CN108611415A - The detection kit and its application method of acquired cell clone - Google Patents
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- CN108611415A CN108611415A CN201810433109.2A CN201810433109A CN108611415A CN 108611415 A CN108611415 A CN 108611415A CN 201810433109 A CN201810433109 A CN 201810433109A CN 108611415 A CN108611415 A CN 108611415A
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Abstract
The present invention relates to the detection kits and its application method of the acquired cell clone of detection after a kind for the treatment of for primary tumor.The detection kit of the acquired cell clone includes the first fluorescence probe reagent, the second fluorescence probe reagent and third fluorescence probe reagent；Wherein, in the first fluorescence probe reagent the sequence of two detection probes respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2, the sequence of two detection probes is respectively such as SEQ ID NO in the second fluorescence probe reagent:3 and SEQ ID NO:Shown in 4, the sequence of detection probe such as SEQ ID NO in third fluorescence probe reagent:Shown in 5.The detection kit and its application method of the acquired cell clone can be used for the ACC situations to sample of bone marrow, especially SACC situations are detected tracks with follow-up, testing result can be in conjunction with clinical endpoint, pathologic index, streaming parting index, important evidence and reference are provided for the clinical early diagnosis of tMDS and tAML, clinician is preferably instructed to carry out early diagnosis intervention and treatment to treatment correlation myeloma.
Description
Technical field
The present invention relates to molecular biology fields, a kind of detection kit more particularly, to acquired cell clone and its
Application method.
Background technology
The traditional therapy of primary tumor is broadly divided into radiotherapy, chemotherapy or both together, and chemotherapy is mainly wrapped
Include alkanisation agent therapy, purine analogue therapy, anthraquinone therapy, Rituximab therapy etc..Traditional treatment is often to patient
Normal cell generates toxic effect and causes serious damage, easily leads to treatment correlation myelocytome (therapy-related
Myeloid neoplasm, tMN) generation, such as treat correlation myelodysplastic syndrome (therapy-related
Myelodysplastic syndrome, tMDS) and treatment correlation acute myelocytic leukemia (therapy-related
Acute myeloid leukemia, tAML), it generally betides after primary tumor is treated in 10 years, the middle position phase is 5-7,
According to the ASSOCIATE STATISTICS in the U.S., 10 years cumulative life-incidences are about 8%-10%.As the morning to primary tumor finds, morning is controlled
It treats and the use of effectively new tumor therapeutic agent scheme, the survival period of the tumour patient through treatment extends, treat correlation marrow
The case of cytoma increases therewith.In the U.S., the early diagnosis and therapy to treating correlation myelocytome, which early has been cited as, to be faced
Bed neoplastic disease Routine Management.In recent years, treatment correlation myelocytome also begins to be concerned in China, is more and more faced
The attention of bed doctor and virologist, because treatment correlation myelocytome is refractory, the death rate of patient is high and survival period is short.
Short life cycle caused by the characteristics for the treatment of correlation myelocytome is intractable and drug resistance, thus treat related
The early diagnosis and intervention of property myelocytome, there is very important clinical meaning.Treatment correlation myelocytome at this stage
Diagnosis basis includes mainly clinical endpoint, pathologic index, streaming parting index and cytogenetics parting index.Due to treatment
Correlation myelocytome is a kind of symptom of complexity, and when clinical diagnosis needs the testing result comprehensive analysis in conjunction with many indexes,
The testing result of single index often can only be used as intermediate diagnostic result or reference result to be used for assistant analysis.
Acquired cell clone (acquired cytogenetic clone, ACC, also known as acquired cytogenetics gram
Grand or acquired cellular genome clone etc.) it is a kind of cytogenetics parting index.When abnormal cytogenetics clone still locates
When low-level, cellular genome (cytogenomic) change is still within silence state, without causing clinical phenotypes swollen
The generation of tumor lacks pathology cellular morphology and changes and lack any clinical phenotypes, new under this normal clinical state is obtained
It obtains property cell clone and is known as the acquired cell clone of silence type (silent acquired cytogenetic clone, SACC)；
When new acquired cell clone develops to certain level, relevant abnormal cell genome functions are activated and cause to control
The generation of correlation myelocytome is treated, as shown in Figure 1, this clone is known as the acquired cell clone (active of activated form
Acquired cytogenetic clone, AACC).Claim to the period AACC occurs since being treated primary tumor
The phase occurs for ACC.SACC generation phase ratio AACC the generation phase it is long, the former for 80 months and the latter is 47 months.From SACC
The process of AACC is developed to, the variation of blood routine shows that hemoglobin and platelet count reduce, marrow followed by occurs first
Cell number changes.
In cytogenetics parting index, the World Health Organization (WHO) proposed myeloproliferative disorder synthesis in 2008
Levy the preliminary judgement Main Basiss of (MDS)：No. 7 chromosome long arm missings, No. 5 chromosome long arm missings and No. 13 chromosome long arms
Missing, is abbreviated as respectively：Del (7q), del (5q) and del (13q)；Or each autocorrelative No. 7, No. 5 and No. 13 chromosome
Monomer is abbreviated as respectively：- 7, -5 and -13；Or other rare abnormal cell genetic clones.
The mechanism of carcinogenesis of chromosome abnormality includes：The change of genome functions；Chromosome abnormality leads to related gene expression
It is abnormal：Rise or reduces.The clinical assessment standard of the acquired cell clone of silence type (SACC) is：1) presence of ACC is confirmed；
2) there is ACC, but it is different under bone marrow morphology assessment Morphology of Bone Marrow Patient cells' surface antibody autoimmunity do not occur
Often；3) there is ACC, but without any clinical manifestation for the treatment of correlation myeloma.
Invention content
Based on this, it is necessary to provide a kind of detection being used for the acquired cell clone of detection after primary tumor is treated
Kit and its application method.
A kind of detection kit of acquired cell clone, including the first fluorescence probe reagent, the second fluorescence probe reagent
And third fluorescence probe reagent；Wherein, the first fluorescence probe reagent and the second fluorescence probe reagent are double-colored glimmering
Light probe reagent, the third fluorescence probe reagent are one-color fluorescence probe reagent, and two in the first fluorescence probe reagent
The sequence of detection probe is respectively such as SEQ ID NO:1 and SEQ ID NO:Shown in 2, two in the second fluorescence probe reagent
The sequence of detection probe is respectively such as SEQ ID NO:3 and SEQ ID NO:It detects and visits shown in 4, in the third fluorescence probe reagent
The sequence of needle such as SEQ ID NO:Shown in 5.
In one of the embodiments, the detection kit of the acquired cell clone further include in following solution extremely
Few one kind：Fixer, colchicine solution, KCl solution, Jim Sa mother liquor, trypsin solution, SSC washing lotions, NP40 lysates,
DAPI dyeing liquors and pepsin solution.
It is 1 that the fixer, which is volume ratio, in one of the embodiments,:3 glacial acetic acid and the mixed liquor of methanol.
The concentration of the colchicine solution is 20 μ g/ml in one of the embodiments,.
The concentration of the KCl is 0.075mol/L in one of the embodiments,.
The concentration of the pancreatin is 0.5wt% in one of the embodiments,.
A kind of application method of the detection kit of acquired cell clone, which is characterized in that include the following steps：
Sample of bone marrow cell is fixed on the slide of precooling；
Respectively using the first fluorescence probe reagent, the second fluorescence probe reagent and third fluorescence probe reagent to being fixed
Sample of bone marrow cell carries out fluorescence in situ hybridization；
Results of hybridization is detected using fluorescence detector.
The application method of the detection kit of the acquired cell clone further includes to solid in one of the embodiments,
Fixed sample of bone marrow cell carries out the step of G band analyses.
Further include the slide to being fixed with sample of bone marrow cell before fluorescence in situ hybridization in one of the embodiments,
It is pre-processed successively, Protease Treatment and the step of dehydration.
The application method of the detection kit of the acquired cell clone further includes to obtaining in one of the embodiments,
Sample of bone marrow the step of carrying out closed culture, harvesting the sample of bone marrow cell.
The detection kit and its application method of above-mentioned acquired cell clone can be used for the ACC situations to sample of bone marrow,
Especially SACC situations are detected tracks with follow-up, and testing result can be in conjunction with clinical endpoint, pathologic index, streaming point
Type index provides important evidence and reference for the clinical early diagnosis of tMDS and tAML, preferably instructs clinician to treatment
Correlation myeloma carries out early diagnosis intervention and treatment.
Description of the drawings
Fig. 1 is acquired cell clone and treatment correlation myelocytome a situation arises schematic diagram, wherein ACC：It obtains
Property cytogenetics clone；SACC：The acquired cytogenetics clone of silence type；tMN：Treat correlation myelocytome；Active
ACC：The acquired cytogenetics clone of activated form.
Fig. 2 is the flow of the acquired cytogenetics clone tracking of silence type, treatment correlation myelocytome early diagnosis, figure
In, SACC:The acquired cytogenetics clone of silence type；AACC:The acquired cytogenetics clone of activated form；tMN:Treat correlation
Myelocytome；Clinical test checks in fact：Include mainly pathology bone marrow examination, periphery blood routine examination, streaming inspection etc..
Fig. 3 is that the G of No. 5 chromosome long arms missing shows band.
Fig. 4 is No. 5 chromosome FISH, wherein slightly bright No. 7 chromosomes of representative, slightly dark No. 5 chromosomes of representative.
Fig. 5 is that the G of No. 7 chromatid deletions shows band.
Fig. 6 is No. 7 chromosome FISH, wherein slightly bright No. 7 chromosomes of representative, slightly dark No. 5 chromosomes of representative.
Specific implementation mode
To facilitate the understanding of the present invention, below with reference to relevant drawings to invention is more fully described.In attached drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.Keep the understanding to the disclosure more thorough on the contrary, purpose of providing these embodiments is
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article and belong to the technical field of the present invention
The normally understood meaning of technical staff is identical.Used term is intended merely to description tool in the description of the invention herein
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the Listed Items of pass.
It is specific embodiment part below.
One, CYTOGENETIC ANALYSIS OF ONE
(1) collection, inoculation, culture of sample of bone marrow, harvest
1. starting to operate the ultraviolet lamp in front opening Biohazard Safety Equipment, sterilize 30 minutes.
2. checking sample, and sample is moved into and carries out inoculation operation in Biohazard Safety Equipment.The multiple marks of each sample print
Label, to be attached to culture bottle, centrifuge tube, suction pipe, on plastic test tube.
3. melting culture medium again：Take out the culture medium (DMEM of -20 DEG C of preservations11965092), melt at 37 DEG C
Rewarming dries culture medium body, posts label, moves into Biohazard Safety Equipment.
4. checking sample and culture medium, infrared sterilamp is opened, culture bottle Jiao Gai is sterilized on sterilamp.Every
In culture bottle of the bottle containing 5ml culture mediums, it is inoculated with 0.5ml sample of bone marrow, totally 2 bottles, is gently shaken up.It is placed in order after verification
It cultivates in box, marks, indicate the information such as inoculation date, time.
5. culture：Cell culture is closed culture, is assigned in two incubators after each sample inoculation and independently cultivates
24-48 hours, cultivation temperature was 37 DEG C.
6. 1.5-2 hours before cell harvest, sample is taken out from incubator, moves into Biohazard Safety Equipment, it is each to cultivate
The colchicine of a concentration of 20 μ g/ml of 0.1ml is added in bottle, after gently shaking up, puts back in incubator and continues to cultivate.
7. opening exhausting cabinet fan and light, culture bottle is taken out from incubator, is put into exhausting cabinet.To needing in exhausting cabinet
The sample for carrying out cell harvest is ranked up, and label is posted to centrifuge tube, suction pipe needed for experiment, will be trained with corresponding suction pipe
The cell piping and druming for supporting bottle is uniform, and is moved into the centrifuge tube of 15ml.
8. being centrifuged 5 minutes with 2000-2200r/min, supernatant is abandoned, is added the 0.075mol/L's preheated at 37 DEG C
KCl solution 8ml, mix well, 23-30 minutes hypotonic in 37 DEG C of water baths.
9. fixer 2ml mixings are added after hypotonic, and (fixer is methanol and glacial acetic acid by volume 3:1 mixing), with
2000-2200r/min is centrifuged 10 minutes, abandons supernatant, the fixer of 8ml is added, gently shakes up, obtain cell suspension, fixed
45 minutes to 1 hours stood overnight.
10. being centrifuged 10 minutes with 2000-2200r/min, supernatant is abandoned, 8ml fixers gently mixing is added, centrifuges again
10 minutes, supernatant is abandoned, suitable fixer is added and is gently uniformly mixed so as to obtain cell suspension.
11. taking out the cleaning slide being pre-chilled in 4 DEG C of ice water, slide is indicated, slide is placed in drop sheet, inspection
Look into the sequence that the experiment number of slide and slide are placed on plate.
12. cell suspension about 0.3ml is drawn on the cleaning slide that 20-25cm height drips in precooling with suction pipe, slide
Each drop of head, body, tail.The slide dripped is placed in 75 DEG C of oven for baking 3 hours, remains to dye.
(2) G bands analysis
1. 3 are indicated respectively pancreatin, physiological saline, dye liquor dye vat be first placed in 37 DEG C of water baths and preheat 20 minutes.
2.0.025% pancreatin configures：The pancreatin for taking a pipe 3.5ml 0.5%, is added in the PBS buffer solution of 70ml, fully
After mixing, it is placed in water bath for use in the dye vat for indicating pancreatin.
3. the preparation of physiological saline：It takes physiological saline 70-100ml to be placed in water bath in the dye vat for indicating physiological saline to wait for
With.
4. Giemsa stain configures：10-15ml Jim Sa mother liquors are taken, are added in 70ml PBS buffer solution, after mixing well,
It is placed in water bath for use in the dye vat for indicating dye liquor.
5. aobvious band, dyeing：According to the aobvious band and dyeing time of preliminary experiment, aobvious band and dyeing are carried out to clinical sample.Generally
Think, bathozone is the chromosome segment rich in A-T, referred to as These positive bands；Shallow band is the chromosome segment rich in C-G, referred to as negative
Band.
6. in each sample, the medium cell of about 20 clearly high quality is chosen as analysis object.
When making marrow karyotyping, pay special attention to the exception for finding No. 13 No. 5 chromosomes, No. 7 chromosome grades chromosome,
Such as whether there is the case where monomer or clone.
Two, fluorescence in situ hybridization (FISH) is tested
(1) slide pre-processes
1. the glass dye vat for filling 50ml deionized waters is preheated to 80 DEG C with water-bath.
2. 10 minutes during slide is immersed into deionized water.
3. pure water rinsing slide is used in glass dye vat at room temperature 3 minutes.
(2) Protease Treatment
1. slide is taken out from glass dye vat, the edge of paper handkerchief is used in combination to absorb extra moisture.
2. the glass dye vat for filling enough protein enzyme solutions is preheated to 37 DEG C in water bath, it is ensured that the temperature of preheating is not
It to be more than 37 DEG C, otherwise protease will inactivate.
3. slide is immersed in protein enzyme solution 15 minutes.
4. using pure water rinsing slide in glass dye vat 3 minutes, and air-dry.
(3) it is dehydrated
1. being rinsed with the ethyl alcohol of a series of a concentration of 70%, 85% and 100%.
2. air-drying.
(4) hybridize
1. preparing FISH probe
1.1, which test the main FISH probe used, includes：
1.2 are coated in suitable probe mixture on slide, covered, blend rubber glue sealing.
Slide is put on ThemoBrite instruments 73 DEG C by 1.3 to be denaturalized 3 minutes.
1.4 on ThemoBrite instruments 37 DEG C overnight.
2. post-hybridization washing
The dye vat for filling 2 × SSC/0.3%NP40 is preheated to 73 DEG C at least 30 minutes by 2.1 in water bath.
2.2 prepare another dye vat for filling 2 × SSC/0.3%NP40 at room temperature, remove the rubber glue on slide
Water and coverslip.
2.3 are preheated to the slide immersion after removal in 73 DEG C of dye vat, rock 1-3 seconds, glass is taken out after impregnating 2 minutes
Piece.
2.4 immerse slide transfer into the dye vat of room temperature, rock 1-3 seconds, are taken out after impregnating 1 minute, be then protected from light wind
It is dry.
2.5 are dyed with DAPI.
Machine carries out fluorescence signal analysis on 2.6.
Three, the determination of acquired cytogenetics clone
1. being analyzed with cytogenetic methods
20 medium cell chromosomes are analyzed, it is found that two or more cells have abnormal structure sex chromosome to change
Become, either finds that three or three or more cells have abnormal number sex chromosome to change；It reuses FISH and confirms acquisition
Property cytogenetics clone.
2. corresponding using related chromosome when not can confirm that abnormal clone using traditional cytogenetic methods analysis
FISH probe carry out fish analysis
The fish analysis of each probe, 200 Interphase cells (interphase) of number, with the boundary of each FISH probe
It is worth (cutoff) to judge, it was demonstrated that abnormal cell genetic clones exist.No. 5, No. 7, No. 13 chromosome abnormality clone's determinations are divided
Dividing value is：No. 5 chromosome monosomy/No. 5 chromosome long arm missings：(EGR1/D5S23,D5S721：5.2%/4.8%)；No. 7 dyes
Colour solid monomer/No. 7 chromosome long arm missing：(D7S522/CEP7：5.6%/5.4%)；No. 13 chromosome monosomy/No. 13 dyeing
Body deletion of long arm：(RB1：4.5%/5.2%)
3. carrying out report analysis to results of the G with dyeing and FISH experiments with the Intemational Nomenclature system of human chromosomal.
Four, other clinical endpoints detect
1. blood routine examination
Paying special attention to peripheral blood hemoglobin concentration reduces and platelet count reduction variation
2. pathology bone marrow analysis
Pay particular attention to the increased variation of bone marrow cell
Five, the acquired cytogenetics clone tracking of silence type, treatment correlation myelocytome early diagnosis, detailed process is such as
Shown in Fig. 2.
Six, experimental result
It is tested by the sample to No. 5 chromosome long arm missings and No. 7 chromatid deletions, G shows band and FISH knots
Fruit as shown in Fig. 3, Fig. 4, Fig. 5 and Fig. 6, is consistent respectively with expection.
Clinical practice proves that the median 80 months after primary tumor chemotherapy of patients/radiotherapy in the treatment can live in marrow
Inspection finds SACC.After SACC is found, clinical tumor doctor and clinical labororatory must track follow-up in periodically every 6 months, pay attention to
Treat the generation of correlation myelocytome clinical manifestation.The hemochrome of periphery blood routine examination reduces or platelet count reduces,
Pathology bone marrow cell finds that cell number raising is that the acquired cytogenetics clonal shift of silence type is lost for the acquired cell of activated form
The tendency for passing clone, to treat the generation of correlation myelocytome.The acquired cytogenetics clone of partial silence type can be certainly
It goes out.
Since treatment correlation myelocytome is usually refractory, tumour medicine is typically easy to generate drug resistance and therapeutic effect
Therefore difference finds that the acquired cytogenetics clone of silence type, clinician can be directed to the clinical condition pair of patient immediately in time
Treatment correlation myelocytome, which is given, intervenes.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
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Claims (10)
1. a kind of detection kit of acquired cell clone, which is characterized in that including the first fluorescence probe reagent, the second fluorescence
Probe reagent and third fluorescence probe reagent；Wherein, the first fluorescence probe reagent and the second fluorescence probe reagent are equal
For Two Colour Fluorescence probe reagent, the third fluorescence probe reagent is one-color fluorescence probe reagent, and first fluorescence probe
The sequence of two detection probes is respectively such as SEQ ID NO in reagent:1 and SEQ ID NO:Shown in 2, the second fluorescence probe examination
The sequence of two detection probes is respectively such as SEQ ID NO in agent:3 and SEQ ID NO:Shown in 4, the third fluorescence probe reagent
The sequence of middle detection probe such as SEQ ID NO:Shown in 5.
2. the detection kit of acquired cell clone as described in claim 1, which is characterized in that further include in following solution
At least one：Fixer, colchicine solution, KCl solution, Jim Sa mother liquor, trypsin solution, SSC washing lotions, NP40 cracking
Liquid, DAPI dyeing liquors and pepsin solution.
3. the detection kit of acquired cell clone as claimed in claim 2, which is characterized in that the fixer is volume
Than being 1:3 glacial acetic acid and the mixed liquor of methanol.
4. the detection kit of acquired cell clone as claimed in claim 2, which is characterized in that the colchicine solution
Concentration be 20 μ g/ml.
5. the detection kit of acquired cell clone as claimed in claim 2, which is characterized in that the concentration of the KCl is
0.075mol/L。
6. the detection kit of acquired cell clone as claimed in claim 2, which is characterized in that the concentration of the pancreatin is
0.5wt%.
7. a kind of application method of the detection kit of acquired cell clone as described in any one of claim 1-6,
It is characterized in that, includes the following steps：
Sample of bone marrow cell is fixed on the slide of precooling；
Respectively using the first fluorescence probe reagent, the second fluorescence probe reagent and third fluorescence probe reagent to being fixed marrow
Sample cell carries out fluorescence in situ hybridization；
Results of hybridization is detected using fluorescence detector.
8. the application method of the detection kit of acquired cell clone as claimed in claim 7, which is characterized in that further include
The step of G band analyses are carried out to fixed sample of bone marrow cell.
9. the application method of the detection kit of acquired cell clone as claimed in claim 7, which is characterized in that in fluorescence
Further include being pre-processed successively, at Protease Treatment and dehydration to being fixed with the slide of sample of bone marrow cell before in situ hybridization
The step of reason.
10. the application method of the detection kit of acquired cell clone as claimed in any one of claims 7-9, feature
It is, further includes the step of carrying out closed culture to the sample of bone marrow of acquisition, harvest the sample of bone marrow cell.
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