CN103509181A - New ionizing radiation damage resistance function of polyethyleneimine compounds, and its application - Google Patents
New ionizing radiation damage resistance function of polyethyleneimine compounds, and its application Download PDFInfo
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- CN103509181A CN103509181A CN201210202531.XA CN201210202531A CN103509181A CN 103509181 A CN103509181 A CN 103509181A CN 201210202531 A CN201210202531 A CN 201210202531A CN 103509181 A CN103509181 A CN 103509181A
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Abstract
The invention relates to a new ionizing radiation damage resistance function of polyethyleneimine (PEI) compounds, and its application. The PEI mainly has two structures comprising a linear structure (the molecular formula is (C2H5N)x) and a branched structure (the molecular formula is (C2H5N)x[-N(C2H6N)C2H4-]y), wherein each of x and y is an integer not lower than 1. The PEI can specifically activate a cell TLR5 acceptor mediated NF-kappaB signal channel. Results of experiment researches show that the PEI is weakly toxic to cells and animals, can substantially activate the cell TLR5 acceptor mediated NF-kappaB signal channel and display a dosage dependency, and can substantially reduce the mortality of ionizing radiation damaged mice and prolong the animal survival time. The new ionizing radiation damage resistance function of the PEI can be used for researching medicines for preventing and treating ionizing radiation damages and damages caused by radiotherapy.
Description
Technical field:
The present invention relates to the new function of ionizing radiation damage protection effect and the application thereof of polyethyleneimine: aminated compounds.
Background technology:
Along with the widespread use in military, science and technology and daily life such as nuclear energy, electromagnetic energy, it is increasing that people contact the chance of radiation, for the control medicine of radiation injury, also become one of major fields of current biomedical research.Radiation injury control medicine is to give treatment to protection ionization damage to cause that medical science endangers one of the most effective and direct means, and contact front and back all can be used and act on different.The ionization radiation injury control medicine of most study mainly contains: sulfocompound, hormones, plant amedica, cytokine class and some newtype drugs based on mechanism of radiation damage research.Mostly there is limitation in the material with radiation injury preventive and therapeutic effect having been reported so far, as protective effect not significantly, toxicity cannot be applied to more greatly clinical etc.Therefore, develop special, efficient, low toxicity, there is the antiradiation injury medicine of independent intellectual property right, to accelerating existing radiation injury protective drug upgrading, the actual demand of the threat of satisfied reply core terror and the protection of tumour patient radiation-induced damage etc. is significant.
(Toll-like Receptor 5, TLR5) belongs to TLR receptor family to Toll sample acceptor 5, is the acceptor that a class plays a significant role in natural immune system identification, mainly at intestinal epithelial cells, monocyte, scavenger cell and 1 expressed by dendritic cells.TLR5 belongs to pattern recognition receptors, and downstream main signal path is NF-κ B path, and known its unique natural agonist is bacterial flagellin (flagellin) at present.Experimentation on animals proves, the fiagellin structure of take has the dual function of significant radiological protection and tumor radiotherapy protection as basic polypeptide drugs CBLB502.
Polymine (polyethylenimine, PEI) is a kind ofly to have compared with the compound of high-cation electric density, claims again polyethylene imine.CAS:9002-98-6,73 ℃-75 ℃ of melting ranges, 250 ℃ of boiling points, can be water-soluble, current commercial molecular weight product greatly about 600-250000 not etc., the synthetic PEI that can also obtain other molecular weight in chamber by experiment.PEI mainly contains line style (molecular formula (C2H5N) x) and branched (molecular formula (NHCH2CH2-) * [N (CH2CH2NH2) CH2CH2-] y) two kinds of structure formations (wherein x and y are more than or equal to 1 integer).In recent years its structure is transformed, obtained the PEI molecule of superelevation branch.PEI is used as cationic flocculant, pollutant treatment agent etc. at industrial circle conventionally; In the application of biomedicine field, be mainly the carrier as gene transfection, be a kind of be hopeful very much to obtain clinical application gene therapy transport vehicle; Also have report, branched polymine (PEI) separately or the conventional antibacterials (as amphotericin B, fluconazole and polymyxin B) different classes of with three kinds combine Candida albicans had to good anti-mycotic efficiency.
Our seminar is devoted to the research in the fields such as host anti-virus innate immune reaction and TLR signal transduction for many years, successfully cloned people TLR5 full length gene, set up the NF-κ B Pathway Activation agent screening cell model of TLR5 mediation, utilize this model, screened and obtained a kind of polyethyleneimine: aminated compounds of new ionizing radiation damage protection effect, for researching and developing ionization radiation injury protection and medicine and tumour patient radiotherapy, caused that the protection of damage and medicine provide technical foundation.
Summary of the invention:
The object of the invention is: based on TLR5 receptor stimulant, can activate NF-κ B and host's innate immune reaction, thereby the mechanism of performance radiate protective action provides a kind of TLR5 of activation acceptor, has the compound of ionization radiation injury provide protection.
Specifically; the present invention has set up the NF-κ B Pathway Activation agent screening cell model of TLR5 mediation; utilize this model to screen; find that polyethyleneimine: aminated compounds PEI has the characteristic that activates TLR5 acceptor; and there is provide protection to being subject to the mouse of ionization radiation injury; be a kind of novel ionizing radiation-resistant damage compound, can develop becomes Novel electric and from radiation injury protection and medicine and tumour patient radiotherapy, causes protection and the medicine of damage.
In the present invention, the outstanding advantages of PEI molecular radiation provide protection is embodied in: (1) high efficiency: by reporter gene luciferase assays method, find, PEI molecule can activate TLR5 acceptor in 0.1-1.0 μ M concentration range, TLR5 downstream NF-κ B reporter gene uciferase activity is significantly raise, and present dose-dependently; For the C57BL/6J mouse of accepting total dose 8Gy radiation exposure, PEI can significantly improve tested mouse mean body weight, reduces the rear mortality ratio of irradiation, extend dead mouse survival time in 5-8mg/kg dosage range.(2) security: in cell levels experiment, PEI when working concentration is 0.01-1.0 μ M to HEK293T cell without remarkable toxicity, only when 5 μ M, HEK293T cell is shown to certain toxicity; In the experiment of integral animal level, abdominal injection PEI dosage does not cause tested dead mouse while being 8mg/kg.(3) being easy to get property: PEI molecule is a kind of synthetics, abundant raw material, finished product easily obtain, price is suitable, have avoided the expensive ,Yao source of biotechnology medicine and natural plant rare etc. not enough.
Accompanying drawing explanation:
Fig. 1 is PEI molecular structure of chemistry (A.PEI structural formula; B. the branched PEI structural representation of superelevation).
Fig. 2 is the toxicity of PEI to HEK293T cell.
Fig. 3 is that PEI activates the receptor-mediated NF-κ of TLR5 B signal path.
Fig. 4 is PEI, and to the provide protection of ionization radiation injury mouse, (A.PEI can suppress the Mouse Weight that ionizing rays causes and alleviate; B.PEI can reduce the mortality ratio of ionization radiation injury mouse and extend the survival time of ionization radiation injury mouse).
Embodiment:
One, the foundation of the NF-κ B Pathway Activation agent screening cell model of TLR5 mediation:
Extract CaCo-2 cell total rna, through RT-PCR method, obtain hTLR5 full-length gene, be cloned in pcDNA3.1-V5/HisB carrier, obtain pcDNA3.1-V5/HisB-hTLR5 recombinant plasmid, through enzyme, cut, express and check order and identify that this recombinant plasmid is correct.
By pcDNA3.1-V5/HisB-hTLR5 and NF-κ B luciferase reporter gene, sea pansy Luci reporter gene (TK) transfection simultaneously HEK293T cell, adopt two fluorescence detections, by the uciferase activity of NF-κ B reporter gene, reflect the activation situation of hTLR5 gene; Utilizing this cell model to screen has the compound of lateral reactivity, small peptide and natural drug etc.
Two,, by above-mentioned model discrimination, screen PEI and there is TLR5 receptor agonist activity, and observe PEI ionization radiation injury mouse is had to provide protection.Specific implementation process is as follows:
Embodiment mono-: the cell levels toxicity assessment of PEI
1.HEK293T cell cultures
HEK293T cell, in the DMEM substratum containing 10% foetal calf serum and 200 μ g/ml, is cultivated in 37 ℃, 5%CO2 incubator.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, and cell is inoculated to 96 orifice plates (1 * 10
4cells/well) 37 ℃, 5%CO2 cultivation.
The preparation of 2.PEI working fluid
Branched PEI is purchased from Sigma company (Cat.408727), light scattering method (LS) is surveyed its weight-average molecular weight (Mw) and is about 25000, gel permeation chromatography (GPC) records its viscous modulus (M η) and is about 10000,73 ℃-75 ℃ of melting ranges, 250 ℃ of boiling points, room temperature lower density 1.03g/ml.The accurate PEI 10.0mg that claims, is dissolved in 4ml containing in dual anti-DMEM nutrient solution, obtains PEI100 μ M storing solution; With being diluted to working concentration containing dual anti-DMEM nutrient solution, carry out cell experiment.
3.MTT detects
Be seeded to cell cultures in 96 orifice plates 24 hours, after growing to 40-60% cell and converging, add the DMEM nutrient solution containing different concns PEI (0.01-5 μ M), within 6 hours, Hou Mei hole adds MTT10 μ l/100ul nutrient solution, lucifuge is cultivated after 4 hours and is added formanzan lysate (100 μl/ hole), continue to hatch 4 hours, through multiple labeling inspection enzyme-linked immunosorbent assay instrument, 570nm detects absorbance.
4. result
When concentration is 0.01-0.5 μ M to HEK 293T cell without remarkable toxicity, only when 5 μ M, HEK293T cell is shown to certain toxicity.As shown in Figure 2.
Embodiment bis-: the activation of PEI to the NF-κ B path of TLR5 mediation
1.HEK293T cell cultures
HEK293T cell, in the DMEM substratum containing 10% foetal calf serum and 200 μ g/ml, is cultivated in 37 ℃, 5%CO2 incubator.Observation of cell growth conditions is good, is cultured to logarithmic growth after date, and cell is inoculated in to 24 orifice plates (0.5ml/ hole, about 3 * 10
6individual cell), CO
2overnight incubation in incubator.
The preparation of 2.PEI working fluid
Accurately weighed PEI 10.0mg, is dissolved in 4ml containing in dual anti-DMEM nutrient solution, obtains PEI100 μ M storing solution; With being diluted to working concentration containing dual anti-DMEM nutrient solution, carry out cell experiment.
3. Relative luciferase activity assay method detects the activation situation of the NF-κ B path of TLR5 mediation
By NF-κ B luciferase reporter gene and the common transfection HEK293T of PEI, proceed to sea pansy Luci reporter gene (TK) as internal reference simultaneously; After transfection 24 hours, add the DMEM nutrient solution containing PEI (0.1-1.0 μ M) or Flagellin (1 μ g/ml), continue to cultivate 6 hours, utilize luciferase reporter gene to measure kit measurement uciferase activity.
4. result
PEI molecule can activate TLR5 acceptor in 0.1-1.0 μ M concentration range, TLR5 downstream NF-κ B reporter gene uciferase activity is significantly raise, and present dose-dependently.As shown in Figure 3.
Embodiment tri-: the provide protection of PEI to ionization radiation injury mouse
1. ionization radiation injury Establishment of mouse model
Select the male 7-8 of specific-pathogen free (specific pathogen free, SPF) level C57BL/6J mouse in age in week, body weight (20 ± 2) g ，You Military Medical Science Institute's experimentation on animals center provides.Animal is according to body weight random packet: irradiation control group (IRmock), and PEI low dose group (PEI 5mg/kg), PEI high dose group (PEI 8mg/kg), PEI high dosage does not irradiate group (PEI8mg/kg mock).Irradiation control group mouse peritoneal injecting normal saline 20ml/kg, PEI low dose group mouse peritoneal injection PEI-normal saline solution 5mg/kg, PEI high dose group mouse peritoneal injection PEI-normal saline solution 8mg/kg, administration after 30 minutes these three groups of mouse accept the 60Co gamma-ray irradiation that total dose is 8Gy simultaneously, after irradiation, normally raise; PEI high dosage is not accepted x ray irradiation x after not irradiating group administration.
2. the observation of ionization radiation injury mouse
Mouse is accepted, after the damage of 60Co gamma-rays, normally to raise and observe 30 days, and every day, the mean body weight of mouse respectively organized in record, and mortality ratio and the survival time of mouse respectively organized in record.
3. result
Be subject to after the damage of 60Co gamma-rays, PEI processes Mouse Weight and declines seldom, and mortality ratio is significantly lower than irradiation control group, and dead mouse survival time is longer than irradiation control group; PEI integral level toxicity is less, and 8mg/kg dosage does not cause animal dead.As shown in Figure 4.
Claims (6)
1. a compound with ionization radiation injury protective effect, is characterized by: chemical name is polymine, and English name is polyethylenimine, referred to as PEI.
2. compound claimed in claim 1, is characterized by: linear polyethylene imine compound and branched polyethyleneimine: aminated compounds, linear polyethylene imine compound molecular formula is (C
2h
5n) x, branched polyethyleneimine: aminated compounds molecular formula is (NHCH
2cH
2-) x[-N (CH
2cH
2nH
2) CH
2cH
2-] y, wherein x and y are more than or equal to 1 integer.
3. compound claimed in claim 1, is characterized by: can obtain by the method for chemosynthesis, but be not limited to this method.
4. compound claimed in claim 1, is characterized by: can the receptor-mediated NF-κ of special activating cells TLR5 B signal path, and be a kind of TLR5 receptor stimulant.
5. compound claimed in claim 1, is characterized by: having ionizing radiation-resistant damaging action, is a kind of novel ionization radiation injury repellent compound, can be used for developing protection and the medicine of ionization radiation injury.
6. compound claimed in claim 1, is characterized by: can be used for developing the control medicine that tumour patient radiotherapy causes damage.
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Citations (3)
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|---|---|---|---|---|
| US20080164439A1 (en) * | 2007-01-10 | 2008-07-10 | Xinggao Fang | Textiles treated with hyperbranched polyethyleneimine derivatives for odor control properties |
| CN101638484A (en) * | 2009-08-24 | 2010-02-03 | 中国科学院长春应用化学研究所 | Polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine copolymer, preparation method thereof and application thereof |
| CN101812179A (en) * | 2009-07-14 | 2010-08-25 | 中国科学院长春应用化学研究所 | Reticular poly-beta-urethane/amide graft polyethyleneimine copolymer, preparation method and application in gene delivery |
-
2012
- 2012-06-19 CN CN201210202531.XA patent/CN103509181A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080164439A1 (en) * | 2007-01-10 | 2008-07-10 | Xinggao Fang | Textiles treated with hyperbranched polyethyleneimine derivatives for odor control properties |
| CN101812179A (en) * | 2009-07-14 | 2010-08-25 | 中国科学院长春应用化学研究所 | Reticular poly-beta-urethane/amide graft polyethyleneimine copolymer, preparation method and application in gene delivery |
| CN101638484A (en) * | 2009-08-24 | 2010-02-03 | 中国科学院长春应用化学研究所 | Polyethylene glycol monomethyl ether-poly 2-methyl-carboxyl propylene carbonate graft polyethyleneimine copolymer, preparation method thereof and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JUAN R. CUBILLOS-RUIZ等: "Polyethylenimine-based siRNA nanocomplexes reprogram tumor-associated dendritic cells via TLR5 to elicit therapeutic antitumor immunity", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| 祁鲁梁等: "《水处理药剂及材料实用手册》", 31 December 2006 * |
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Application publication date: 20140115 |