CN105983108A - Applications of novel VEGFR-3 receptor imaging agent <99m>Tc-HYNIC/EDDA-TMVP1 in tumor diagnosis - Google Patents
Applications of novel VEGFR-3 receptor imaging agent <99m>Tc-HYNIC/EDDA-TMVP1 in tumor diagnosis Download PDFInfo
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Abstract
The invention discloses applications of a novel VEGFR-3 receptor imaging agent <99m>Tc-HYNIC/EDDA-TMVP1 in tumor diagnosis, relates to a novel tumor molecule imaging agent for the nuclear medicine, and in particular relates to polypeptide marked by radionuclide <99m>Tc and aiming at VEGFR-3 molecules. The high expression of VEGFR-3 in many tumor neonatal lymphatics and neonatal blood vessels shows the application prospect of VEGFR-3 in the tumor molecule diagnosis, but at present, the number of imaging agents aiming at VEGFR-3 is very limited, and the defects of high background and poor targeting effect exist, while TMVP1 (with the core sequence of LARGR) can be specifically bound to VEGFR-3, in the scheme, TMVP1 is marked by the radionuclide for the first time, and the imaging effects of TMVP1 for tumor in various tumor models are evaluated. Natural polypeptide has a short half-life period in the body, while in the scheme, cyclization is carried out on TMVP1, and therefore, the stability of TMVP1 in the body is increased. The polypeptide TMVP1 marked by radionuclide <99m>Tc (<99m>Tc-HYNIC/EDDA-E-G-Cyclic (CGLARGRGC)) can target to the tumor tissue with high expression of VEGFR-3, and can be used for carrying out SPECT imaging on the tumor.
Description
One, technical field
One, technical field
The present invention relates to a kind of novel VEGFR-3 receptor developer99mTc-HYNIC/EDDA-TMVP1 application in diagnosing tumor, this medicine includes LARGR polypeptide, bifunctional chelating agent, altogether part and radionuclide99mTc.The structure of described polypeptide is E-G-Cyclic (CGLARGRGC), and this polypeptide cyclization mode is cysteine disulfide bond cyclization；Described bifunctional chelating agent is HYNIC (6-Hydrazinonicotinic acid)；Described part altogether is EDDA (Ethylenediamine-N, N '-diacetic acid)；Described radionuclide is99mTc.In sum, the structural formula of this novel VEGFR-3 receptor developer is:99mTc-HYNIC/EDDA-E-G-Cyclic(CGLARGRGC).The described field of this invention relates to the novel tumor molecular imaging agent of a kind of Nuclear Medicine Department, particularly relates to the radionuclide nucleic for VEGFR-3 molecule99mThe polypeptide of Tc labelling.
Two, background technology
In recent years, malignant tumor has become the primary disease threatening human health, and 2014 " report of world's cancer " China new diagnosis cases of cancer is 3,070,000, accounts for the 21.8% of whole world sum.Number of cancer deaths about 2,200,000, account for the 26.9% of whole world number of cancer deaths.Current entity tumor patient therapeutic modality, still based on excision, is aided with chemotherapy, radiotherapy and molecular targeted therapy, and affect cancer patient's therapeutic effect and prognosis the most key be early find, early diagnosis and early intervening.The superfine impact of existing CT/MR/B is learned and is checked the diagnosis being the most well applied to tumor, but the accuracy of its diagnosis depends on whether doctor has rich experience and sturdy theoretical knowledge, and its Differential Diagnosis innocent and malignant tumour remains weak point.Therefore, search out a species specific diagnostic method or diagnostic agent is still that the target of the unremitting pursuit of each researcher.
The super theoretical basis to diagnosing tumor of CT/MR/B is density and the mechanicalness behavior of tumor growth of tumor tissues.In recent years, oncobiology research it turned out tumor cell to be had and differs from Normocellular biological characteristics, such as unstability and the change etc. of protein expression profiles of genome, scientists develops a kind of new diagnostic method for some specific surface molecular albumen (integrin receptor, EGF-R ELISA, inhibin receptor etc.) of tumor cell high expressed: tumor cells diagnoses.The molecular diagnosis agent and the medicine that have been applied to clinic are broadly divided into monoclonal antibody, recombiant protein, little signaling pathway molecule inhibitor and polypeptide, and wherein polypeptide is little because of having molecular weight, internal metabolism soon and non-immunogenicity and interested to researchers.At present octreotide and rgd peptide are the most classical molecular diagnosis of clinical practice and the polypeptide for the treatment of: octreotide labelling radionuclide comes into the clinical practice stage,99mTc-EDDA/HYNIC-TOC can be good at diagnosing neurosecretion type tumor such as cancer of pancreas, cerebral glioma；Rgd peptide can be in conjunction with integrin receptor αvβ3And αvβ 5 grade (it is specificity overexpression in tumor neogenetic blood vessels), by rgd peptide labelling radionuclide99mTc、18F and111In etc. can be specific to tumor imaging, the rgd peptide (NC100692) of 99m Tc labelling and [18F] Galacto-RGD also come into clinical three the phases experiment.Polypeptide derivative has become following new diagnostic medicine and the key areas of curative drug research and development.
The vasculolymphatic formation of tumor neogenetic is to promote Tumor cell dissemination and lymphatic metastasis the key link, particularly branch mode cervical cancer, breast carcinoma and gastric cancer etc. based on lymphatic metastasis.The lymph node dissection to be carried out that the tumor patient such as breast carcinoma and cervical cancer is conventional clinically, to improve the survival rate of patient, the course for the treatment of determining the chemotherapy after operation in patients and scheme and the prognosis of assessment patient.Substantial amounts of research proves before Nasopharyngeal neoplasms to lymph node, lymph node microenvironment has occurred and that and changes the migration (seed soil theory) promoting tumor cell, wherein in lymph node, the substantial increase of microenvironment VEGFC promotes the formation (cancer research, 2006) of a large amount of Lymphangiogenesis.Based on Lymphangiogenesis at neoplasm metastasis and the importance sent out, scientists is wished to develop a kind of for the vasculolymphatic developer of tumor neogenetic, 2010Utilize VEGFR-3 monoclonal antibody mF4-3IC1 labelling radionuclide111In can image (Nuclear Medicine and Biology 37 (2010) 957-964) to the primary tumor of tumor and micro-Metastatic Lymph Nodes SPECT/CT, within 2007, utilizes VEGFR-3 scFv antibody labeling131Iodine images (ONCOLOGY REPORTS 18:933-941,2007) to tumor.But owing to antibody molecule amount is big, metabolism in vivo is mainly through liver etc., and the background such as the such as spleen of distribution in vivo, lung regulating liver-QI is higher, and imaging results is not good enough.And polypeptide molecular weight is little, internal metabolism mainly has good prospect through advantages such as kidney and immunogenicity are low in tumor imaging application.But it is the fewest now for vasculolymphatic polypeptide developer, domestic The Fourth Military Medical University filters out a polypeptide (CSDSWHYWC) combining VEGFR-3 receptor, imaging results the best (2010, Third Military Medical University's journal) after labelling radionuclide.In sum, the most also there is no a kind of preferably targeting Lymphangiogenesis radionuclide image agent.
VEGFR-3 albumen is a kind of tyrosine kinase receptor (FLT4), its part is VEGF-C and VEGF-D, all expressing at all of endotheliocyte at the early development VEGFR-3 of embryo, but be as the maturation of embryo, its expression is only limited in lymphatic endothelial cells.The most multinomial research confirms that VEGFR-3 plays an important role in the generation of Lymphangiogenesis and maintenance, and directly related with the propagation of tumor and Invasion and Metastasis, can be as one of lymphatic vessel mark.The up-to-date research display VEGFR-3 about VEGFR-3 is not only at lymphatic expression, and the blood capillary at tumor neogenetic is expressed and also raised, and can promote tumor neovasculature formation.About VEGFR-3 tumor cell whether express still suffer from so far dispute, some documents report VEGFR-3 high expressed in a lot of tumor tissues, especially in the breast carcinoma based on lymph metastasis, gastric cancer and cervical cancer etc., but within 2008, it is published in the article on " cancer cell " magazine " VEGFR-3 Expression Is Restricted to Blood and Lymphatic Vessels in Solid Tumors " research and confirms that VEGFR-3 expression is only limitted to Lymphangiogenesis and blood vessel, do not express at tumor cell." the In vivo imaging of lymphatic vessels in development that Sagrario Ortega in 2012 is published on PNAS, wound healing, inflammation, and tumor metastasis " prove that the also height of the VEGFR-3 specificity in the expression of Lymphangiogenesis, the especially sentinel node in neoplasm metastasis generating process and trace neoplasm metastasis lymph node is expressed further.In sum, although whether VEGFR-3 expresses in tumor still suffers from dispute, but its height in new vessels and Lymphangiogenesis is expressed the clearest and the most definite, especially VEGFR-3 can express all show that VEGFR-3 can be as the targeted molecular of tumor diagnosis and therapy as sentinel node and the trace neoplasm metastasis lymph node height of one of vasculolymphatic marker molecule with neoplasm metastasis.
TMVP1 (LARGR) is that this laboratory filters out the novel tumor targeting peptides containing five amino acid by bacterial flagellum peptide storehouse display technique, its receptor is Lymphangiogenesis marker molecule VEGF R3 (VEGFR-3, also FLT4 is become), the experimentation of early stage shows, TMVP1 can specific combine with VEGFR-3, and targeting is incorporated into tumor neogenetic lymphatic vessel and the tumor tissues of high expressed VEGFR-3.TMVP1 is connected apoptosis peptide (KLAKLAK) 2, it is possible to the formation of suppression Lymphangiogenesis and the growth of tumor.
One good developer should possess advantages below: good stability in vivo, not by vivo protein enzymatic degradation, the organ background beyond target organs is low, and in making illness position body, imaging results is good.Most of natural polypeptide half-life in vivo is shorter, amicine (somatostatin) polypeptide that such as clinical practice is classical, only about three minutes its naturally occurring form half-life in vivo, in order to improve polypeptide metabolism in vivo stability, slow down internal various protease system to the degraded of polypeptide and increase the polypeptide drugs half-life in vivo, most of natural polypeptidess need through structure of modification repeatedly to reach the level of clinical practice.The transformation of polypeptide structure is had following several method: 1. polypeptide cyclization, limit the motility of polypeptide core sequence；2. D type aminoacid is increased；3. the modification of polypeptide amino acid includes methylating, refines and hydroformylation etc..The core sequence of TMVP1 is LARGR, and according to above theoretical basis, the present invention carries out structure of modification to it makes it the most stable and the half-life is longer.Up to now, the polypeptide tracer being applied to clinic is mainly radionuclide,99mTC is the widest radionuclide of nuclear medicine application, and its utility ratio reaches 80%.99mThe Tc half-life only has 6 hours, has range short safety advantages of higher.So the present invention is by improved LARGR polypeptide marker radionuclide99mTc, application nuclear medicine SPECT instrument carries out primary tumors and micrometastasis lymph node imaging to the Lymphangiogenesis of high expressed VEGFR-3 molecule.
It is contemplated that develop a kind of novel VEGFR-3 molecular receptor developer99mTc-HYNIC/EDDA-TMVP1, utilizes this developer can image primary tumors and Metastatic Lymph Nodes SPECT, increases early stage primary tumor and the diagnosis of sentinel node of malignant tumor.The blank of tumor neogenetic lymphatic vessel polypeptide developer has been filled up in this invention, improves the SPECT imaging results to primary tumors and Metastatic Lymph Nodes.
Summary of the invention:
Based on above technical background and the medical demand of VEGFR-3 acceptor molecule developer, the present invention a kind of carries out the improved structural formula of polypeptide structure, labelling radionuclide by disclosing on the basis of the peptide T MVP1 (LARGR) containing five amino acid99mStructural formula, preparation method and the application in primary tumors and metastasis diagnose after Tc.This preparation method is utilized to be available for the tumor developer of VEGFR-3 acceptor molecule99mTc-HYNIC/EDDA-TMVP1, this developer can target tumor tissues and the Metastatic Lymph Nodes of VEGFR-3 high expressed.Utilize Nuclear Medicine Department's SPECT instrument, this developer can be to including the tumor VEGFR-3 molecular imagings such as cervical cancer, breast carcinoma, ovarian cancer, melanoma and hepatocarcinoma, and then reach primary tumors and the diagnosis of micrometastasis lymph node and provide theoretical foundation for follow-up TMVP1 derivatives for treatment tumor, weak point crucial in current tumor cells diagnostic techniques and practice can be solved.
Invention achieves following effect:
1. by polypeptide marker radionuclide99mTc: radionuclide99mTc launches γ photon, and Clinical practice SPECT instrument diagnoses various diseases has become one of conventional diagnostic techniques of medical institutions.The present invention uses bifunctional chelating agent HYNIC and is total to part EDDA by polypeptide marker radionuclide99mTc, artificial synthetic polypeptide HYNIC-E-G-Cyclic (CGLARGRGC), utilize this laboratory existing polypeptide marker radionuclide99mThe ripe scheme of Tc is by TMVP1 labelling radionuclide99mTc.After labelling success, its structural formula is:
99mTTc-HYNIC/EDDA-E-G-Cyclic(CGLARGRGC)
3. experiment in vitro shows99mTc-HYNIC/EDDA-TMVP1 in the serum of normal saline, cysteine and normal person can stable existence for 18 hours, and it can be specific binding with lymphatic endothelial cells and vascular endothelial cell.Utilizing slow virus after process LAN VEGFR-3 in tumor cell, this newtype drug can be specific mutually affine with it.The above results confirms not affecting its specificity combined with VEGFR-3 after the structure of modification of polypeptide,99mTc-HYNIC/EDDA-TMVP1 can be as VEGFR-3 acceptor molecule developer.
4. use this novel VEGFR-3 acceptor molecule developer can the nude mice by subcutaneous tumor model SPECT such as cervical cancer, breast carcinoma, ovarian cancer, melanoma and hepatocarcinoma be imaged, subcutaneous tumors imaging is clear, its imaging results is relevant with tumor tissues VEGFR-3 expression, and this developer is through renal metabolism, remaining organ uptake ratio is low.
Four, accompanying drawing explanation:
Fig. 1: experimental design process figure.
Fig. 2:99mThe chemical constitution of Tc-HYNIC/EDDA-TMVP1.
Fig. 3:99mTc-HYNIC/EDDA-TMVP1 stability test in vitro.
Fig. 4:99mTc-HYNIC/EDDA-TMVP1 and azygos vein endotheliocyte compatibility test.
Fig. 5: different time points 4T1 tumor model SPECT imaging after intravenous injection developer.
Fig. 6: 4T1 subcutaneous tumors model99mTc-HYNIC/EDDA-TMVP1 and 400 μ g TMVP1 joint injection competitive binding SPECT imaging.
Fig. 7: various subcutaneous tumors animal model tumors SPECT image.
Fig. 8:99mTc-HYNIC/EDDA-TMVP1 is the distribution situation of each organ in 4T1 tumor-bearing mice.
Expression of tumor tissue situation in Fig. 9: VEGFR-3 various tumor models.
Four, detailed description of the invention:
1. the synthesis of polypeptide
Synthetic HYNIC-E-G-Cyclic (CGLARGRGC) polypeptide, purity is 98%, by its molecular weight of RP-HPLC and MS technology for detection and quality.
2. the radioisotope labeling of polypeptide
Naturally cool to room temperature
3. desalting and purifying scheme
Use Sep-pak vac C-18 reversed-phase column purification (being purchased from waters company of the U.S.).Cross post solution successively: 5mL dehydrated alcohol, 5mL normal saline, 5mL air, reaction mixture, 5mL normal saline.200 μ L 70% dehydrated alcohol eluted product, each of eluting solution is put in the EP pipe of 1.5ml respectively, detects radioactivity and the radio-chemical purity of each.
Use this tagging scheme and purification schemes successfully by radionuclide99mTc is tagged on HYNIC-E-G-Cyclic (CGLARGRGC).More than 99% is all reached after before purification.
4. radio-chemical purity detection
The radio-chemical purity of three kinds of developing solvents detection synthetic products of iTLC-SG application:
1., developing solvent acetone determines to be not bound with what polypeptide dissociated99mTc measures (Rf=1.0)
2., 0.1M citric acid (PH=5.0) determines99mTc-Coligand is with free99mThe amount (Rf=1.0) of Tc
3., methanol: ammonium acetate=1: 1 (v/V) determines99mTc-colloid (Rf=0)
99mTc-HYNIC/EDDA-TMVP1 mobility in three kinds of developing solvents is 0.0,0.3,0.7-1.0 respectively,
Experimental result:99mTc-HYNIC/EDDA-TMTP1 labeling effciency more than 99%, free99mTc、99mTc-coligand、99mTc-colloid is almost nil.
5. vitro stability test:
1. the stability in normal saline: 1mci99mTc-HYNIC/EDDA-TMVP1 uses thin layer chromatography iTLC-SG to detect its stability after placing 1h, 3h, 6h and 18h in normal saline.
2. the stability in cysteine: 1mci99mTc-HYNIC/EDDA-TMVP1 adds in 1mL cysteine solution (1mg/mL), uses thin layer chromatography iTLC-SG to detect its stability after incubated at room temperature 1h, 3h, 6h and 18h.
3. the stability in normal blood group: by 1mci99mTc-HYNIC/EDDA-TMVP1 adds in 1ml normal human serum, 37 DEG C hatch 1h, 3h, 6h and 18h, after having arrived incubation time, sucking-off 250 μ L sample serum and 750 μ L methanol/acetonitrile (1: 1) mixing respectively, fully after mixing, 3000rpm is centrifuged 3 minutes, takes supernatant and detects its radio-chemical purity.
Experimental result:99mTc-HYNIC/EDDA-TMVP1 stability in normal saline, in cysteine and normal serum is preferable, the most still reaches 86.94%, 94.07% and 96.99% after 18.
6. the affine test of cell
With ECM culture medium culturing people's azygos vein endotheliocyte HUVEC (deriving from ATCC), when cell density reaches 80%, counting after trypsin digestion cell, seed cells in 24 holes and (control cell density about 70%), second day by 1 μ ci99mTc-HYNIC/EDDA-TMVP1 joins in adherent cell orifice plate, is transferred in Pasteur's pipe by every hole supernatant respectively after hatching 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours, and washes twice with ice-cold cell PBS.It is subsequently adding 500ul 0.1M NaOH and cell dissociation gets off to be transferred to a new Pasteur pipe, count with r-counts.In order to avoid non-specific binding, secondary orifices is set by unlabelled99mTc-HYNIC/EDDA-TMVP1 joins in orifice plate together with the polypeptide of labelling, detects its cellular uptake rate.
Experimental result: prolongation over time, HUVEC cellular uptake99mTc-HYNIC/EDDA-TMVP1 dosage increases, and 6 little %AD constantly reach 3%.The picked-up that cell is described is specific picked-up, and HUVEC cell VEGF expression R-3, according to theoretical foundation99mThe receptor of Tc-HYNIC/EDDA-TMVP1 is VEGFR-3, and it can combine with high expressed VEGFR-3 cell.
7. foundation and the SPECT of tumor model images
Set up cervical cancer, ovarian cancer and breast carcinoma subcutaneous tumor model:
(1) tumor cell is prepared
Cell cervical cancer tumer line C-33A, HeLa and SiHa and the ovarian cancer cell line SKOV3 DMEM culture medium culturing containing 10% serum, (37 DEG C, 5%CO2Incubator is cultivated), mouse mammary carcinoma cell line 4T1 and human breast cancer cell line SKBR3 RPMI 1640 culture medium culturing.When cell grows to 80% with 0.25% trypsinization, after terminating digestion with complete medium, cell is collected in 15mL centrifuge tube, 800rpm is centrifuged 5 minutes, cell PBS is resuspended for supernatant discarded, 800rpm is centrifuged 5 minutes again, cell PBS is resuspended for supernatant discarded, and cell counting calculates cell density.
(2) subcutaneous tumors model
BALB/c-nude nude mice and the common BALB/C in 3-4 week buy in Beijing HFK Bio-Technology Co., Ltd., raise in Tongji Medical College, Huazhong Science and Technology Univ.'s animal medicine center SPF level Animal House.1 × 10 is inoculated respectively at every BALB/c-nude nude mice axillary fossa fat pad7Individual SKOV3,1 × 107Individual SiHa, 5 × 106Individual C-33A or 3 × 106Individual HeLa cell, after about inoculating 20-27 days, nude mice by subcutaneous tumor grows to 1-1.5cm2.By 1 × 106Individual 4T1 cell is inoculated at the right front mammary gland original position of common BALB/C mouse, about 1-2 week tumor growth to 1-1.5cm2。
(3) SPECT imaging
According to preparation method synthesizing and purifying of the present invention99mTc-HYNIC/EDDA-TMVP1, this medicine of 1mci is passed through tail vein injection in tumor-bearing mice, anaesthetize with 3% pentobarbital sodium respectively at different time points, the tumor-bearing mice anaesthetized is carried out SPECT instrument low energy high-resolution rate collimator and gathers anteposition imaging (acquisition parameter: matrix 128 × 128, window width 20%, amplification 4 times, energy peak 140Kev, static state gather 10 minutes).CBA take by99mThe TMVP1 of Tc-HYNIC/EDDA-TMVP1 and 400 μ g is injected into SPECT imaging in tumor-bearing mice simultaneously.
SPECT images experimental result: 1) 1mei99mTc-HYNIC/EDDA-TMVP1 tail vein injection enters in 4T1 breast cancer in situ model, and along with the prolongation of injection time, tumor locus imaging is more and more obvious, and the when of 3 hours, 4 hours and 5 hours, imaging results is preferably (such as Fig. 5).2), after in the TMTP1 of 400 μ g and developer are injected into 4T1 tumor-bearing mice simultaneously, competitive binding group mouse tumor model position imaging results is the most far short of what is expected compared with experimental group.CBA SPECT image results proves that this developer is specific (Fig. 6) to the imaging of tumor.3)99mTc-HYNIC/EDDA-TMVP1 can to kinds of tumors model visualization such as cervical cancer subcutaneous tumors model (C33A, SiHa and HeLa) and Ovarian Cancer Model (SKOV3 and A2780), but this developer can not be to hepatocarcinoma HepG2 subcutaneous tumors model visualization (Fig. 7).
7. distribution in vivo test
Subcutaneous tumors model is set up according to the method described above, 20 μ ci's99mTc-HYNIC/EDDA-TMVP1 passes through tail vein in 15 4T1 subcutaneous tumors mices, every three one group, blood the execution of cervical vertebra detachment is taken by eyeball venous sinus respectively at 60min, 2h, 4h and 6h, the tumor of every mice, the heart, liver, spleen, lung, kidney, stomach, small intestinal, large intestine, muscle and brain being taken out and weigh, r-counts counts its radioactivity.Take three 1mci99mTc-HYNIC/EDDA-TMVP1 is dissolved in 100 μ L normal saline respectively, loads and seals tubule decay correction after injection.Calculate percent dose rate %ID/g often organizing each organ of mouse, graphpad prism analytic statistics experimental result.
Distribution in vivo result of the test:99mTc-HYNIC/EDDA-TMVP1 mainly drains through urinary system, and its kidney is 23.7%ID/g 2 hours percent dose rates, is more than 20 times of other organs.It 2 hours is that 0.65%ID/g, T/B and T/M are respectively 2.73 and 4.24 (Fig. 8) in the distribution of 4T1 tumor
8. VEGFR-3 expression in its tumor tissues of tissue chemical analysis.
By fixing for the tumor tissues formaldehyde of tumor-bearing mice and specimens paraffin embedding slices, histochemistry step is as follows:
1. paraffin section dehydration, the roasting sheet of paraffin section 68 DEG C 2 hours, proceed to immediately in environmental protection dewaxing liquid 1 10 minutes, it is immersed in successively in following solution: environmental protection dewaxing liquid 210 minutes, dehydrated alcohol 5 minutes, 95% ethanol 5 minutes, 80% ethanol 5 minutes, 75% ethanol 5 minutes, is finally putting into groupization with in PBS.
2. PBS washes 3 × 5 minutes.
3. 3% hydrogen peroxide 37 DEG C hatches 30 minutes
4. PBS washes 3 × 5 minutes.
5. citric acid antigen repair liquid height fire boils low fire reparation 10 minutes.Naturally cool to room temperature.
6. PBS washes 3 × 5 minutes.
7. 5% lowlenthal serum 37 DEG C is closed 1 hour.
8. VEGFR-3 antibody 1:100 dilutes (biolegend clone9D9F9) 4 DEG C of overnight incubation.
9. PBS washes 3 × 10 minutes.
10. biotin labeling goat antirabbit 37 DEG C hatches 30 minutes.
PBS washes 3 × 10 minutes.
Horseradish peroxidase-labeled chain poison avidin 37 DEG C hatches 30 minutes.
PBS washes 3 × 10 minutes.
DAB develops the color, lucifuge incubated at room 5min.
Lignum Sappan uniformly dyeing core 10 minutes, 1% hydrochloride alcohol decolouring, 3% ammonia returns indigo plant, examines under a microscope its nuclear targeting situation.
Dehydration, cuts into slices and is soaked into solution successively: 75% ethanol 5 minutes, 80% ethanol 5 minutes, 95% ethanol 5 minutes, dehydrated alcohol 5 minutes, environmental protection dewaxing liquid 210 minutes, environmental protection dewaxing liquid 110 minutes.Dry
Resinene mounting.
Take pictures under Leika microscope and observe tumor tissues VEGFR-3 expression.
VEGFR-3 group result: SPECT imaging obvious tumor tissues (such as 4T1 and HeLa) VEGF expression R-3 is many, and images unconspicuous tumor tissues such as HepG2 and express relatively low.
Claims (2)
1. a novel VEGFR-3 receptor developer99mTc-HYNIC/EDDA-TMVP1, including core sequence LARGR polypeptide, is characterized in that LARGR
Polypeptide passes through cysteine disulfide bond cyclization, utilizes bifunctional chelating agent 6-hydrazinonicotinic acid (HYNIC) and is total to part EDDA (EDDA)
E-G-Cycli c (CGLARGRGC) is connected radionuclide99mTc。
The most according to claim 1,99mTc-HYNIC/EDDA-TMVP1 is applied to the diagnosis of tumor, it is characterized in that utilizing99mTc-HYNIC/EDDA-TMVP1
High expressed VEGFR-3 tumor tissues can be targeted and tumor is carried out SPECT imaging.
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| CN114044804A (en) * | 2021-10-29 | 2022-02-15 | 厦门大学附属第一医院 | TMTP1 polypeptide ligand radioactive probe and preparation method and application thereof |
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| CN103804470A (en) * | 2014-01-22 | 2014-05-21 | 深圳市奥尼克斯基因技术有限公司 | Acquisition and application of novel polypeptide TMVP1 of specificity targeting tumoral lymphatic vessel |
| CN104208727A (en) * | 2013-08-10 | 2014-12-17 | 深圳市奥尼克斯基因技术有限公司 | Preparation and applications of novel tumor developing agent (99m) TC-HYNIC/EDDA-TMTP1 |
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| CN104208727A (en) * | 2013-08-10 | 2014-12-17 | 深圳市奥尼克斯基因技术有限公司 | Preparation and applications of novel tumor developing agent (99m) TC-HYNIC/EDDA-TMTP1 |
| CN103804470A (en) * | 2014-01-22 | 2014-05-21 | 深圳市奥尼克斯基因技术有限公司 | Acquisition and application of novel polypeptide TMVP1 of specificity targeting tumoral lymphatic vessel |
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| CN114044804A (en) * | 2021-10-29 | 2022-02-15 | 厦门大学附属第一医院 | TMTP1 polypeptide ligand radioactive probe and preparation method and application thereof |
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