CN109481667A - Transthyretin is inhibiting the application in neonate tumour blood vessel - Google Patents

Transthyretin is inhibiting the application in neonate tumour blood vessel Download PDF

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Publication number
CN109481667A
CN109481667A CN201811530055.8A CN201811530055A CN109481667A CN 109481667 A CN109481667 A CN 109481667A CN 201811530055 A CN201811530055 A CN 201811530055A CN 109481667 A CN109481667 A CN 109481667A
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transthyretin
blood vessel
cancer
application
cell
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肖振
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Shanghai Kashu Biomedical Technology Co Ltd
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Shanghai Kashu Biomedical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cardiology (AREA)
  • Hematology (AREA)
  • Vascular Medicine (AREA)
  • Oncology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Marine Sciences & Fisheries (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses transthyretins to inhibit the application in neonate tumour blood vessel, belongs to biochemistry, molecular biology and medical domain.The present invention demonstrates the effect of TTR angiogenesis inhibiting by tumor neogenetic blood vessels in vitro models, the results show that TTR, which acts on huve cell, can obviously inhibit 90% vascularization.

Description

Transthyretin is inhibiting the application in neonate tumour blood vessel
Technical field
The present invention relates to transthyretins to inhibit the application in neonate tumour blood vessel, belongs to biochemistry, molecule Biology and medical domain.
Background technique
Angiogenesis (Angiogenesis), which refers to from vein after existing capillary or capillary, to be developed and is formed New blood vessel specifically includes that activation phase blood vessel basement membrane degradation；Activation, proliferation, the migration of vascular endothelial cell；It rebuilds and is formed New blood vessel and rete vasculosum is the complex process of a different kinds of molecules for being related to various kinds of cell.Vascularization is to promote vascularization The complex process of the factor and inhibiting factor coordinative role, the two is in equilibrium state under normal circumstances, once this balance is broken Vascular system can be activated, make angiogenesis excessively or vascular system is inhibited to make vascular deterioration.Angiogenesis mechanism is complicated, participates in simultaneously Promote the factor of angiogenesis also numerous, macrophage quantity obviously increases in EMT peritoneal fluid, the TNF-α and IL-8 of secretion It can promote the proliferation of vascular endothelial cell, transforming growth factor-β (TGF-β), thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF), heparitinase, angiogenin (angs), osteogenin (OPN), Cycloxygenase (COX-2), hypoxia inducible The factor -1, laminin (LN), placenta growth factor (PLGF), Survivin, hematopoietin (Epo) both participate in EMT vascularization process (De Palma M.Nat.Rev.Cancer 2,017 08；17(8)457-474).
The related disease of pathologic neovascularization includes tumor neogenetic blood vessels (Li X.Science 2018Mar23；359 (6382) 1335-1336), cerebral ischemia new vessels (Chang J.Nat.Med.2017；23 (4) 450-460), the new green blood of kidney Manage (Amin EM.Cancer Cell 2011；20 (6) 768-780), diabetes new vessels (Hu J.Nature 2017 1214；552 (7684)) etc..New vessels are produced as a complicated biological process, involve a variety of growth factors and its message Receptor is transmitted, and targeting single molecule possibly in message transmission path (signaling cascade) can not be to disease (example Such as cancer) in uncontrolled angiogenesis effective clinical treatment is provided.Therefore, development can the several keys of collaborative combination blood vessel The newborn factor will constantly be increased with the demand of the innovative treatments of effective angiogenesis inhibiting and disease process.Nowadays blood is treated Pipe is newborn mainly to utilize targeted drug anti-vegf therapy, however the individual insensitivity and drug resistance clinic for targeted drug are still Do not capture method effectively.
Summary of the invention
The first purpose of the invention is to provide transthyretins in the drug that preparation inhibits neonate tumour blood vessel Application.
In one embodiment of the invention, the Genbank accession number of the transthyretin is CAG33189.1。
In one embodiment of the invention, the effective dose of the transthyretin is per unit >=4mmol.
In one embodiment of the invention, the effective dose of the transthyretin is >=4mmol/L.
In one embodiment of the invention, the transthyretin acts on tumor neogenetic blood vessels outgrowth factor VEGF。
In one embodiment of the invention, the neonate tumour blood vessel includes liver cancer, lung cancer, bladder cancer, breast cancer, The carcinoma of the rectum, osteosarcoma, gastric cancer, cancer of pancreas, the angiogenesis of leukaemia, lymthoma, myeloma blood cancer.
In one embodiment of the invention, the neonate tumour blood vessel hemangioma, the concurrent tissue blood vessel of hemangioma are new It is raw, angiomatosis, hemangioblastoma, non-malignant vascular proliferative disease.
The present invention is also claimed the transthyretin that Genbank accession number is CAG33189.1 and is preparing new green blood Application in terms of pipe outgrowth factor VEGF inhibitor.
Advantages of the present invention and effect: present invention discover that TTR, which acts on huve cell, can obviously inhibit 90% Vascularization；It acts in rat and mouse model, new vessels is effectively inhibited to generate up to 90%.
Detailed description of the invention
Fig. 1 is the chemical structural drawing (A) of TTR；And the cell activity (B) under difference TTR concentration；
Fig. 2 is huve cell transition graph；Wherein, A is that the normal huve cell 48 of DMEM in high glucose culture is small When, microscopically observation photograph takes 5 visuals field to carry out the mobility that cytometer figures cell at random；After B is exogenous addition TTR Microscopically observation photograph, take 5 visuals field to carry out cytometer at random and figure the mobility of cell；
Fig. 3 is that huve cell is tested at pipe, wherein L is that low sugar DMEM culture medium simulates blood vessel under normal environment New life, H are angiogenesis under DMEM in high glucose culture；T is that the blood vessel after cultivating exogenous addition TTR under DMEM in high glucose environment is new It is raw.
Specific embodiment
Embodiment 1
As shown in Figure 1, TTR structure is is centrosymmetric, axial symmetry；Monomer includes 147 amino acid, encodes the Gene of TTR ID is 7276.TTR corresponds to GenbanK accession number is the amino acid sequence of CAG33189.1.
Human microvascular endothelial cell (mvec) is acted on using the TTR of mtt assay detection various concentration under DMEM in high glucose condition of culture Cell viability after (3000/ hole).The results show that group of cells proliferation compares after 48h, 0 μm of ol/L group A value for (0.40 ± 0.03), 4 μm of ol/L group A values are (0.17 ± 0.02).4 μm of ol/L groups are higher than 0 μm of ol/L group, difference to the inhibiting effect of proliferation Statistically significant (t=15.47, P=0.0001) (Fig. 1).4 μm of ol/L group cell Proliferations reduce compared with 0 μm of ol/L group 57.4%.It can be seen that TTR has inhibiting effect to the proliferation of human microvascular endothelial cell (mvec).
The migration experiment of 2 huve cell of embodiment
It is separately added into the 600 μ l of DMEM in high glucose culture medium containing 0 μM and 4 μM TTR in each hole of 24 orifice plates, is placed in simultaneously The cell transwell that aperture is 8.0 μm, small interior 200 μ l of addition contain 1 × 104A Human umbilical vein endothelial cells are trained without FBS Support base culture.It is inhaled after 48h and abandons room culture medium up and down, cleaned upper chamber inner cell with cotton swab, 0.4g/L poly first is added in 24 orifice plates 600 μ l room temperature of aldehyde fixes 30min, inhales and abandons paraformaldehyde, and PBS rinses the cell back side twice, and 600 μ of crystal violet is added in 24 orifice plates L, room temperature dye 20min, and PBS rinses the cell back side twice after suction, and microscopically observation photograph takes 5 visuals field to carry out thin at random Born of the same parents count.
48h Cell migration assay the results show that 0 μm of outside transport number of ol/L group Human umbilical vein endothelial cells be (227 ± 14) a, 4 μm of outside transport numbers of ol/L group human umblilical vein endothelial are that (140 ± 7) are a.The outside transport number of cell compares between two groups, 4 μ Than 0 μm ol/L group of mol/L is 150 ± 3 low, difference statistically significant (t=6.75, P=0.0005) (Fig. 2).It can be seen that TTR pairs The migration of Human umbilical vein endothelial cells has inhibition.
3 huve cell of embodiment is tested at pipe
The Matrigel matrigel that -20 DEG C save is placed in 4 DEG C of refrigerator overnights, when it melts into red colloidal fluid completely It can be used when body.Matrigel glue, every 150 μ l of hole are added into 48 orifice plates of pre-cooling with the pipette tips of pre-cooling.On ice gently Culture plate is shaken, Matrigel glue is made to be laid in board bottom.48 orifice plates are then placed in 37 DEG C of incubator 30min, make Matrigel Gelling is solid.Long to when 80%~90% fusion when cell, replacement serum free medium starvation is for 24 hours.PBS is cleaned cell 3 times, is added 0.25% trypsin digestion cell, volume are limited with covering bottom of bottle, are jiggled body, are come into full contact with pancreatin with cell, and 37 DEG C It is incubated for several minutes.Cellular morphology is observed under inverted microscope, when discovery cytoplasm retraction, space between cells increases, but cell is still When non-levitating, complete medium is added immediately and terminates digestion.It blows and beats culture bottle bottom repeatedly with culture solution, completely falls off cell, Whole liquid are collected into centrifuge tube, cell counting board counts.Room temperature centrifugation, 1000rpm, 5min.It discards supernatant, is added new Culture solution, piping and druming are centrifuged, 1000rpm, 5min at cell suspension, isometric packing into four centrifuge tubes again.Each pipe discards Supernatant is added isometric each group conditioned medium and cell suspension is made.With 3 × 104The density in a/hole, which is inoculated in, has completed glue Culture plate in, every group sets 2 multiple holes, 37 DEG C, is incubated for 8h in 5%CO2 incubator.Cell is observed using inverted microscope, and Image is acquired with photographic system.Using ImagePro-Plus software analysis image, the number for forming lumen of vessels is calculated.
As a result as shown in figure 3, L is normal glucose concentration DMEM culture medium, H is DMEM in high glucose culture medium, and T is DMEM in high glucose training Base+TTR is supported, lumen state is presented in normal huve cell blood vessel, and the angiogenesis after exogenous addition TTR is pressed down System, using Matrigel matrigel can Fiber differentiation in vitro huve cell angiogenesis, form luminal structure.It is interior 4h initially forms luminal structure to chrotoplast after incubation, and 8h can establish the external model of stable vascularization.High sugar+TTR Group (50 ± 1) substantially reduces (P=0.013, < 0.05) than high sugar group lumen number (500 ± 20), and difference is statistically significant.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Claims (8)

  1. The transthyretin that 1.Genbank accession number is CAG33189.1 is in the drug that preparation inhibits neonate tumour blood vessel Application.
  2. 2. application according to claim 1, which is characterized in that the effective dose of the transthyretin is per unit ≥4mmol。
  3. 3. application according to claim 1 or 2, which is characterized in that the effective dose of the transthyretin be >= 4mmol/L。
  4. 4. any application according to claim 1~3, which is characterized in that the Genbank of the transthyretin is stepped on Record number is CAG33189.1.
  5. 5. application according to any one of claims 1 to 4, which is characterized in that the transthyretin acts on tumour Neovascularization resulting factor Ⅴ EGF.
  6. 6. applying according to claim 1 or 5, which is characterized in that the neonate tumour blood vessel includes liver cancer, lung cancer, wing Guang cancer, breast cancer, the carcinoma of the rectum, osteosarcoma, gastric cancer, cancer of pancreas, the angiogenesis of leukaemia, lymthoma, myeloma blood cancer.
  7. 7. applying according to claim 1 or 5, which is characterized in that the neonate tumour blood vessel hemangioma, hemangioma are concurrent Tissue blood vessel is newborn, angiomatosis, hemangioblastoma, non-malignant vascular proliferative disease.
  8. The transthyretin that 8.Genbank accession number is CAG33189.1 is preparing neovascularization resulting factor Ⅴ EGF inhibition Application in terms of agent.
CN201811530055.8A 2018-12-14 2018-12-14 Transthyretin is inhibiting the application in neonate tumour blood vessel Pending CN109481667A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101102758A (en) * 2004-09-15 2008-01-09 奥德威研究院 Thyroid hormone analogs for promoting angiogenesis
CN101855553A (en) * 2007-06-29 2010-10-06 科里罗吉克系统公司 The predictive marker of oophoroma
US20160137725A1 (en) * 2014-10-15 2016-05-19 Cell Signaling Technology, Inc. Methylation and Acetylation Sites

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101102758A (en) * 2004-09-15 2008-01-09 奥德威研究院 Thyroid hormone analogs for promoting angiogenesis
CN101855553A (en) * 2007-06-29 2010-10-06 科里罗吉克系统公司 The predictive marker of oophoroma
US20160137725A1 (en) * 2014-10-15 2016-05-19 Cell Signaling Technology, Inc. Methylation and Acetylation Sites

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EBERT,L. ET AL.: ""TTR [Homo sapiens] ACCESSION NO：CAG33189"", 《GENBANK DATABASE》 *
JUN SHAO, YONG YAO: "Transthyretin represses neovascularization in diabetic retinopathy", 《 MOLECULAR VISION》 *
RAQUEL J. NUNES ET AL.,: "Transthyretin Proteins Regulate Angiogenesis by Conferring Different Molecular Identities to Endothelial Cells", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 *
邵珺: "转甲状腺素蛋白在糖尿病视网膜病变新生血管生成中的阻遏作用及机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 *
邵珺等: "高糖缺氧环境下转甲状腺素蛋白对视网膜血管内皮细胞的影响", 《中华眼底病杂志》 *

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Application publication date: 20190319