CN101988091A - In-situ hybridization detection kit and detection method for PTEN gene, and application of kit - Google Patents

Abstract

The invention relates to an in-situ hybridization detection kit for a PTEN gene. The kit comprises a hybridization probe and a marker, wherein the hybridization probe has a sequence shown as SEQ ID NO.1. The invention also provides an in-situ hybridization detection method for the PTEN gene. In addition, the invention provides application of the kit to the preparation of medicaments for detecting cancers. The invention has the advantages that: the kit has the characteristics of high sensitivity and specificity; and the detection method is convenient and easy to operate and can be universally used and popularized in hospitals of district-level or above.

Description

A kind of hybridization in situ detection kit of PTEN gene and detection method thereof and application

[technical field]

The present invention relates to a kind of test kit, specifically about a kind of hybridization in situ detection kit and detection method and application of PTEN gene

[background technology]

Cancer also is malignant tumour.Tumour is meant body under the effect of various tumorigenesis factor, the cellular abnormality hyperplasia of local organization and the local lump that forms.Innocent tumour is removed clean easily, does not generally shift, does not recur, and organ, tissue are had only extruding and blocking action.But the 26S Proteasome Structure and Function of all right disorganize of malignant tumour, organ causes downright bad hemorrhage concurrent infection, and the patient finally may the death owing to the organ failure.

The fundamental unit of cancerous lesion is a cancer cells.Have neonatal cell after the aging death of human body cell and replace it, to keep body function.As seen, human body overwhelming majority cells can hyperplasia, but this hyperplasia is limited, and the hyperplasia of cancer cells then is endless, and this makes the intravital nutritive substance of patient by mass consumption.Simultaneously, cancer cells can also discharge multiple toxin, makes human body produce a series of symptoms.If find and treat untimely, it also can be transferred to whole body growth and breeding everywhere, causes at last that human body is become thin, unable, anaemia, poor appetite, heating and organ function are impaired etc.

There is the human Use immunohistochemistrySP SP to detect Rb2/p130 and PTEN in 30 routine normal endometrial tissues, 32 routine simple form Swiss cheese hyperplasias, 31 routine complexity Swiss cheese hyperplasias, 36 routine atypical hyperplasias, the 64 example endometrial carcinoma tissues, the expression of KI-67.Rb2/p130 and the pten protein positive expression in normal endometrial tissue, simple form Swiss cheese hyperplasia, complexity Swiss cheese hyperplasia, atypical hyperplasia, uterine endometrium cancerous tissue successively decreases successively as a result, and the positive expression rate of KI-67 increases progressively successively, Rb2/p130 and PTEN positive expression rate are starkly lower than uterus and simple property hyperplasia of normal proliferative phase in carcinoma of endometrium and the atypical hyperplasia, difference all has significance, and (P is equal,＜0.01) trigenic expression is relevant with the clinical pathological stages of cancer.Recent Phosphoric acid esterase and the tensin homologue gene (phosphatase and tension homolog deleted on chromosome ten.PTEN) of discovering i.e. No. 10 chromosome deletion of PTEN gene, it is a kind of cancer suppressor gene of discovering by STECK etc. in 1997 with phosphatase activity, be called MMAC1 (muted in multiple advanced cancers) or TEP1 again, this gene is positioned at karyomit(e) 10q23.3 " on, total length 2095bp.Discover that at present the PTEN gene has certain effect in natural death of cerebral cells migration and tumour take place, develop.Even finding is in the recent period also needed to be positioned in the nucleus by the normal PTEN that produces of cell, to guarantee tumor suppression function completely.What order was complete studies have shown that in dissimilar cancers, PTEN can leave nucleus, by hemocyte unusual in the acute myeloid leukemia is analyzed discovery, wherein PTEN flees from out from nucleus, therefrom observed another kind of tumor-inhibiting factor PML, PML is the one of the main reasons that causes acute myeloid leukemia, and losing of the PML factor is the root that causes PTEN to flee from from cell.By discovering that further PML factor resistance met a kind of activity of the HAUSP of being called enzyme, this kind of enzyme extracellular of under normal circumstances PTEN being led.

Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics, so far, we might do more accurate early diagnosis on gene level, form the early prediction diagnosis that (during mono-clonal) just can accomplish gene level in canceration early stage or cancer cells.Adopt nucleic acid hybridization in situ technology for detection PTEN gene more than detecting the pten protein sensitivity, and, more detect the generation and the PTEN gene-correlation of cancer in the time of morning.PTEN gene order NM-000314, CDS:1032 ... 2243.

Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark, under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.

[summary of the invention]

The objective of the invention is provides a kind of purposes of hybridization in situ detection kit of PTEN gene at deficiency of the prior art.

One purpose more of the present invention is that a kind of hybridization in situ detection kit of PTEN gene is provided.

Another purpose of the present invention is that a kind of in situ hybridization detection method of PTEN gene is provided.

For achieving the above object, the technical scheme taked of the present invention is:

The application of a kind of hybridization in situ detection kit of PTEN gene in preparation detection Cancerous disease medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence is shown in SEQ ID NO.1.

The RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

Described marker is selected from radionuclide or non-radioactive marker.

Described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

Described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

Described non-radioactive marker is preferably from digoxin.

Described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

A kind of hybridization in situ detection kit of PTEN gene comprises hybridization probe, marker, and wherein said hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is:

A kind of in situ hybridization detection method of PTEN gene, this method may further comprise the steps:

A, the hybridization probe in the test kit is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

The condition that forms hybridization complex in the described a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour, and described substrate is selected blood cell sample or histocyte sample for use.

The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein Za Jiao concrete steps comprise:

Instrumentation:

1). sample to be measured is put into reactive tank;

2). instrument discards liquid automatically, adds Digestive system automatically;

3). instrument discards liquid automatically, and the back is fixing automatically;

4). instrument discards liquid automatically, automatically prehybridization (42 ℃);

5). instrument discards liquid automatically, cleans automatically;

6). instrument discards liquid automatically, automatically hybridization (42 ℃);

7). instrument discards liquid automatically, cleans automatically;

8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);

9). instrument discards liquid automatically, cleans colour developing automatically;

10). take out the mounting microscopy.

The invention has the advantages that:

1, test kit provided by the invention has characteristics highly sensitive, high specificity.

2, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.

3, clinical meaning of the present invention is that more early stage tracking detects cancer generation dynamic process, and the detection and the screening implement that are used for cancer prevention medical science.Diagnostic kit of the present invention is with other detects the oncofetal protein mark clinically, and the medical imaging inspection has remarkable difference.The present invention can detect the PTEN unconventionality expression on gene level, before medical imaging inspection and other inspection is not found occupancy cancer focus, before the cancer biochemical indicator does not produce unusually, also do not form before the lump, can accomplish the information acquisition of above abnormal gene expression early, give real prognosis early diagnosis of clinical cancer sufferer.So just might implement early diagnosis, early prevention, the early treatment of cancer, might from the source, thoroughly effect a radical cure the cancer foul disease.

[description of drawings]

Fig. 1 is a uterus cancer patient PTEN genetic expression picture in the embodiment of the invention.

Fig. 2 is an ovarian cancer patient P TEN genetic expression picture in the embodiment of the invention.

Fig. 3 is a normal people PTEN genetic expression picture in the embodiment of the invention.

[embodiment]

Below in conjunction with accompanying drawing the specific embodiment of the invention is elaborated.

Embodiment 1

A kind of hybridization in situ detection kit of PTEN gene comprises hybridization probe, marker, synergistic agent, and wherein, described hybridization probe sequence is shown in SEQ ID NO.1.The hybridization probe digoxigenin labeled.Other liquid and sample in the test kit are composed as follows:

Digestive system 100 μ l/ manage 1 pipe/box colourless transparent liquid

Protection liquid 100 μ l/ pipe 1 pipe/box colourless transparent liquid

Prehybridization solution 1300 μ l/ manage 2 pipe/box colourless transparent liquids

Justice hybridization solution 10 μ l/ pipe 1 pipe/box colourless transparent liquid

Antisense hybridization solution 10 μ l/ manage 1 pipe/box colourless transparent liquid

Confining liquid 1000 μ l/ manage 1 pipe/box colourless transparent liquid

Alkaline phosphatase enzyme antibody 1 μ l/ manages 1 pipe/box colourless transparent liquid

Developer A 175 μ l/ manage 1 pipe/box yellow liquid

Developer B 320 μ l/ manage 1 pipe/box colourless transparent liquid

Light yellow or the colourless transparent liquid of damping fluid I 10x 90ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid II 10x 80ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid III 10x 20m/ bottle 3 bottle/boxes

Light yellow or the colourless transparent liquid of damping fluid IV 10x 90ml/ bottle 1 bottle/box

Stationary liquid 90ml/ bottle 1 bottle/box colourless transparent liquid

6/box of positive control sample

Mentioned reagent composition explanation: (all reagent are available from SIGMA)

1, Digestive system: the 20mg/ml Proteinase K, the 100mg Proteinase K adds DEPC-H ₂O 5ml;

2, the glycine of protection liquid: 0.2g adds 1 * damping fluid I of 1ml;

3, prehybridization solution: 1 * damping fluid II 7.5ml

50×D?3ml

10mg/ml?yest?t-RNA?750ul

11mg/ml?SALMON?TESTES?DNA 682ul

0.04M?EDTA?3ml

50％?formamide?15ml

4, the bloking of confining liquid: 0.03g (buying from Roche Holding Ag) adds 1ml 1 * damping fluid III;

5,10x damping fluid I:(PH7.1-7.4)

NaCl?80g

Na ₂HPO ₄.12H ₂O 360g

KCl 2g

KH ₂PO ₄?2g

Add tri-distilled water to 1l, and autoclaving;

6,10x damping fluid II:(PH7.0)

NaCl 175.3g

Trisodium Citrate 88.2g

Several of HCl

Add tri-distilled water to 1l, and autoclaving;

7, damping fluid III:(PH7.9)

Tris 121.1g

NaCl?87.66g

About HCl 60ml

Add tri-distilled water to 1l, and autoclaving;

8, damping fluid IV:

1M Tris-HCl (PH9.5): Tirs 121.1g adds about HCl 3ml, adds water 900ml, transfers PH to 9.5, adds water to 1l, and autoclaving;

1M NaCl:NaCl 58.44 adds water to 1l, and autoclaving;

0.5M MgCl ₂: 101.65g MgCl ₂.6H ₂O adds water to 1l, and autoclaving;

9, stationary liquid: Paraformaldehyde 96 40g adds 1 * damping fluid I to 1l, and heat (about 50-60 degree) is stirred to dissolving a little;

10, developer A:NBT 1g adds 70%DMF11.44ml;

11, developer B:BCIP 1g adds 100%DMF30ml.

Test kit of the present invention can many person-portions use or person-portion use.

Embodiment 2

A kind of PTEN gene hybridization in situ detection method and test kit thereof are used

One, sample disposal

1, with the centrifuge tube of 10ml, dress 4.5ml lymphocyte separation medium, again the 3ml anticoagulation is slowly added contain lymphocyte separation medium (blood: in centrifuge tube lymphocyte separation medium=1: 1.5), the centrifugal 10min of 2000r/min;

2, draw the middle layer white corpuscle to another centrifuge tube, in this pipe, add 1 * damping fluid I of about twice again, mixing, the centrifugal 10min of 1500g/min;

3, abandon supernatant. precipitation adds 1 * damping fluid I of about twice, mixing, the centrifugal 10min of 1500g/min;

4, abandon supernatant, and test tube mouth excess liquid is gone with the tissue suction.Again precipitation is made suspension, drop in push jack on the slide, seasoning.(hospital with good conditionsi can use the pelleter film-making.) 3ml blood, can do 4 slice, thin pieces;

5, with 40ml 4% stationary liquid, in glass jar, fixedly 30min uses 1 * damping fluid I to wash 5min again.Every cylinder can be put 16;

6, sample can be kept at-20 ℃, or continues to do experiment.

Two, reagent in the test kit is mixed with working concentration

1, with 10 * damping fluid I with tri-distilled water by being diluted to 1 * damping fluid I at 1: 10;

2, with 20 * damping fluid II with tri-distilled water by being diluted to 2 * damping fluid II at 1: 10;

By being diluted to 0.2 * damping fluid II at 1: 100; By being diluted to 0.1 * damping fluid II at 1: 200;

3, with 10 * damping fluid III with tri-distilled water by being diluted to 1 * damping fluid III at 1: 10;

4,10 * damping fluid IV with tri-distilled water by be diluted at 1: 10 * damping fluid IV (get 1#, 2#, each 10ml of 3#, add water to 100ml both can).

Three, experimental procedure:

1, gets two of every person's samples to be checked, two of (other two give over to check with) and positive control samples (test at every turn and do a pair of positive control);

2, in glass jar, add Digestive system (Digestive system 100ul adds 1 * damping fluid 199.9ml, is working concentration) 20ml.37 ℃ of water-bath preheatings 10 minutes.Put 16 slides into, handle 12min, use 1 * damping fluid I to wash 5min again for 37 ℃;

3, wash 10min with 0.2% protection liquid (protection liquid 1ml adds 1 * damping fluid I99ml and is working concentration), tri-distilled water is washed 5min, and above process is all carried out at glass jar.The slide seasoning;

4, slide is put into the box of preserving moisture, add prehybridization solution 20ul/ sheet. covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;

5, take out slide, discard cover glass, slide is put into glass jar, with 70%, 90%, 95% ethanol is respectively washed 2min, seasoning;

6, slide is put into the box of preserving moisture, two of every patient specimens, one adds just hybridization solution 20ul/ sheet, and another adds antisense hybridization solution 20ul/ sheet, covered, the lid box of tightly preserving moisture is placed on 16-24h in 42 ℃ of constant water bath box;

7, take out slide, discard cover glass, slide is put into glass jar

In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II

In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II

In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;

8, wash 30s with 1 * damping fluid III, take out slide, seasoning;

9, slide is put into the box of preserving moisture, add 0.5%l confining liquid (the 1ml confining liquid adds 5ml1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture, at room temperature act on 30min;

10, take out slide, III washes 30s with 1 * damping fluid, seasoning;

11, slide is put into the box of preserving moisture, add alkaline phosphatase enzyme antibody (adding 1.8ml 1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture and at room temperature act on 30min;

12, take out slide, wash 3 times, each 15min with 1 * damping fluid III;

13,1 * damping fluid IV washes 2min, adds developer (developer A73.3ul, developer B157.5ul are added among 30ml1 * damping fluid IV, mixing), more than the room temperature lucifuge 12h;

14, wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.

Four, the result judges

100-300 cell of counting calculates the per-cent of catching the purple cell under light microscopic.

What the positive control sample added the antisense hybridization solution should catch purple more than 80%.

All add the negative internal reference of just hybridization solution should be colourless.

The cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, method with immunohistochemical methods develops the color again, under light microscopic, observe existence and the location of mRNA,, judge the expression amount of goal gene according to painted cell count.

The inventive method is a nucleic acid hybridization in situ technology commonly used at present, and this method is used for determining the prognosis of cancer by detecting the PTEN gene expression amount in the substrate cell.Clinical study shows, the PTEN gene is low the expression in cancer, because the PTEN gene is at normal people's high expression level, and the low expression explanation cancer of PTEN gene, PTEN expression of gene degree is relevant with the prognosis of cancer, belongs to negative correlation, expresses that more to hang down prognosis poorer.Thereby obtain the diagnostic message of cancer.As mentioned above, when detecting PTEN genetic expression, measurable experimenter is a cancerous condition.The embodiment of the invention is sampled as: 5 of uterus carcinoma patients, 5 of ovarian cancer patients, 5 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result represents that all cancer patients PTEN genes are not expressed the cell dye-free.Normal control group PTEN gene has overexpression, cell dyeing.Concrete outcome is asked for an interview Fig. 1, Fig. 2 and Fig. 3.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

SEQUENCE?LISTING

＜110〉Rui bends biotechnology (Shanghai) Co., Ltd.

＜120〉a kind of hybridization in situ detection kit of PTEN gene and detection method thereof and application

<130>/

<160>1

<170>PatentIn?version?3.3

<210>1

<211>5572

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

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ggcattatat?atattatata?tataaatata?tattatacat?actctcctta?ctttatttca 4680

gttaccatcc?ccatagaatt?tgacaagaat?tgctatgact?gaaaggtttt?cgagtcctaa 4740

ttaaaacttt?atttatggca?gtattcataa?ttagcctgaa?atgcattctg?taggtaatct 4800

ctgagtttct?ggaatatttt?cttagacttt?ttggatgtgc?agcagcttac?atgtctgaag 4860

ttacttgaag?gcatcacttt?taagaaagct?tacagttggg?ccctgtacca?tcccaagtcc 4920

tttgtagctc?ctcttgaaca?tgtttgccat?acttttaaaa?gggtagttga?ataaatagca 4980

tcaccattct?ttgctgtggc?acaggttata?aacttaagtg?gagtttaccg?gcagcatcaa 5040

atgtttcagc?tttaaaaaat?aaaagtaggg?tacaagttta?atgtttagtt?ctagaaattt 5100

tgtgcaatat?gttcataacg?atggctgtgg?ttgccacaaa?gtgcctcgtt?tacctttaaa 5160

tactgttaat?gtgtcatgca?tgcagatgga?aggggtggaa?ctgtgcacta?aagtgggggc 5220

tttaactgta?gtatttggca?gagttgcctt?ctacctgcca?gttcaaaagt?tcaacctgtt 5280

ttcatataga?atatatatac?taaaaaattt?cagtctgtta?aacagcctta?ctctgattca 5340

gcctcttcag?atactcttgt?gctgtgcagc?agtggctctg?tgtgtaaatg?ctatgcactg 5400

aggatacaca?aaaataccaa?tatgatgtgt?acaggataat?gcctcatccc?aatcagatgt 5460

ccatttgtta?ttgtgtttgt?taacaaccct?ttatctctta?gtgttataaa?ctccacttaa 5520

aactgattaa?agtctcattc?ttgtcaaaaa?aaaaaaaaaa?aaaaaaaaaa?aa 5572

Claims (10)

1. the application of the hybridization in situ detection kit of a PTEN gene in preparation detection Cancerous disease medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence is shown in SEQ ID NO.1.

2. application according to claim 1 is characterized in that: the RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

3. application according to claim 1 and 2 is characterized in that: described marker is selected from radionuclide or non-radioactive marker.

4. application according to claim 3 is characterized in that: described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

5. application according to claim 3 is characterized in that: described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

6. application according to claim 5 is characterized in that: described non-radioactive marker is preferably from digoxin.

7. application according to claim 1 and 2 is characterized in that: described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

8. the hybridization in situ detection kit of a PTEN gene comprises hybridization probe, marker, it is characterized in that, described hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

9. the in situ hybridization detection method of a PTEN gene is characterized in that, this method may further comprise the steps:

A, the hybridization probe in the described test kit of claim 8 is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

10. detection method according to claim 9 is characterized in that: the condition that forms hybridization complex in a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour, and described substrate is selected blood cell sample or histocyte sample for use.

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