CN109248322A - Target the CKIP-1 RNAi compound and its preparation method and application of macrophage - Google Patents
Target the CKIP-1 RNAi compound and its preparation method and application of macrophage Download PDFInfo
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Abstract
The invention belongs to biomedicine fields, and in particular to target CKIP-1 RNAi compound of macrophage and its preparation method and application.The CKIP-1 RNAi compound connects combination by electrostatic interaction with CKIP-1 RNAi by fusogenic peptide, and the fusogenic peptide is connect with T9 peptide by V6 peptide and formed, and the nucleotide sequence of the CKIP-1 RNAi is as shown in SEQ ID NO.4.It is combined to obtain the CKIP-1 RNAi compound again with CKIP-1 RNAi connection by the synthesis of the fusogenic peptide, purifying.The CD11b receptor of CKIP-1 RNAi compound special target Macrophage Surface of the invention, high-efficiency transfection enters macrophage simultaneously, inhibit the expression of CKIP-1 and the activation of macrophage, and then inhibit by the inflammatory reaction after macrophage-mediated cerebral hemorrhage, and then can be used for preparing treatment by the anti-inflammatory drug after macrophage-mediated cerebral hemorrhage, there is good development and application prospect in cerebral hemorrhage treatment field.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to it is a kind of target macrophage CKIP-1 RNAi compound and
Preparation method and application.
Background technique
Cerebral hemorrhage (intracerebral hemorrhage, ICH) is clinically common cerebrovascular disease, is lacked effective
Treatment method, case fatality rate and disability rate are higher.The secondary neurotrosis of cerebral hemorrhage after a few days is the critical period of aggravation.
The mechanism of secondary neurotrosis is complicated, may relate to the formation of perihematoma brain edema, perihematoma metabolic disorder, perihematoma
The variation etc. of local cerebral blood flow (rCBF).Correlative study shows that macrophage-mediated inflammatory reaction is secondary in cerebral hemorrhage
Play a significant role in neurotrosis.It is now recognized that the inflammatory reaction of hemotoncus ingredient excitation has weight in secondary neurotrosis
It acts on and is concerned.The inflammatory cell activations such as perihematoma macrophage after ICH, release TNF-α, IL-1 etc. cause it is scorching because
Son, blood-brain barrier disruption, the inflammatory cells such as peripheral blood mononuclear macrophage are assembled to hemotoncus peripheral region in addition, thus induce inflammation
Cascaded amplification reaction eventually leads to secondary brain injury and neurologic impairment aggravation.
CKIP-1 initially as casein kinase 2 (CK2 kinases) binding protein and be found.Research shows that CKIP-1 tune
The form of ganglion cell, differentiation, the generation of apoptosis and tumour.CKIP-1 can be by lipopolysaccharides (LPS) induction in human monocyte cell line
It adjusts, and t cell activation can be inhibited in vitro by negative regulation NF- κ B, this prompt CKIP-1 may be in the adjusting of immune system
Play critical function.Recently studies have shown that CKIP-1 takes part in the polarization process of macrophage Ml/M2, CKIP-1 can inhibit M2
It polarizes and M1 is promoted to polarize.
Therefore, inhibit, interfere the expression of CKIP-1 in macrophage in the inflammation damnification of macrophage-mediated cerebral hemorrhage
Field has extremely wide application prospect.
Summary of the invention
In view of this, the compound can be special one of the objects of the present invention is to provide a kind of CKIP-1 RNAi compound
Different targeting macrophage inhibits the activation of macrophage, and then inhibits by macrophage-mediated central nervous system disease
Occur or deteriorates.
In order to achieve the above objectives, the invention provides the following technical scheme:
The CKIP-1 RNAi compound for targeting macrophage, targets the CKIP-1 RNAi compound of macrophage, special
Sign is that the CKIP-1 RNAi compound connects combination by electrostatic interaction with CKIP-1 RNAi by fusogenic peptide, described to melt
Conjunction peptide, which is connect by V6 peptide with T9 peptide, to be formed, and the amino acid sequence of the V6 peptide is as shown in SEQ ID NO.1, the amino of the T9 peptide
Acid sequence is as shown in SEQ ID NO.2, and the nucleotide sequence of the CKIP-1 RNAi is as shown in SEQ ID NO.4.
Further, the fusogenic peptide is made of 2 V6 peptides and 1 T9 peptide connection, successively by the c-terminus of V6 peptide, T9 peptide
Aminoterminal and the aminoterminal of V6 peptide be directly connected to.
Further, the amino acid sequence of the fusogenic peptide is as shown in SEQ ID NO.3.
Further, the mass ratio of the CKIP-1 RNAi and fusogenic peptide is 4:1.
The second object of the present invention is the provision of the CKIP-1 RNAi compound that macrophage is targeted described in one kind
Preparation method.
In order to achieve the above objectives, the invention provides the following technical scheme:
The preparation method of the CKIP-1 RNAi compound of the targeting macrophage, comprising the following steps:
1) synthesis of fusogenic peptide: the synthesis of the fusogenic peptide is on Peptide synthesizer and carries out, according to the fusogenic peptide
Amino acid sequence extends peptide chain one by one from c-terminus to aminoterminal as shown in SEQ ID NO.3；
2) purifying of fusogenic peptide: the resin containing peptide chain, which is transferred to cutting liquid and is stirred to react at room temperature, makes peptide chain from resin
On be cleaved, then filter, collect filtrate, low pressure is evaporated at room temperature, after residue deionized water dissolving, the hydraulic fluid phase in
Chromatograph is purified and is identified；
3) preparation of CKIP-1 RNAi compound: under vortex conditions, to CKIP-1 RNAi and citric acid-sodium citrate
In the aqueous solution of buffer, the fusion peptide solution of isometric concentration is added dropwise, after being added dropwise, continues to be vortexed, then stand to get institute
The CKIP-1 RNAi compound stated.
Further, cutting liquid described in step 2) is made of ethylenediamine tartrate, trifluoroacetic acid and deionized water, described
The volume ratio of ethylenediamine tartrate, trifluoroacetic acid and deionized water is 1:38:1.
Preferably, the time of mesoscale eddies is 60-90 minutes.
Preferably, the time of standing is 30-60 minutes.
CKIP-1 RNAi compound one kind that the third object of the present invention is the provision of the targeting macrophage is answered
With, can be used for preparing treatment by the anti-inflammatory drug after macrophage-mediated cerebral hemorrhage, have well in cerebral hemorrhage treatment field
Development and application prospect.
In order to achieve the above objectives, the invention provides the following technical scheme:
CKIP-1 RNAi compound inflammation damnification macrophage-mediated after being used to prepare encephalemia it is anti-inflammatory
Application in drug.
The fourth object of the present invention, which is the provision of, a kind of treats the anti-of macrophage-mediated inflammation damnification after encephalemia
Scorching drug.
In order to achieve the above objectives, the invention provides the following technical scheme:
The anti-inflammatory drug includes the CKIP-1 RNAi compound and pharmaceutically acceptable carrier or helps
Agent.
The administration route of the anti-inflammatory drug includes: oral administration, drug administration by injection, rectally, inhalation etc..
The drug dosage can according to specifically used dosage regimen, administration route, treated illness it is tight
Weight degree and the difference of subject's individual and change, can be determined by methods known in the art are conventional.
The beneficial effects of the present invention are:
1) V6 peptide is positively charged small peptide, can polymerize to form densification with negatively charged RNAi by electrical neutralization
Particle improves cell to the uptake ratio of RNAi, enhances transfection efficiency.CD11b receptor is in central nervous system mainly in macrophage
Expressed on cell, from the ligand polypeptide T9 of CD11b receptor N-terminal can block CD11b receptor combination physiological chemotactic because
The chemotaxis that son is formed, but itself do not cause Chemotaxis, intracellular signal transduction and cell natural activity are not influenced.
2) CKIP-1 RNAi compound of the present invention can pass through the guiding role of T9 peptide, special target macrophage table
Face specificity MCP-1 receptor, while T9 peptide and MCP-1 receptor acting, CKIP-1 RNAi high-efficiency transfection enters macrophage.
3) CKIP-1 RNAi compound of the present invention significantly inhibits the expression of CKIP-1 and the activation of macrophage, and then presses down
System is gone out by the inflammatory reaction after macrophage-mediated cerebral hemorrhage, such as cellular inflammation factor IL-8 and TNF-α, the significant brain that mitigates
Brain edema after blood, and improve nervous function.
Therefore, after CKIP-1 RNAi compound of the invention can be used for preparing treatment by macrophage-mediated cerebral hemorrhage
Anti-inflammatory drug, have good development and application prospect in cerebral hemorrhage treatment field.
Detailed description of the invention
Attached drawing 1 is the CKIP-1 RNAi compound of the transmission electron microscope identification in embodiment 1.
Attached drawing 2 is that the CKIP 1 of CKIP-1 RNAi compound erythrocyte cracked liquid stimulating expression of macrophage in embodiment 2 is expressed
It influences.
Attached drawing 3 is CKIP-1 RNAi compound in embodiment 2 to thin to macrophage after erythrocyte cracked liquid stimulating expression of macrophage
The influence of intracrine IL-8 and TNF-α.
Attached drawing 4 is the detection that CKIP-1 RNAi compound influences cerebral hemorrhage mouse brain edema in embodiment 2.
Attached drawing 5 is detection of the CKIP-1 RNAi compound to cerebral hemorrhage mouse Nerve function effect in embodiment 2.
Specific embodiment
Illustrated embodiment is in order to which preferably the present invention will be described, but is not that the contents of the present invention are limited only to institute
For embodiment.So those skilled in the art according to foregoing invention content to embodiment carry out it is nonessential improvement and
Adjustment, still falls within protection scope of the present invention.
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to of the invention excellent
Embodiment is selected to be described in detail.Test method without specific conditions in preferred embodiment, usually according to normal condition,
Such as in Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brooker etc. write, and Huang Peitang etc. is translated, Science Press, 2002)
The condition, or according to the normal condition proposed by manufacturer.
The preparation of 1 fusogenic peptide of embodiment
Fusogenic peptide is successively directly connected to by the aminoterminal of the aminoterminal and V6 peptide of the c-terminus of V6 peptide and T9 peptide, amino
Acid sequence is SEQ ID NO.3.
The synthesis of fusogenic peptide carries out on AB-431A type Peptide synthesizer, using standard Fmoc scheme.With 0.25mmol pairs
Hydroxymethyl phenoxy methylated polystyrene (HMP) resin makes peptide chain according to the amino acid sequence of fusogenic peptide H9-V9 for initial resin
Extend one by one from c-terminus to aminoterminal, after peptide chain synthesizes, the resin containing peptide chain is transferred to cutting liquid (by tartaric acid
Ethylenediamine 0.25mL, trifluoroacetic acid 9.5mL and deionized water 0.25mL composition) in, be stirred to react makes peptide chain from resin at room temperature
On be cleaved, then with G6 glass sand hourglass filter, collect filtrate, low pressure is evaporated at room temperature, residue deionized water dissolving
Afterwards, it uses100 type medium pressure liguid chromatograph of explorer is purified, and chromatographic column is C18 column, and mobile phase A is quality
The trifluoroacetic acid aqueous solution that score is 0.1%, Mobile phase B is the trifluoroacetic acid acetonitrile solution that mass fraction is 0.1%, binary line
Property gradient elution, the volume fraction of Mobile phase B rises to 50%, flow velocity 1mL/min by 10% in 0~15 minute, collects
The eluent of fusogenic peptide is freeze-dried to get fusogenic peptide, the solution that concentration is 3mg/mL is made with deionized water dissolving, uses hole
Diameter is 0.20 μm of filtering with microporous membrane degerming, -70 DEG C freeze it is spare.
The eluent for taking fusogenic peptide carries out Purity with 600 type reverse-phase HPLC instrument of Delta, and chromatographic column is
Symmetry C18 column, mobile phase A are the trifluoroacetic acid aqueous solution that mass fraction is 0.1%, and Mobile phase B is that mass fraction is
0.1% trifluoroacetic acid acetonitrile solution, binary linear gradient elution, the volume fraction of Mobile phase B is in 0~15 minute by 10%
60% is risen to, flow velocity 1mL/min.As a result it is calculated through areas of peak normalization method, the purity of fusogenic peptide is 98 ± 1.5%.Separately
The eluent for taking fusogenic peptide carries out molecular weight identification with 2000 LC/MS/MS type mass spectrograph of API, the results show that fusogenic peptide
Molecular weight measured value is consistent with theoretical value.
The preparation of 2 CKIP-1 RNAi compound of embodiment
The nucleotide sequence of CKIP-1 RNAi are as follows: 5'-CCTGAGTGACTATGAGAAG-3'(SEQ ID No.4).In whirlpool
It is slow to the citric acid-sodium citrate for being 0.1mol/L containing the CKIP-1 RNAi and concentration that concentration is 200 μ g/mL under the conditions of rotation
In the aqueous solution of fliud flushing, the fusion peptide solution that isometric concentration is 50 μ g/mL is added dropwise, after being added dropwise, continues to be vortexed 60 minutes,
30 minutes CKIP-1 RNAi compounds to get targeting macrophage are stood again.
By freshly prepared CKIP-1 RNAi compound drop on 200 mesh copper mesh, adsorbs 3 minutes, is blotted with blotting paper,
It dries 30 seconds, with acetic acid uranium aqueous solution negative staining 30 seconds that mass fraction is 1%, is blotted, dried 30 seconds with blotting paper, 80kV transmission
Electronic Speculum observation.As a result as shown in Fig. 1, gained CKIP-1 RNAi compound is in uniform subcircular particle, most
Particle aspect is less than 25nm.
Meanwhile according to above-mentioned same procedure, CKIP-1 RNAi is substituted with negative control (PBS), it is compound that negative control is made
Object.
The preparation of 3 CKIP-1 RNAi compound of embodiment
The nucleotide sequence of KIP-1 RNAi are as follows: 5'-CCTGAGTGACTATGAGAAG-3'(SEQ ID No.4).In whirlpool
It is slow to the citric acid-sodium citrate for being 0.1mol/L containing the CKIP-1 RNAi and concentration that concentration is 200 μ g/mL under the conditions of rotation
In the aqueous solution of fliud flushing, the fusion peptide solution that isometric concentration is 50 μ g/mL is added dropwise, after being added dropwise, continues to be vortexed 90 minutes,
60 minutes CKIP-1 RNAi compounds to get targeting macrophage are stood again.
By freshly prepared CKIP-1 RNAi compound drop on 200 mesh copper mesh, adsorbs 3 minutes, is blotted with blotting paper,
It dries 30 seconds, with acetic acid uranium aqueous solution negative staining 30 seconds that mass fraction is 1%, is blotted, dried 30 seconds with blotting paper, 80kV transmission
Electronic Speculum observation.As a result as shown in Fig. 1, gained CKIP-1 RNAi compound is in uniform subcircular particle, most
Particle aspect is less than 25nm.
Meanwhile according to above-mentioned same procedure, CKIP-1 RNAi is substituted with negative control (PBS), it is compound that negative control is made
Object.
The preparation of 4 CKIP-1 RNAi compound of embodiment
The nucleotide sequence of KIP-1 RNAi are as follows: 5'-CCTGAGTGACTATGAGAAG-3'(SEQ ID No.4).In whirlpool
It is slow to the citric acid-sodium citrate for being 0.1mol/L containing the CKIP-1 RNAi and concentration that concentration is 200 μ g/mL under the conditions of rotation
In the aqueous solution of fliud flushing, the fusion peptide solution that isometric concentration is 50 μ g/mL is added dropwise, after being added dropwise, continues to be vortexed 75 minutes,
45 minutes CKIP-1 RNAi compounds to get targeting macrophage are stood again.
By freshly prepared CKIP-1 RNAi compound drop on 200 mesh copper mesh, adsorbs 3 minutes, is blotted with blotting paper,
It dries 30 seconds, with acetic acid uranium aqueous solution negative staining 30 seconds that mass fraction is 1%, is blotted, dried 30 seconds with blotting paper, 80kV transmission
Electronic Speculum observation.As a result as shown in Fig. 1, gained CKIP-1 RNAi compound is in uniform subcircular particle, most
Particle aspect is less than 25nm.
Meanwhile according to above-mentioned same procedure, CKIP-1 RNAi is substituted with negative control (PBS), it is compound that negative control is made
Object.
Embodiment 5 targets the anti-inflammatory effect research of the CKIP-1 RNAi compound of macrophage
1, the detection of macrophage CKIP-1 after erythrocyte splitting object is handled
The macrophage or control macrophage that CKIP-1 RNAi compound is incubated for is added in 10 μ l erythrocyte cracked liquids,
37 DEG C, for 24 hours, with the expression of WB method detection erythrocyte cracked liquid stimulating expression of macrophage CKIP-1.Result of study is as shown in Fig. 2,
" 1 " is the CKIP-1 protein expression of control group macrophage, and " 2 " are the CKIP-1 albumen table of CKIP-1 RNAi group macrophage
It reaches.The expression for the macrophage CKIP-1 that CKIP-1 RNAi compound is incubated for is considerably less than control macrophage.
2, the detection of the inflammatory factor of macrophage after erythrocyte splitting object is handled
By macrophage in monolayer cultivation in 6 orifice plates to area coverage about 30%, plasma-free DMEM medium 2mL is changed to,
And 100 μ L of CKIP-1 RNAi compound is added, it cultivates 4 hours, then change to complete DMEM culture medium 2mL, cultivates 48 hours, receive
Collect cell, washed and be resuspended with PBS, obtains the macrophage of CKIP-1 RNAi compound transfection.By 10 μ l erythrocyte cracked liquids
Macrophage or control macrophage is added, 37 DEG C, for 24 hours, is secreted with ELISA method detection erythrocyte cracked liquid stimulating expression of macrophage
The ability of IL-8 and TNF-α.Result of study is as shown in Fig. 3, secreted by the macrophage of CKIP-1 RNAi compound transfection
IL-8 and TNF-α considerably less than control group macrophage.
3, the detection of the brain edema of cerebral hemorrhage mouse
The 25 self whole bloods of μ l are injected into mouse Basal ganglia, after ten minutes, and it is multiple in same area injection CKIP-1 RNAi
Close 100 μ L of object or control group compound.After 3 days, mouse is put to death, obtains brain tissue, and detects the brain water of mouse with dry and wet weight method
Swollen situation.Result of study is as shown in Fig. 4, and compared with the control group, 1 RNAi compound of CKIP can significantly mitigate cerebral hemorrhage mouse
Brain edema.
4, the detection of the nervous function of cerebral hemorrhage mouse
The 25 self whole bloods of μ l are injected into mouse Basal ganglia, after ten minutes, and it is multiple in same area injection 1 RNAi of CKIP
Close 100 μ L of object or control group compound.After 3 days, with Neuroscore method, the nervous function of mouse is observed.Result of study is such as
Shown in attached drawing 5, compared with the control group, 1 RNAi compound of CKIP can significantly improve the nervous function of mouse.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Sequence table
<110>Yongchuan Hospital Affiliated to Chongqing Medical University
<120>the CKIP-1 RNAi compound and its preparation method and application of macrophage is targeted
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<170> PatentIn Version 3.5
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Claims (10)
1. targeting the CKIP-1RNAi compound of macrophage, which is characterized in that the CKIP-1RNAi compound is by fusogenic peptide
Combination is connected by electrostatic interaction with CKIP-1RNAi, the fusogenic peptide is connect with T9 peptide by V6 peptide and formed, the ammonia of the V6 peptide
Base acid sequence is as shown in SEQ ID NO.1, and the amino acid sequence of the T9 peptide is as shown in SEQ ID NO.2, the CKIP-
The nucleotide sequence of 1RNAi is as shown in SEQ ID NO.4.
2. CKIP-1RNAi compound according to claim 1, which is characterized in that the fusogenic peptide is by 2 V6 peptides and 1
T9 peptide connection composition, is successively directly connected to by the aminoterminal of the c-terminus of V6 peptide, the aminoterminal of T9 peptide and V6 peptide.
3. CKIP-1RNAi compound according to claim 2, which is characterized in that the amino acid sequence of the fusogenic peptide is such as
Shown in SEQ ID NO.3.
4. CKIP-1RNAi compound according to claim 1, which is characterized in that the CKIP-1RNAi and fusogenic peptide
Mass ratio is 4:1.
5. the preparation method of the CKIP-1RNAi compound of macrophage is targeted described in claim 1-4 any one, including
Following steps:
1) synthesis of fusogenic peptide: the synthesis of the fusogenic peptide is on Peptide synthesizer and carries out, according to the amino of the fusogenic peptide
Acid sequence extends peptide chain one by one from c-terminus to aminoterminal as shown in SEQ ID NO.3；
2) purifying of fusogenic peptide: the resin containing peptide chain, which is transferred to cutting liquid and is stirred to react at room temperature, splits peptide chain from resin
Solution is got off, and is then filtered, and collects filtrate, low pressure is evaporated at room temperature, after residue deionized water dissolving, uses medium pressure liquid chromatography
Instrument is purified and is identified；
3) preparation of CKIP-1RNAi compound: under vortex conditions, to CKIP-1RNAi and citric acid-sodium citrate buffer solution
Aqueous solution in, the fusion peptide solution of isometric concentration is added dropwise, after being added dropwise, continues to be vortexed, then stand to get described
CKIP-1RNAi compound.
6. according to the method described in claim 5, it is characterized in that, cutting liquid described in step 2) is by ethylenediamine tartrate, three
Fluoroacetic acid and deionized water composition, the volume ratio of the ethylenediamine tartrate, trifluoroacetic acid and deionized water are 1:38:1.
7. according to the method described in claim 5, it is characterized in that, the time of step 3) mesoscale eddies is 60-90 minutes.
8. according to the method described in claim 5, it is characterized in that, the time stood in step 3) is 30-60 minutes.
9. CKIP-1RNAi compound described in claim 1-3 any one is macrophage-mediated after being used to prepare encephalemia
Inflammation damnification anti-inflammatory drug in application.
10. a kind of anti-inflammatory drug for treating macrophage-mediated inflammation damnification after encephalemia, which is characterized in that described is anti-inflammatory
Drug includes CKIP-1RNAi compound described in claim 1 and pharmaceutically acceptable carrier or auxiliary agent.
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Citations (1)
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| WO2015027895A1 (en) * | 2013-08-26 | 2015-03-05 | 苏州瑞博生物技术有限公司 | Nucleic acid, pharmaceutical composition and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015027895A1 (en) * | 2013-08-26 | 2015-03-05 | 苏州瑞博生物技术有限公司 | Nucleic acid, pharmaceutical composition and uses thereof |
| CN105473164A (en) * | 2013-08-26 | 2016-04-06 | 苏州瑞博生物技术有限公司 | Nucleic acid, pharmaceutical composition and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李定 等: "《计算机辅助 药物设计基础》", 31 March 2018, 西北农林科技大学出版社 * |
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Application publication date: 20190122 |