CN103784977B - Ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) and its preparation method and application - Google Patents
Ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) and its preparation method and application Download PDFInfo
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- CN103784977B CN103784977B CN201410081629.3A CN201410081629A CN103784977B CN 103784977 B CN103784977 B CN 103784977B CN 201410081629 A CN201410081629 A CN 201410081629A CN 103784977 B CN103784977 B CN 103784977B
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Abstract
The present invention relates to gene ultrasonic targeting transfection field, be specifically related to a kind of ultrasonic microbubble for the ultrasonic targeting transfection of C-myc peptide nucleic acid(PNA) and preparation method thereof, described ultrasonic microbubble is mounted with peptide nucleic acid(PNA), described peptide nucleic acid(PNA) is peptide nucleic acid(PNA) prepared by the antisensenucleic acids of C-myc proto-oncogene, and the nucleotide sequence of described antisensenucleic acids is as follows: 5 '-AGCGAGGATATCTGGAAGAAATTCGAG-3 '.The preparation method of the ultrasonic microbubble of described targeting transfection peptide nucleic acid(PNA), comprises the steps: peptide nucleic acid(PNA) prepared by the antisensenucleic acids adding C-myc proto-oncogene when preparing ultrasonic microbubble.The present invention adds quantitative PNA when preparing ultrasonic microbubble in albumin solution, and make it be coated in microvesicle wall albumin substrate and form the part of microvesicle wall construction, targeting transfection under ultrasonic field effect, transfection efficiency is high, and transfection efficiency can reach more than 30%.
Description
Technical field
The present invention relates to gene ultrasonic targeting transfection field, be specifically related to ultrasonic microbubble of a kind of targeting transfection peptide nucleic acid(PNA) and its preparation method and application.
Background technology
C-myc proto-oncogene is one of important member of myc gene family, is the immediate early gene of inducing after multiple factors stimulated growth.It is that one can translocation genes, is again the scalable gene that one kind of multiple materials regulate.After C-Myc synthesizes in endochylema, form oligomer with other albumen, then transfer in core, and be attached in specific DNA sequence, thus transcribing of the many target genes of Activation and inhibition, cause the change of Growth of Cells and differentiation, play its physiological regulation function and vicious transformation effect.Be that one can make cell infinite multiplication, obtain immortalization function, promote fissional gene.Myc gene participates in apoptosis, kinds of tumors develops, between intimal smooth muscle cells early evoking, differentiation, propagation and migration and endo cell, the synthesis of substrate regulates with piling up after blood vessel injury.
The in vitro study of antisense oligonucleotide targeted inhibition tumor, smooth muscle cell proliferation Related oncogene has obtained great progress, is applied to that limited in body mainly it is very easily made its half-life extremely short by nuclease degradation as genomic medicine; Enter specific target cell more difficult and less.Therefore, many scholars wish the ability increasing its opposing nuclease by carrying out chemical modification to it; Carry by virus and increase its transfection specificity etc.But the peptide nucleic acid(PNA) of the design and synthesis such as the Nielsen of University Copenhagen Denmark (PNA) is known dna or the most successful analog of RNA.
PNA is the similar molecule of DNA or RNA of a class synthetic, is the phosphoric acid DNA (deoxyribonucleic acid) in DNA or RNA molecule or ribose phosphate backbone are replaced by the N-2-ethyl glycine unit (peptide chain) having amido link to be connected of repeated arrangement with nucleic acid molecule differ part.PNA in conjunction with DNA or RNA sequence, can form stable double-spiral structure by the form identification of Watson-Crick base pairing.Because PNA is not electronegative, and there is not electrostatic repulsion between DNA and RNA, the stability thus combined and specificity all greatly improve; Be different from the hybridization between DNA or DNA, RNA, the hybridization of PNA and DNA or RNA affects by hybridization system salinity hardly, DNA/DNA or DNA/RNA is much better than with the hybridization ability of DNA or RNA molecule, show very high hybridization stability, excellent distinguished sequence identification ability, not by nuclease and protease hydrolysis, and the cotransfection that can be connected with aglucon enters cell, its cytotoxicity is lower.These are all the advantages not available for other oligonucleotide.Can be used as gene probe, and the research such as gene function, gene expression regulation can be used for the form of antisense sequences.
In medicinal study, at present, it is on the basis of the first generation, second filial generation antisense agent, is built and the third generation antisense agent of final synthetic by Computer Design.According to the metabolic stability of PNA, mainly use it for the antisense drug research field of inhibition of gene expression, abroad several pharmacy and biotech company, the companies such as ISIS, PE all drop into great effort and are engaged in developmental research; The hybridization stability excellent according to itself and DNA, PNA is widely used in again DNA molecular identification and manipulation.
Although antisense PNA has the feature such as high specificity, good stability, and may in the copying of gene, the expression of transcribing and the link such as translation suppresses or lowers target gene, reach the object of disease therapy, but Problems existing not easily enters cell as medicinal PNA at present, raising tissue and cell enter nucleus to people's amount of taking the photograph of antisense PNA and antisense PNA is further affect it to be widely used in clinical key issue always.
The development of the development of contrast-enhanced ultrasound technique and the microvesicle acoustic contrast of portability gene, makes ultrasonic from a kind of clinical diagnosis instrument, enters into treatment field.The two is combined the treatment for tumor or cardiovascular disease etc., for these patients provide a safety, efficiently therapy approach.Microbubble contrast agent Combining diagnosis Ultrasound mediated gene targeting delivery is a kind of novel, noinvasive, efficient, easy easily universal technology.Ultrasonic trigger destruction microvesicle method is considered to the target gene treatment means that can be used as certain organs.Its ultimate principle is: (1) acoustic contrast agent is the good carrier of gene, and compared with viral vector, contrast agent microbubble has larger capacity, portability antisense oligonucleotide, any segment DNA, and even whole chromosome.(2) contrast agent reduces ultrasonic cavitation thresholding, ultrasonic irradiation can destroy contrast agent microbubble at particular space (focal zone) and special time, produce cavitation effect and sonochemical effect, make target cell (comprising vascular endothelial cell and histiocyte) Dilated intercellular space around, membrane permeability increases, the transient keyhole formation of cell surface (acoustic horn effect), the shock wave simultaneously produced during microbubble ruptures, micro-acoustic streaming, as a kind of driving force, impels the gene that discharges from microvesicle to enter target cell by the blood capillary of breaking and endotheliocyte gap.
Because PNA is water solublity, enter cell more difficult, people adopted liposome latter end labeling method to carry it into cell in the past, but cellular uptake amount is limited, played a role body is more difficult; Apply efficient liposome such as cationic-liposome etc. and may produce cytotoxicity.
Ultrasonic microbubble especially with albumin be raw material prepare ultrasonic microbubble tool hypotoxicity, reduced immunogenicity, low aggressivity, organ specificity, can repeated application and the suitability widely, as the carrier carrying therapeutic gene, and with ultrasonic trigger destruction microbubble technique to have carried out certain research to target tissues such as tumor, cardiac muscle, blood vessel, skeletal muscle.The amount that these microvesicles carry DNA from 10g to 2mg not etc.; Alone ultrasonic irradiation can make the transfection efficiency of naked DNA to vascular endothelial cell improve 10 times, as conbined usage microbubble contrast agent, then the transfection efficiency of gene can be made to improve 3000 times.First prepare microvesicle and then it is connected with gene or plasmid mostly when preparing ultrasound microbubble gene carrier in the past, combination stability and bearing capacity meter more difficult to estimate, the amount of targeting rotaring redyeing gene and the more difficult control of effect.
Summary of the invention
In order to solve the defect that in above-mentioned prior art, PNA and ultrasonic microbubble exist, the object of this invention is to provide a kind of ultrasonic microbubble for the ultrasonic targeting transfection of C-myc peptide nucleic acid(PNA), namely in albumin solution, quantitative PNA is added when preparing ultrasonic microbubble, make it be coated in microvesicle wall albumin substrate and form the part of microvesicle wall construction, under ultrasonic field effect, targeting transfection enters nucleus.
Technical scheme of the present invention is for providing a kind of ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA), described ultrasonic microbubble is mounted with peptide nucleic acid(PNA), described peptide nucleic acid(PNA) is peptide nucleic acid(PNA) prepared by the antisensenucleic acids of C-myc proto-oncogene, and the nucleotide sequence of described antisensenucleic acids is as follows: 5 '-AGCGAGGATATCTGGAAGAAATTCGAG-3 '.
Another technical scheme of the present invention, for providing a kind of preparation method of ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA), comprises the steps: peptide nucleic acid(PNA) prepared by the antisensenucleic acids adding C-myc proto-oncogene according to claim 1 when preparing ultrasonic microbubble in albumin solution.
Preferably, the preparation method of the ultrasonic microbubble of above-mentioned targeting transfection peptide nucleic acid(PNA), specifically comprises the steps:
A, get the 5% bovine serum albumin liquid 10ml comprising sucrose, peptide nucleic acid(PNA) prepared by the antisensenucleic acids adding the C-myc proto-oncogene as claimed in claim 1 of 2mg synthesis, mix to obtain liquid A, the quality percentage by volume of described sucrose in bovine serum albumin liquid is 10%;
B, use oxygen and fluorocarbon gas saturated liquid A successively, after supersonic generator process, namely obtain ultrasonic microbubble.
Preferably, in the preparation method of the ultrasonic microbubble of above-mentioned targeting transfection peptide nucleic acid(PNA), concentration 0.8-1.8 × 10 of described ultrasonic microbubble
9individual/ml.
Preferably, in the preparation method of the ultrasonic microbubble of above-mentioned targeting transfection peptide nucleic acid(PNA), the flow of described oxygen and fluorocarbon gas is 6ml.min
-1.
Preferably, in the preparation method of the ultrasonic microbubble of above-mentioned targeting transfection peptide nucleic acid(PNA), described supersonic generator process is specially: supersonic generator output 180W, and frequency is process 1min under 20kHz condition.
Another technical scheme of the present invention is for providing the application of a kind of ultrasonic microbubble of above-mentioned targeting transfection peptide nucleic acid(PNA) in the medicine of preparation treatment C-myc proto-oncogene relevant disease.
Beneficial effect of the present invention: the present invention considers that PNA belongs to peptide class from structure, its physical behavior is similar to albumin and not charged, and do not produce physical and chemical effect between albumin, with water-soluble, therefore, in albumin solution, adding quantitative PNA when preparing ultrasonic microbubble, making it be coated in microvesicle wall albumin substrate and forming the part of microvesicle wall construction, targeting transfection under ultrasonic field effect, transfection efficiency is high.In one embodiment of the present of invention, prepare iliac artery neointimal hyperplasia model, design and synthesis is for the peptide nucleic acid(PNA) of rabbit C-mycmRNA proto-oncogene, ultrasonic microbubble is prepared as raw material with albumin, targeting transfection tunica intima under ultrasonic field effect, transfection is higher, and transfection efficiency can reach more than 30%.
Accompanying drawing explanation
Fig. 1: HRP-Streptavindin and the positive SMC of the few c-mycPNA transfection of trueblueTM dyeing display tunica intima antisense, 100 ×;
Fig. 2: HRP-Streptavindin and trueblueTM dyeing display tunica intima antisense few c-mycPNA transfection positive cell, 400 ×;
Fig. 3: after blood vessel injury, 1 week blood vessel wall obviously thickens through elastic fibers dyeing display inner membrance, visible thrombosis in tube chamber, 200 ×;
Fig. 4: the visible more PCNA reacting positive cell of tunica intima of immunohistochemical staining display hypertrophy, 200 ×.
Detailed description of the invention
By describing technology contents of the present invention, structural feature in detail, realized object and effect, accompanying drawing is coordinated to be explained in detail below in conjunction with embodiment.
Embodiment
1. the present invention is used for preparation and the transfection thereof of the ultrasonic microbubble of the ultrasonic targeting transfection of C-myc peptide nucleic acid(PNA)
The peptide nucleic acid(PNA) design of 1.1C-mycmRNA proto-oncogene:
Relative analysis protokaryon and eucaryon animal comprise the cDNA sequence of virus (v-myc), mice, rat, rabbit, sheep, cat, Canis familiaris L., pig and people c-myc proto-oncogene, find that it starts 133 to 177, downstream from transcribing initiation codon totally 45 base sequences are identical.According to peptide nucleic acid(PNA) (PNA) sequence following (totally 27 bases) of nucleic acid probe design principle design for c-mycmRNA: 136 to 162,5,-AGCGAGGATATCTGGAAGAAATTCGAG-3, synthesized by Beijing AudioCodes Bioisystech Co., Ltd, its 5 ' aminoterminal is with biotin molecule labelling.
The preparation of the ultrasonic microbubble of 1.2 years above-mentioned PNA
Aseptic preparation is containing 10% (g.ml
-1) 5% (g.ml of sucrose
-1) bovine serum albumin liquid 10ml, be placed in lid 50ml plastic centrifuge tube; Add the PNA of 2mg above-mentioned steps synthesis and fully mix; Use the liquid (flow: 6ml.min of oxygen and the saturated above-mentioned mixing of fluorocarbon gas successively
-1), about 10min, supersonic generator process 1min(condition: 180W, fixed frequency 20kHz); Adjustment microbubble concentration 0.8-1.8 × 10
9individual/ml, PNA content is 0.2mg/ml, saves backup airtight at the microvesicle 4 DEG C of preparation.Get part microscopic examination microvesicle form and count; Zetasizer3000 instrument measures size and surperficial Zeta potential.
1.3 blood vessel modelings and ultrasonic targeting transfection
Purebred new zealand rabbit 22 (male, 2.3-2.5kg), is divided into grouping at random:
(1) experimental group (n=12): through femoral arteriography, sacculus strips off side iliac artery inner membrance and causes inner film injury and model of hyperplasia.Liver is selected to be ultrasonic development contrast organ.
Postoperative two-dimensional ultrasound injects through auricular vein and carries PNA albumin microsphere infusion 5ml (containing PNA1mg) and observe the obvious enhancing of liver development after stomach wall observation liver develops, percutaneous capable Local Damaged vascular treatment ultrasonic irradiation (supersonic frequency 1MHz, intensity 1.5W/cm immediately
2, 6min) and observe liver development and progressively return to normal.
(2) matched group (n=10): sacculus strips off after inner membrance through vein input normal saline and the ultrasonic treatment of equal conditions.
1.4 draw materials and pathologic finding
Postoperative 1 week gas embolism puts to death animal, and treating excess syndrome tests paraffin embedding after iliac artery blood vessel gross examination of skeletal muscle, routine pathology biopsy, and specimens paraffin embedding slices does that HE, geneva are trichroism, elastic fibers dyeing; True color Pathologic image analysis instrument (Leica) directly carries out tunica intima morphometry; Immunohistochemical staining.
The 1.5 in-situ transesterification potencies of gene detect
Paraffin section dewaxes through dimethylbenzene, graded ethanol aquation, PBS and SSC washes and the HRP-Streptavindin (Jing Mei bio-engineering corporation) 37 DEG C adding dilution is hatched, trueblueTM dyeing and Orcein counterstaining is added, the transparent and mounting of dimethylbenzene after 1 × biotinwashsolusion washes.Positive products is aeruginous granular precipitates in endochylema or nucleus.Count the positive cell number shared by each section of tremulous pulse cross-sectional vessel wall 200 cells, calculate positive percentage as transfection efficiency.
1.6 immunohistochemical staining
Adopt labelling streptavidin biotin method (LSAB method) and press test kit description operation.I antibody is little mouse-anti SMC
α-actin monoclonal antibody (DACO, 1:200 dilute) and little mouse-anti PCNA monoclonal antibody (DACO, 1:100 dilute) step new company by Fujian to be provided.Count the PCNA positive cell number shared by 200 cells in each section of tremulous pulse cross section inner membrance, ask its meansigma methods as actual count.
2 interpretations of result:
Experimental data is all with SPSSforWindow11.0 process, and data acquisition χ ± s represents, group difference adopts variance analysis.P < 0.05 is for statistically to have significance.
The physical characterization of 2.1 ultrasonic microbubbles
The ultrasonic microbubble garden spherical vesicles shape of preparation, size is comparatively consistent, good dispersion, diameter 2-5 μm.After preserving 2 weeks through 4 DEG C, microbubbles coalesce do not occur, break, morphology does not change, counting and freshly prepd microbubble solution difference with insignificance, to good thermal stability (40 DEG C, 30min).
2.2 targeting turn PNA effect
Matched group blood vessel wall has no PNA transfection positive cell.Experimental group is the visible a small amount of positive cell of layer in the blood vessel, and positive cell is mainly distributed in the inner membrance (referring to Fig. 1 and Fig. 2) of hypertrophy, and counting average out to (67.25 ± 25.10), transfection efficiency is 33.63%.
2.3 blood vessel wall pathology and SABC change
Within after balloon injured iliac artery 1 week, see endothelial loss, intimal thickening, endo cell showed increased, tube chamber narrows, visible thrombosis (referring to Fig. 3).SABC display most cells anti alpha-actin antibody response is positive, reflects that these proliferative cells are SMC; PCNA is positive for the display of part cell, shows these hyperplasias comparatively active (referring to Fig. 4).Pathomorphism is measured and the SABC positive counts and antisense PNA sees the following form 1 to these Index Influences.Visible antisense PNA obviously suppresses intimal thickening and endo cell to express PCNA.Table 1 is the impact (χ ± s) of antisense PNA on inner film thickness, area and PCNA expression in 1 week after blood vessel injury, wherein * P < 0.05; * P < 0.01.
Table 1
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention and accompanying drawing content to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (6)
1. the ultrasonic microbubble of a targeting transfection peptide nucleic acid(PNA), it is characterized in that, described ultrasonic microbubble is mounted with peptide nucleic acid(PNA), described peptide nucleic acid(PNA) is peptide nucleic acid(PNA) prepared by the antisensenucleic acids of C-myc proto-oncogene, and the nucleotide sequence of described antisensenucleic acids is as follows: 5 '-AGCGAGGATATCTGGAAGAAATTCGAG-3 '.
2. a preparation method for the ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA), is characterized in that, comprises the steps: peptide nucleic acid(PNA) prepared by the antisensenucleic acids adding C-myc proto-oncogene according to claim 1 when preparing ultrasonic microbubble in albumin solution.
3. the preparation method of the ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) according to claim 2, is characterized in that, comprise the steps:
A, get the bovine serum albumin liquid 10ml that the quality percentage by volume comprising sucrose is 5%, peptide nucleic acid(PNA) prepared by the antisensenucleic acids adding the C-myc proto-oncogene as claimed in claim 1 of 2mg synthesis, mix to obtain liquid A, the quality percentage by volume of described sucrose in bovine serum albumin liquid is 10%;
B, use oxygen and fluorocarbon gas saturated liquid A successively, after supersonic generator process, namely obtain described ultrasonic microbubble.
4. the preparation method of the ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) according to claim 3, is characterized in that, concentration 0.8-1.8 × 10 of described ultrasonic microbubble
9individual/ml.
5. the preparation method of the ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) according to claim 3, is characterized in that, the flow of described oxygen and fluorocarbon gas is 6ml.min
-1.
6. the preparation method of the ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) according to claim 3, is characterized in that, described supersonic generator process is specially: supersonic generator output 180W, and frequency is process 1min under 20kHz condition.
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| CN101850124B (en) * | 2010-04-13 | 2012-05-30 | 姬尚义 | Albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system and preparation method thereof |
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| CN101850124B (en) * | 2010-04-13 | 2012-05-30 | 姬尚义 | Albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| cutrona G et al..effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal.《nat biotechnol.》.2000,第18卷(第3期), * |
| 荆慧等.超声介导靶向微泡造影剂促进c-myc反义基因在人肝癌细胞的表达.《中华超声影像学杂志》.2009,第18卷(第9期), * |
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