CN1213056C - Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease - Google Patents
Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease Download PDFInfo
- Publication number
- CN1213056C CN1213056C CN 02123678 CN02123678A CN1213056C CN 1213056 C CN1213056 C CN 1213056C CN 02123678 CN02123678 CN 02123678 CN 02123678 A CN02123678 A CN 02123678A CN 1213056 C CN1213056 C CN 1213056C
- Authority
- CN
- China
- Prior art keywords
- pai
- asodn
- dna
- antisense oligonucleotide
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a structure design of plasminogen activator inhibitor PAI-1 genes and synthetic antisense oligonucleotide (ASODN) for inhibiting the expression of the PAI-1 genes of human bodies. The present invention designs 27 pieces of antisense oligonucleotide complementary with a region 5', a translation region and a region 3'of PAI-1mRNA, makes activity sieving and evaluation and discovers that human umbilical vein endothelial cells cultivated in vitro by the oligonucleotide in the specific region of the PAI-1mRNA can effectively inhibit the expression of the PAI-1 genes for the first time. The present invention relates to the sequence and the structure of the antisense oligonucleotide of the PAI-1 genes and new medicines for treating thrombus diseases and reducing the damage of the thrombus diseases.
Description
The present invention relates to the biotechnology pharmaceutical field, specifically a kind of antisense oligonucleotide (antisense oligodeoxynucleotide at the PAI-1 gene, ASODN) sequential structure and become the treatment thrombotic diseases reduces the newtype drug of the diseases related hazardness of thrombus.
Thrombotic diseases is a class high incidence, high disability rate, high mortality disease, comprises disseminated inravascular coagulation (DIC) that myocardial infarction, cerebral thrombosis, venous thrombosis, pulmonary infarction, multiple serious disease (infection, wound etc.) late period is concurrent etc.Domestic data shows: dead number summations such as the myocardial infarction that is caused by thromboembolism, cerebral infarction, lung infraction occupy first of other disease; U.S.'s data shows: the death toll that thromboembolism causes is more than 5 times of cancer mortality number.The most effective treatment means of thrombotic diseases is that thrombolysis is handled.At present clinical thrombolytic drug commonly used is a plasminogen activator class medicine (as urokinase, tissue plasminogen activator etc.), though clinical application has obtained more satisfactory effect, also demonstrates easily shortcoming such as cause bleeding, the transformation period lacks, cost an arm and a leg.Clinical in recent years and fundamental research finds that all it is an independent hazard factor (Hasenstab D who causes thrombotic diseases that PAI-1 raises, etal.Arterioscler Thromb Vasc Biol 2000,20 (3): 853-859.Anderson JL, et al.J Am CollCardiol.1999,34 (6): 1778-1783.), prompting is started with from reduction PAI-1 might filter out thrombolytic drug.
But still not having this type of medicine at present comes out.
ASODN can combine with target gene mRNA behind transfered cell and form the DNA-RAN heterozygote, and sealing or inhibition target gene duplicate or express, thereby reduces the expression level of its " downstream " product-albumen or enzyme.ASODN is in malignant tumour at present, disease of viral infection (hepatitis, influenza etc.), obtained more satisfactory curative effect in the experimental therapy of some heredopathia and other abnormal gene expression associated diseases, and compare with the traditional treatment medicine and to show out long action time, the effect high specificity, advantages such as toxic side effect is little, since the first antiviral antisense drug of in August, 1998 drugs approved by FDA enters the bed application, external existing a collection of antisense drug enters that the clinical II-III phase tests or clinic trial (Joensen H, Ann Med.2000,32 (1): 31-33.Morris MJ, et al.Clin Cancer Res.2002,8 (3): 679-683.Corey DR.Telomerase inhibition, oligonucleotides, and clinical trials.Oncogene 2002,21 (4): 631-637).
People PAI-1mRNA by reports such as David Ginsenberg is 2786bp, utilizes information biology software can simulate the secondary structure of PAI-1mRNA, as the important evidence of design ASODN.But because PAI-1mRNA is difficult to prediction at intracellular state, can therefore designed ASODN near PAI-1mRNA and hybridize with sealing with it or reduce PAI-1 and express and still remain experiment in vivo and vitro and do further screening and evaluation.
The objective of the invention is to seek differential high efficient novel thrombolytic antisense drug, promptly PAI-1mRNA being expressed has the ASODN of better retarding effect and can improve its bioactive chemically modified derivative.
To the effect that of the present invention, people PAI-1mRNA full length sequence according to reports such as David Ginsberg among the Gene Bank, the secondary structure of reference computers prediction, 27 the sulfo-ASODN (seeing Table 1) that adopted computer aided design (CAD), it is synthetic and carry out thio-modification when synthetic to adopt PE company to produce ABI8909 type automatic dna synthesizer.Standby through the reverse purification column of Micro Pure II (Solid Phase Sciences) purifying.Adopt the Human umbilical vein endothelial cells (HUVEC) of cultivating that above-mentioned ASODN is carried out the external activity screening and estimates.Result proof have 5 ASODN be PAI86, PAI508, PAI588, PAI1265, PAI1316 etc. genetic expression has specific dose-dependent inhibition effect to PAI in concentration is 0.25-1 μ mol/L concentration range, point out them to can be used for the treatment of thrombotic diseases (as cerebral thrombosis, myocardial infarction, atherosclerosis, venous thrombosis etc.).
Table 1:27 bar sulfo-Antisensedigonucleotsequence sequence and character
Serial number name action target spot character length (nt) sequence (5 '-3 ')
1 PAI86 67-86 A 20 GAC?ATC?TGC?ATC?CTG?AAG?TT
2 PAI76 57-76 A 20 TCC?TGA?AGT?TCT?CAG?AGG?TA
3 PAI166 147-166 A 20 CGT?AGG?ATG?GGG?GAT?GGT?GC
4 PAI218 199-218 A 20 TGC?GCC?ACC?TGC?TGA?AAC?AC
5 PAI339 320-339 A 20 CTT?GAA?TCC?CAT?AGC?TGC?TT
6 PAI382 363-382 A 20 TGT?ACA?GAT?GCC?GGA?GGG?CG
7 PAI508 489-508 A 20 TGA?CCG?TGC?TCC?GGA?ACA?GC
8 PAI588 569-588 A 20 GAT?CAT?ACC?TTT?TGT?GTG?TG
9 PAI660 641-660 A 20 GTT?GAA?GTA?GAG?GGC?ATT?CA
10 PAI775 756-775 A 20 TAT?AGT?TFA?ACT?TGT?TGG?TC
11 PAI881 862-881 A 20 GGC?ACC?TCT?TTT?TCA?TAA?GG
12 PAI1132 1113-1132 A 20 CGT?TCA?CCT?CGA?TCT?TCA?CT
13 PAI1207 1188-1207 A 20 CCA?TGA?TGA?TCT?CCT?CGG?GG
14 PAI1265 1246-1265 A 20 CCC?ATG?AAA?AGG?ACT?GTT?CC
15 PAI1316 1297-1316 A 20 TTT?GTC?CCA?GAT?GAA?GGC?GT
16 PAI1294 1275-1294 A 20 TTC?CCC?AGG?GTC?AGG?GTT?CC
17 PAI1557 1538-1557 A 20 AGG?CGT?CAC?CGT?CTG?GTT?TG
18 PAI1808 1789-1808 A 20 TGC?CAC?AGT?GGA?CTC?TGA?GA
19 PA19051 1886-1905 A 20 GGG?GAG?GGA?GAT?GGC?CAG?GC
20 PAI2078 2059-2078 A 20 ATA?TGA?TAA?ATA?TTT?AGG?TA
21 PAI2195 2176-2195 A 20 GGC?TGG?ACT?TCC?TGA?GAT?AC
22 PAI2469 2450-2469 A 20 AAA?GTT?CTG?TCC?TGG?TAG?GT
23 PAI2502 2483-2502 A 20 CCA?ATG?CGG?CTG?TGA?GTC?AC
24 PAI2614 2595-2614 A 20 AAA?GAT?TAT?CTA?AGG?TAG?TT
25 PAI1103 1084-1103 A 20 CCC?CTC?CCC?ACC?AAG?AGA?TT
26 PAI 53-68 A 16 GAG?GGC?TGG?AGA?CAT?C
27 PAI 53-68 S 16 GAG?GGC?TGA?AGA?CAT?C
Annotate: A:ASODN:S:scramble
According to the present invention, ASODN at PAI-1mRNA 3 ' district (PAI1265), 5 ' district (PAI86) and translation district (PAI508, PAI588, PAI1316) can specificity suppress the PAI-1 expression of gene, might become the novel biological engineering medicine of treatment thrombotic diseases.
According to the present invention, be the nuclease resistance that strengthens antisense oligonucleotide, bioavailability, and tissue target tropism etc., the present invention has comprised the various chemically modifieds of PAI86, PAI508, PAI588, PAI1265, PAI1316, for example directly modification of thio-modification, semi-lactosi, lipophilic molecules or the quick target liposomes modification of PH etc.
According to the present invention, the oligonucleotide of invention and modifier thereof can be made into the reagent of parenterai administration by means known in the art.
According to the present invention, the treatment of oligonucleotide of the present invention and modifier thereof is formed can use independent effective constituent or composition forms comprises other antisense oligonucleotide of associating and derivative thereof.
According to the present invention, treatment of the present invention is formed, comprise pharmacokinetics, pharmacokinetics, mode of administration, route of administration, the receptor's of certain drug age, body weight, hepatic and renal function state, character, degree and the treatment time limit etc. of disease according to different situations, with an amount of dosed administration.
According to the present invention, enforcement of the present invention has important society and economic benefit to the treatment of the thrombotic diseases of serious harm human health with prevention.
In conjunction with subordinate list specific implementation method of the present invention is described
Table 1:27 bar Antisensedigonucleotsequence sequence and character
The effective ASODN sequence of table 2:5 bar, action target spot and target gene local structure
Table 3:5 bar ASODN is to the retarding effect of PAI-1Ag
Table 4:5 bar ASODN is to the active retarding effect of PAI-1
Table 5:5 bar ASODN is to the active influence of t-PA
Embodiment:
1.ASODN design with synthetic
People PAI-1mRNA full length sequence according to reports such as David Ginsberg among the Gene Bank, the secondary structure of reference computers prediction, 27 the sulfo-ASODN (seeing Table 1) that adopted computer aided design (CAD) adopt the ABI8909 type automatic dna synthesizer of PE company synthetic and carry out thio-modification when synthetic.Behind synthetic the finishing, 55 ℃ of cuttings of strong aqua and deprotection be after 15 hours, through the reverse purification column of Micro Pure II (Solid Phase Sciences) purifying, ultraviolet quantitatively after, vacuum-drying ,-20 ℃ of preservations are standby.
2.ASODN cell transfecting
To contain 10% calf serum and 100
U/ ml penicillin, the DMEM nutrient solution of 100 μ g/ml Streptomycin sulphates is at 37C, 5%CO
2Cultivate HUVEC in the incubator.With 48 porocyte culture plate (NUNC) inoculating cells, every hole inoculation 1 * 10
4Individual cell (0.5ml), after treating that cell 50%-60% merges, the cell culture fluid that contains Lipofectin with serum-free, antibiotic-free carries out sulfo-ASODN cell transfecting, concrete steps are referring to the Lipofectin working instructions, every ASODN is provided with four kinds of concentration of 0.1,0.25,0.5,1.0 (μ mol/L) respectively, same concentration is respectively established 3 holes, and transfection is after 6 hours, transfection liquid is changed to contain 10% calf serum and antibiotic DMEM nutrient solution is cultivated.Because under the normal circumstances, PAI-1 content is extremely low in the cell culture fluid, be difficult for measuring, the also high level of PAI-1Ag when simulating thrombosis simultaneously, each hole of experimental group all adds 0.5ng/ml TGF-β
1Express to increase PAI-1Ag as inductor.Set up TGF-β simultaneously
1(the C group, control group only adds 0.5ng/ml TGF-β to group in the enchylema
1), Lipofectin organizes (only adding Lipofectin in the enchylema) and blank group (not adding any composition in the enchylema), each group cultivate 24 respectively, behind the 48h, the collecting cell supernatant is used for PAI-1Ag, activity and t-PA determination of activity.
3.ASODN activity rating
Adopt indexs such as PAI-1Ag, PA1-1 activity, t-PA activity to carry out the ASODN evaluated biological activity.
(1) PAI-1Ag measures the ELISA method that adopts:
Get cell conditioned medium liquid 25 μ l, dilution buffer liquid 75 μ l add in the 96 hole enzyme plates, under the 37C condition, hatched 1 hour with the PAI-1 monoclonal antibody that is coated on the enzyme plate, the back adds two of peroxidase labelling and resists, 37C was hatched 1 hour, with dcq buffer liquid flushing three times, every hole adds the 0.1mlTMB chromogenic substrate, hatch every hole adding 0.1ml stop buffer after 10 minutes under the room temperature, be to measure each hole optical density(OD) on many marks determinator of 450nm at wavelength, according to standard substance that test kit provides, preparation optical density(OD)-PAI-1Ag typical curve, and (actual value is: table intermediate value * extension rate 4) to calculate this PAI-1Ag of test sample of institute level according to this.
(2) the chromophoric substrate development process is adopted in the PAI-1 determination of activity:
With cell conditioned medium 25 μ l to be measured, damping fluid 25 μ l, and 5 * 10
-2IU/ml t-PA standard substance 50 μ l add in the 96 hole enzyme plates, 25C placed after 20 minutes, every hole adds the mixture of 100 μ l chromophoric substrates, covalency thing, Profibrinolysin, insulation is after 160 minutes in the wet box of 37C, add 30 μ l stop buffer termination reactions, be to measure each hole optical density(OD) on many marks determinator of 405nm at wavelength, according to standard substance that test kit provides, the active typical curve of preparation optical density(OD)-PAI-1, and (actual value is: table intermediate value * extension rate 4) to calculate this PAI-1 of test sample of institute activity according to this.
(3) the chromophoric substrate development process is adopted in the t-PA determination of activity:
With acidifying cell conditioned medium 100 μ l to be measured, add in the 96 hole enzyme plates, every hole adds the mixture of 100 μ l chromophoric substrates, covalency thing, Profibrinolysin, insulation is after 160 minutes in the wet box of 37C, adding 30 μ l stop buffer termination reactions, is to measure each hole optical density(OD) on many marks determinator of 405nm at wavelength, according to standard substance that test kit provides, the active typical curve of preparation optical density(OD)-t-PA, and (actual value is: table intermediate value * extension rate 2) to calculate this t-PA of test sample of institute activity according to this.
(4) result
A.ASODN suppresses ordered sequence and the dose-dependently that PAI-1Ag expresses
In 27 designed ASODN, there are 5 to be that PAI86, PAI508, PAI588, PAI1265, PAI1316 have obvious retarding effect to PAI-1Ag.24h and control group after the transfection (C group, i.e. TNF group) are compared: PAI508, PAI1265 are at concentration 〉=0.25 μ M; PAI86, PAI1316 when concentration is 1 μ M, express equal tool retarding effect to PAI-1Ag at concentration 〉=0.5 μ M and PAI588, are dose-dependently.48h after the transfection, PAI86, PAI508, PAI1265, PAI1316 ASODN be at concentration 〉=0.25 μ M, and PAI588 expresses PAI-1Ag and all to show retarding effect (table 3) when concentration 〉=0.5 μ M.
B.ASODN inhibition active ordered sequence of PAI-1 and dosage, time-dependent manner
24h compares with the C group after the ASODN transfection: PAI508, PAI1316 are at concentration 〉=0.25 μ M, and PAI1265 is 0.5 μ M at concentration 〉=0.5 μ M, PAI86 in concentration, and the PAI-1 activity all significantly was lower than control group when PAI588 concentration was 1 μ M.48h:PAI86, PAI588, PAI1265ASODN be at concentration 〉=0.25 μ M after the transfection, and PAI508 is at concentration 〉=0.5 μ M, and PAI1316 all obviously suppresses the PAI-1 activity when concentration 〉=0.1 μ M.Relatively 24, phase point discovery during two of 48h, other 4 ASODN are low than 24h all in the active concentration of 48h appearance obvious inhibition PAI-1 except that PAI508, prompting is along with the prolongation of time, and PAI86, PAI588, PAI1265, PAI1316ASODN strengthen (table 4) gradually to the active retarding effect of PAI-1.
C.ASODN increase active ordered sequence of t-PA and dosage, time-dependent manner
24h and C group is compared after the ASODN transfection: PAI86, PAI508, PAI588, PAI1265 be at concentration 〉=0.25 μ M, and PAI1316 can obviously increase t-PA activity in the cell conditioned medium when concentration 〉=0.1 μ M.48h:PAI86 after the transfection,, PAI588, PAI1265, at concentration 〉=0.25 μ M, PAI508, PAI1316 also obviously increase the t-PA activity when concentration 〉=0.5 μ M, be dose-dependently (table 5).
Above-mentioned activity experiment result shows, PAI86, PAI508, PAI588, PAI1265, PAI1316 can suppress PAI-1Ag in certain dosage and time range expresses, reduces the active and increase t-PA activity of PAI-1, and prompting can be used for the treatment of thrombotic diseases.
The local structure of the effective ASODN sequence of table 2:5 bar, action target spot and target gene
ASODN sequence number action target spot target gene local structure ASODN sequence
PAI86 67-86 multi-branched ring GAC ATC TGC ATC CTG AAG TT
PAI508 489-508 multi-branched ring TGA CCG CCG TGC TCC GGA ACA GC
PAI588 569-588 multiple-branching construction GAT CAT ACC TTT TGT GTG TG
Ring CCC ATG AAA AGG ACT GTT CC in the PAI1265 1246-1265
Ring TTT GTC CCA GAT GAA GGC GT in the PAI1316 1297-1316
The retarding effect that table 3:ASODN expresses PAI-1Ag (ng/mL)
24h 48h
Sequence
C 0.1 0.25 0.5 1.0(uM) C 0.1 0.25 0.5 1.0(uM)
PAI86 173.3±35.1 200.3±60.0 98.30±16.7 52.3±13.1
* 68.3±6.7
* 290.0±57?2 302.7±34.5 150.6±18.8
* 109.3±14.0
* 110.3±11.6
*
PAI508 173.3±35?1 183.0±27.1 63.0±12.2
* 52.0±16.1
* 52.6±7.6
* 290.0±57.2 343.6±33.0 215.0±29.1
* 131.0±27.2
** 154.0±22?0
*
PAI588 173.3±35.1 127.3±27.2 104.6±21.8 104.7±21.8 77.0±10.5
* 290.0±57.2 316.3±29.6 218±29.7 113.6±11.6
* 141.3±16.8
*
PAI1265 173.3±35.1 197.3±17.5 121.3±19.1
* 1037±16.2
* 89.0±19.1
** 290.0±57.2 330?3±34.7 137.5±18.2
* 91.0±18.6
** 61.7±14.4
**
PAI1316 173.3±35.1 193.3±26.5 189.6±23.0 116.0±18.0
* 114.3±20.1
* 290.0±57.2 292.3±18.2 134.7±21.2
* 150.0±21.6
* 104.3±23.7
**
Note：
*P＜0.05，
**P＜0.01?vs?C?at?same?time.
Table 4:ASODN is to PAI-1 activity (* 10-2AU/mL) retarding effect
24h 48h
Sequence
C 0.1 0.25 0.5 1.0(uM) C 0.1 0.25 0.5 1.0(uM)
PAI86 14.3±1.5 12.3±2.1 10.3±1.5 10.3±1.2
* 10.0±1.7 15.0±1.5 12.0±2.6 8.0±1.5
* 9.0±1.0
** 9.0±1.2
**
PAI508 14.3±1.5 11.0±2.6 9.7±1.5
* 9.7±0.8
* 9.0±0.2
* 15.0±1.5 13.0±2.5 10.0±1.7 9.0±1.1
* 8.1±1.1
*
PAI588 14.3±1.5 11.3±3.1 11.7±3.1 9.7±1.1 9.3±1.5
* 15.0±1.5 13.0±2.0 11.1±3.1
* 9.0±1.1
* 9.0±0.6
**
PAI12665 14.3±1.5 11.3±3.1 10.3±1.5 9.7±1.2
** 7.7±0.6
** 15.0±1.5 11.1±0.6 9.0±2.19
* 8.0±1.7
** 7.1±0.6
**
PAI1316 14.3±1.5 13.1±3.6 11.3±2.5
* 9.3±0.6
* 8.3±0.6
* 15.0±1.5 12.1±2.5
* 10.0±1?1
* 8.1±1.2
** 7.1±0.6
**
Note：
*P＜0.05.
**P＜001?vsC?at?same?time.
Table 5:ASODN is to t-PA activity (* 10
-4IU/mL) reinforcing effect
24h 48h
Sequence
C 0.1 0.25 0.5 1.0(uM) C 0.1 0.25 0.5 1.0(uM)
PAI86 4.7±1.2 8.7±2.1 10.7±0.6
* 12.7±0.6
** 13.3±0.6
** 6.7±1.2 8.0±1.1 9.3±0.6
* 11.3±1.5
* 14.7±2.1
**
PAI508 4.7±1.2 9.3±2.1 10.7±1.5
** 12.7±0.6
** 13.3±1.5
** 6.7±1.2 8.0±2.6 10.0±1.1 11.3±0.6
** 13.7±0.6
**
PAI588 4.7±1.2 10.3±1.7 11.3±2.1
* 11.3±1.5
** 13.3±1.5
** 6?7±1.2 8.3±1.7 10.0±2.7
** 11.3±1.5
** 14.0±1.0
**
PAI1265 4.7±1.2 9.3±1.5 10.3±1.3
* 10.7±1.5
* 12.7±0.6
** 6.7±1.2 8.3±2.6 10?7±1.5
* 13.3±1.5
** 14.0±2.0
**
PAI1316 4.7±1.2 9.7±1.5
* 12.7±2.1
* 11.6±1.2
* 13.0±1.0
** 6.7±1.2 8.3±1.2 10.0±1.0 12.0±1.7
** 12.7±2.1
**
Note：
*P＜0.05.
**P＜0.01?vs?C?at?same?time.
Sequence table
＜110〉Institute of Radiation Medicine, Academy of Military Medical Sciences, PLA
＜120〉structure and the purposes of the antisense oligonucleotide of treatment thrombotic diseases
<130>
<150>02123678.x
<151>2002-07-28
<160>27
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
<213>
<400>1
tcctgaagtt?ctcagaggta 20
<210>2
<211>20
<212>DNA
<213>
<400>2
cgtaggatgg?gggatggtgc 20
<210>3
<211>20
<212>DNA
<213>
<400>3
tgcgccacct?gctgaaacac 20
<210>4
<211>20
<212>DNA
<210>10
<211>20
<212>DNA
<213>
<400>10
ggcacctctt?tttcataagg 20
<210>11
<211>20
<212>DNA
<213>
<400>11
cgttcacctc?gatcttcact 20
<210>12
<211>20
<212>DNA
<213>
<400>12
ccatgatgat?ctcctcgggg 20
<210>13
<211>20
<212>DNA
<213>
<400>13
cccatgaaaa?ggactgttcc 20
<210>14
<211>20
<212>DNA
<213>
<400>14
tttgtcccag?atgaaggcgt 20
<210>15
<211>20
<212>DNA
<213>
<213>
<400>4
cttgaatccc?atagctgctt 20
<210>5
<211>20
<212>DNA
<213>
<400>5
tgtacagatg?ccggagggcg 20
<210>6
<211>20
<212>DNA
<213>
<400>6
tgaccgtgct?ccggaacagc 20
<210>7
<211>20
<212>DNA
<213>
<400>7
gatcatacct?tttgtgtgtg 20
<210>8
<211>20
<212>DNA
<213>
<400>8
gttgaagtag?agggcattca 20
<210>9
<211>20
<212>DNA
<213>
<400>9
tatagtttaa?cttgttggtc 20
<400>15
ttccccaggg?tcagggttcc 20
<210>16
<211>20
<212>DNA
<213>
<400>16
aggcgtcacc?gtctggtttg 20
<210>17
<211>20
<212>DNA
<213>
<400>17
tgccacagtg?gactctgaga 20
<210>18
<211>20
<212>DNA
<213>
<400>18
ggggagggag?atggccaggc 20
<210>19
<211>20
<212>DNA
<213>
<400>19
atatgataaa?tatttaggta 20
<210>20
<211>20
<212>DNA
<213>
<400>20
ggctggactt?cctgagatac 20
<210>21
<211>20
<212>DNA
<213>
<400>21
aaagttctgt?cctggtaggt 20
<210>22
<211>20
<212>DNA
<213>
<400>22
ccaatgcggc?tgtgagtcac 20
<210>23
<211>20
<212>DNA
<213>
<400>23
aaagattatc?taaggtagtt 20
<210>24
<211>20
<212>DNA
<213>
<400>24
cccctcccca?ccaagagatt 20
<210>25
<211>16
<212>DNA
<213>
<400>25
gagggctgga?gacatc 16
<210>26
<211>16
<212>DNA
<213>
<400>26
gagggctgaa?gacatc 16
<210>27
<211>20
<212>DNA
<213>
<400>27
gacatctgca?tcctgaagtt 20
Claims (3)
1. but a specificity is in conjunction with the antisense oligonucleotide of people PAI-1 mRNA, and its sequence is selected from:
1).PAI86： 5’-GAC?ATC?FGC?ATC?CTG?AAG?TT-3’
2).PAI508：?5’-TGA?CCG?TGC?TCC?GGA?ACA?GC-3’
3).PAI588：?5’-GAT?CAT?ACC?TTT?TGT?GTG?TG-3’
4).PAI1265：5’-CCC?ATG?AAA?AGG?ACT?GTT?CC-3’
5).PAI1316：5’-TTT?GTC?CCA?GAT?GAA?GGC?GT-3’。
2. contain Antisensedigonucleotsequence sequence in the claim 1 and the pharmaceutical composition of pharmaceutically acceptable carrier.
3. the antisense oligonucleotide in the claim 1 is used to prepare the purposes of antithrombotic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02123678 CN1213056C (en) | 2002-07-08 | 2002-07-08 | Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02123678 CN1213056C (en) | 2002-07-08 | 2002-07-08 | Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1467217A CN1467217A (en) | 2004-01-14 |
| CN1213056C true CN1213056C (en) | 2005-08-03 |
Family
ID=34142445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02123678 Expired - Fee Related CN1213056C (en) | 2002-07-08 | 2002-07-08 | Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1213056C (en) |
-
2002
- 2002-07-08 CN CN 02123678 patent/CN1213056C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1467217A (en) | 2004-01-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1310907C (en) | Heterocyclic compound and antitumour agent containing the same as active ingredient | |
| CN1033971C (en) | 9-[(substituted glycyl) amido]-6-demethyl-6-deoxytetracyclines | |
| CN1258604C (en) | Reagent box used for detecting non pathogenic or pathogenic A type influenze virus H5 subtype virus | |
| CN101035777A (en) | Modulators of hcv replication | |
| CN1550501A (en) | Oligopolynucleotide of inhibiting activity of necrosin in human tumor | |
| CN1355790A (en) | Method of inhibiting amyloid protein aggregation and imaging amyloid deposits using isindoline derivatives | |
| CN1289331A (en) | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof | |
| CN1366881A (en) | Method for curing some cancers by using estrogenic excitomotor and agonist | |
| CN1630535A (en) | Inhibition of STAT-1 | |
| CN1236769C (en) | Inhibitor for inhibiting cell adhesion | |
| CN1643136A (en) | Method of proliferating human hepatocytes and method of obtaining human hepatocytes | |
| CN1812986A (en) | Furazanobenzimidazoles | |
| CN1240439C (en) | Genetic switch medicine for treating tumor | |
| CN1198649C (en) | Drug composition containing PGD antagonists for curing inthing | |
| CN1213056C (en) | Structure and uses of autisense oligodeoxynucleotide for treating thrombus disease | |
| CN1744892A (en) | Breast cancer resistance protein (BCRP) inhibitor | |
| CN1594354A (en) | Oleanolic acid couple derivatives and their pharmaceutical use | |
| CN1144586C (en) | 2-(1-hydroxyethyl)-5-hydroxynaphtho{2,3-B}furan-4, 9-dione and antitumor agents comprising this compound | |
| CN1033631A (en) | Epipodophyllotoxin glucoside 4 '-acyl derivative | |
| CN1580259A (en) | SiRAN and expression carrier for inhibiting human VEGF gene expression and their pharmaceutical use | |
| CN1069488A (en) | Hexahydrobenzene is [f] quinolinone also | |
| CN1277634A (en) | New tissue-specific calpaines, their production and their use | |
| CN1271213C (en) | Chip for detecting meningitis, its preparation method and application | |
| CN1842590A (en) | Method of differentiation from embryo-stem cell of primate to hematogenous cell | |
| CN1310922C (en) | Fused bicyclic pyridine derivative as tachykinin receptor antagonist |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20050803 |