CN111410694A - Anti-glioma polypeptide molecule and application thereof - Google Patents
Anti-glioma polypeptide molecule and application thereof Download PDFInfo
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- CN111410694A CN111410694A CN202010226824.6A CN202010226824A CN111410694A CN 111410694 A CN111410694 A CN 111410694A CN 202010226824 A CN202010226824 A CN 202010226824A CN 111410694 A CN111410694 A CN 111410694A
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- glioma
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Abstract
The invention discloses an anti-glioma polypeptide molecule, which consists of a leader peptide TAT and a PICK1, and also discloses an application of the anti-glioma polypeptide molecule in preparing an anti-glioma drug, and an application of the anti-glioma polypeptide molecule in a nude mouse subcutaneous glioma model.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an anti-glioma polypeptide molecule and application thereof.
Background
Glioma is the most common primary malignant tumor of the nervous system, accounts for more than 45 percent of tumors of the central nervous system, and seriously threatens human health. The world health organization divides the disease into 4 grades, wherein the I grade and the II grade are low-grade gliomas, the III grade and the IV grade are high-grade malignant gliomas (neuroblastoma), the incidence rate of the malignant gliomas is 3/10000, but the malignant gliomas have the characteristics of strong infiltration capacity, easy invasion, high recurrence rate and mortality, and the average life cycle of patients is only 12-15 months. At present, malignant glioma mainly adopts treatment modes such as surgical resection, radiotherapy, chemotherapy and the like, but the prognosis of a patient is still poor, and the survival time is short. With the development of brain tumor research, how to accurately and targetedly treat the brain glioma becomes a significant problem. Aiming at the existing problems, a molecule or a medicine capable of accurately targeting brain glioma is urgently needed, which becomes an important research direction for effectively treating brain tumor and is one of important ways for solving the existing problems.
Disclosure of Invention
The invention aims to provide an anti-glioma polypeptide molecule to solve the problems in the prior art, the polypeptide molecule has a targeting effect, and the PICK1 gene in most brain gliomas is low-expressed and is combined with intracellular T L R4 through penetrating cell membranes to cause endocytosis of the brain gliomas, so that inflammatory pathways of the brain gliomas are blocked, and the occurrence and development of gliomas are influenced.
The invention also aims to provide the application of the anti-glioma polypeptide molecule in the preparation of anti-glioma drugs, and the polypeptide molecule can accurately target glioma, so that the problem that glioma cannot be accurately targeted at present is solved, and the diagnosis of glioma or the guidance of clinical medication is realized.
The invention also provides application of the anti-glioma polypeptide molecule in a nude mouse subcutaneous glioma model, and verifies that the polypeptide molecule can accurately target the brain glioma and inhibit the growth of brain glioma cells on an animal level.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an anti-glioma polypeptide molecule which is formed by connecting a leader peptide TAT and a PICK1 through an amide bond. The polypeptide is synthesized by Kinsley Biotechnology GmbH, and the chemical structure characteristics of the polypeptide molecule are shown in figure 1 and figure 2.
Preferably, the leader TAT amino acid sequence is YGRKKRRQRRR and the PICK1 amino acid sequence is YGRKKRRQRRRVPGKVT L QKDAQ.
The invention also provides application of the anti-glioma polypeptide molecule in preparing an anti-glioma drug, wherein the polypeptide molecule penetrates through a cell membrane to be combined with intracellular T L R4, so that the polypeptide molecule is endocytosed, and an inflammatory pathway is blocked to inhibit glioma.
Preferably, the polypeptide molecule is used to inhibit glioma cell C6 migration.
Preferably, the polypeptide molecule is used to inhibit the proliferation of glioma cell C6.
The invention also provides application of the anti-glioma polypeptide molecule in a nude mouse subcutaneous glioma model, and the anti-glioma polypeptide molecule is used for inhibiting growth of glioma cells in the nude mouse subcutaneous glioma model.
The invention discloses the following technical effects:
the invention discloses a PICK1 which is a multifunctional binding protein, is closely related to central nervous system diseases, pain, insulin secretion and tumor development and development, and PICK1 can be combined with a phospholipid bilayer and the C-terminal of a plurality of transmembrane receptors to regulate the expression and endocytosis on the cell membrane of the receptors, PICK1 has low expression in brain glioma tissues and maintains glioma cell inflammatory reaction through a T L R4/MyD 88/NF-kB pathway, while the invention designs a synthesized TAT-PICK1 polypeptide by taking a TAT leader peptide as a penetrating peptide, wherein the polypeptide can penetrate the cell membrane to be combined with intracellular T L R4 to cause partial endocytosis, thereby blocking an inflammatory pathway dependent on MyD88 and influencing the development and development of glioma, and the polypeptide can quickly transduce and activate transcription factors which are already inactivated in tumor cells and activate signal pathways related to transcription factors, thereby inhibiting the growth of tumor, providing guidance for the research related to the fields of drug guidance and the development of TAT-PICK1 which can penetrate the cell membrane and can be used for the targeted therapy of glioma.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a synthetic high performance liquid chromatogram of a TAT-PICK1 polypeptide of the invention;
FIG. 2 is a synthetic mass spectrum of a TAT-PICK1 polypeptide of the invention;
FIG. 3 is a graph showing the results of the TAT-PICK1 polypeptide of the present invention inhibiting migration of glioma cells C6; a: staining results of the control group; b: the dyeing result of the experimental group; c: the number of cells in the control and experimental groups;
FIG. 4 is a graph showing the results of the TAT-PICK1 polypeptide of the present invention on the inhibition of proliferation of glioma cells C6;
FIG. 5 shows the effect of TAT-PICK1 polypeptides of the present invention on inhibiting tumor growth in a nude mouse subcutaneous graft tumor model.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
TAT-PICK1 purity was analyzed by analytical high performance liquid chromatography (flow rate: 1 ml/min). The method specifically comprises the following steps:
the model of the analytical high performance liquid chromatograph: agilent 1200, type of chromatography column used: angilenteclipse XDB-C18 Analytical,5um,4.6X150006 Dm.
The chromatographic operation conditions are linear gradient elution, eluent consists of mobile phase A liquid and mobile phase B liquid, wherein the mobile phase A liquid is trifluoroacetic acid aqueous solution (water is used as a solvent) with the volume percentage concentration of 0.1%, the mobile phase B liquid is trifluoroacetic acid acetonitrile solution (acetonitrile is used as the solvent) with the volume percentage concentration of 0.1%, the elution time is 12min totally, the elution flow rate is 1m L/min, and the ultraviolet detection wavelength is 220 nm.
The detection result of the analytical high performance liquid chromatograph shows that: the purity of the obtained TAT-PICK1 was 97.2%.
Example 2
TAT-PICK1 chemical structure was characterized by MA L DI-TOF mass spectrum, and TAT-PICK1 mass spectrum characterization results are shown in FIG. 2 molecular weight (Mw): 2825.30, and other three peaks also demonstrated that TAT-PICK1 molecular weight was consistent.
Example 3
TAT-PICK1 polypeptide molecule inhibits migration of glioma cells C6
The effect of TAT-PICK1 polypeptide on the migration ability of glioma cells C6 was examined using a Transwell plate (Corning Corp., product # 3422). Cell migration experiments were performed according to the Transwell plate instructions. The main operation steps are as follows:
1) the glioma cells C6 and TAT-PICK1 polypeptide which are digested and counted are diluted to 20 ten thousand/m L by serum-free culture solution DMEM (final concentration is 30uM), 200u L is taken and added into the upper chamber of the Transwell, 600u L complete cell culture solution is added into the lower chamber of the Transwell to serve as an experimental group, and serum-free culture solution containing TAT leader peptide is used as a negative control group.
2) In CO 25% CO at 37 ℃ in an incubator2Culturing for 24h, scraping the cells in the upper Transwell chamber with a cotton swab, fixing with 4% paraformaldehyde for 30min, washing with PBS buffer for 1 time, dyeing with 0.1% crystal violet for 30min, washing with PBS buffer for 1 time, and counting the number of migrated cells under microscope.
The experimental results are shown in fig. 3, and the results show that: TAT-PICK1 polypeptide can obviously inhibit the function of C6 migration of glioma cells.
Example 4
The TAT-PICK1 polypeptide inhibits glioma cell C6 proliferation.
The inhibition effect of TAT-PICK1 polypeptides with different concentrations on the proliferation of glioma cells C6 is detected by a CCK-8 kit (Biyuntian, Cat. C0038). The specific operation steps are as follows:
1) glioma cells C6 were plated in 96-well plates with a confluency of around 50%. Three replicate wells were set for each group of cells.
2) After the cells were attached, TAT-PICK1 polypeptide (final concentration of 30uM) was added as an experimental group, and three wells without added polypeptide were used as a control group.
3) At various time detection points (0, 0.5h, 1h, 1.5h, 2h), 10u L CCK-8 detection reagent was added to each well and incubated at 37 ℃ for 2 h.
4) The OD was measured at 490 nm.
The experimental results are shown in fig. 4, and the results show that: TAT-PICK1 polypeptide can obviously inhibit the proliferation of glioma cell C6.
Example 5
Growth inhibition of TAT-PICK1 on tumors in nude mouse subcutaneous transplantation tumor model
In order to study the effect of TAT-PICK1 polypeptide on tumor growth in an in vivo mouse model, a glioma cell transplantation model mouse was used, TAT-PICK1 was injected periodically around the tumor, and the tumor growth was observed, thereby evaluating the effect of TAT-PICK1 on glioma. The method comprises the following specific operation steps:
1) selecting 5-week-old 18-20g BA L B/C nude mice, inoculating nude mice with cultured glioma cells C6 growing in logarithmic phase, and injecting 1 × 10 per one time6And C6 cells.
2) When the tumor grows to 110mm3Thereafter, the nude mice selected for consistent tumor size were randomly divided into 2 groups of 10 mice each. 6mg/kg of TAT-PICK1 polypeptide was injected peritumorally on days 0, 3, 5, 7, and 9 of the experiment, with the TAT leader peptide as a negative control. Tumor growth was recorded daily.
3) After 10 days of dosing, all nude mice were sacrificed by cervical dislocation and the subcutaneous tumors were photographed.
The experimental results are shown in fig. 5, and the results show that: TAT-PICK1 in a nude mouse subcutaneous transplantation tumor model has obvious inhibition effect on the growth of the tumor after 10 days of action.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (6)
1. An anti-glioma polypeptide molecule consisting of a leader peptide TAT and a PICK1 linked by an amide bond.
2. The glioma-resistant polypeptide molecule of claim 1 wherein the leader peptide TAT amino acid sequence is YGRKKRRQRRR and the amino acid sequence of PICK1 is YGRKKRRQRRRVPGKVT L QKDAQ.
3. Use of the anti-glioma polypeptide molecule of any one of claims 1-2 in the preparation of an anti-glioma drug, wherein said polypeptide molecule penetrates a cell membrane and binds to intracellular T L R4, resulting in its endocytosis, blocking inflammatory pathways, and inhibiting glioma.
4. Use of the anti-glioma polypeptide molecule of claim 3 in the preparation of an anti-glioma medicament, wherein said polypeptide molecule is used to inhibit glioma cell C6 migration.
5. Use of the anti-glioma polypeptide molecule of claim 3 in the preparation of an anti-glioma medicament, wherein said polypeptide molecule is used to inhibit the proliferation of glioma cells C6.
6. Use of an anti-glioma polypeptide molecule of any one of claims 1-2 in a nude mouse subcutaneous glioma model for inhibiting the growth of glioma cells in the nude mouse subcutaneous glioma model.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114989259A (en) * | 2022-05-27 | 2022-09-02 | 成都佩德生物医药有限公司 | Small molecular peptide Ped4 and application thereof |
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| WO2007090094A2 (en) * | 2006-01-27 | 2007-08-09 | The University Of Mississippi Medical Center | Thermally-targeted delivery of medicaments, including doxorubicin |
| WO2008131099A1 (en) * | 2007-04-19 | 2008-10-30 | The Johns Hopkins University | Biological agents active in central nervous system |
| CN102300578A (en) * | 2008-12-01 | 2011-12-28 | 延寿有限责任公司 | Methods And Compositions For Altering Health, Wellbeing, And Lifespan |
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Patent Citations (5)
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|---|---|---|---|---|
| EP1910837B1 (en) * | 2005-07-01 | 2012-03-14 | Arbor Vita Corporation | Methods and compositions for diagnosis and treatment of influenza |
| WO2007090094A2 (en) * | 2006-01-27 | 2007-08-09 | The University Of Mississippi Medical Center | Thermally-targeted delivery of medicaments, including doxorubicin |
| WO2008131099A1 (en) * | 2007-04-19 | 2008-10-30 | The Johns Hopkins University | Biological agents active in central nervous system |
| CN102300578A (en) * | 2008-12-01 | 2011-12-28 | 延寿有限责任公司 | Methods And Compositions For Altering Health, Wellbeing, And Lifespan |
| CN102858985A (en) * | 2009-07-24 | 2013-01-02 | 西格马-奥尔德里奇有限责任公司 | Method for genome editing |
Non-Patent Citations (5)
| Title |
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| JONATHAN G. HANLEY: "PICK1: A multi-talented modulator of AMPA receptor trafficking", 《PHARMACOLOGY AND THERAPEUTICS》 * |
| JUNYU XU: "Structure and Function of PICK1", 《NEUROSIGNALS》 * |
| 孟胜男: "《药剂学》", 31 January 2016, 中国医药科技出版社 * |
| 谢娟 等: "PICK1对人肝癌细胞HepG2增殖的影响", 《安徽医科大学学报》 * |
| 陈娟 等: "PICK1在中枢神经系统中的研究进展", 《湖北科技学院学报（医学版）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114989259A (en) * | 2022-05-27 | 2022-09-02 | 成都佩德生物医药有限公司 | Small molecular peptide Ped4 and application thereof |
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