CN102533855A - Gene-vector composition of targeted microglia as well as preparation method and application of gene-vector composition - Google Patents
Gene-vector composition of targeted microglia as well as preparation method and application of gene-vector composition Download PDFInfo
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Abstract
The invention discloses a gene-vector composition of targeted microglia. The gene-vector composition is formed by combining a CX3CR1shRNA (Ribose Nucleic Acid) interference vector and a gene vector through an electrostatic interaction, wherein the gene vector is conjugate of H9 polypeptide and chitosan; the composition can be successfully targeted to a specific CX3CR1 acceptor molecule on the surface of the microglia under the guiding action of the H9 polypeptide; and while the H9 polypeptide exerts the antagonism for a CX3CR1 acceptor, the CX3CR1shRNA interference vector can be efficiently transfected into the microglia to intervene the expression of the CX3CR, inhibit the activation of the microgli and further inhibit the generation or deterioration of microglia-medicated nervous system diseases. The invention also discloses a preparation method of the gene-vector composition. The preparation method is simple and convenient to operate; the obtained gene-vector composition is nanoparticles with uniformity in size so as to bring convenience to uptake of cells; the gene-vector composition disclosed by the invention can be used for preparing a microglia activator inhibitor and has favorable development and application prospect in the field of treatment on the microglia-medicated nervous system diseases.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of cell targeted gene-carrier complexes that has, also relate to the preparation method and the application of this gene-carrier complexes.
Background technology
Microglia plays a significant role in inflammatory reaction as participating in immunoreactive main effects cell in the cns.As exotic disease substance invasion or have necrocytosis, microglia to be activated rapidly and, discharge the inflammatory factor inducing inflammatory reaction simultaneously with its removing.But there are some researches show that overactive microglia possibly quicken the process of some central nervous system disease, and the CF of its release also can cause further damage to neurocyte.For example, in Alzheimer's disease, find, with the active microglia of cortex same area existence at neuritic plaques place; Find that in Parkinson's disease mainly there is the zone in degeneration---there are a large amount of activated microglia in the black substance; In multiple sclerosis, the aixs cylinder degree of damage in the early lesion is corresponding with the activation degree of microglia.At present, the inflammatory reaction that is mediated by microglia is considered to the mechanism that multiple nervous system disorders takes place or worsens, and activated microglia is through discharging infringement neurone such as the number of mechanisms of urging inflammatory factor, cytotoxin and myelins.Therefore, the activation of appropriate inhibition microglia possibly be the effective way of treatment central nervous system disease.
CX3C Chemokine Receptors 1 (CX3CR1) is mainly expressed on microglia in cns, and its ligands specific CX3CL1 belongs to the δ class chemokine of CX3C family, is mainly secreted by neurone.CX3CL1 can promote chemotactic, propagation, the survival of microglia through the CX3CR1 acceptor, the secretion of the rising of intracellular Ca2+ level and cytokine and metalloprotease, and these reactions can be stoped by anti-CX3CR1 antibody.Therefore,, then can suppress the activation of microglia in theory, and then suppress generation or deterioration by the nervous system disorders of microglia mediation if can effectively disturb the CX3CR1 receptor expression.
RNA disturbs (RNA interference; RNAi) be the mode of a kind of regulate gene expression in most of most eukaryotes; Main through siRNA (Small interfering RNA; SiRNA) reticent specifically target gene expression has advantages such as high efficiency, specificity, has been widely used in research fields such as gene function, intracellular signal transduction pathway, drug target screening, gene therapy.Therefore, can utilize the RNAi technology to disturb the CX3CR1 receptor expression.But the RNAi technology also faces a key issue, is exactly how siRNA to be transmitted the entering target cell effectively.Because siRNA is difficult for the ectogenic exposed nucleic acid of picked-up by nuclease degradation and eukaryotic cell easily; Directly with siRNA transfectional cell inefficiency; Therefore, must select appropriate carriers parcel siRNA, in order to avoid siRNA degrades and can assist the siRNA transfectional cell.Chitosan is a kind of polysaccharide that from the shell of crustaceans biology, extracts, and has excellent biological compatibility and biodegradability, is used widely as pharmaceutical carrier.Chitosan is rich in positive charge, can combine with electronegative siRNA through electrostatic interaction, and parcel siRNA forms chitosan-siRNA nanoparticle, thereby protection siRNA avoids the degraded of nucleicacidase, and improves the picked-up of cell to siRNA.Discover that the siRNA that chitin carrier carries is transfectional cell efficiently, prolong the siRNA transformation period in vivo simultaneously.But chitin carrier is the same with non-viral gene vector with present most virus, all exists to lack cell targeted defective, thereby has limited its application in gene therapy.
US28 is that (human cytomegalovirus HCMV) strides one of film Chemokine Receptors seven times for four of coding to the human cytomegalovirus, has broad-spectrum chemokine and combines actively, and structurally the homology with people source CX3CR1 acceptor is the highest.Discover that the temptation ligand polypeptide H9 that derives from US28 acceptor N-terminal can block the chemotaxis that people source CX3CR1 receptors bind physiological chemokine forms, but itself do not cause the chemotactic motion, do not influence intracellular signal transduction and cell natural activity; It can cause cell surface receptor CX3CR1 internalization, but the acceptor portion of internalization can be recycled to cell surface, to the not obviously influence of physiological function of people source CX3CR1 acceptor.
Summary of the invention
In view of this; One of the object of the invention is to provide a kind of gene-carrier complexes, and this mixture can successful target microglia, disturbs the CX3CR1 receptor expression; Suppress the activation of microglia, and then suppress generation or deterioration by the nervous system disorders of microglia mediation.
For achieving the above object, the present invention provides following technical scheme:
Gene-carrier complexes of target microglia passes through the electrostatic interaction be combined into by CX3CR1 shRNA interference carrier and genophore, and said genophore is the conjugate of H9 polypeptide and chitosan.
Preferably, contain in the said CX3CR1 shRNA interference carrier positive-sense strand shown in SEQ ID No.2, the shRNA sequence of antisense strand shown in SEQ ID No.3.
Preferably, said CX3CR1 shRNA interference carrier is to be carrier is carrier with pSilencer 2.1-U6neo carrier.
Preferably, the conjugate of said H9 polypeptide and chitosan is with 1-ethyl (3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy-succinamide the carboxyl of H9 polypeptide and the amino of chitosan to be carried out coupling.
Two of the object of the invention is to provide a kind of preparation method of said gene-carrier complexes, and is easy and simple to handle, and constant product quality is controlled.
For achieving the above object, the present invention provides following technical scheme:
The preparation method of gene-carrier complexes of said target microglia; Be under the vortex condition; In the phosphate buffer soln of CX3CR1 shRNA interference carrier, splash into genophore solution; Dropwise the continued vortex 20 ~ 60 minutes, left standstill again 20 ~ 60 minutes, promptly get gene-carrier complexes of target microglia.
Preferably, the mass ratio of said CX3CR1 shRNA interference carrier and genophore is 1:2.
Preferably; The preparation method of said CX3CR1 shRNA interference carrier be earlier with positive-sense strand shown in SEQ ID No.2, the 1 pair shRNA single stranded oligonucleotide fragment annealing of antisense strand shown in SEQ ID No.3 forms shRNA double chain oligonucleotide fragment; Again gained shRNA double chain oligonucleotide fragment is connected with the pSilencer 2.1-U6neo carrier of handling through BamH I and Hind III double digestion, promptly gets CX3CR1 shRNA interference carrier.
Preferably, the preparation method of said genophore is dissolved in chitosan in the Tetramethyl Ethylene Diamine damping fluid of pH5, adds 1-ethyl (3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide; Stir; Add the H9 polypeptide again, stirring at room reaction 8 ~ 16 hours, dialysis; Drying, the conjugate that makes H9 polypeptide and chitosan is a genophore.
Three of the object of the invention is to provide the application of said gene-carrier complexes in pharmacy field, thereby more, better drug candidate is provided for the clinical treatment of disease.
For achieving the above object, the present invention provides following technical scheme:
The application of gene-carrier complexes of said target microglia in preparation microglia activation inhibitor.Said microglia activation inhibitor can be used as the medicine of treatment by the nervous system disorders of microglia mediation, and said nervous system disorders by the microglia mediation includes but not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis etc.
Beneficial effect of the present invention is: the gene-carrier complexes that the invention discloses a kind of target microglia; This mixture is through the guide effect of H9 polypeptide; Can successful target microglia surface specific CX3CR1 acceptor molecule; In the antagonistic action of H9 polypeptide performance to the CX3CR1 acceptor, CX3CR1 shRNA interference carrier can be transfected into microglia efficiently, disturbs the expression of CX3CR1; Suppress the activation of microglia, and then suppress generation or deterioration by the nervous system disorders of microglia mediation; The invention also discloses the preparation method of this mixture, easy and simple to handle, products obtained therefrom is uniform nanoparticle, helps the picked-up of cell to this mixture; Mixture of the present invention can be used for preparing the microglia activation inhibitor, as the medicine of treatment by the nervous system disorders of microglia mediation, in the nervous system disease field excellent development application prospect is arranged.
Description of drawings
Fig. 1 identifies the purity of synthetic H9 polypeptide for performance liquid chromatography.
Fig. 2 identifies the molecular weight of synthetic H9 polypeptide for mass spectrum.
Fig. 3 identifies gene-carrier complexes of the present invention for transmission electron microscope.
Fig. 4 is the expression level of CX3CR1 mRNA in the microglia of RT-PCR detection gene of the present invention-carrier complexes transfection, and 1 is blank control group, and 2 is control group, and 3 is experimental group.
Fig. 5 is the expression level of CX3CR1 in the microglia of immunoblotting detection gene of the present invention-carrier complexes transfection, and 1 is blank control group, and 2 is control group, and 3 is experimental group.
Fig. 6 detects the secretion level of cytokine after the microglia activation of gene of the present invention-carrier complexes transfection for ELISA.
Fig. 7 does
51The Cr method for releasing detects the kill and wound level of the microglia of gene of the present invention-carrier complexes transfection to oligodendrocyte.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the preferred embodiments of the present invention are carried out detailed description below.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example; J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press; 2002) described in condition, or the condition of advising according to manufacturer.
One, the preparation of gene-carrier complexes of target microglia
1, the H9 polypeptide is synthetic
The H9 amino acid sequence of polypeptide is VLNAHCALH (SEQ ID No.1).It synthesizes on ABI 431A type solid-phase polypeptide synthesizer and carries out, and adopts standard fluorenylmethyloxycarbonyl (Fmoc) scheme.The initial 0.125mmol that selects for use is to hydroxymethyl phenoxy methylated polystyrene (HMP) resin, according to the H9 amino acid sequence of polypeptide peptide chain extended one by one to aminoterminal from carboxyl terminal, and the consumption of every seed amino acid is 0.5mmol, with the mol ratio of resin be 4:1.Various amino acid whose alpha-amino groups are the Fmoc protection, and all the other side chain protected groups are respectively: Lys (Boc), Ser (tBu), Glu (OtBu), Arg (Pmc), His (Trt), Thr (tBu) and Tyr (tBu).First amino acid is connected on the resin with 4-Dimethylamino pyridine (DMAP); Amino acid whose activation is with I-hydroxybenzotriazole (HOBt) and NSC 57182 (DCC), and the use volume(tric)fraction is 20% piperidines aqueous solution removal Fmoc protection base after the coupling.Polypeptide is synthetic finish after, resin-polypeptide complex is mixed under condition of ice bath in the 10mL cutting liquid (is that EDT 0.25mL, thioanisole 0.5mL, deionized water 0.25mL and trifluoroacetic acid are that TFA 10mL forms by crystallization phenol 0.75g, 1); Stirring at room reaction 2 hours is got off peptide chain cracking from the resin, removes the kinds of protect base simultaneously; Filter the removal resin with the G4 glass sand hourglass, use 1mL TFA, 5 ~ 10mL methylene dichloride to wash reaction flask, resin and funnel repeatedly, merging filtrate and washing lotion successively; Under normal temperature low pressure, be evaporated to 1 ~ 2mL, add 50mL precooling ether sedimentation polypeptide, 4 ℃ of placements are spent the night; The G6 glass sand hourglass filters; Vacuum is drained, and promptly gets the polypeptide bullion, and-20 ℃ of preservations are subsequent use.
The polypeptide bullion is processed the solution that concentration is 20mg/mL with DMSO 99.8MIN. (DMSO) dissolving; After via hole diameter is the filtering with microporous membrane of 0.45 μ m, upward carry out purifying with the SOURCE gel column at AKTA explorer 100 type medium pressure liguid chromatographs (Sweden Amerssham Bioscienc company).Mobile phase A is that 10% ethanol and volume(tric)fraction are that 0.1% TFA forms by volume(tric)fraction, and Mobile phase B is that 90% ethanol and volume(tric)fraction are that 0.1% TFA forms by volume(tric)fraction.Type of elution is a gradient elution: earlier with 1.5 column volumes of mobile phase A wash-out; Use 8 column volumes of mixed solution wash-out (volume(tric)fraction that Mobile phase B accounts for mixed solution increases to 80% by 0% gradually in 8 column volumes) of mobile phase A and Mobile phase B again, use 0.5 column volume of mixed solution wash-out (volume(tric)fraction that Mobile phase B accounts for mixed solution increases to 100% by 80% gradually in 0.5 column volume) of mobile phase A and Mobile phase B again.Collect polypeptide solution at the main peak place, lyophilize promptly gets the pure article of polypeptide, and with the DMSO dissolving ,-20 ℃ of preservations are subsequent use.
The pure article of polypeptide are identified purity with Delta 600 type high pressure liquid chromatographs.The result is as shown in Figure 1, and the purity of synthetic polypeptide reaches more than 90%.The pure article of polypeptide are identified molecular weight with API 2000 LC/MS type electro-spray ionization mass spectrographs.The result is as shown in Figure 2, and the molecular weight of synthetic polypeptide conforms to the theoretical molecular of H9 polypeptide.
2, the preparation of H9 polypeptide-chitosan conjugate
With 1-ethyl (3-dimethylaminopropyl) phosphinylidyne diimine (EDC) and N-hydroxy-succinamide (NHS) is that coupling agent carries out coupling: the 0.5g chitosan is dissolved in Tetramethyl Ethylene Diamine (TEMED) damping fluid of 50mL pH5.0; Add 0.5g EDC.HCl and 0.5g NHS, stirred 2 hours, add the H9 polypeptide that molar weight is equivalent to amino of chitosan molar weight 50% again; Stirring at room reaction 12 hours; Dialysis, lyophilize promptly gets H9 polypeptide-chitosan conjugate.
3, the structure of CX3CR1 shRNA interference carrier
Principle of design according to disclosed people CX3CR1 gene order (NM_015898) on the NCBI GenBank and short hairpin RNA (shRNA); Filter out public target spot earlier to the RNAi effect of different montage of CX3CR1 mRNA; Again according to this public target spot; Design and synthesize 1 pair of shRNA single stranded oligonucleotide fragment: positive-sense strand: 5 ＇-aatgcttcctgcctctgtgttt-3 ＇ (SEQ ID No.2), antisense strand: 5 '-atccagccattcagtcttggtt-3 ' (SEQ ID No.3); And 1 pair of negative control shRNA single stranded oligonucleotide fragment (not having any coupling): positive-sense strand with human genomic sequence: 5 '-ttctccgaacgtgtacgttt-3 ' (SEQ ID No.4), antisense strand: 5 '-acgtgacacgttcggagaatt-3 ' (SEQ ID No.5).To use the distilled water dissolving to process the solution that concentration is 100 μ mol/L respectively to 1 pair of shRNA single stranded oligonucleotide fragment of CX3CR1 mRNA, and respectively get 5 μ L again and mix, annealing forms shRNA double chain oligonucleotide fragment.With BamH I and Hind III double digestion, enzyme is cut product, and to carry out massfraction be 1% agarose gel electrophoresis, reclaims test kit with glue and reclaim the carrier segments after purifying enzyme is cut with pSilencer 2.1-U6neo carrier (Ambion company).Carrier segments after enzyme cut is connected with shRNA double chain oligonucleotide fragment; Connect product transformed into escherichia coli DH5 α competent cell; Select the amicillin resistance bacterium colony; Amplification cultivation send Wuhan brilliant match biotechnology ltd to check order bacterium liquid, inserts fragment sequence in the recombinant vectors and is the CX3CR1 shRNA interference carrier that successfully makes up with the consistent person of shRNA sequence of design.According to the method described above, adopt 1 pair of shRNA single stranded oligonucleotide fragment, can make up and obtain negative control shRNA interference carrier as negative control.
4, the preparation of gene-carrier complexes
It is that 7.4 phosphate buffered saline buffer (PBS) dissolves and processes the solution that concentration is 500 μ g/mL as 0.1mol/L, pH that the CX3CR1 shRNA interference carrier that success is made up uses concentration.Get this solution 100 μ L; Under vorticity with the speed of 5 μ L/min to wherein splashing into the H9 polypeptide that concentration is 1000 μ g/mL-chitosan conjugate solution 100 μ L; Dropwise the continued vortex 30 minutes, left standstill again 30 minutes, promptly get gene-carrier complexes.Gene-the carrier complexes of prepared fresh being dripped on 200 order copper mesh, adsorbed 3 minutes, blot with thieving paper, dried 30 seconds, is 1% acetic acid uranium aqueous solution negative staining 30 seconds with massfraction, blots with thieving paper, dries the 80kV transmission electron microscope observing 30 seconds.The result is as shown in Figure 3, and gained gene-carrier complexes is uniform subcircular particle, and most particle major diameters are less than 25nm.
According to the method described above, adopt the negative control shRNA interference carrier that successfully makes up, can make negative control gene-carrier complexes.
Two, the active testing of gene-carrier complexes of target microglia
1, gene-carrier complexes transfection microglia
Microglia BV-2 monolayer culture to area coverage in 6 orifice plates is about 30%, change to serum-free DMEM substratum 2ml, and add gene-carrier complexes 100 μ L; Cultivated 4 hours; Change to complete DMEM substratum 2mL again, cultivated collecting cell 48 hours; With PBS washing and resuspended, obtain the microglia of gene-carrier complexes transfection.
According to the method described above, adopt negative control gene-carrier complexes, can obtain the microglia of negative control gene-carrier complexes transfection.In addition, experiment contrasts as irrelevant cell with oligodendrocyte HO, according to the method described above, can obtain the oligodendrocyte HO of gene-carrier complexes or negative control gene-carrier complexes transfection.
2, the expression level of CX3CR1 mRNA in the microglia of RT-PCR detection gene-carrier complexes transfection
Experiment is provided with 2 big groups: microglia group and oligodendrocyte group, each big group is provided with 3 groups again: blank control group (normal cell of untransfected), control group (negative control gene-carrier complexes cells transfected) and experimental group (gene-carrier complexes cells transfected).Get each group's cell; Extract cell total rna with TRIzol reagent; Is cDNA with the total RNA of gained with reverse transcription test kit (QIAGEN company) reverse transcription; Be template with this cDNA again, with upstream primer 5 '-caaacacagcaatccaggca-3 ' (SEQ ID No.6) and downstream primer 5 '-the CX3CR1 gene fragment of long 359 bp of atcactgggcttcaccacttt-3 ' (SEQ ID No.7) pcr amplification, (β-actin) is an internal reference with beta-actin.The pcr amplification parameter is: 95 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 30 seconds then, 58 ℃ of annealing 40 seconds, and 72 ℃ were extended totally 30 circulations 30 seconds; Last 72 ℃ were extended 10 minutes.It is 1% agarose gel electrophoresis that the PCR product carries out massfraction, takes pictures under the uv lamp, according to the absorbancy of each band, utilizes the expression level of CX3CR1 mRNA in the GEL-Pro Analyzer software analysis cell.The result is as shown in Figure 4; Gene-carrier complexes of the present invention can obviously reduce the expression level of CX3CR1 mRNA in the microglia; But can not reduce the expression level of CX3CR1 mRNA in the oligodendrocyte, explain that gene-carrier complexes of the present invention can special target transfection microglia.
3, the expression level of CX3CR1 in the microglia of immunoblotting detection gene-carrier complexes transfection
Experiment is provided with 2 big groups: microglia group and oligodendrocyte group, each big group is provided with 3 groups again: blank control group (normal cell of untransfected), control group (negative control gene-carrier complexes cells transfected) and experimental group (gene-carrier complexes cells transfected).Get each group's cell,, add 400 μ L cell pyrolysis liquids with PBS washing 2 times; Boiling water bath heating 5 minutes, cooled on ice 5 minutes, 1200g, 4 ℃ are centrifugal 10 minutes; Get supernatant and measure total protein concentration, get appearance on the 50 μ g total proteins again, using massfraction is 12% polyacrylamide gel electrophoretic separation 50 minutes under the 200V constant voltage; Afterwards with the protein electrotransfer to nitrocellulose filter, in 4 ℃ of incubated overnight, use IgG antibody (Abcam company) incubated at room 1 hour of horseradish peroxidase-labeled with anti-CX3CR1 antibody again; With luminescent solution effect 5 minutes, put in the magazine and made public 5 minutes, observations after development, washing, the photographic fixing again.The result is as shown in Figure 5; Gene-carrier complexes of the present invention can obviously reduce the expression level of CX3CR1 in the microglia; But can not reduce the expression level of CX3CR1 in the oligodendrocyte, explain that gene-carrier complexes of the present invention can special target transfection microglia.
4, the secretion level of cytokine after the microglia activation of ELISA detection gene-carrier complexes transfection
Blank control group (normal microglia BV-2), control group (microglia of negative control gene-carrier complexes transfection) and experimental group (microglia of gene-carrier complexes transfection) are set.Each is organized cell with 4 * 10
5Plant in 24 orifice plates in/hole; In cell culture fluid, adding LPS (LPS) to final concentration is 10 μ g/mL, stimulates 0.5 hour, discards the cell culture fluid that contains LPS and washed cell afterwards 2 times; Add DMEM substratum 1mL again; Continue to cultivate 24 hours, and drew cell culture fluid, adopt the wherein content of interleukin-1 ' beta ' (IL-1 β) and tumor necrosis factor alpha (TNF-α) of ELISA kit measurement.The result is as shown in Figure 6, and gene-carrier complexes of the present invention can obviously reduce the level of activated microglia secrete cytokines.
5,
51The Cr method for releasing detects the kill and wound level of the microglia of gene-carrier complexes transfection to oligodendrocyte
Blank control group (normal microglia BV-2), control group (microglia of negative control gene-carrier complexes transfection) and experimental group (microglia of gene-carrier complexes transfection) are set.Oligodendrocyte HO used contain the RPMI 1640 substratum vitro culture that massfraction is 10% foetal calf serum, collecting cell, centrifuge washing 2 times uses that to contain massfraction be that to regulate cell density be 1 * 10 for the RPMI1640 substratum of 10% foetal calf serum
6/ mL gets 1mL and puts in the 10mL serum bottle, adds 100 μ Ci Na
51CrO
4, to hatch 90 minutes for 37 ℃, the thorough washing cell is removed unconjugated
51Cr uses that to contain massfraction be that the RPMI 1640 substratum adjustment cell density of 10% foetal calf serum is 1 * 10 again
5/ mL is as target cell.In 96 hole U templates; Every hole is by imitating target than add effector cell's (adding corresponding microglia by dividing into groups) and target cell (oligodendrocyte) for 1:1; Imitate the target ratio for every kind and establish 3 multiple holes, establish maximum release group (using the hydrochloric acid substitution effect cell of concentration) and minimum release group (use contains the RPMI 1640 substratum substitution effect cells that massfraction is 10% foetal calf serum) simultaneously as 2mol/L; 96 orifice plate 500r/min after centrifugal 30 seconds, were hatched 4 hours for 37 ℃, and 1000r/min is centrifugal 10 minutes again, and supernatant 100 μ L are got in each hole, measures PM radioactivity (cpm value) with gamma counter, calculates kill rate, and kill rate is judged to specific killing greater than 15%.The result is as shown in Figure 7, and gene-carrier complexes of the present invention can obviously reduce the kill and wound level of activated microglia to oligodendrocyte, explains that gene-carrier complexes of the present invention can reduce activated microglia to neural destruction.
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
< 110>No.2 Hospital Attached to No.3 Military Medical College, PLA
< 120>gene-carrier complexes of target microglia
<160> 7
<210> 1
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<212> PRT
< 213>artificial sequence
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< 223>H9 polypeptide
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Val Leu Asn Ala His Cys Ala Leu His
1 5
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aatgcttcct?gcctctgtgt?tt 22
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atccagccat?tcagtcttgg?tt 22
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Claims (9)
1. gene-carrier complexes of target microglia is characterized in that, passes through the electrostatic interaction be combined into by CX3CR1 shRNA interference carrier and genophore, and said genophore is the conjugate of H9 polypeptide and chitosan.
2. gene-carrier complexes of target microglia according to claim 1 is characterized in that, contain in the said CX3CR1 shRNA interference carrier positive-sense strand shown in SEQ ID No.2, the shRNA sequence of antisense strand shown in SEQ ID No.3.
3. gene-carrier complexes of target microglia according to claim 1 is characterized in that, said CX3CR1 shRNA interference carrier is to be carrier is carrier with pSilencer 2.1-U6neo carrier.
4. gene-carrier complexes of target microglia according to claim 1; It is characterized in that the conjugate of said H9 polypeptide and chitosan is through 1-ethyl (3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy-succinamide the carboxyl of H9 polypeptide and the amino of chitosan to be carried out coupling.
5. the preparation method of gene-carrier complexes of the described target microglia of claim 1; It is characterized in that; Under the vortex condition, in the phosphate buffer soln of CX3CR1 shRNA interference carrier, splash into genophore solution, dropwised the continued vortex 20 ~ 60 minutes; Left standstill again 20 ~ 60 minutes, and promptly got gene-carrier complexes of target microglia.
6. the preparation method of gene-carrier complexes of target microglia according to claim 5 is characterized in that, the mass ratio of said CX3CR1 shRNA interference carrier and genophore is 1:2.
7. the preparation method of gene-carrier complexes of target microglia according to claim 5; It is characterized in that; The preparation method of said CX3CR1 shRNA interference carrier be earlier with positive-sense strand shown in SEQ ID No.2, the 1 pair shRNA single stranded oligonucleotide fragment annealing of antisense strand shown in SEQ ID No.3 forms shRNA double chain oligonucleotide fragment; Again gained shRNA double chain oligonucleotide fragment is connected with the pSilencer 2.1-U6neo carrier of handling through BamH I and Hind III double digestion, promptly gets CX3CR1 shRNA interference carrier.
8. the preparation method of gene-carrier complexes of target microglia according to claim 5 is characterized in that, the preparation method of said genophore is dissolved in chitosan in the Tetramethyl Ethylene Diamine damping fluid of pH5; Add 1-ethyl (3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, stir, add the H9 polypeptide again; Stirring at room reaction 8 ~ 16 hours; Dialysis, drying, the conjugate that makes H9 polypeptide and chitosan is a genophore.
9. the application of gene-carrier complexes of the described target microglia of claim 1 in preparation microglia activation inhibitor.
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| CN102943086A (en) * | 2012-10-22 | 2013-02-27 | 山东大学 | Application of cationic imidazole Gemini surfactant [Cn-s-Cnim]Br2 in gene transfection |
| CN103028121A (en) * | 2012-12-30 | 2013-04-10 | 中国人民解放军第三军医大学第二附属医院 | miR-21 compound of target microglial cell and preparation method and application of miR-21 compound |
| CN104324369A (en) * | 2014-10-31 | 2015-02-04 | 重庆医科大学附属永川医院 | MiR-223 compound of targeted microglia, as well as preparation method and application thereof |
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| CN103028121A (en) * | 2012-12-30 | 2013-04-10 | 中国人民解放军第三军医大学第二附属医院 | miR-21 compound of target microglial cell and preparation method and application of miR-21 compound |
| CN103028121B (en) * | 2012-12-30 | 2014-06-11 | 中国人民解放军第三军医大学第二附属医院 | miR-21 compound of target microglial cell and preparation method and application of miR-21 compound |
| CN104324369A (en) * | 2014-10-31 | 2015-02-04 | 重庆医科大学附属永川医院 | MiR-223 compound of targeted microglia, as well as preparation method and application thereof |
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