g/L, L-glutaminate: 0.6g/L, NaHCO3:2g/L, penicilline g potassium: 0.1g/L, Vetstrep: 0.08g/L, Ox blood serum: 200ml/L, phytoh(a)emagglutinin PHA:50-200mg/L, water for injection add to 1L.
6. a method of chromosome preparation is characterized in that, may further comprise the steps:
Step 1, medium preparation prepares substratum according to the said method of claim 1;
Step 2; Cell cultures goes into human peripheral blood 0.3ml-0.4ml kind in the said substratum to cultivate, and is the Colchiceinamidum of 0.1ug/ml stopping cultivating preceding 2.5 hours adding concentration; Make cell stop at metaphase, cultivate the back harvested cell through the 68-72 hour cell;
Step 3, the preparation cell suspension passes through the hypotonic 40min of KCL hypotonic medium of 0.075mol/L again, pre-fix after three times fixing, the preparation cell suspension is used for Chromosome Preparation;
Step 4, film-making promptly prepares chromosome specimen; Beat even and fine born of the same parents' suspension with dropper, draw to drip to the cleaning of soaking through frozen water from the 20-30cm eminence on a small quantity and do not have on the slide of fat, every drips 2-3 and drips; Drip off the back on spirit lamp flame back and forth for several times, make its drying, make chromosome specimen;
Step 5 is carried out chromosome banding analysis or non-apparent band analysis according to the diagnosis needs with above-mentioned chromosome specimen.
7. a kind of method of chromosome preparation according to claim 6 is characterized in that, said cell cultures may further comprise the steps:
The first step, blood sampling, human vein is got blood, anticoagulant heparin;
Second step, plant blood, prepare the karyomit(e) substratum, return to room temperature, the plug that the syringe needle of syringe is passed culturing bottle adds the 0.3-0.4ml whole blood in every flask culture base, shake up immediately, puts into 37 ℃ of constant incubators,
The 3rd step, cell cultures, respectively shake up once every day sooner or later behind the kind blood, stopped cultivating in the 3rd day after the cell cultures, and whole incubation time is 68-72h;
The 4th step, detention metacinesis phase, 2.5h adds Colchiceinamidum before stopping cultivating, and the Colchiceinamidum final concentration is that 0.1ug/ml shakes up, and continues to put into 37 ℃ of constant incubators, continues to be cultured to 68h-72h;
In the 5th step, harvested cell takes out substratum from 37 ℃ of constant incubators, shakes up, and pours culture into conical centrifuge tube, 1000 commentaries on classics/min, and centrifugal 10min after the centrifugal end, inhales and removes supernatant, only keeps about blood preparation 1ml.
8. according to claim 6 or 7 described a kind of method of chromosome preparation, it is characterized in that said detention metacinesis may further comprise the steps mutually:
The first step, hypotonic, in above-mentioned centrifuge tube, add temperature in advance to 0.075mol/L KCL hypotonic medium liquid 6～8ml of 37 ℃, with the evenly rearmounted 37 ℃ of hypotonic 40min of water-bath of dropper piping and druming;
Second step pre-fixed, according to Glacial acetic acid min. 99.5: methyl alcohol=1: 3 preparation stationary liquid, after the hypotonic end, add stationary liquid 1ml along centrifugal tube wall, behind the piping and druming mixing, 1000 commentaries on classics/min, centrifugal 10min;
The 3rd step, fixing for the first time, inhale and remove supernatant, add the 7ml stationary liquid along centrifugal tube wall, with placing more than 30 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 4th step, fixing for the second time, inhale and remove supernatant, keep 1.5ml.Add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
The 5th step, fixing for the third time, outwell supernatant, add the 7ml stationary liquid, with placing more than 15 minutes 1000 commentaries on classics/min, centrifugal 10min behind the dropper piping and druming mixing;
In the 6th step, the preparation of cell suspension and preservation are inhaled and to be removed supernatant, and it is an amount of to add stationary liquid, beat evenly gently, process the suitable cell suspension of concentration.
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| CN106701937A (en) * | 2016-12-20 | 2017-05-24 | 云南师范大学 | Method for detecting instability of human cell mitotic chromosomes in vitro |
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| CN109580299A (en) * | 2017-09-28 | 2019-04-05 | 上海新培晶医学检验所有限公司 | Bone marrow cell chromosome flaking method |
| CN109856330A (en) * | 2019-01-29 | 2019-06-07 | 南京农业大学 | A method of chrysanthemum tip of a root Mitotic phase is improved by artificial regulatory |
| CN112577803A (en) * | 2020-12-10 | 2021-03-30 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754690A (en) * | 2016-12-06 | 2017-05-31 | 河北意和医学检验所有限公司 | A kind of chromosome culture medium of quick results medium cell and application |
| CN106701937A (en) * | 2016-12-20 | 2017-05-24 | 云南师范大学 | Method for detecting instability of human cell mitotic chromosomes in vitro |
| CN109580299A (en) * | 2017-09-28 | 2019-04-05 | 上海新培晶医学检验所有限公司 | Bone marrow cell chromosome flaking method |
| CN109856330A (en) * | 2019-01-29 | 2019-06-07 | 南京农业大学 | A method of chrysanthemum tip of a root Mitotic phase is improved by artificial regulatory |
| CN109856330B (en) * | 2019-01-29 | 2021-05-28 | 南京农业大学 | Method for improving mitotic phase of chrysanthemum root tip through artificial regulation |
| CN112577803A (en) * | 2020-12-10 | 2021-03-30 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
| CN112577803B (en) * | 2020-12-10 | 2021-11-09 | 中国水产科学研究院珠江水产研究所 | Method for preparing turtle chromosome specimen by using peripheral blood cells |
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