CN104313023B - The gene probe of alveolar soft part sarcoma and its kit application - Google Patents
The gene probe of alveolar soft part sarcoma and its kit application Download PDFInfo
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Abstract
The present invention relates to the gene probe of alveolar soft part sarcoma and its kit application.The cloned sequence that gene probe of the present invention is selected is that RP11 634L10, RP11 51H16 and RP11 475F12 are combined respectively, and CTD 2311N12, RP11 416B14, CTD 2522M13, CTD 2312C1 and CTD 2248C21 combinations.Instant invention overcomes RT PCR and cell method of karyotype analysis be cumbersome and time-consuming and RNA degradeds and a lot of other factors limit RT PCR application, and Accurate Diagnosis can not be obtained, the defects of influenceing prognosis and post surgery treatment.Accuracy rate of the present invention is high, specificity is high, success rate is high, fluorescence signal is strong, it is simple to operation, it can be applicable in paraffin section, the scope of detection sample is expanded, the new method of accurately and reliably easy diagnosis alveolar soft part sarcoma is established, has started the beginning using FISH detection alveolar soft part sarcomas.
Description
Technical field
The invention belongs to the application field of fluorescence in situ hybridization probe, the more particularly to gene of alveolar soft part sarcoma is visited
Pin and its kit application.
Background technology
Alveolar soft part sarcoma is that a kind of origin is not clear, is apt to occur in children and young rare pernicious soft tissue sarcoma,
Cardinal symptom shows as the soft mass slowly grown, spy in the tumor tissues of clear and definite alveolar soft part sarcoma be present
The non-equilibrium transposition of specific gene, i.e.,：der(17)t(X；17)(p11.2；Q25), TFE3 telomeres side base is caused at Xp11.2 because of warp
Transposition merges to form ASPL-TFE3 fusions to 17q25, and with the centromere side of 17q25ASPL genes after duplication.Most glands
Easily there is DISTANT METASTASES IN after discovery and treatment in alveolar soft part sarcoma patient, and insensitive to chemicotherapy, and overall prognosis is poor,
5 years, 10 years, 20 years survival rates be 60%, 38%, 15% respectively, therefore Accurate Diagnosis is to alveolar soft part sarcoma patient's
Index for diagnosis and successive treatment have considerable meaning.
Before making the present invention, the diagnosis of alveolar soft part sarcoma relies primarily on Histopathological Characteristics, tumour under light microscopic
Pai Lie Cheng Testis shapes or acinus shape, the fibroid interval that surrounding is not often waited by width wrap, and oncocyte is rounded or polygonal, kytoplasm
Abundant be translucent empty balloon-shaped or eosinophil granule shape, core is not of uniform size, and monokaryon or double-core, kernel is big and obvious, most feature
Endochylema in bar-shaped crystalline solid occurrence probability only in 20%-80% or so.The typical alveolar soft part sarcoma of tissue morphology compared with
Easily diagnosis, Partial tumors tissue lack typical morphologic features, then need and Chromaffionoma, GCT, XP11.2
Transposition/TFE3 Gene Fusion correlation kidneys and melanoma etc. mutually differentiate, especially in rare site of pathological change, transfer stove, thin
It is more difficult to clarify a diagnosis in the case that needle biopsies tissue is less.Alveolar soft part sarcoma forms ASPL-TFE3 fusion bases
Cause, cause the overexpression of TFE3 albumen, therefore the detection of TFE3 SABCs has necessarily special to alveolar soft part sarcoma
Property, but other tumours such as XP11.2 transpositions/TFE3 Gene Fusion correlations kidney, granular cell tumor, adrenocortical carcinoma,
Also the positive findings of TFE3 SABCs occurs in the tissue such as Perivascular epithelioid cell tumor and high-level myxofibrosarcoma,
So TFE3 SABCs can only be as the auxiliary foundation of diagnosis alveolar soft part sarcoma.
For alveolar soft part sarcoma, its specific gene alteration type is the important evidence of its diagnosis, only
There are RT-PCR and cell karyotyping accurate could reach diagnostic purpose, just must be swollen in excision but carry out karyotyping
After tumour cell is carried out into cell culture to m period after knurl, caryogram point is carried out to tumour cell using technologies such as the aobvious bands of G
Analysis, this method it is cumbersome and it is time-consuming be not suitable in clinical position for application.Likewise, consider alveolar soft part sarcoma
Rare property and PCR experiment processes complexity be can not routinely using RT-PCR to alveolar soft part sarcoma tissue carry out
The detection of ASPL-TFE3 fusions, and RNA can only be extracted from paraffin section when suspecting alveolar soft part sarcoma, RNA
Degraded and a lot of other factors limit RT-PCR application, and technical sophistication is not suitable for extensive use.
The patient that these limitations result in some doubtful alveolar soft part sarcomas can not obtain Accurate Diagnosis, influence clinic
Doctor is to alveolar soft part sarcoma patient prognosis and post surgery treatment.At present need badly one kind can quickly, economy, Accurate Diagnosis
The method of alveolar soft part sarcoma.
The content of the invention
The purpose of the present invention is the defects of being directed to above-mentioned diagnostic method, there is provided a kind of gene of alveolar soft part sarcoma is visited
Pin and its kit application.
The technical scheme is that：
The gene probe of alveolar soft part sarcoma, it is mainly characterized by the cloned sequence point of gene probe selection
It is not that RP11-634L10, RP11-51H16 and RP11-475F12 are combined, and CTD-2311N12, RP11-416B14, CTD-
2522M13, CTD-2312C1 and CTD-2248C21 are combined.
Wherein RP11-634L10, RP11-51H16 and RP11-475F12 combination is to be positioned at No. 17 chromosome ASPL
On gene, three is labeled as identical red fluorescent.
Wherein CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-2248C21 group
Conjunction is positioned on X chromosome TFE3 genes, and mark is green florescent signal.
The cloned sequence being positioned on No. 17 chromosome ASPL genes, RP11-634L10, RP11-51H16 and
It must be completely covered between ASPL genes and adjacent cloned sequence and exist on a small quantity between this 3 cloned sequences of RP11-475F12
Fragment is overlapping.
The cloned sequence being positioned on X chromosome TFE3 genes, CTD-2311N12, RP11-416B14, CTD-
TFE3 genes and homonymy must be completely covered in 2522M13, CTD-2312C1 and CTD-2248C21 this 5 cloned sequence combination
Exist between adjacent cloned sequence a small amount of fragment it is overlapping or adjacent between at a distance of being no more than 10Kb.
The present invention another technical scheme be：
The gene probe kit of alveolar soft part sarcoma, the kit by probe hybridization solution and 4 ', 6- diamidino-
2-phenylindone counterstain forms, and it is mainly characterized by：
(1) RP11-634L10, RP11-51H16 and RP11-475F12 group being positioned on No. 17 chromosome ASPL genes
Close, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2312C1 and CTD-2248C21 are combined, labeled as green florescent signal；
(2) probe hybridization solution is mixed in proportion with Human Cot-1DNA, hybridization buffer, purified water by the probe
Configuration is formed, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6- diamidino -2-phenylindone counterstain are mainly used in nuclear targeting.
RP11-634L10, RP11-51H16 and RP11-475F12 group being positioned on No. 17 chromosome ASPL genes
Close, must be completely covered between ASPL genes and adjacent cloned sequence that a small amount of fragment to be present overlapping between this 3 cloned sequences,
Three is labeled as identical red fluorescent；Be positioned at CTD-2311N12, RP11-416B14 on X chromosome TFE3 genes,
CTD-2522M13, CTD-2312C1 and CTD-2248C21 are combined, and X dyeing must be completely covered in the combination of this 5 cloned sequence
Exist between body TFE3 genes and the adjacent cloned sequence of homonymy a small amount of fragment it is overlapping or adjacent between be apart no more than
10Kb, above-mentioned probe and Human Cot-1 DNA, hybridization buffer, purified water are mixedly configured into probe hybridization solution in proportion.
The present invention also has a kind of technical scheme to be：
The gene probe kit application of alveolar soft part sarcoma, it is mainly characterized by soft in diagnosis acinus shape
Application in sarcomatous tissue.
Advantages of the present invention and effect are in the distinctive ASPL- in using FISH technology detection alveolar soft part sarcoma
TFE3 fusions in gene level so that diagnose the tumour, and the accuracy rate height of gene probe application, specificity are high, success rate
Height, fluorescence signal is strong, simple to operation, can be applicable in paraffin section, has expanded the scope of detection sample, has established accurately and reliably
The new method of simplicity diagnosis alveolar soft part sarcoma, has started the beginning using FISH detection alveolar soft part sarcomas.
Brief description of the drawings
Fig. 1 --- gene probe station-keeping mode schematic diagram.
Fig. 2 --- male's granular cell carcinoma negative control case application schematic diagram.
Fig. 3 --- women granular cell carcinoma negative control case application schematic diagram.
Fig. 4 --- male's alveolar soft part sarcoma application schematic diagram.
Fig. 5 --- women alveolar soft part sarcoma application schematic diagram.
Fig. 6 --- male's alveolar soft part sarcoma application schematic diagram.
Fig. 7 --- women alveolar soft part sarcoma application schematic diagram.
Embodiment
The present invention technical thought be：
FISH be using be marked with fluorescein probe specificity with chromosome and (or) gene loci phase
With reference to by the type of fluorescence microscope fluorescence signal, so as to detect the method for chromosome and corresponding gene change, having
Safety, quick, economy, high sensitivity, detection signal is strong, hybrid specificities are high, can show the advantages that multiple color simultaneously, and
It compensate for the defects of conventional method can not diagnose to Interphase cells, complex karyotype cell and microdeletion.Meanwhile fluorescence
Hybridization in situ technique can apply to carry out retrospective study on the sample of FFPE, greatly reduce detection to studying sample
Requirement.Based on the fast development of fluorescence in situ hybridization technique in recent years, the present invention proposes according to the principle of FISH
A kind of gene probe and its kit application for being used to diagnose alveolar soft part sarcoma.
Below by embodiment, the invention will be further elaborated.
The gene probe that the present invention uses is different from prior art, and it is soft to belong to first Application FISH technology detection acinus shape
Sarcomatous tissue, it is based on the research to alveolar soft part sarcoma gene alteration feature and to TFE3 genes and ASPL genes
The scheme that the comprehensive analysis of specific BAC cloned sequences binding site filters out.
In the present invention, BAC cloned sequences on ASPL genes are RP11-634L10 (fragment length is about 172Kb),
RP11-51H16 fragment lengths are about 166Kb) and RP11-475F12 (fragment length is about 185Kb) combination；On TFE3 genes
BAC cloned sequences for CTD-2311N12 (fragment length is about 210Kb), RP11-416B14 (fragment length is about 182Kb),
CTD-2522M13 (fragment length is about 182Kb), CTD-2312C1 (fragment length is about 210Kb) and CTD-2248C21 (pieces
Segment length is about 88Kb) combination.These fragments are bacterial artificial chromosome clone (BAC clones), and it is in human chromosomal
On positioning it is disclosed.
Select two groups totally 8 cloned sequences have some following advantage：(1) most cloned sequence sizes are substantially optimum
Mark lengths 150-200Kb or so, not only signal intensity is similar each other for such cloned sequence, and is not in because clone
The strength difference that fragment is different in size to cause signal is larger；(2) a small amount of overlapping sequences or phase be present between homonymy adjacent segment
Away from no more than 10Kb so that same color signal is mutually continuous, is not in the fracture of homonymy signal and going out for independent signal
It is existing, reduce the probability of erroneous judgement；(3) two groups of cloned sequences complete gene selected by covering, can be with alveolar soft part sarcoma
Form single fusion signal.
BAC cloned sequence mark fluorescents, fluorescence microscope is easy to use, it is convenient directly perceived, meanwhile, if desired using it
His detection technique can also be marked as corresponding detection label.
The effect of the present invention：
The present invention is according to the characteristics of the group translocation of alveolar soft part sarcoma, using FISH principle,
The gene probe of not homochromy fluorescence labeling is used at TFE3 genes and ASPL genes respectively, is cut into slices by detecting paraffin-embedded tissue
On fusion or separation signal, judge whether tumor tissues ASPL-TFE3 genes occur the non-equilibrium transposition of specific gene and melt
Close, accuracy rate of diagnosis is improved, to judge that patient's prognosis and postoperative adjuvant therapy provide accurate information and guidance.Set in probe
After the completion of meter, it is applied to the detection for the alveolar soft part sarcoma clarified a diagnosis, should according to our experimental result
The accuracy rate of probe application is high, specificity is high, success rate is high, and experimental signal is strong, simple and quick, can especially be cut in paraffin
Applied in piece, expanded the scope of detection sample, new method and instrument are provided for Accurate Diagnosis alveolar soft part sarcoma.
Embodiment 1：
Step 1：The preparation of gene probe：
Pass through http://genome.ucsc.edu/ searches bacterium people corresponding to X chromosome TFE3 genes and ASPL genes
Work chromosome (BAC clones), corresponding BAC cloned sequences are selected respectively, control BAC cloned sequences selected by homonymy completely to cover phase
For gene, and homonymy fragment has the overlapping of certain sequence between each other.The BAC chosen by above-mentioned requirements on ASPL genes is cloned
Fragment is RP11-634L10 (chr17：79796813-79969288, fragment length are about 172Kb), RP11-51H16
(chr17：79931578-80097452, fragment length are about 166Kb) and RP11-475F12 (chr17：80032268-
80216871, fragment length is about 185Kb) combination；BAC cloned sequences on TFE3 genes are CTD-2311N12 (chrX：
48713289-48923401, fragment length are about 210Kb), CTD-2522M13 (chrX：48420425-48602847, fragment
Length is about 182Kb), RP11-416B14 (chrX：48580872-48763198, fragment length are about 182Kb), CTD-
2312C1(chrX：48959513-49170367, fragment length are about 210Kb), CTD-2248C21 (chrX：49154756-
49242991, fragment length is about 88Kb).And in http://projects.tcag.ca/cgi-b i n/efi sh/
Index.cgi carries out specific analysis, the specificity of clearly selected BAC fragments on chromosome to selected BAC clones.This
Gene probe station-keeping mode in invention is as shown in Figure 1.Corresponding BAC clones are purchased from Invitrogen companies, BAC is cloned
Plasmid is extracted after culture, using nick-translation, 3 BAC cloned sequences of ASPL genes are used
Tetramethylrhodamine-5-dUTP is labeled as red fluorescence, and the BAC cloned sequences of TFE3 genes are used
Fluorescein-12-dUTP is labeled as green fluorescence, and both ends color can exchange.In order to detect the correctness of structure clone,
The cloned sequence of each mark fluorescent signal is individually dripped into piece (by the peripheral blood cells of healthy human body by training in cell respectively
Support, colchicine processing is hypotonic, obtains the suspension containing a large amount of mitotic cells after fixed and is made) on to carry out hybridization positioning real
Test, the positioning of cloned sequence on chromosome is observed on mitosis metaphase cell.Above-mentioned 10 groups of cloned sequences pass through respectively
Checking, can be accurately positioned on ASPL genes and TFE3 genes, finally by cloned sequence mark purification after with Human
Cot-1DNA, hybridization buffer, purified water are configured to probe hybridization solution, -20 DEG C of lucifuge freezen protectives in proportion.
In next step, the method that the probe of preparation is used to FISH, is alveolar soft part in clinical diagnosis
Its diagnosis effect is verified in sarcoma and granular cell carcinoma.
Step 2：FISH process：
(1) histotomy pre-processes：The tumor tissue section previously prepared is baked into piece in 65 ± 1 DEG C of insulating boxs to stay overnight, taken
Xylene soak 30mi n are put into after going out, place into soaked in absolute ethyl alcohol 10min.It is subsequently placed into 100%, 85%, 70% gradient
Each 3min of rehydration in ethanol and purified water, it is put into purified water 100 ± 5 DEG C and boils piece 20min.After section taking-up is dried, in sample
100ul stomach cardia enzyme liquids, digestion 5mi n is added dropwise in region.Rapid be put into 2 × SSC solution washs 5min, is finally putting into room temperature
70%th, 85%, 100% graded ethanol is dehydrated each 3min successively, and taking-up is dried.(2) histotomy and probe co-variation：Add 10 μ l
Probe hybridization solution to dry 22x22mm cover glasses on, by step (1) treat histotomy sample face lower cover in lid glass
On piece, light cap slide is uniformly distributed hybridization solution after reversion, with rubber cement along cover glass edge mounting.Slide is placed on 85
5min is denatured in ± 1 DEG C of thermal station, is put into the hybridizing box of preheating rapidly, 37 ± 1 DEG C of lucifuges are incubated overnight.(3) washed after hybridizing
Wash and redye：In 37 ± 1 DEG C of water bath, preheating 2xSSC solution, 0.1%NP-40/2xSSC solution.By step (2) processing
The histotomy crossed removes rubber cement and cover glass, is put into 2xSSC solution and washs 10min, places into 0.1%NP-40/
5min is washed in 2xSSC solution, then 70% ethanol dehydration 3min, dark place are dried at room temperature.10 μ l DAPI counterstains are added dropwise extremely
It is on another dry 22x22mm cover glass, the histotomy sample previously dried is face-down, target area is contacted lid glass
Piece, light cap slide removes bubble removing after reversion, and dark place is deposited to be seen.
Result judgement：
Excited in darkroom with the color optical filterings of 0lympus B × 5I fluorescence microscopes DAPI/FIFC/TexasRed tri-, 100
Times thing Microscopic observation FISH fluorescence signals, the fish analysis software analysis image provided with Imstar companies, assess whole section
Probe hybridisation events.
Using fluorescence microscope it is observed that two kinds of signal types：(1) red green separation signal：TFE3 genes are not occurring
With in the tumor tissues of ASPL group translocations, because X chromosome and No. 17 chromosomes are apart from each other, then distinguishing respectively on chromosome
Mark red and green signals independently occur, and the quantity of red and green signals depends on X chromosome and No. 17 chromosome numbers in nucleus, normally
Due to two X chromosomes and 2 No. 17 chromosomes+2 greens of 2 danger signals just occur, and male only goes out in women
The green conjunction signal of existing 2 danger signals+1；(2) single fusion signal：When TFE3 telomere side bases in alveolar soft part sarcoma occur
During because non-equilibrium transposition occurs with No. 17 chromosome ASPL gene telomeres sides, X chromosome structure does not have any change, and new 17
Number chromosome then by ASPL genes centromere side with transposition and Lai TFE3 telomeres side base because forming, because red and green signals mark covers
Covered the full section of TFE3 genes and ASPL genes, thus cause to marked after transposition the ASPL genes centromere side of danger signal with
The TFE3 telomeres side base of green be marked because mutually adjoining in position, red green continuous signal is formed or yellow signal is (red
The effect of green double-colored superposition).Women alveolar soft part sarcoma patient may occur in which 1 fusion+2 green of danger signal of signal+1
Signal, represent respectively on the ASPL genes and replicated and next X chromosome in the nucleus on 1 No. 17 autosome
There occurs non-equilibrium transposition to merge for TFE3 genes, while remains 2 normal X chromosomes and 1 normal No. 17 chromosome.Man
Property patient 1 fusion+1 danger signal of green+1 of signal occurs, represent in the nucleus 1 No. 17 often dyeing respectively
ASPL genes on body and TFE3 genes that are replicated and coming are merged there occurs non-equilibrium transposition and 1 normal X chromosome
With 1 normal No. 17 chromosome.
Each section is at least observed 100 in the region that nuclear targeting is clear, hybridization signal is stronger and not weighed mutually
Folded nucleus and there are clear and definite FISH signals just to can determine that FISH detections are effective.There is fusion letter in neoplastic cell nuclei more than 20%
Number, it is determined as that the non-equilibrium transposition FISH detections of ASPL-TFE3 are positive, alveolar soft part sarcoma can be diagnosed as；Only occur red
Green separation signal or signal are not up to threshold value, then are determined as that the non-equilibrium transposition FISH detections of ASPL-TFE3 are negative, it is impossible to diagnose
For alveolar soft part sarcoma.
As a result：
We are detected to 10 alveolar soft part sarcomas and granular cell carcinoma sample using the gene probe, wherein
5 have been diagnosed as alveolar soft part sarcoma, 5 granular cell carcinomas.If there is fusion signal and reached in patient's pathological section
Positive criteria is diagnosed to FISH, that is, is diagnosed as alveolar soft part sarcoma.In 5 alveolar soft part sarcomas all
There is signal and reach positive threshold value, the non-equilibrium transposition FISH detections of ASPL-TFE3 are positive, meet alveolar soft part sarcoma
Diagnosis；And granular cell carcinoma histotomy does not detect to separate signal, it is judged as feminine gender.Fig. 2 is male's granular cell carcinoma
Negative control case ,+1 green of 2 danger signals is seen in tumor tissue cell's core, 2 is represented respectively and does not occur
ASPL and No. 17 chromosomes and 1 normal X chromosome of TFE3 genic balance transpositions；Fig. 3 is that women granular cell carcinoma is negative
Case is compareed ,+2 greens of 2 danger signals are seen in tumor tissue cell's core, 2 is represented and ASPL and TFE3 does not occur
No. 17 chromosomes and 2 normal X chromosomes of genic balance transposition；Fig. 4 is male's alveolar soft part sarcoma case, swollen
1 fusion green of danger signal+1 of signal+1 is seen in tumor tissue nucleus, 1 fusion signal is represented in No. 17 dyeing
For body there occurs ASPL and the non-equilibrium transposition of TFE3 genes, 1 danger signal represents 1 No. 17 that transposition does not occur into the cell
Autosome, 1 green represent 1 normal X chromosome；Fig. 5 is women alveolar soft part sarcoma case, in tumour
1 fusion green of danger signal+2 of signal+1 is seen in histocyte core, represents that there occurs ASPL in No. 17 chromosomes
With the non-equilibrium transposition of TFE3 genes, while also retain 1 normal No. 17 chromosome and 2 normal X chromosomes；Fig. 6 is
Male's alveolar soft part sarcoma, 1 fusion green of danger signal+1 of signal+1 is seen in tumor tissue cell's core,
Represent respectively and ASPL and the non-equilibrium transposition of TFE3 genes, and 1 No. 17 dyeing that transposition does not occur occur in No. 17 chromosomes
Body and 1 X chromosome；Fig. 7 is women alveolar soft part sarcoma, and 1 fusion signal+1 is seen in tumor tissue cell's core
+ 2 greens of danger signal, represent ASPL and No. 17 chromosomes of the non-equilibrium transposition of TFE3 genes occurs respectively, and 1
Normal No. 17 autosomes and 2 normal X chromosomes.
Evaluation：
Using FISH technology, autonomous Design gene probe detection alveolar soft part sarcoma is domestic first Application.At me
Cloned sequence size used is relatively uniform on the gene probe that designs, each cloned sequence marking signal intensity is more similar,
The probe signals of homonymy mark occur without interruption, and the more general probe of signal intensity is strong, and specificity is high, fully take into account and are applied to examine
The reliability and validity of disconnected reagent and probe when being prepared into diagnostic kit, meet the requirement applied to clinical diagnosis.
Using fluorescence in situ hybridization technique, had using gene probe diagnosis alveolar soft part sarcoma accurate, quick, economic and successful
The high characteristic of rate, greatly optimize the diagnostic method of alveolar soft part sarcoma.It can be seen that utilizing the spy from accompanying drawing 2-3
It is negative that pin, which detects all granular cell carcinoma FISH results, and soft group of the probe in detecting acinus shape is shown in accompanying drawing 4-7
Knit and FISH detections are successfully carried out in the tumor tissue section of sarcoma, signal, while FISH testing results and typical disease occur
It is consistent to manage diagnostic result.The experimental result display present invention is in the high advantage of high specificity and sensitiveness, while the probe is hybridized to
Power is high, and signal is clear, and brightness is high, meets the requirement for being prepared into diagnostic kit.
Embodiment 2：Alveolar soft part sarcoma diagnostic kit
The gene probe kit of alveolar soft part sarcoma, the kit by probe hybridization solution and 4 ', 6- diamidino-
2-phenylindone counterstain forms, it is characterised in that：
(1) RP11-634L10, RP11-51H16 and RP11-475F12 group being positioned on No. 17 chromosome ASPL genes
Close, labeled as red fluorescent；It is positioned at CTD-2311N12, RP11-416B14, CTD- on X chromosome TFE3 genes
2522M13, CTD-2312C1 and CTD-2248C21 are combined, labeled as green florescent signal；
(2) probe hybridization solution is mixed in proportion by the probe and Human Cot-1 DNA, hybridization buffer, purified water
Configuration is closed to form, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6- diamidino -2-phenylindone counterstain are mainly used in nuclear targeting.
(4) being positioned on No. 17 chromosome ASPL genes described in the gene probe kit of alveolar soft part sarcoma
RP11-634L10, RP11-51H16 and RP11-475F12 are combined, and ASPL genes must be completely covered between this 3 cloned sequences
And have that a small amount of fragment is overlapping, and three is labeled as identical red fluorescent between adjacent cloned sequence；It is positioned at X dyeing
CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-2248C21 are combined on body TFE3 genes,
The combination of this 5 cloned sequence must be completely covered between X chromosome TFE3 genes and the adjacent cloned sequence of homonymy exist it is few
Apart it is no more than 10Kb between the fragment of amount is overlapping or adjacent.By above-mentioned probe and Human Cot-1DNA, hybridization buffer,
Purified water is mixedly configured into probe hybridization solution in proportion.
Detection kit application method of the present invention is as follows：
1. the sample comprising tumor tissues is fixed through 10% neutral formalin, 3um tissues are prepared into after FFPE
Section；
Stayed overnight 2. section is placed in into 65 ± 1 DEG C of insulating boxs and bakes piece, xylene soak 30min is put into after taking-up, places into nothing
Water-ethanol soaks 10min.Each 3mi n of rehydration in 100%, 85%, 70% graded ethanol and purified water are subsequently placed into, are placed into pure
100 ± 5 DEG C are boiled piece 20min in change water；
3. after taking-up are dried, 100ul pepsin reaction solutions being added dropwise in sample areas, digest 5min, it is put into 2 rapidly ×
SSC washs 5min, is finally putting into the graded ethanol of room temperature 70%, 85%, 100% and is dehydrated each 3mi n successively, taking-up is dried；
4. the probe hybridization solution of the 10 μ l present invention is added to overturn sample chips to drying on 22x22mm cover glasses, make sample court
Under, cover rapidly, light cap slide is uniformly distributed hybridization solution after reversion, with rubber cement along cover glass edge mounting, completely
The edge that covering cover glass contacts with slide；
5. slide is placed in 85 ± 1 DEG C of thermal station, 5min is denatured, slide is put into the hybridizing box of preheating rapidly, kept away
Light, 37 ± 1 DEG C of overnight incubations；
6. 2xSSC solution, 0.1%NP-40/2xSSC solution to be put into 37 ± 1 DEG C of water-bath and be preheated, removal is cut
Rubber cement and cover glass on piece, slide is put into 2xSSC solution and washs 10min, then is transferred to 0.1%NP-40/2xSSC
5min is washed in solution；Then the ethanol dehydration 3min of room temperature 70%；
After 7. taking-up slide dries in the dark, it is added dropwise on 10 μ l DAPI counterstains to dry 22x22mm cover glasses, instead
Turn sample chips, contact the target area of cover glass slide, gently pressed after reversion, avoid producing bubble, deposit in the dark, use
Fluorescence microscope fluorescence signal.Each section is at least seen in the region that nuclear targeting is clear, hybridization signal is stronger
Observe the nucleus of 100 non-overlapping copies and there are clear and definite FISH signals just to can determine that FISH detections are effective.It is effective when being determined as
FISH detection section in, red and green signals individually occur or signal be not up to 20% threshold value, then be determined as that ASPL-TFE3 is non-
Balanced translocation FISH detections are negative, it is impossible to can be diagnosed as alveolar soft part sarcoma；When being determined as cutting for effective FISH detection
In piece, there is fusion signal in the neoplastic cell nuclei for having more than 20%, then is determined as the non-equilibrium transposition FISH detections sun of ASPL-TFE3
Property, alveolar soft part sarcoma can be diagnosed as.
Claims (7)
1. the gene probe of alveolar soft part sarcoma, it is characterised in that the cloned sequence that gene probe is selected is RP11- respectively
634L10, RP11-51H16 and RP11-475F12 combine, and CTD-2311N12, RP11-416B14, CTD-2522M13,
CTD-2312C1 and CTD-2248C21 combinations.
2. the gene probe of alveolar soft part sarcoma according to claim 1, it is characterised in that wherein RP11-
634L10, RP11-51H16 and RP11-475F12 combination are positioned on No. 17 chromosome ASPL genes, and three is labeled as identical
Red fluorescent.
3. the gene probe of alveolar soft part sarcoma according to claim 1, it is characterised in that wherein CTD-
2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-2248C21 combination are to be positioned at X chromosome
On TFE3 genes, mark is green florescent signal.
4. the gene probe of alveolar soft part sarcoma according to claim 2, it is characterised in that be positioned at No. 17 dyeing
Cloned sequence on body ASPL genes, must between this 3 cloned sequences of RP11-634L10, RP11-51H16 and RP11-475F12
It must be completely covered between ASPL genes and adjacent cloned sequence that a small amount of fragment to be present overlapping.
5. the gene probe of alveolar soft part sarcoma according to claim 3, it is characterised in that be positioned at X chromosome
Cloned sequence on TFE3 genes, CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-
The combination of 2248C21 this 5 cloned sequence must be completely covered between TFE3 genes and the adjacent cloned sequence of homonymy exist it is few
Apart it is no more than 10Kb between the fragment of amount is overlapping or adjacent.
6. the gene probe kit of alveolar soft part sarcoma, it is characterised in that the kit by probe hybridization solution and 4 ',
6- diamidinos -2-phenylindone counterstain composition：
(1) RP11-634L10, RP11-51H16 and the RP11-475F12 being positioned on No. 17 chromosome ASPL genes
Combination, labeled as red fluorescent；Be positioned at CTD-2311N12, RP11-416B14 on X chromosome TFE3 genes,
CTD-2522M13, CTD-2312C1 and CTD-2248C21 are combined, labeled as green florescent signal；
(2) probe hybridization solution is by the probe and HumanCot-1DNA, hybridization buffer, purified water mixed configuration in proportion
Into, it is necessary to -20 DEG C of lucifuge freezen protectives；
(3) 4 ', 6- diamidino -2-phenylindone counterstain are mainly used in nuclear targeting.
7. the gene probe kit of alveolar soft part sarcoma according to claim 6, it is characterised in that：It is positioned at 17
RP11-634L10, RP11-51H16 and RP11-475F12 combination on number chromosome ASPL genes, between this 3 cloned sequences
It must be completely covered between ASPL genes and adjacent cloned sequence that a small amount of fragment to be present overlapping, three is red labeled as identical
Fluorescence signal；It is positioned at CTD-2311N12, RP11-416B14, CTD-2522M13, CTD- on X chromosome TFE3 genes
X chromosome TFE3 genes and homonymy must be completely covered in 2312C1 and CTD-2248C21 combinations, the combination of this 5 cloned sequence
Exist between adjacent cloned sequence a small amount of fragment it is overlapping or adjacent between at a distance of being no more than 10Kb, by above-mentioned probe and
HumanCot-1DNA, hybridization buffer, purified water are mixedly configured into probe hybridization solution in proportion.
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Non-Patent Citations (5)
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| Age-related genetic and epigenetic chromosomal changes: A twin study;Kimberly Jones;《Virginia Commonwealth University》;20041231;正文第94页表12 * |
| Feasibility of Differential Diagnosis of Kidney Tumors by Comparative Genomic Hybridization of Fine Needle Aspiration Biopsies;Joana Vieira et al;《GENES, CHROMOSOMES & CANCER》;20101231;第49卷;第936右栏最后1段—937页左栏第1段 * |
| High resolution array CGH and gene expression profiling of alveolar soft part Sarcoma;Shamini Selvarajah et al;《clin cancer res》;20140203;摘要以及第1522页右栏第3段 * |
| Newly Designed Break-Apart and ASPL-TFE3 Dual-FusionFISH Assay Are Useful in Diagnosing Xp11.2 TranslocationRenal Cell Carcinoma and ASPL-TFE3 Renal Cell Carcinoma;Xiancheng Chen， et al;《Medicine》;20150531;第94卷(第19期);1-8页 * |
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