CN101850124B - Albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system and preparation method thereof - Google Patents
Albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of thrombus prevention medicament, and discloses an albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system. The albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system comprises carrier recombination tPA gene plasmid albumin nanometer particles and sucrose albumin ultrasonic microbubbles; and the preparation method comprises the following steps of: (1) preparing the carrier recombination tPA gene plasmid albumin nanometer particles; (2) preparing the sucrose albumin ultrasonic microbubbles; and (3) preparing the albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system by using the carrier recombination tPA gene plasmid albumin nanometer particles and the sucrose albumin ultrasonic microbubbles. In the invention, the recombination tPA gene plasmid is constructed, the albumin particles and the ultrasonic microbubbles are selected as carriers, and the carriers are transduced into organism cells to obtain effectiveness and generation of continuous tPA so as to achieve the function of resisting coagulation for a long time and preventing formation of thrombus, thereby the albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system is safe without wound.
Description
Technical field
The present invention relates to the thrombus prevention and cure drug world, relate in particular to a kind of albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system and preparation method thereof.
Background technology
Under the normal physiological situation, blood coagulation-anticoagulant and fibrinolytic-anti-fibrinolytic mechanism is balance mutually, guarantees blood proper flow in vivo, neither forms thrombosis, also is unlikely to hemorrhage.Thrombosis and thromboembolism mechanism unbalance make thrombosis form inadequately or form after can not be removed efficiently, cause tissue or organ ischemia/necrosis; On the other hand, anticoagulant/fibrinolytic excessively also can cause hemorrhage generation.
(tissue-type Plasminogen Activator is the main activity factor of fibrinolytic system tPA) to tPA, is the important anticoagulant composition of body.It is synthetic by vascular endothelial cell, under the especially fibrinous effect of various stimulations, is released into blood, acts on plasminogen and makes it convert plasmin promotion fibrin degradation into.Clone successfully from the cDNA of tPA, people can make up its retroviral vector and obtain the proteic high expressed of tPA at external transduction endotheliocyte.Zoopery confirms, the endotheliocyte of this transduction is attached at sacculus rack surface or arterial bridge anastomotic stoma can prevents behind the stenting or the generation of vascular bypass postoperative thrombosis and restenosis, obviously improves the long-term effect of performing the operation.People's Recomposed tPA has been applied to the thromboembolism treatment of coronary heart disease acute myocardial infarction, cerebral embolism, lung infraction etc. and has obtained produce effects as biological preparation, but because the not seen widespread use that costs an arm and a leg.
Albumin is the more a kind of nano material of Recent study, its good biocompatibility, and biodegradable, no immunogenicity and cytotoxicity, gene transfering efficiency is higher.Because it has accurate nanostructured and the characteristic of surface with inner portability molecule, make it to become the carrier of a good DNA transfered cell.
Gene imports intravital mode and is mostly the genes of interest direct injection in target tissue, adopts in the cardiac muscle direct injection or cardiac catheter to inject DNA or adenovirus vector like cardiovascular disease more.Chinese invention patent CN200610033187.0 discloses a kind of gene suture, and this suture is the surgery dacron suture that is loaded with high expressed tPA gene plasmid, and this gene suture directly is sutured in cardiac muscular tissue, can eliminate or avoid the formation of sludged blood.Chinese invention patent CN200910106773.7 discloses the effective expression that all obtains tPA with chitosan nanometer tPA genophore of having done feedstock production and direct injection in cardiac muscle.But the method for above-mentioned two patents record has wound property, limits its clinical practice.
Summary of the invention
For addressing the above problem, the object of the present invention is to provide a kind of safe, effective, noninvasive albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system.
Second purpose of the present invention is to provide a kind of method for preparing albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system.
For realizing above-mentioned purpose, the present invention adopts following technical scheme: a kind of albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system comprises and carries Recomposed tPA gene plasmid albumin nano granular and sucrose albumin ultrasonic microbubble.
Said Recomposed tPA gene plasmid contains Recomposed tPA gene, promoter, terminator and selection markers expression casette.
Said Recomposed tPA gene is by 1686 base compositions, and its base sequence is shown in SEQ ID:NO.1.
Said albumin is a bovine serum albumin.
Sucrose albumin ultrasonic microbubble content is 0.8-1.8 * 10 in the target system
9Individual/ml, Recomposed tPA gene plasmid content is 0.1-0.3mg/ml.
The method for preparing of albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system may further comprise the steps:
1) Recomposed tPA gene plasmid albumin nano granular is carried in preparation;
2) preparation sucrose albumin ultrasonic microbubble;
3) prepare albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system with carrying Recomposed tPA gene plasmid albumin nano granular and sucrose albumin ultrasonic microbubble.
Step 1) is specially after hatching in the Recomposed tPA gene plasmid adding bovine serum albumin solution; In the presence of ultrasonic field, splash into ethanol water; Milky occurs and dropwise splash into 0.5% glutaraldehyde solution until solution; Stir, leave standstill under the room temperature, obtain carrying Recomposed tPA gene plasmid albumin nano granular.
Step 2) be specially the bovine serum albumin liquid that sterile preparation contains sucrose, and place container, use oxygen and fluorocarbon gas saturated liquid successively, supersonic generator is handled, and obtains oxygen-fluoro-propane sucrose albumin ultrasonic microbubble.
The albumin nano granular that step 3) is specially step 1) preparation adds to step 2) in the albumin microvesicle of preparation; Add glutaraldehyde; Hatch under 4 ℃ carry out crosslinked; With normal saline washing, centrifugal, layering, come-up foam suspension is albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system.
The present invention makes up the Recomposed tPA gene plasmid and selects albumin nano granular and ultrasonic microbubble is a carrier, and its transduction body cell is obtained the generation of effective and lasting tPA, and can reach long-term anticoagulant and prevent thrombotic effect, and safety, noinvasive;
The present invention adopts one-step method preparation to carry a gene albumin nano granular, and this method is wrapped in the plasmid physical package in the albumin nano granular, its major advantage be carry by the transgenic amount higher; Medicament slow release and transgenic time are longer; Have preferably enzyme protection and prevent DNA enzyme degradation in the body; Be designed to targeting vector easily, be applied to the interior research of body or be prepared into the Nano medication application have more superiority;
The present invention adds 10% sucrose in albumin liquid in preparation ultrasonic microbubble process, avoid sucrose albumin liquid viscosity strong, ultrasonic difficulty and the bigger problem of institute's energy requirement during preparation, and microvesicle has good stability;
The microvesicle that contains perfluorocarbon is added with minor amounts of oxygen simultaneously and can further promotes microvesicle in stability in blood, thereby obtains stronger myocardial visualization effect;
Adopt ultrasonic triggering to destroy albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system; The albumin nano granular targeting location of carrying genes plasmid is discharged and the target gene expression enhancing, and this method is a kind of simple and easy effective targeted nano gene transfer new technique.
Description of drawings
Fig. 1 is that Recomposed tPA gene plasmid pSecTag2B-tPA makes up flow chart;
Fig. 2 carries Recomposed tPA gene plasmid albumin nano granular Zetasizer3000 appearance results of grain size analysis figure;
Fig. 3 is agarose gel electrophoresis DNA retardance experiment and DNA enzyme protection experimental result picture;
Fig. 4 is the following 400 times of enlarged drawings of albumin ultrasonic microbubble and albumin nano granular crosslinked back ordinary optical microscope;
Fig. 5 is cardiac ultrasonic iconography change figure before and after the ultrasound wave targeted therapy, before the A input microvesicle; The heart development strengthens behind the B input microvesicle; After the C ultrasonic therapy;
Fig. 6 is that the liver ultrasonic image is learned change figure before and after the ultrasound wave targeted therapy, before the A input microvesicle; The liver development strengthens behind the B input microvesicle; After the C ultrasonic therapy;
Fig. 7 is the myocardial cell increase figure of the genetically modified cardiac muscular tissue of ultrasonic targeting, and endochylema is red to be dyed, HE 200 *;
Fig. 8 is the loose figure of the genetically modified skeletal muscle tissue myocyte of ultrasonic targeting, and endochylema is red to be dyed, and nucleus increases, HE 200 *;
Fig. 9 A is the tPA of matched group cardiac muscular tissue negative antibody reaction SABC figure as a result, and Fig. 9 B experimental group tPA of cardiac muscular tissue antibody positive reaction SABC is figure as a result, 200 *;
Figure 10 A is the tPA of matched group cardiac muscular tissue negative antibody reaction SABC figure as a result, and Figure 10 B is the tPA of experimental group cardiac muscular tissue antibody positive reaction SABC figure as a result, SABC 200 *;
Figure 11 is experimental group hepatocyte tPA antibody positive reaction SABC figure as a result, 400 *;
Figure 12 is the chondrocyte tPA of experimental group costicartilage basic unit antibody positive reaction SABC figure as a result, 400 *;
Figure 13 is experimental group medullary cell tPA antibody positive reaction SABC figure as a result, 400 *.
The specific embodiment
Albumin nanometer of the present invention-ultrasonic microbubble carrier tissue type plasminogen activator gene target system method for preparing mainly may further comprise the steps:
1, make up Recomposed tPA gene plasmid pSecTag2B-tPA, following embodiment 1 can specify;
2, adopt above-mentioned Recomposed tPA gene plasmid and albumin, carry out the preparation of albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system, following embodiment 2 can specify.
The preparation of embodiment 1 Recomposed tPA gene plasmid pSecTag2B-tPA
1, main agents
A, plasmid pSecTag2B, available from American I nvitrogen biotech firm,
B, competent cell E.Coli JM109, available from Promega company,
C, restricted enzyme HindIII, KpnI, BamHI and XhoI, available from precious biological engineering company limited,
D, T4-DNA ligase, available from New England Biolabs company,
E、QIAquick?Gel?Extraction?Kit、QIAGEN?PCR?Product
Purification Kit is available from QIAGEN company.
2, method
Three EST:tPA-1 of synthetic shown in SEQ ID NO.2, tPA-2 shown in SEQ ID NO.3 and tPA-3 shown in SEQ ID NO.4, connect cloning vehicle respectively, be prepared into plasmid pOTB6, pOTB7 and pCMV-SPORT6.
Adopt three couples of primer tPA-1F (SEQ ID NO.5) and tPA-1R (SEQ ID NO.6), tPA-2F (SEQ ID NO.7) and tPA-2R (SEQ ID NO.8), tPA-3F (SEQ ID NO.9) and tPA-3R (SEQ ID NO.10) increase respectively above-described said plasmid pOTB6, pOTB7 and pCMV-SPORT6; The PCR program of amplification tPA-1 and tPA-3 is: 94 ℃ of preparatory degeneration 4min; 94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 1min; Totally 25 circulations, last 72 ℃ are extended 5min; The PCR program of amplification tPA-2 is: 94 ℃ of preparatory degeneration 4min, and 94 ℃ of 45sec, 60 ℃ of 45sec, 72 ℃ of 1min, totally 25 circulations, last 72 ℃ are extended 5min.
Products therefrom is digested with three couples of restricted enzyme HindIII and KpnI, KpnI and BamHI, BamHI and XhoI respectively behind QIAGEN PCR Product Purification Kit purification; Use restricted enzyme HindIII and XhoI digested plasmid pSecTag 2B simultaneously, purification process is seen QIAGEN PCR Product Purification Kit description.
Above-mentioned enzyme action product is carried out agarose gel electrophoresis; QIAquick Gel ExtractionKit reclaims target DNA fragment; Purification process is seen QIAquick Gel Extraction Kit description; Connect above-mentioned withdrawal product with the T4-DNA ligase, construct Recomposed tPA gene plasmid pSecTag2B-tPA, make up flow process and see Fig. 1.
The preparation of embodiment 2 albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system
The Recomposed tPA gene plasmid pSecTag2B-tPA and the albumin that utilize embodiment 1 to make prepare albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system, may further comprise the steps:
1, Recomposed tPA gene plasmid albumin nano granular is carried in the one-step method preparation, is specially: the 100mg bovine serum albumin adds the dissolving of 5ml distilled water, with 0.1N HCI adjustment pH value to 5.5.The 1mg Recomposed tPA gene plasmid that has made up is added in the above-mentioned albumin solution; In the presence of ultrasonic field, slowly splash into ethanol water (ethanol: water=2: 1) after hatching 30min; Milky occurs and dropwise splash into 0.5% glutaraldehyde solution 30ul until solution; Leave standstill 2h under the agitation as appropriate, room temperature.After decompression made remaining ethanol volatilization, the centrifugal 30min of gained solution 17000r/min was to remove free albumin and unnecessary cross-linking agent, and gained is precipitated as and carries the gene albumin nano granular.Deposition with distilled water resuspended and with ultrasound wave (condition: 180W, fixed frequency 20kHz 30sec) are dispersed into emulsion state, store for future use under 4 ℃.
Get part and prepare scanning electron microscope BIAO and BEN observation nanoparticle form, do envelop rate detection and gel retardation assasy, and measure size and surperficial Zeta potential with the Zetasizer3000 appearance.The scanning electron microscope result shows that albumin nano granular is spherical, the even and good dispersion of size.The grain size analysis of Zetasizer3000 appearance shows that albumin particle diameter size is normal distribution, and the mean diameter size is 132.0nm, and maximum particle diameter is 152.4nm, and minimum grain size is 49.1nm.Polydispersity index is average 0.33, sees Fig. 2.Surface Zeta is average+31.32-+41.42mV.The envelop rate of sample detects with ultraviolet spectrophotometer, measures the total DNA content that adds.The nanometer solution of parcel behind the gene is centrifugal and measure free DNA in the supernatant, and envelop rate (%)=(W total-W trip)/W is total * 100% (W is total: addition; W trip: the DNA amount of recording in the supernatant).Measure the result and show envelop rate average out to 73.58%, meet requirement of experiment.
0.9% agarose gel electrophoresis result is as shown in Figure 3, visible behind albumin nanometer parcel DNA block fully and in well, fail to move (DNA: albumin is 1/100).By the degraded after DNase I digestion of albumin nano granular parcel, electrophoresis does not see that band manifests to DNA.The DNA of parcel nanoparticle then remains intact, and is blocked fully in well, shows that albumin nano granular is to the protective effect of DNA tool.
2, preparation oxygen-fluoro-propane sucrose albumin ultrasonic microbubble, be specially: sterile preparation contains 5% (g.ml of 10% sucrose
-1) bovine serum albumin liquid 10ml; And placed and cover the 50ml plastic centrifuge tube, use oxygen and fluorocarbon gas saturated liquid (flow: 6ml.min successively
-1), about 10min, supersonic generator is handled 1min (condition: 180W, fixed frequency 20kHz); 4 ℃ airtight preservation is subsequent use down with the microvesicle for preparing.
Get part microscopic examination microvesicle form, measure size and counting; The Zetasizer3000 appearance is measured size and surperficial Zeta potential.Ordinary optical microscope shows the glycoprotein ultrasonic microbubble garden spherical vesicles shape of preparation, and size is consistent, good dispersion, and the minimum 0.9 μ m of diameter, maximum 9.8 μ m, 95% above microvesicle diameter 2-5 μ m can satisfy blood capillary ultrasonic contrast demand.The albumin microvesicle that contains 10% sucrose is preserved in the morphology in 30 days through 4 ℃ and is not changed, and to good thermal stability (40 ℃, 30min).
3, prepare albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system with carrying gene albumin nano granular and oxygen-fluoro-propane sucrose albumin ultrasonic microbubble; Be specially: the albumin nano granular that will once prepare (containing the 1mg plasmid) adds in the 5ml albumin microvesicle, adds to hatch 2h under 4 ℃ of the 50% glutaraldehyde 10ul solution and carry out crosslinked (the glutaraldehyde solution final concentration is 0.1%).With normal saline washing, centrifugal, layering, centrifugal speed 200 commentaries on classics/min, 1min gets come-up foam suspension and promptly gets albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system.The adjustment microbubble concentration makes and contains microvesicle 0.8-1.8 * 10 in the 5ml target system
9Individual/ml, gene plasmid content is 1mg.Preserve subsequent use down for 4 ℃.Significant change does not also take place in character such as the crosslinked back of albumin ultrasonic microbubble and albumin nano granular form, size, sees Fig. 4.
1. material: 25 of purebred NZws, male, body weight 2.3-2.5kg, Nanfang Medical Univ's animal center provides.
2. main agents: goat-anti buman tPA polyclonal antibody, the anti-sheep polyclonal antibody of rabbit, bovine serum albumin, tPA ELISA detection kit are provided by the U.S. biological engineering company limited of crystalline substance.Perfluoropropane gas (Halocarbon-218) is provided by the prompt sharp company in Foshan.
3. key instrument: supersonic generator is a Ningbo new sesame bio tech ltd product; Therapeutic ultrasound appearance (US-700 is German product).
4. method:
4.1 rabbit is with 5% Nembutal vein anesthetic (20-25mg/kg); Observe internal organs such as the heart, liver develops with two-dimensional ultrasound.Inject year tPA gene nanometer albumin microsphere infusion 5ml and observe obvious enhancing of relevant internal organs development through auricular vein.Divide into groups: (1) experimental group (n=16): further divide the heart group 6 examples, liver group 6 example and muscle groups 4 examples.Vein inject to carry behind the tPA gene nanometer albumin microsphere infusion 5ml immediately through the front wall over against heart antetheca and hepatic region, through ultrasonic irradiation 30min such as the left back lower limb adductor muscles of skin and observe and microvesicle is smashed (two-dimensional ultrasound observation), supersonic frequency 1MHz; Intensity 1.5W/cm
2(2) matched group (n=9): simple microvesicle group 3 examples, inject a year tPA gene nanometer albumin microsphere infusion 5ml through auricular vein; Without ultrasonic irradiation; Nano gene group 3 examples, vein input albumin nanometer tPA gene and ultrasonic irradiation; Blank group 3 examples, vein input 5ml normal saline and ultrasonic therapeutic.The ultrasonic therapeutic condition is the same.The 2 groups of target organs in back are liver.The postoperative normal diet is fed, 4 weeks of observation period.
4.2 pathology and immunohistochemical observation: the animal via routine is put to death respectively after raising and observe 4 weeks; The thoracic wall costicartilage tissue that dirty, liver, adduction of the hip joint muscular tissue and the ultrasonic field of coring passed through; 4% paraformaldehyde is fixed, FFPE, conventional section and HE dyeing pathologic finding; The dyeing of SABC method detects the expression of tPA gene in each tissue and cell indirectly, and the positive reaction cell is a yellowish-brown graininess deposition in the cytoplasm.Get lungs, renal tissue as contrast.Finish to get respectively blood testing D-dimer content and detect blood tPA content with observing before the animal Rhizoma Atractylodis Macrocephalae with the ELISA method
5. result
5.1 ultrasonoscopy and targeted therapy: the target tissue that ultrasonoscopy heart, liver etc. are set after rabbit ear edge vein inject to carry tPA gene nanometer albumin microsphere infusion 5ml, observe with inject before compare the obviously enhancing of developing of relevant internal organs.Behind ultrasonic therapy 30min, organize video picture return back to the injection before level.The simple microvesicle group of non-ultrasonic therapeutic, nano gene group and the ultrasonoscopy of blank group do not have obvious change, see Fig. 5 and Fig. 6.
5.2 tissue pathologies change and tPA gene expression: the myocardial cell that ultrasonic targeting is handled, Skeletal Muscle Cell and hepatocyte equal-volume increase, the cytoplasm engrain, and nucleus increases, and Skeletal Muscle Cell nuclear increases.The visible cavity of part cell cytosol.Tissue and cell without ultrasonic irradiation are not seen obvious change; Control tissue such as nephridial tissue, lung tissue etc. are not seen obviously unusual, see Fig. 7 and Fig. 8.Parenchyma in the visible heart in ultrasonic 4 week of targeting transfection back, liver, skeletal muscle tissue and the osseous tissue is expressed tPA antigen.The positive cardiac muscle of tPA antigen presentation is with Skeletal Muscle Cell and is dispersed in distribution, and these cell volumes increase, and the endochylema band reduces or disappears and is fine particulate, the positive reaction a little less than part Interstitial cell and little blood vessel wall are visible.The hepatocyte of tPA reacting positive is and is dispersed in or radial distribution, mainly is distributed in the lobules of liver portal area, and other cell is not seen positive reaction.Positive reacting cells mainly is distributed in cartilage germinal layer cell and myeloid element and part Interstitial cell (Fig. 9,10,11,12) in the osseous tissue.
5.3 blood D-dimer and tPA content: blood D-dimer and tPA content detection result are as shown in table 1 before and after the targeting transgenic, and the index of 2 reactions of 4 all blood fibrinolytic all significantly increases (table 1) behind the transgenic.
Table 1: the rabbit targeting changes before the tPA gene art and postoperative 4 all blood D-dimers and tPA content (μ g/L, x ± s)
* 0.025＜P＜0.01, * * P＜0.01 is compared with postoperative before the art
Blood tPA content increases and the D-dimer content increases obtaining to detect when target tissue is expressed tPA, has reacted the tPA anticoagulating active and has obviously strengthened.
SEQUENCE?LISTING
< 110>Ji, the Shangyi
Season, army
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cccaagctta?tggatgcaat?gaagagaggg 30
<210>6
<211>27
<212>DNA
< 213>artificial sequence
<400>6
ggggtaccac?ggtaggctga?cccattc 27
<210>7
<211>26
<212>DNA
< 213>artificial sequence
<400>7
ggggtaccca?cagcctcacc?gagtcg 26
<210>8
<211>30
<212>DNA
< 213>artificial sequence
<400>8
cgggatccag?caggagctga?tgagtatgcc 30
<210>9
<211>26
<212>DNA
< 213>artificial sequence
<400>9
cgggatcctc?tctgccgccc?actgct 26
<210>10
<211>26
<212>DNA
< 213>artificial sequence
<400>10
ccctcgaggc?ggtcgcatgt?tgtcac 26
Claims (9)
1. albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system is characterized in that: comprise and carry Recomposed tPA gene plasmid albumin nano granular and sucrose albumin ultrasonic microbubble.
2. albumin nanometer according to claim 1-ultrasonic microbubble carrier tissue type plasminogen activator gene target system is characterized in that: said Recomposed tPA gene plasmid contains Recomposed tPA gene, promoter, terminator and selection markers expression casette.
3. albumin nanometer according to claim 2-ultrasonic microbubble carrier tissue type plasminogen activator gene target system is characterized in that: said Recomposed tPA gene is by 1686 base compositions, and its base sequence is shown in SEQ ID:NO.1.
4. according to any described albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system of claim 1-3, it is characterized in that: the albumin in said year Recomposed tPA gene plasmid albumin nano granular is a bovine serum albumin.
5. according to any described albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system of claim 1-3, it is characterized in that: sucrose albumin ultrasonic microbubble content is 0.8-1.8 * 10 in the target system
9Individual/ml, Recomposed tPA gene plasmid content is 0.1-0.3mg/ml.
6. the method for preparing of albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system may further comprise the steps:
1) Recomposed tPA gene plasmid albumin nano granular is carried in preparation;
2) preparation sucrose albumin ultrasonic microbubble;
3) prepare albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system with carrying Recomposed tPA gene plasmid albumin nano granular and sucrose albumin ultrasonic microbubble.
7. the method for preparing of albumin nanometer according to claim 6-ultrasonic microbubble carrier tissue type plasminogen activator gene target system; It is characterized in that: step 1) is specially after hatching in the Recomposed tPA gene plasmid adding bovine serum albumin solution; In the presence of ultrasonic field, splash into ethanol water, milky occurs and dropwise splash into 0.5% glutaraldehyde solution, stir until solution; Leave standstill under the room temperature, obtain carrying Recomposed tPA gene plasmid albumin nano granular.
8. the method for preparing of albumin nanometer according to claim 6-ultrasonic microbubble carrier tissue type plasminogen activator gene target system; It is characterized in that: step 2) be specially the bovine serum albumin liquid that sterile preparation contains sucrose; And place container; Use oxygen and fluorocarbon gas saturated liquid successively, supersonic generator is handled, and obtains oxygen-fluoro-propane sucrose albumin ultrasonic microbubble.
9. the method for preparing of albumin nanometer according to claim 6-ultrasonic microbubble carrier tissue type plasminogen activator gene target system; It is characterized in that: the albumin nano granular that step 3) is specially step 1) preparation adds to step 2) in the albumin ultrasonic microbubble of preparation; Add glutaraldehyde; Hatch under 4 ℃ and carry out crosslinkedly, with normal saline washing, centrifugal, layering, come-up foam suspension is albumin nanometer-ultrasonic microbubble carrier tissue type plasminogen activator gene target system.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103784977A (en) * | 2014-03-06 | 2014-05-14 | 杨建安 | Ultrasound micro-bubbles of targeted transfection peptide nucleic acid and preparation method and application thereof |
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| WO2014065513A1 (en) * | 2012-10-25 | 2014-05-01 | 서강대학교 산학협력단 | Ultrasound contrast medium in which nanoparticles containing drug are combined, and preparation method therefor |
| KR101595795B1 (en) * | 2014-03-19 | 2016-02-22 | (주)아이엠지티 | Dual-Purpose PAT/Ultrasound Contrast Agent with Nanoparticles Including Drug and Method for Preparing the Same |
| CN104096245B (en) * | 2014-07-18 | 2017-03-29 | 重庆医科大学 | Parcel carries lipid ultrasonic microvesicle of medicine albumin nano granular and preparation method thereof |
| CN104436323B (en) * | 2014-11-13 | 2016-06-01 | 季军 | The biological support of a kind of load t-PA gene and its preparation method |
| CN113713126A (en) * | 2021-09-24 | 2021-11-30 | 江苏省人民医院（南京医科大学第一附属医院） | Nano ultrasonic contrast agent for heart targeted gene delivery and preparation method and application thereof |
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| CN101537191B (en) * | 2009-04-21 | 2011-05-11 | 姬尚义 | Recomposed tPA gene-chitosan nanoparticle complex and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103784977A (en) * | 2014-03-06 | 2014-05-14 | 杨建安 | Ultrasound micro-bubbles of targeted transfection peptide nucleic acid and preparation method and application thereof |
| CN103784977B (en) * | 2014-03-06 | 2015-11-18 | 杨建安 | Ultrasonic microbubble of targeting transfection peptide nucleic acid(PNA) and its preparation method and application |
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| CN101850124A (en) | 2010-10-06 |
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