The molecular probe of metastatic prostate cancer early prediction, test kit and this molecular probe
Application
Technical field
The present invention relates to the application of a kind of molecular probe, test kit and this molecular probe is and in particular to before a kind of transitivity
The application of the molecular probe, test kit and this molecular probe of row adenocarcinoma early prediction.
Background technology
Carcinoma of prostate (Prostate cancer, PCa) is seriously to threaten one of malignant tumor of men's health, its morbidity
Rate ranks first in American-European countries's height, and mortality rate occupies second.In recent years, increase with China's economic development, aged tendency of population and
Dietary structure changes, and the M & M of PCa increases year by year.Up-to-date cancer statistical data display PCa becomes within 2015
For urinary system male reproductive system M & M highest, rate of increase malignant tumor the fastest, metastatic prostate cancer patient 5
The survival rate only 34% in year.PCa accelerates to affect quality of life and the life expectancy of China's elderly men.Because PCa is widely different
Different biological behaviour and the pathogenesis of complexity, with preoperative prostate specific antigen (Prostate-specific
Antigen, PSA), the PCa risk assessment set up of GS (Gleason Score) scoring and clinical TNM stage can not be accurately anti-
Reflect PCa individual patients biological information.Traditional view is thought that only tumor breaks through peplos and is just started to be in progress to whole body, and PCa is early
Phase just can occur micrometastasis.Therefore, find the biomarker closely related with PCa transfer extremely urgent.In recent years, with
Tumor aetiology and the progress of molecular biology, people progressively recognize the occurrence and development of tumor be a multi-path,
Too many levels, multi-level, high complexity but controlled cascade reaction process, and the activity phase closely with promotion sensitivity gene and antioncogene
Close.Detection peripheral blood in circulating tumor cell (circulating tumor cells, CTCs) monitoring tumor recurrence transfer,
Determine that the aspects such as tumor cells feature and judging prognosis have significant application value.
The essence of CTCs is to be present in all kinds of tumor cells of peripheral circulation, sheds into periphery by primary tumor or metastasis
Blood.CTCs in peripheral blood can occur apoptosis actively or passively, and only fraction CTCs can survive and then pass through blood
The mode of transfer develops into new metastasis.The appearance of CTCs occurs early than neoplasm metastasis, points out the cancer metastasiss may be by
CTCs leads to.But there is apoptosis in most of CTCs in peripheral circulation, or swallowed, and only minority develops into metastasis.Inspection
Survey this part and can form the CTCs number of metastasis and play an important role in Urology Surgery oncotherapy.Research finds the shape of CTCs
Become relevant with epithelial cell-mesenchymal transformation mechanism (epithelial-mesenchymal transition, EMT).CTCs is enriched with
Effect directly decides the sensitivity of subsequent detection, and current conventional method is divided into based on cellular biochemistry characteristic according to principle
Concentration method and the concentration method based on cytophysicss characteristic.
Concentration method based on cellular biochemistry characteristic mainly has immunomagnetic beadses concentration method (immunomagnetic
Separation, IMS) and adhesion concentration method.Wherein, IMS is a kind of efficient, highly sensitive CTCs enrichment method.CTCs table
The specific monoclonal antibody that face epithelial adherence molecule (EpCAM) can be connected to magnetic bead with chain combines, and so that CTCs is had magnetic and can be detained
In magnetic field.By immunomagnetic beadses concentration method and conventional sense technology (such as immunocytochemical method, flow cytometry RT-PCR etc.)
Combining, being greatly improved the concentration of CTCs in peripheral blood sample thus improving recall rate.IMS need to be by CTC surface EpCAM's
Expression, thus lead to the method to be only applicable to limited cancer kind.The affinity interaction based on CTCs and capture surface for the adhesion concentration method,
Capture surface obtains the affinity with CTCs by certain biochemical modification.The method can be static or conventional
Carry out separation and concentration under continuous microflow control chip mode.In static separation pattern, sample is incubated one first on capture surface
The section time.Subsequently, nonadherent cell is rinsed, and CTCs will be attracted to capture surface.Due to metastatic carcinoma cell more
Tend to invade the Collagen tissues such as bone matrix, therefore this method is used to capture the CTCs with higher aggressive.
Concentration method based on cytophysicss characteristic mainly have membrane filtration concentration method, Graded Density concentration method and
OncoQuick concentration method.Wherein, the principle of membrane filtration concentration method is to be filtered based on cell size and mechanical performance.By blood
CTC can be enriched with by specimen by the filter membrane in 5-10 μm of aperture.To the concentration effect of circulating tumor microemboli (CTM) than based on immunity
The concentration method learning principle is advantageous, but has fraction tumor cell and lose because diameter is less than filter sizes, leads to the method
Sensitivity low.The principle of Graded Density concentration method is based on cell density difference various in blood, with layering liquid by tumor
Cell is separated with other cellular layers with mononuclear cell.This method is simple to operate, but the blood specimen amount that this separation method requires is relatively
Greatly (usual 15-30ml), and sensitivity and specificity all relatively low.OncoQuick concentration method utilizes a kind of built-in porous barrier
50ml Special test tube, before being centrifuged, the whole blood of 15-30ml is laid on perforated membrane, by centrifugation by tumor cell from
Separate and then reach the purpose of CTC enrichment in other cells.The method is possible to lose a small amount of cell in separation process,
Once the tumor cell that blood anticoagulant not exclusively, is present in small blood clot may be settled down to the bottom of Graded Density liquid
Portion.
At present, the detection method of carcinoma of prostate mainly has imaging examination method, serological detection method and pathological biopsy monitoring.
Imaging examination for prostate cancer diagnosis mainly has the expansion technique based on ultrasonic, MR and PET-CT.Ultrasonic technique is
The most ancient most widely used technology of prostate cancer diagnosis.B ultrasonic can preferably distinguish prostate zone, because peripheral zone under B ultrasonic
Echo is stronger than central zone and transitional zone.Black and white ultrasonic technique can differentiate low echo focus, the ultrasonic skill of per rectum black and white well
Art stochastic system punctures combines basis (Hodge K K, the Mcneal J that prostate biopsy diagnosis has been established in low echo focus puncture
E,Stamey T A.Ultrasound guided transrectal core biopsies of the palpably
abnormal prostate.[J].J Urol,1989,142(1):66-70;Hodge KK,McNeal JE,Terris MK,
Stamey TA.Random systematic versus directed ultrasound guided transrectal
core biopsies of the prostate.[J]J Urol.1989;142(1):71–74.).But B ultrasonic and black and white are ultrasonic
In technology, some benign diseases such as prostatitis, prostatic hyperplasia also can be shown as low echo focus.Around early prostate cancer
Substantial amounts of optimum body of gland echo cover it can be led to be shown as equal echo and be difficult to detect by come (Ukimura O, Faber K,
Gill IS.Intraprostatic targeting.Curr Opin Urol.2012;22(2):97–103.).According to statistics,
60% ultrasonic focus are benign lesions, the carcinoma of prostate of 21-47% fail to pinpoint a disease in diagnosis in first aspiration biopsy (Loch T,
Eppelmann U,Lehmann J,Wullich B,Loch A,Stockle M.Transrectal ultrasound
guided biopsy of the prostate:random sextant versus biopsies of sono-
morphologically suspicious lesions.World J Urol.2004;22(5):357–360;Singh H,
Canto EI,Shariat SF,et al.Predictors of prostate cancer after initial
negative systematic 12core biopsy.J Urol.2004;171(5):1850–1854;Taira AV,
Merrick GS,Galbreath RW,et al.Performance of transperineal template-guided
mapping biopsy in detecting prostate cancer in the initial and repeat biopsy
setting.Prostate Cancer Prostatic Dis.2010;13(1):71–77.).Additionally, stochastic system puncture side
Method, neither tumour-specific is not patient personalized diagnostic method, is diagnosed to be clinical insignificant carcinoma of prostate sometimes,
Sometimes underestimate Gleason scoring (Taira AV, Merrick GS, Galbreath RW, the et of carcinoma of prostate again
al.Performance of transperineal template-guided mapping biopsy in detecting
prostate cancer in the initial and repeat biopsy setting.Prostate Cancer
Prostatic Dis.2010;13(1):71–77;Zaytoun OM,Moussa AS,Gao T,Fareed K,Jones
JS.Ofce based transrectal saturation biopsy improves prostate cancer
detection compared to extended biopsy in the repeat biopsy population.J
Urol.2011;186(3):850–854;Isariyawongse BK,Sun L,Banez LL,et al.Signifcant
discrepancies between diagnostic and pathologic Gleason sums in prostate
cancer:the predictive role of age and prostate-specifc antigen.Urology.2008;
72(4):882–886.).Doppler ultrasound can detect blood flow signal, and Tumor Growth is to meet nutritional need usually to send out
Raw regional flow increases.But it is limited in that, doppler ultrasound is for diameter<0.1mm blood vessel resolution is extremely limited
(Ukimura O,Faber K,Gill IS.Intraprostatic targeting.Curr Opin Urol.2012;22
(2):97 103.), and tumour growth is exactly based on the new vesselses of 10 50 μm of diameter.Therefore, doppler ultrasound is detected
Be also the higher carcinoma of prostate of relatively late Gleason rank.Ultrasonic contrast can detect angiogenesis during tumour progression
Microvessel density change (Russo G, the Mischi M, Scheepens W, de la Rosette causing with new vesselses disorder
JJ,Wijkstra H.Angiogenesis in prostate cancer:onset,progression and
imaging.BJU Int.2012;110(11pt C):E794 E808.), but there is limitation to the resolution of big blood vessel.Many ginsengs
Number nuclear magnetic resonance, NMR (mpMRI) comprises to dissect sequence T2 weighting picture and at least two functional sequence Diffusion weighted imaging and dynamic arthrography increases
Strong picture.MpMRI has preferable resolution, can differentiate histological difference identification carcinoma of prostate, carcinoma of prostate first visit, recurrence,
By stages and transfer monitoring plays a significant role.But its expensive enormous costs limits it and extensively applies, nor can carry out
Real-time visualization, technical operation is also required to substantial amounts of training and learning.MpMRI merges ultrasonic technique and combines both advantages, front
More accurate in the diagnosis of row adenocarcinoma, but increased the expense of mpMRI, its training difficulty is also more difficult compared with mpMRI.Therefore,
Large-scale application can not be enabled.Based on the image technology of PET, there is the metabolism targeting PET, also 18- that 18F-FDG represents at present
The report sub- targeting PET that the propagation targeting PET that F ACBC represents, PSMA represent.PET is less for carcinoma of prostate first visit, clinical
On be mainly used in by stages, biochemical recurrence and local recurrence and metastasis detection.According to the difference of targeting index, PET can essence
Really arrive each body of gland information so that clear and definite by stages, but its shortcoming is somewhat expensive and radioactive exposure.Serologic detection is applied to
Prostate cancer diagnosis, the research of branch prediction are reported numerous.PSA is the most biomarker of research report, and it removes as often
Rule prostate cancer screening molecule beyond, also have treatment evaluate and Index for diagnosis important function (Smith MR,
Kabbinavar F,Saad F,et al.Natural history of rising serum prostate-specifc
antigen in men with castrate nonmetastatic prostate cancer.J Clin Oncol.2005;
23(13):2918–2925;A contemporary prognostic nomogram for men with hormone-
refractory metastatic prostate cancer:a TAX327study analysis.Clin Cancer
Res.2007;13(21):6396–6403.).After treatment, PSA level is closely related with disease overall survival.Basic PSA and dynamic
PSA change such as PSA doubling time, PSA speedup etc. is closely related with carcinoma of prostate recurrence and transfer.(Smith MR,
Kabbinavar F,Saad F,et al.Natural history of rising serum prostate-specifc
antigen in men with castrate nonmetastatic prostate cancer.J Clin Oncol.2005;
23(13):2918–2925;D’Amico AV,Cote K,Lofredo M,Renshaw AA,Schultz
D.Determinants of prostate cancer-specifc survival after radiation therapy
for patients with clinically localized prostate cancer.J Clin Oncol.2002;20
(23):4567–4573;Pound CR,Partin AW,Eisenberger MA,Chan DW,Pearson JD,Walsh
PC.Natural history of progression after PSA elevation following radical
prostatec-tomy.JAMA.1999;281(17):1591–1597;Vickers AJ,Brewster SF.PSA
velocity and doubling time in diagnosis and prog-nosis of prostate cancer.Br
J Med Surg Urol.2012;5(4):162–168;Chun FK,Briganti A,Gallina A,et
al.Prostate-specifc antigen improves the ability of clinical stage and biopsy
Gleason sum to predict the pathologic stage at radical prostatectomy in the
new millennium.Eur Urol.2007;52(4):1067–1074;Schellhammer PF,Chodak G,
Whitmore JB,Sims R,Frohlich MW,Kantof PW.Lower baseline prostate-specifc
antigen is associated with a greater overall survival beneft from sipuleucel-
T in the Immunotherapy for Prostate Adenocarcinoma Treatment(IMPACT)
trial.Urology.2013;81(6):1297–1302).But PSA only has organ specificity, does not have tumour-specific,
Many benign diseases also can cause the dynamic change of PSA.Additionally, many research report PCA3 and ERG also with the sending out of carcinoma of prostate
Raw progress is closely related, monitors the Preventive that the change of its serum expression can predict carcinoma of prostate.However, these blood
The clear specificity learning mark and/or sensitivity is not very preferable.Main cause may is that existing blood serum tumor markers
It the transfer of carcinoma of prostate is not crucial driving gene.The transfer of tumor is a multi-step, be related to multi-signal logical
The biological process on road, the tumour serum mark studied at present may be limited only to play in a certain step or a certain path to be made
With, but the regulating and controlling effect in multi signal passage way network does not obtain and knows.The shortcoming that pathological biopsy monitors carcinoma of prostate transfer is then
Biopsy wound is big, punctures inconvenient in deep focus.The puncture of biopsy obtains and typically requires iconography means auxiliary, therefore
Puncture and be also only limitted to focus that iconography can detect that is to say, that puncture can not make diagnosis prediction to early stage micrometastasis.And
And, biopsy possesses the complication risk such as certain infection, bleeding.It is reported that, 2.67% prostate biopsy patient is tight because puncturing
Related complication hospitalization (Gaylis F, the Nasseri R, Fink L, et al.Prostate Biopsy of weight
Complications:A Dual Analysis.[J].Urology,2016,93:135-140.).
There is following defect in the detection method of above-mentioned carcinoma of prostate:Common iconography means, by obtaining anatomy letter
Breath and metabolic information, size to tumor (>The tumor focus of 0.2cm) and the metabolism of tumor be estimated, and then judge swollen
The present situation of tumor, then, this generally requires can be found when tumor possesses certain volume, accordingly, there exist certain stagnant
Property afterwards, in addition, because the safety issue of radiation, nor is used for multiple times for a long time.And serology aspect, it passes through swollen
Tumor protein marker reflecting the state of tumor, so specificity and sensitivity are also relatively low.As for pathology means, can be accurate
Acquisition tumor gene information, then, it relies on the tumor tissues sample of invasive because for late period have no idea obtain
Exist certain restricted for the patient of sample of tissue.
Therefore, it is necessary to find the new detection method of carcinoma of prostate.There are some researchs with regard to CRMPs at present.
There are five members of CRMP1-5 in CRMPs family.Document report CRMP1 low expression prompting disease in some subbreed of lung carcinoma cell
Poor prognosis, with progression of disease (P=0.010), lymphatic metastasiss (P=0.043), early postoperation recurrence (P=0.030), existence
Time, short (P=0.016) was relevant.Hereafter, he is found that the Novel isomeric LCRMP-1 of CRMP-1 again.Sequence analysis show outside it
The nitrogen end sequence of aobvious son 1 is different from CRMP-1, but the carbon teminal sequence of exon 2-14 is identical.Further investigation revealed that
LCRMP-1 expression is also related to the Clinical Outcome of Patients with Non-small-cell Lung and lymphatic metastasiss.LCRMP-1 passes through to strengthen pseudopodium
The ability forming promotion tumor invasiveness can be by CRMP-1 antagonism, and this phenomenon prompting LCRMP-1 and CRMP-1 is adjusting cancer
In cell invasion and transfer, there is contrary function.On the one hand, LCRMP-1 strengthens pseudopodium and is formed, and stablizes actin, Jin Erzeng
Strong cell migration, aggressivity.This process needs the highly conserved nitrogen end regional stability of LCRMP-1 and WAVE-1 to participate in.It is also
May be by acting on Rho family protein Cdc42.Cdc42 albumen is related to actin restructuring.On the other hand, CRMP1 passes through
Interrupt LCRMP1 and WAVE-1 to interact and suppress Nasopharyngeal neoplasms (Pan S H, Chao Y C, Chen H Y, et
al.Long form collapsin response mediator protein-1(LCRMP-1)expression is
associated with clinical outcome and lymph node metastasis in non-small cell
lung cancer patients.[J].Lung Cancer,2010,67(1):93-100;Shih J Y,Yang S C,Hong
T M,et al.Collapsin response mediator protein-1and the invasion and
metastasis of cancer cells.[J].J Natl Cancer Inst,2001,93(18):1392-1400;Pan S
H,Chao Y C,Hung P F,et al.The ability of LCRMP-1to promote cancer invasion by
enhancing filopodia formation is antagonized by CRMP-1.[J].J Clin Invest,
2011,121(8):3189-3205.).(Hiroshima Y, Nakamura F, Miyamoto H, the et such as Hiroshima
al.Collapsin Response Mediator Protein 4Expression is Associated with Liver
Metastasis and Poor Survival in Pancreatic Cancer.[J].Ann Surg Oncol,2013,
Suppl 3:S369-78. research) finds, the expression of long subtype C RMP4 increases the clinical prognosis poor with cancer of pancreas hepatic metastasess
Relevant, show as promotion sensitivity gene.(Quinn C C, Chen E, Kinjo T G, et al.TUC-4b, a such as Quinn CC
novel TUC family variant,regulates neurite outgrowth and associates with
vesicles in the growth cone.[J].J Neurosci,2003,23(7):2815-2823.) research also send out
Now, the long hypotype (TUC-4B CRMP4b) of the CRMP4 of 75kDa and short subtype C RMP4a (TUC-4A) also have in axon growth
There are reverse functions.Therefore, a possible explanation is exactly that CRMP4 has splicing isomer, body during cancer of pancreas hepatic metastasess
Show the characteristic of the long hypotype of CRMP4b.(Duplan L, Bernard N, the Casseron W.et such as Laure Duplan
al.Collapsin Response Mediator Protein 4a(CRMP4a)Is Upregulated in
Motoneurons of Mutant SOD1Mice and Can Trigger Motoneuron Axonal Degeneration
and Cell Death.J Neurosci.2010,13;30(2):785-96.) exist also by proteomics report CRMP4
Two kinds of hypotypes of CRMP4a and CRMP4b.4a type is different from 4b amino acid N end 126aa region in amino acid N end 13aa region
Coding, belongs to chain isomerism.And confirm that CRMP4a forward direction can be adjusted and inspire mouse mutant SOD1 motor neuron axons degeneration.
Content of the invention
It is an object of the invention to providing one kind to have high accuracy, sensitivity in place of overcoming the deficiencies in the prior art
Molecular probe and test kit with specific metastatic prostate cancer early prediction.
Another object of the present invention is to providing the application of the molecular probe of above-mentioned metastatic prostate cancer early prediction.
For achieving the above object, the technical scheme that the present invention takes is:A kind of metastatic prostate cancer early prediction point
Sub- probe, it comprises LCRMP4 probe, the sequence such as SEQ ID NO of described LCRMP4 probe:Shown in 8.
LCRMP4 be subside reaction mediating proteins 4 (collaspsin response mediator protein 4,
CRMP4 isomer).127 aminoacid of the N-terminal of LCRMP4 are different from CRMP4, and CRMP4 is in addition to 13 aminoacid of N-terminal, 14-
570aa is identical with the 128-684aa of LCRMP4.LCRMP4 probe being capable of specific difference CRMP4 long Short interfering RNA isomery
Body, specific detection encodes the mRNA of LCRMP4 albumen.Our research shows, CRMP4 is in normal prostate tissue and limitation
Property PCa tissue in express higher, and no differential expression between two groups, expression in transitivity PCa tissue substantially reduces, vitro invasion
Experiment can suppress the invasive ability of PCa cell after confirming CRMP4 transfection, and confirms that in clinical tissue sample CRMP4 exists
Transcription and the downward of protein expression level, point out CRMP4 may be PCa nm-23.We further study show that,
The isomer LCRMP4 of CRMP4 plays the oncobiology function contrary with CRMP4 in carcinoma of prostate, before that is, LCRMP4 promotes
The growth of row adenocarcinoma and transfer.In prostate gland cancer cell, overexpression LCRMP4 is obviously promoted cell proliferation growth, and can tie up
The prostate cancer cell line LNCaP holding hormonal independent significantly increases under eunuchism.Additionally, cell migration and Matrigel
Display overexpression LCRMP4 is obviously promoted the migration invasive ability of prostate gland cancer cell.Further study show that, LCRMP4 promotes
The potential mechanism of prostate cancer growth and transfer is the classical β-catenin signal path of regulation and control, and this regulating and controlling effect is not
Depend on the activation of WNT part.Additionally, AR receptor is the important albumen of carcinoma of prostate generation, progress, LCRMP4 can promote AR
To nucleus internal shift so promote AR target gene transcriptional expression.Nude mice zoopery also demonstrates LCRMP4 and promotes prostate
Cancer growth and the effect of transfer.
LCRMP4 is and the carcinoma of prostate closely related gene of transfer, the height of its expression and the classification of carcinoma of prostate and
Metastasis degree is closely related.As metastatic prostate cancer early prediction probe when testing result have compared with high precision
Property, Sensitivity and Specificity.
As the preferred implementation of the molecular probe of metastatic prostate cancer early prediction of the present invention, described molecule
Probe also comprises epithelium mark, interstitial mark and CD45 probe;Described epithelium mark by EpCAM probe, CK8 probe,
CK18 probe and CK19 probe composition, described interstitial mark is made up of Vimentin probe and Twist probe;Wherein, described
The sequence of EpCAM probe such as SEQ ID NO:Shown in 1, the sequence such as SEQ ID NO of CK8 probe:Shown in 2, the sequence of CK18 probe
Row such as SEQ ID NO:Shown in 3, the sequence such as SEQ ID NO of CK19 probe:Shown in 4, the sequence such as SEQ of Vimentin probe
ID NO:Shown in 5, the sequence such as SEQ ID NO of Twist probe:Shown in 6, the sequence such as SEQ ID NO of CD45 probe:Shown in 7.
Twist is a kind of highly conserved basic Helix. ring. helix transcription factor family important member, and in animals and human beingses
Play key regulatory effect in the fetal development of class.During Embryonic Development in Animal, Twist is as transcription factor in inducing cell
Play a major role during mobile and tissue is moulding.Recent research indicate that, Twist plays pass in the conversion regulation and control of epidermis one mesenchymal cell
Key acts on, and Twist overexpression plays a significant role in the generation of tumor, Invasion and Metastasis, angiogenesis and drug resistance of tumor cell.
Twist can cause the generation of tumor by number of ways, finds Twist expression in the tumor of multiple human epitheliums source at present
Increase, and close with Prognostic Significance.
In addition, present invention also offers a kind of test kit of metastatic prostate cancer early prediction, for realizing this purpose, this
Inventing the technical scheme taken is:A kind of test kit of metastatic prostate cancer early prediction, it comprises above-mentioned transitivity prostatitis
The molecular probe of adenocarcinoma early prediction.
LCRMP4 play in carcinoma of prostate tumor promotion and with carcinoma of prostate shift closely related, use it for transitivity
In the test kit of carcinoma of prostate early prediction, the test kit obtaining can also carry out early prediction to metastatic prostate cancer, and
Result has high accuracy, Sensitivity and Specificity.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described test kit
For hybridization in situ detection kit, described LCRMP4 probe, EpCAM probe, CK8 probe, CK18 probe, CK19 probe,
Vimentin probe, Twist probe and CD45 probe are all with label.It is highly preferred that described test kit is RNA in situ hybridization
Detection kit.
In the original location in hybridization check test kit, above-mentioned probe both functions as hybridization probe.RNA hybridization in situ technique is to use
A kind of hybridization in situ technique of rna expression in the probe in detecting cell such as cRNA or oligonucleotide and tissue.Its ultimate principle is:?
Under conditions of cell or tissue structure keeps constant, with the known RNA nucleotide fragments of labelling, join by base in nucleic acid hybridization
To principle, combine (hybridization) with corresponding genetic fragment in cell or tissue to be measured, crossbred (Hybrids) warp being formed
In optical microscope or its intracellular corresponding mRNA, rRNA and tRNA molecule of observed under electron microscope after chromogenic reaction.RNA
The field of hybridization in situ technique application is far beyond DNA hybridization in situ technique.Especially can determine in terms of gene analysiss and diagnosis
Property, positioning and quantitative analyses, it has also become maximally effective molecular pathology technology, analyzing low abundance and rare mRNA table simultaneously
Reach aspect and there is advantage.RNA hybridization in situ technique using CTCs detects LCRMP4 expression, being capable of Accurate Prediction prostate
Cancer patient shifts, and its sensitivity reaches 95.5%, and specificity reaches 91.7%.Can be early with the CTCs liquid Biopsy of less wound
Phase prediction carcinoma of prostate transfer;And the method uses blood drawing detection, the little few intercurrent disease of wound, can detect clinical image
Learn the tumor micrometastasis that can not detect.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described label
For radionuclide or non-radioactive marker;It is highly preferred that described non-radioactive marker is biotin, digoxin, alkalescence
Phosphatase, horseradish peroxidase or fluorescein.Label can be selected according to the usual manner of the art, when with fluorescence
When element is as label, what epithelium mark, interstitial mark, CD45 probe and LCRMP4 probe were carried is different colours
Fluorescent labeling.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described test kit
Also comprise agent, Digestive system, hybridization buffer, pre- amplifier working solution, amplifier working solution, nitrite ion and redye liquid.
As the more preferably embodiment of the test kit of metastatic prostate cancer early prediction of the present invention, described digestion
Liquid is made up of PBS (phosphate buffer) and digestive enzyme;Described hybridization buffer comprises TritonX and Methanamide;Described pre-expansion
Increase working solution and become with pre- amplification probe groups by expanding buffer solution;Described amplifier working solution is by expanding liquid and amplification probe forms;
Described nitrite ion is made up of colorbuffer and colour developing probe;Described liquid of redying is 4', 6- diamidino -2-phenylindone.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described pre- amplification
Probe is by SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11 and SEQ ID NO:Sequence shown in 8 forms;Described amplification
Probe is by SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14 and SEQ ID NO:Sequence shown in 8 forms;Described aobvious
Color probe is by SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17 and SEQ ID NO:Sequence shown in 8 forms.Wherein,
SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:15 is epithelium mark (EpCAM probe, CK8 probe, CK18 probe
With CK19 probe) DNA probe, SEQ ID NO:10、SEQ ID NO:13 and SEQ ID NO:16 is interstitial mark
The DNA probe of (Vimentin probe and Twist probe), SEQ ID NO:11、SEQ ID NO:14 and SEQ ID NO:17 are
The DNA probe of CD45.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described amplification is delayed
Rush solution, amplification liquid and colorbuffer and all comprise following components:Horse serum 30% (w/w), 1.5% sodium lauryl sulphate
(w/w) pH value of, 3mM Tris-HCl, described Tris-HCl is 8.0.
As the preferred implementation of the test kit of metastatic prostate cancer early prediction of the present invention, described test kit
Also comprise cleaning mixture.
The present invention adopts the method for in situ hybridization to metastatic prostate cancer early prediction, using the method for in situ hybridization
When, the specific using method of mentioned reagent box is:(1) gather peripheral blood, carry out erythrocyte splitting, be then centrifuged for and fix,
After be dehydrated, obtain testing sample;(2) gained testing sample is carried out hydrophilic and be permeabilized, then digested, Ran Houyu
Probe hybridization, amplification;(3) colour developing and microscopy.
Finally, present invention also offers above-mentioned molecular probe is used for the reagent of metastatic prostate cancer early prediction in preparation
Application in box.
As the preferred implementation of application of the present invention, described test kit is hybridization in situ detection kit.
Compared with prior art, beneficial effects of the present invention are:The invention provides a kind of new metastatic prostate cancer
When the probe groups of early prediction and test kit, this probe groups and test kit carry out early prediction to metastatic prostate cancer, accurately
Property, Sensitivity and Specificity are all higher.
The test kit of metastatic prostate cancer early prediction of the present invention can be hybridization in situ detection kit, especially RNA
Hybridization in situ detection kit.The present invention passes through CTCs detection technique (Surexam benefit is kind), is lived using liquid in molecular pathology
Procuratorial organ's formula, CTC and its typing in dynamic monitoring tumor patient blood;Design the LCRMP4 gene closely related with transitivity PCa
Detection probe, detects the expression of LCRMP4 gene in CTCs using multiple rna in situ hybridization detection technique, and statistical analysiss set up art
Front prediction PCa patient whether there is transfer.RNA hybridization in situ technique using CTCs detects LCRMP4 expression, Neng Gouzhun
Really prediction patients with prostate cancer transfer, its sensitivity reaches 95.5%, and specificity reaches 91.7%.Can be with the CTCs liquid of less wound
Body Biopsy early prediction carcinoma of prostate shifts.
Additionally, the present invention also has the advantage that:1. present invention detection sample is blood samples of patients, can be controlled according to conditions of patients
Treatment situation multi collect specimen, it is to avoid traditional tissue penetration, wound is little, few intercurrent disease, and the detection method of the present invention
Minimally invasive, safety, dynamic monitoring, can detect transfer invisible for clinical imageology.2. the detection method of the present invention from
Dynamicization, convenient and swift:CTC extracts and adopts automation equipment, being capable of batch operation detection.
Brief description
Fig. 1 is the flow chart detecting LCRMP4 expression using the RNA hybridization in situ technique of CTCs;
Fig. 2 is CTCs identification and classifying method testing result figure;
The result figure that Fig. 3 expresses in CTCs for LCRMP4;
Fig. 4 is CTCs row RNA in situ hybridization detection prediction carcinoma of prostate (lymph node/bone) transfer ROC analysis result figure;
Fig. 5 is CTCs row RNA in situ hybridization detection prediction carcinoma of prostate lymph metastasis ROC analysis analysis result figure;
Fig. 6 is CTCs row RNA in situ hybridization detection prediction prostate cancer with osseous metastasis ROC analysis analysis result figure.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below will be with RNA in situ hybridization detection CTCs
The expression of LCRMP4 gene, as a example statistical analysiss preoperative prediction PCa patient whether there is transfer, and combines accompanying drawing to present invention work
Further illustrate.
In following embodiments, LCRMP4 probe is used in situ hybridization detection, but it is noted that LCRMP4 probe also may be used
In test kit for other metastatic prostate cancer early predictions;Meanwhile, in the following embodiments, use fluorescence in situ
The method of hybridization, therefore the label on hybridization probe is fluorescein;But label is not limited to fluorescein, we can be according to reality
Border needs, and the detection according to adopting selects the label being suitable for.
Embodiment 1
A kind of embodiment of the probe groups of metastatic prostate cancer early prediction of the present invention, before transitivity described in the present embodiment
The probe groups of row adenocarcinoma early prediction comprise epithelium mark, interstitial mark, CD45 probe and LCRMP4 probe;Described epithelium
Mark is made up of EpCAM probe, CK8 probe, CK18 probe and CK19 probe, and described interstitial mark is by Vimentin probe
With Twist probe composition;Wherein, the sequence of described EpCAM probe such as SEQ ID NO:Shown in 1, the sequence such as SEQ of CK8 probe
ID NO:Shown in 2, the sequence such as SEQ ID NO of CK18 probe:Shown in 3, the sequence such as SEQ ID NO of CK19 probe:Shown in 4,
The sequence of Vimentin probe such as SEQ ID NO:Shown in 5, the sequence such as SEQ ID NO of Twist probe:Shown in 6, CD45 probe
Sequence such as SEQ ID NO:Shown in 7, the sequence such as SEQ ID NO of LCRMP4 probe:Shown in 8.The particular sequence of described probe
As shown in table 1 below.
Table 1
Embodiment 2
A kind of embodiment of the test kit of metastatic prostate cancer early prediction of the present invention, before transitivity described in the present embodiment
The test kit of row adenocarcinoma early prediction is hybridization in situ detection kit, and this test kit comprises:Hybridization probe, saturating agent, digestion
Liquid, hybridization buffer, pre- amplifier working solution, amplifier working solution, nitrite ion, redye liquid and cleaning mixture;
Wherein, described hybridization probe is probe groups described in embodiment 1, and described LCRMP4 probe, EpCAM probe, CK8 visit
All with label on pin, CK18 probe, CK19 probe, Vimentin probe, Twist probe and CD45 probe;Epithelium mark
Thing (EpCAM probe, CK8 probe, CK18 probe and CK19 probe) redly fluorescent labeling;(Vimentin visits interstitial mark
Pin and Twist probe) band green fluorescent label;LCRMP4 probe band purple fluorescence labelling;CD45 probe band blue-fluorescence labelling.
Described Digestive system is made up of PBS and digestive enzyme;
Described hybridization buffer consists of the following composition:10% dextran sulfate, 10mmol/L NaCl, 50% formyl
Amine, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidine quinoline, 0.2%FICOLL, 5mmol/L EDETATE SODIUM, 0.1%Triton
X-100、50mmol/L Tris-HCl;
Described pre- amplifier working solution is become with pre- amplification probe groups by expanding buffer solution;Described amplifier working solution is by expanding liquid
Form with amplification probe, described nitrite ion is made up of colorbuffer and colour developing probe;Wherein, described pre- amplification probe is by SEQ
ID NO:9、SEQ ID NO:10、SEQ ID NO:11 and SEQ ID NO:Sequence shown in 8 forms;Described amplification probe is by SEQ
ID NO:12、SEQ ID NO:13、SEQ ID NO:14 and SEQ ID NO:Sequence shown in 8 forms;Described colour developing probe is by SEQ
ID NO:15、SEQ ID NO:16、SEQ ID NO:17 and SEQ ID NO:Sequence shown in 8 forms;Described amplification buffer solution,
Amplification liquid and colorbuffer all comprise following components:Horse serum 30% (w/w), sodium lauryl sulphate 1.5% (w/w), 3mM
The pH value of Tris-HCl, described Tris-HCl is 8.0;Described SEQ ID NO:9~SEQ ID NO:17 particular sequence is shown in Table
2.
Described liquid of redying is 4', 6- diamidino -2-phenylindone (DAPI).
Table 2
SEQ ID NO:9 |
CGCAGCCTCAGCC |
SEQ ID NO:10 |
GACGGTCGGCGTT |
SEQ ID NO:11 |
GCGCGCTGTAGGG |
SEQ ID NO:12 |
CCCAGACCCTACC |
SEQ ID NO:13 |
GTCACCGCTCCAC |
SEQ ID NO:14 |
AGGCGAGGGGAGA |
SEQ ID NO:15 |
CTACAAACAAACAATATT |
SEQ ID NO:16 |
CTTCTCAATAACTAACAT |
SEQ ID NO:17 |
CTTTATACCTTTCTTTCA |
Embodiment 3
The present embodiment adopts test kit described in embodiment 2, the RNA hybridization in situ technique detection LCRMP4 expression using CTCs
Level, to be predicted to metastatic prostate cancer.The present embodiment utilizes the RNA hybridization in situ technique of CTCs to detect LCRMP4 table
The flow process reaching level is as shown in Figure 1.
Comprise the following steps that:
First, clinical sample Collecting and dealing
A, collection peripheral blood:
1st, EDTA anticoagulant blood-collecting pipe is used to gather 5mL peripheral blood, every patient's collection 2 pipe (public 10mL);
2nd, after taking a blood sample, reverse blood taking tube fully mixes for more than 5 times and preserves after 4 DEG C, and the holding time is less than 4 hours;
3rd, record patient's reference numeral, with linker, the blood in blood taking tube is proceeded to Sample preservation pipe and (split containing erythrocyte
Solution liquid).
B, erythrocyte splitting:
1st, Sample preservation pipe is turned upside down and mix 10 times, (15-30 DEG C) standing 30min cracking of room temperature, room temperature is less than 15
DEG C when pyrolysis time extend to 1 hour;
2nd, the erythrocyte of lysis at room temperature, as processed in time, can be stored in 4 DEG C, and the holding time is less than 8 hours.
C, centrifugation and fixation:
1st, sample is centrifuged 5min with 1850rpm (600 × g), abandons supernatant;
2nd, sequentially add 4mLPBS and 1mL fixative (for the fixation of sample preprocessing), be vortexed and mix, room temperature stands
8min.
D, sucking filtration are fixed again:
1st, check whether filter is intact, if having filter membrane, filter is fixed on vacuum discrimination plate;
2nd, open vacuum pump, make negative pressure rise to -0.06MPa (this step completes during sample is fixing);
3rd, the liquid in Sample preservation pipe is transferred in filter, opens two-way valve, by sample sucking filtration to filter
In filter membrane on, close valve after completing to filter, in filter, add 1mL sample fixative, fixing 1h.
E, dehydration and preservation:Use 1mL50% dehydrant, 100% dehydrant immersion treatment successively, soak 2min, place every time
After reason terminates, put samples into -20 DEG C of refrigerators and can preserve two weeks, or put into -80 DEG C of refrigerators and can preserve 6 months.
2nd, CTCs sample typing detection
A, hydrophilic and saturatingization:
1st, 400uL100% ethanol, 70% ethanol, 50% ethanol immersion treatment, each 2min are used successively;
2nd, add 1-2mLPBS to wash three times, soak 2min every time;
3rd, filter membrane is tipped upside down in the saturating agent of 100uL, incubated at room 5min;
4th, add 1-2mLPBS to wash three times, soak 2min every time.
B, digestion:
1st, filter membrane is tipped upside down on the Digestive system (590uL PBS+10uL digestive enzyme) of 100uL, incubated at room 1h;
2nd, add 1-2mL PBS to wash three times, soak 2min every time.
C, probe hybridization:
1st, filter membrane is tipped upside down on the hybridization solution (84uL hybridization buffer+16uL hybridization probe) of 100uL, 40 DEG C of incubations
3h;
2nd, add 1-2mL cleaning mixture (i.e. probe cleaning mixture) to wash three times, soak 2min every time.
D, amplification:
1st, filter membrane is tipped upside down on pre- amplifier working solution (the pre- amplification probe of 90uL amplification buffer+10uL), 40 DEG C of incubations
30min;
2nd, add 1-2mL cleaning mixture (i.e. probe cleaning mixture) to wash three times, soak 2min every time;
3rd, filter membrane is tipped upside down on the amplifier working solution (90uL expands liquid+10uL amplification probe) of 100uL, 40 DEG C of incubations
30min;
2nd, add 1-2mL cleaning mixture (i.e. probe cleaning mixture) to wash three times, soak 2min every time.
E, colour developing and microscopy:
1st, by filter membrane tip upside down on 100uL nitrite ion (90uL colorbuffer+10uL colour developing probe on, 40 DEG C incubation
30min;
2nd, add 1-2mL cleaning mixture (i.e. probe cleaning mixture) to wash three times, soak 2min every time;
3rd, filter membrane is face-up placed on microscope slide, is cut off along iron ring internal ring filter membrane with blade.
4th, plus 10uL redyes liquid, cover the lid plectrum of 18mm × 18mm, direct microscopy or put into -20 DEG C of Refrigerator stores.
3rd, CTCs microscopy flow chart
A, start:
Open computer;Open microscope switch (switch of microscope lower left);Open fluorescence power supply;Start software (left
Key double-clicks desktop " Metafer4 " software)
B, 20 times of scannings:
Left button clicks setup, selects surexam01-CTC-20 × program to shoot;1 and 3 regions are selected after the completion of shooting
Cell, " Reject Undefined " removes unselected cell.(every agreement that contracts a film or TV play to an actor or actress 5 minutes)
C, 40 times of DAPI shoot:
Left button clicks setup, selects surexam01-CTC-40 ×-DAPI program to shoot;After the completion of 40 times of DAPI shoot
Select core stricture of vagina clearly cell, " Reject Undefined " removes unselected cell.(every agreement that contracts a film or TV play to an actor or actress 2 minutes)
D, 40 times of fluorescence signals shoot:
Left button clicks setup, selects surexam01-CTC-40 × -4F program to shoot;(each cell about 40 seconds)
E, Image Adjusting:
Open the result of shooting in Review software, click cell by right key, in Colors option adjustment cell brightness,
Contrast.
F, result judgement:
CTCs microscopy result judgement list carries out result judgement, CTCs criterion:1st, nucleus are clear and have core stricture of vagina;
2nd, danger signal point or green point are more than or equal to 7, and are less than 7 for white signal point.
G, image output:
By clicking on Export Cell Gallery, output epitheliated type, mixed type, interstitial type and LCRMP4 are each respectively
The picture of expression in type CTCs.CTCs identification and classifying method testing result are as shown in Figure 2.In Fig. 2, DAPI represents 4', 6- bis-
Amidino groups -2-phenylindone;E marker represents epithelium mark, by EpCAM probe, CK8 probe, CK18 probe and CK19 probe
Composition;M marker represents interstitial mark, is made up of Vimentin probe and Twist probe;CD45 is leukocyte mark.
H, interpretation of result:
According to CTCs quantity and type in testing result, draw CTCs quantity type change block diagram, statistical analysiss
LCRMP4 expression in various CTC, calculates LCRMP4 expression signal number.Collect clinical patients information, by ROC curve
Relation between LCRMP4 expression and transfer in analysis CTCs.
The result that LCRMP4 expresses in CTCs is as shown in Figure 3.In Fig. 3, A represents that LCRMP4 no expresses, B and C represents
LCRMP4 expresses (purple).
CTCs row RNA in situ hybridization detection prediction carcinoma of prostate (lymph node/bone) transfer ROC analysis result is as shown in Figure 4;
CTCs row RNA in situ hybridization detection prediction carcinoma of prostate lymph metastasis ROC analysis analysis result is as shown in Figure 5;CTCs row RNA is former
Position hybridization check prediction prostate cancer with osseous metastasis ROC analysis analysis result is as shown in Figure 6.From fig. 4, it can be seen that RNA in situ hybridization is glimmering
Sensitivity 95.5% during light number prediction carcinoma of prostate (lymph node/bone) transfer threshold values 0, specificity 91.7%;As seen from Figure 5, RNA
Sensitivity 83.3% during Fluorescence In Situ Hybridization number prediction carcinoma of prostate lymphatic metastasiss threshold values 0, specificity 91.7%;Can by Fig. 6
See, sensitivity 80% during RNA Fluorescence In Situ Hybridization number prediction prostate cancer with osseous metastasis threshold values 5, specificity 94.7%.
Last should be noted that above example is only in order to illustrate technical scheme rather than to present invention guarantor
The restriction of shield scope, although being explained in detail to the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.