CN100368424C - Tumor inhibiting antisense nucleotide - Google Patents
Tumor inhibiting antisense nucleotide Download PDFInfo
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- CN100368424C CN100368424C CNB2006100292024A CN200610029202A CN100368424C CN 100368424 C CN100368424 C CN 100368424C CN B2006100292024 A CNB2006100292024 A CN B2006100292024A CN 200610029202 A CN200610029202 A CN 200610029202A CN 100368424 C CN100368424 C CN 100368424C
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Abstract
The present invention belongs to the field of biological technology and medical technology, and is one kind of antisense nucleotide. The antisense nucleotide has the structural general expression of 5'-CTC CCA GCG TGC GCC AT Z-3', and is preferably thio antisense nucleotide. It is proved that the antisense nucleotide of the present invention has effect higher than other Bcl-2 inhibiting medicines and less toxic side effect.
Description
Technical field
The invention belongs to biotechnology or medical field; More specifically, the present invention relates to can be used for regulating the antisense nucleotide of Bcl-2 expression level, and its production and use.
Background technology
Tumour is the first disease that threatens the human life, and the method for traditional treatment tumour comprises chemotherapy, radiation therapy and cutting operation, shortcoming such as it is low all to exist specificity, and side effect is big.Many weakly tumour patients finally are not to die from cancer, but die from chemotherapy, the huge side effect of radiotherapy.Excision only is applicable to solid tumor, and the operation meeting brings huge misery to the patient, also might impel tumour to worsen.In addition, tumour also is the big problem that people run in the process of treatment cancer to chemotherapeutics generation resistance easily.
For these reasons, this area researchist is seeking new oncotherapy target spot always hardy, in the hope of developing the specificity height, and the cancer therapy drug that side effect is little, and Bcl-2 albumen just is considered to have the antitumor target spot of potentiality like this.
Bcl-2 albumen is a member of Bcl-2 protein family, plays key effect in apoptotic regulation process.It promotes the factor " grappling " by the apoptosis that the BH3 hydrophobic domains will belong to same protein family, thereby the restriction latter plays a role, and " startup person " cytochrome C etc. of having avoided the endogenous cell apoptosis is from Intramitochondrial release and the final apoptosis that stops.At present, a received hypothesis is: " being the survivin of representative and with Bcl-2; Bax is that the apoptosis of representative promotes the ratio of the factor in cell to determine whether cell moves towards apoptosis " with Bid, it serves to show the importance of Bcl-2 in apoptosis regulation.
At present, known most of tumour cell comprises lung cancer, liver cancer, and mammary cancer, colorectal carcinoma or the like has all been found the high expression level of Bcl-2.It has been generally acknowledged that the high expression level of this important survivin makes tumour cell escape set apoptosis process, infinite multiplication.In addition, think clinically that the high expression level of the Bcl-2 also generation with drug resistance of tumor is relevant.Therefore, the downward modulation of the Bcl-2 expressing quantity in the tumour cell will have the apoptosis of tumor cells of promotion and increase its effect to chemotherapy drug susceptibility.
But the enforcement of Bcl-2 function mainly relies on the interaction between the albumen, but not with the combining of small molecules part, the micromolecular difficulty of developing the specific Bcl-2 of influence protein binding function is bigger.
Therefore, this area presses for further at gene level and protein level further investigation Bcl-2, in the hope of finding the specificity height, the Bcl-2 inhibition medicine that side effect is little, thereby the prevention, improvement or the treatment that are Bcl-2 relative disease, particularly tumour provide new approach.
Summary of the invention
The object of the present invention is to provide a kind of antisense nucleotide that is used for prevention or treatment Bcl-2 relative disease, particularly tumour.
In a first aspect of the present invention, a kind of antisense nucleotide is provided, it is characterized in that described antisense nucleotide has the structure shown in the general formula (I):
5’-CTC CCA GCG TGC GCC AT Z-3’ (I)
Wherein, Z is selected from: C or CC.
In a preference of the present invention, at least 2 phosphate bonds are by thio-modification in the sequence of described antisense nucleotide.
In another preference of the present invention, all phosphate bonds are all by thio-modification in the sequence of described antisense nucleotide.
In another preference of the present invention, described antisense nucleotide is used to prepare the medicine of prevention or treatment Bcl-2 relative disease.
In another preference of the present invention, described Bcl-2 relative disease comprises: tumour.
In the preferred preference of the present invention, described tumour includes, but is not limited to: lung cancer, liver cancer, mammary cancer, cervical cancer, colorectal carcinoma, hemopoietic system cancer, endocrine system cancer, or lymphoglandula cancer, and the not clear cancer in various former position.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition contains the described antisense nucleotide of significant quantity, and pharmaceutically acceptable carrier.
In another preference of the present invention, also can contain in the described pharmaceutical composition: taxol, fludarabine, cytosine arabinoside, daunorubicin, cis-platinum, 5 FU 5 fluorouracil, Dx, Etoposide, asparaginase, prednisone or methotrexate.
In a third aspect of the present invention, a kind of method of extracorporeal suppression tumor cell growth is provided, described method comprises: described tumour cell contacted with described antisense nucleotide, thus the growth of inhibition tumour cell.
On the other hand, the present invention also provides the method for a kind of prevention or treatment Bcl-2 relative disease, comprises the described antisense nucleotide that gives the safe and effective amount of experimenter.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the mechanism of action synoptic diagram of antisense drug and conventional medicament.
Fig. 2 has shown when the preparation antisense drug, has been a kind of step of sulfo-deoxynucleotide chain with deoxynucleotide chain thio-modification.
Fig. 3 A has shown the structural representation after phosphate bond is by sulfo-; Fig. 3 B has shown the structural formula of the base of four kinds of deoxynucleotides.
Fig. 4 has shown the growth-inhibiting effect (3 experiment get average, different medication groups carried out the variance analysis of completely random data, P＜0.05) of antisense antisense nucleotide medicine of the present invention to human tumor cells HT-29.
Fig. 5 has shown that the colony experiment detects the tumour cell colony and forms inhibiting rate.
Fig. 6 has shown that antisense nucleotide KS0604 and KS0605 have tangible raising tumour cell HT-29 and the H446 effect to chemotherapeutics Etoposide susceptibility.
Embodiment
The inventor unexpectedly finds a kind of antisense nucleotide of preventing or treating the Bcl-2 relative disease through extensive and deep research.Described antisense nucleotide is compared with the various medicines of existing treatment Bcl-2 relative disease, has obviously more excellent prevention or result of treatment.Finished the present invention based on this.
As used herein, " Bcl-2 " relative disease is meant and gene or the relevant disease of proteic expression of Bcl-2, includes but not limited to cell hyperplastic disease (as tumour).
The antisense nucleotide medicine
As used herein, " antisense nucleotide " is called " antisense oligonucleotide (AS-ONs, antisense-oligonucleotides) " or " antisense drug " again, is meant that length is about the dna molecular and the analogue thereof of 15-22 base, can with the mRNA complementation.According to Nucleotide hybridization principle, described antisense nucleotide can be hybridized with special genes, activates endogenous nucleic acid enzyme (being generally RNase H) degraded mRNA or blocking-up translation process, thereby causes cell related protein horizontal down-regulation.The synoptic diagram of antisense drug action principle is seen Fig. 1, and it is different from traditional medicine.
In the present invention, described " antisense nucleotide " also comprises the modified antisense nucleotide that adopts as based on the acquisition of means such as nucleic acid chains backbone modification technology, described modification does not change the activity of antisense nucleotide substantially, more preferably, described modification can improve the activity or the result of treatment of antisense nucleotide.For example, the described thio-modification that is modified to, or carry out alkyl in 2 ' position of ribose and modify.
Up to now, those skilled in the art have just attempted comprising N-myc, Raf, and survivin, MDM-2 or the like is as the target spot of antisense drug.But, because first-generation antisense drug is simple deoxynucleotide chain, solvability, cellular uptake abilities etc. are all not ideal enough, and easily by the endogenous nuclease degradation, therefore, can not reach good therapeutic action.The s-generation antisense drug that goes out based on the modification technique development of nucleic acid chains skeleton is in solubility, and aspects such as nuclease-resistant degraded have improvement greatly, and is easy to a large amount of syntheticly, can satisfy clinical required.The backbone modification method of oligonucleotide has multiple, comprises the sulfo-method, is that a kind of step of sulfo-deoxynucleotide chain is seen Fig. 2 with deoxynucleotide chain thio-modification.This method is that the Sauerstoffatom of the phosphate bond on the DNA skeleton is alternative with sulphur atom, makes medicine have good water-solubility, and can resist nuclease degradation.In the clinical trial, adopt method administrations such as subcutaneous, muscle and intravenous injection, can be distributed in each organ and tissue except that cerebral tissue, can combine, mainly from urine, discharge with plasma proteins.Theoretically, the antisense molecule that obtains according to antisense technology can be used for treating any disease that is caused by genetic expression or genetically deficient, such as virus infection, cancer and inflammation.Although antisense technology is in theory quite perfect, also be faced with challenge in actual applications, be not that all antisense molecules can both play good effect.
The inventor with the mRNA of Bcl-2 as template, but designed specificity and this mRNA bonded antisense nucleotide; Particularly, the antisense base sequences general formula that the present invention relates to following (wherein, A, G, C, T represent VITAMIN B4 respectively, guanine, cytosine(Cyt), four kinds of bases of thymus pyrimidine, the structural formula of these bases is referring to shown in Fig. 3 B):
5’-X CTC CCA GCG TGC GCC AT Y-3’
Wherein, X is selected from: blank, T, TT or GTT;
Y is selected from: C, CC or CCT.
In addition, can carry out suitable modification, improve its Bcl-2 inhibition effect, purposes such as raising is active, increase is stable, reduction toxicity, prolong half-life thereby reach to antisense nucleotide provided by the invention.Should be understood that and anyly can keep most of or whole active modifications of described antisense nucleotide to be included among the present invention.
In a kind of optimal way of the present invention, at least 2 phosphate bonds are by thio-modification in the sequence of described antisense nucleotide.Phosphate bond is seen Fig. 3 by the structural representation of sulfo-.
In a kind of preferred mode of the present invention, all phosphate bonds are all by thio-modification in the sequence of described antisense nucleotide.
In a kind of optimal way of the present invention, relate to the combination of 12 kinds of antisense base sequences among the present invention with identical sequence skeleton 5 '-CTCCCA GCG TGC GCC AT-3 ', see table 1 for details.
Table 1
| Title | Sequence |
| KS0601 KS0602 KS0603 KS0604 KS0605 KS0606 KS0607 KS0608 KS0609 KS0610 KS0611 KS0612 | 5’-TCT CCC AGC GTG CGC CAT C-3’(SEQ ID NO：1) 5’-TCT CCC AGC GTG CGC CAT CC-3’(SEQID NO：2) 5’-TCT CCC AGC GTG CGC CAT CCT-3’(SEQ ID NO：3) 5’-CTC CCA GCG TGC GCC ATC-3’(SEQ ID NO：4) 5’-CTC CCA GCG TGC GCC ATC C-3’(SEQ ID NO：5) 5’-CTC CCA GCG TGC GCC ATC CT-3’(SEQ ID NO：6) 5’-TTC TCC CAG CGT GCG CCA TC-3’(SEQ ID NO：7) 5’-TTC TCC CAG CGT GCG CCA TCC-3’(SEQ ID NO：8) 5’-TTC TCC CAG CGT GCG CCA TCC T-3’(SEQ ID NO：9) 5’-GTT CTC CCA GCG TGC GCC ATC-3’(SEQ ID NO：10) 5’-GTT CTC CCA GCG TGC GCC ATC C-3’(SEQ ID NO：11) 5’-GTT CTC CCA GCG TGC GCC ATC CT-3’(SEQ ID NO：12) |
In preferred mode of the present invention, described antisense base sequences general formula is as follows:
5’-CTC CCA GCG TGC GCC AT Z-3’(I)
Wherein, Z is selected from: C or CC.
Also promptly, described antisense nucleotide has the sequence shown in SEQ ID NO:4 or the SEQ ID NO:5; Preferred, all phosphate bonds are all by thio-modification in the described antisense nucleotide.The inhibition effect to Bcl-2 of the antisense nucleotide of these two kinds of sulfo-forms is unusual ideal, is far superior to have with it the antisense nucleotide of other kind of one or more base differences.
The purposes of antisense nucleotide medicine
The inventor discovers, uses above-mentioned antisense nucleotide (preferably sulfo-antisense nucleotide), can suppress the expression of Bcl-2, thereby can reach the purpose of prevention or treatment Bcl-2 relative disease.In optimal way of the present invention, described antisense nucleotide suppresses tumor cell proliferation by the proteic expression of Bcl-2 in the regulate tumor cell.
Antisense nucleotide of the present invention also can strengthen the susceptibility of tumour cell to chemotherapeutics.Because the drug-fast generation of tumour is closely related with Bcl-2 albumen, the antisense drug that the present invention relates to can reduce the level of Bcl-2 in the cell, strengthen its susceptibility to chemotherapeutics, thereby reduce the usage quantity of chemotherapeutics, chemotherapy is reduced greatly to the injury that the patient causes, and this is that conventional medicament can't be realized.
Described chemotherapeutics includes, but is not limited to: taxol, fludarabine, cytosine arabinoside, daunorubicin, cis-platinum, 5 FU 5 fluorouracil, Dx, Etoposide, asparaginase, prednisone or methotrexate.
The method that suppresses the Bcl-2 relative disease
The present invention also provides a kind of method of the Bcl-2 of inhibition relative disease, and described method comprises: adopt antisense nucleotide of the present invention, make it to hybridize with the mRNA of Bcl-2, thus the expression of inhibition Bcl-2.
In a kind of optimal way of the present invention, described Bcl-2 relative disease is a tumour.Therefore, the invention provides a kind of method that suppresses tumour, described method comprises: adopt antisense nucleotide of the present invention, suppress the expression of Bcl-2 in the tumour, make tumour cell generation apoptosis, thereby reach the purpose that suppresses tumour.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and described pharmaceutical composition contains the described antisense nucleotide of significant quantity, and pharmaceutically acceptable carrier.
The composition of described " pharmaceutically acceptable " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is promptly arranged.
Described " significant quantity " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
Described " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.This term refers to some medicament carriers like this: they itself are not necessary activeconstituents, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable vehicle in N.J.1991) at Remington ' s Pharmaceutical Sciences.Acceptable carrier can contain liquid on combination of traditional Chinese medicine is learned, as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, as weighting agent, lubricant, glidant, wetting agent or emulsifying agent, pH buffer substance etc.
The significant quantity of antisense nucleotide of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (for example by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of antisense nucleotide, metabolism, transformation period etc.; The disease that the patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Usually, when antisense nucleotide of the present invention every day gives with the dosage of about 0.001-100mg/kg (preferably 0.01-20mg/kg) the weight of animals, can obtain gratifying effect, preferably give with the dosage that separates for 2-4 time every day, or with the slowly-releasing form administration.For most of large mammal, the total dose of every day is about 0.005-100mg, preferably is about 0.008-50mg.Can regulate this dosage replys so that optimal treatment to be provided.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
Any suitable route of administration all is fine, and includes but not limited to: oral, intravenous injection, subcutaneous injection, muscle give, topical administration, implantation, slowly-releasing give, give etc. in the tumour; Preferably, described administering mode is that non-enteron aisle gives.
In a kind of mode of the present invention, also can contain the therapeutical agent of other Bcl-2 relative diseases in the described pharmaceutical composition.Described therapeutical agent is such as tumor therapeutic agent.
In addition, pharmaceutical composition of the present invention can with other the medication combined medication of Bcl-2 relative disease; Perhaps also can use in conjunction with other treatment means.
Major advantage of the present invention is:
1. the target spot of conventional medicament is generally protein, and the action target spot of this medicine is the mRNA of apoptosis supressor Bcl-2.The medicine that the present invention relates to is antisense nucleotide, particularly antisense thio oligonucleotide, and through proof, it is even more ideal that it suppresses effect of drugs than existing other Bcl-2.
2. drug specificity is high and have a broad spectrum.Antisense drug utilizes the nucleic acid hybridization principle to combine with the mRNA particular sequence high specific of target protein.Bcl-2 albumen is high expression level in the various human tumour cell, and relevant with the anti-apoptosis of tumour cell, so this medicine has the potentiality of the multiple cancer of treatment.
3. drug dose is little, and side effect is little.The drug effect that the present invention relates to be positioned at more upstream of cell physiological active, so drug dose is littler in gene level.The parent of antisense drug is human genetic material DNA, does not contain big toxophore, and toxic side effect is little.
4. can strengthen the susceptibility of tumour cell to chemotherapeutics.Before address, the drug-fast generation of tumour is closely related with Bcl-2 albumen, the antisense drug that the present invention relates to can reduce the level of Bcl-2 in the cell, strengthen its susceptibility to chemotherapeutics, thereby reduce the usage quantity of chemotherapeutics, chemotherapy is reduced greatly to the injury that the patient causes, and this is that conventional medicament can't be realized.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.
The production of embodiment 1 antisense drug
Antisense drug uses the AKTA oligopilot DNA/RNA Synthesizer of GE Amersham company synthetic, the preparation of agents useful for same and synthetic operation all carry out according to the instrumentation handbook, and wherein synthetic used carrier is the PS dC 30HL of Amersham company.Purifying and demineralising process use the AKTA ion-exchange purification system of Amersham company to finish equally, and operation is abideed by the instrument handbook and carried out.
The main production process of antisense nucleotide is summarized as follows:
1, require up-to-standard acetonitrile according to the AKTA oligopilot DNA/RNA Synthesizer of GE Amersham company, deprotecting regent, monomer solution, sulfo-and closed reagent are installed on the corresponding site of synthesizer;
2, use the synthetic Controlling System coding control of AKTA Oligopilot Unicorn4.0 synthesizer to carry out synthetic automatically;
3, reinforcing yin essence ion exchange column (cylinder: Fineline100, filler: purifying Source 30Q), reverse-phase chromatographic column (cylinder: Fineline 100, filler: desalination Soucre 15RPC) that synthetic crude product uses Amesham to provide;
4, the desalination product concentrates through rotary evaporation;
5, the product dilution is finite concentration, and vacuum lyophilization is white powder (a GLZ-0.4 type vacuum freeze drier), encapsulates 4 ℃ of preservations.
Whole process of production is all carried out in hundred grades of clean areas.
Synthetic sample detects by HPLC, and purity reaches more than 95%, and impurity mainly comprises: the oligonucleotide sequence (N-x sequence) that length is shorter than purpose segment, NaCl etc.
Synthesizing of the sulfo-antisense nucleotide that the external pharmacodynamics that is used for embodiment 2 detects
Four sequences that the present invention relates to have been synthesized according to the production method of antisense drug, synthetic simultaneously known positive control medicine G3139 and K22 and as the crucial base mispairing sequence (TM of negative control, two-basemismatch, the mispairing of two crucial bases promptly can produce bigger influence to drug effect), the related drugs sequence sees Table 2.
Synthetic sulfo-antisense base sequences among table 2 embodiment
| Title | Sequence |
| KS0601 KS0602 KS0604 KS0605 G3139 K22 TM | 5’-TCT CCC AGC GTG CGC CAT C-3’(SEQ ID NO：1) 5’-TCT CCC AGC GTG CGC CAT CC-3’(SEQ ID NO：2) 5’-CTC CCA GCG TGC GCC ATC-3’(SEQ ID NO：4) 5’-CTC CCA GCG TGC GCC ATC C-3’(SEQ ID NO：5) 5’-TCT CCC AGC GTG CGC CAT-3’(SEQID NO：13) 5’-TCC CAG CGT GCG CCA TCC-3’(SEQ ID NO：14) 5’-CTC CCA GCA TGT GCC AT-3’(SEQID NO：15) |
In the present embodiment, selecting human colon cancer cell HT-29 (available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) is subjects.In the cell bank behind the frozen HT-29 cell recovery cell culture incubator cultivate and go down to posterity that 1 week, to detect cell state good in the back, begin experiment.
Make up the cell culture system of 2ml volume in six orifice plates, wherein every hole comprises 500 in HT-29 cell, does blank not add medicine, and TM does negative control, and G3139 and K22 are as positive control.Antisense drug and feminine gender, the ultimate density of positive control drug are 5 μ M.Cultivate in the cell culture incubator to take out after 15 days and abandon nutrient solution, write down every porocyte colony number.
Statistics is seen Fig. 4.The result shows that antisense drug of the present invention has the activity of obvious suppression human tumor cells HT-29 propagation.The inhibition degree of four kinds of antisense sequences there are differences, wherein, KS0604, the inhibiting rate that KS0605 two sequence cell cluster form can reach more than 50%, suppress effect and be better than control drug G3139 and the K22 that has one or more base differences with it, also be better than the antisense nucleotide KS0601 and the KS0602 that have one or more base differences with it.
Embodiment 4 antisense nucleotide of the present invention are to the influence of human tumor cells Bcl-2 protein expression
Use the method for protein immunization marking experiment (Western Blot), detect antisense drug to human lung carcinoma cell H446 (available from institute of tumour hospital of Chinese medicine institute), human cervical carcinoma cell Hela (available from Shanghai Sheng Ke institute of the Chinese Academy of Sciences), the restraining effect that human colon cancer cell HT-29 human tumor cells Bcl-2 such as (available from Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) expresses.
Cultivated for 1 week behind the above-mentioned cell recovery, check that cell state just often begins experiment.
Cell inoculation is in 6 orifice plates, every hole 15 * 10
4Individual cell adds antisense drug and control drug to final concentration 1 μ M when waiting to grow to the 60-70% full scale, continue to cultivate after 48 hours lysing cell and obtain total protein extracting solution and be used for experiment.The protein extract dilution is 1 μ g/ μ L, sample 20 μ L on the electrophoresis.
Western Blot experimental implementation is abideed by " molecular cloning experiment guide " (second edition, the immunologic assay of immobilized protein Science Press's publication) partly carries out, use the electrophoresis of BIO-RAD in the experiment and change the film system, relevant damping fluid also illustrates preparation according to " molecular cloning experiment guide " (second edition, Science Press publishes).
Developed film with the quantized value of band gray scale as protein expression, is 100% calculating protein expression inhibiting rate with blank after image scanning system scanning.
Experimental result sees Table 3.The antisense drug of 1 μ M dosage can reduce the level of the Bcl-2 of above-mentioned vitro culture human tumor cells in various degree, and wherein KS0604 and KS0605 reach more than 60% the inhibiting rate of the Bcl-2 expression of HT-29 and H446 cell.
The restraining effect that table 3 sulfo-antisense nucleotide medicine is expressed different tumour cell Bcl-2
Detect by external pharmacodynamics this antisense nucleotide medicine, obtain as drawing a conclusion: several sulfo-antisense oligonucleotide medicines that the present invention relates to have the effect of the Bcl-2 expression level that reduces the various human tumour cell, and the downward modulation of this protein level has suppressed the propagation of human tumor cells.
The experiment of embodiment 5 colonies detects the tumour cell colony and forms inhibiting rate
The experimental procedure of taking is as follows:
The tumour cell HT-29 trysinization of logarithmic phase, the blood counting chamber counting;
Diluting cells is also implanted in the six new orifice plates several 400 of every porocyte, final volume 1ml, overnight incubation; Grouping adding next day is subjected to the reagent thing and system is mended is final volume 2ml;
4, to cultivate after 7 days in the incubator and take out, low-power microscope is meter colony number down;
Processing data is calculated colony and is formed inhibiting rate.
The results are shown in Figure 5.As can be known from the results, KS0604 significantly is better than K22 and G3139 to the colony formation inhibition effect of tumour cell HT-29, and KS0604 can reach 45% inhibiting rate when 10uM dosage, and the inhibiting rate of K22 and G3139 is between 30%-35% under the same concentrations.
Therefore, the effect of KS0604 vitro inhibition tumour is high more than 10 percentage points with respect to G3139 and K22.Because the cost of medicine is high, the raising of relatively limited effect may realize the reduction significantly of dosage, that is the reduction significantly of cost.
The detection of embodiment 6 drug susceptibilities
The experimental procedure of medicaments insensitive property testing is as follows:
1, adding final concentration in tumour cell (HT29 or H446 are all available from institute of tumour hospital of the Chinese medicine institute) nutrient solution of logarithmic phase is the antisense drug of 10uM, acts on 48 hours, does not add antisense drug group (solvent control; Solvent Control) and negative medicine (2 base mispairings; Two-base Mismatch) group in contrast;
2, peptic cell and counting are implanted aseptic 96 orifice plates, 5000 every holes of cell, and add certain density Etoposide, use nutrient solution that the system polishing is 200uL;
3, cultivate and adopt mtt assay to detect cytoactive after 72 hours.
The results are shown in Figure 6.As can be known from the results, KS0604 and KS0605 have tangible raising tumour cell HT-29 and the H446 effect to chemotherapeutics Etoposide susceptibility.Especially in test at the H446 cell, medicine effect of enhanced sensitivity under Etoposide 10uM and the 30uM concentration is the most obvious, antisense drug pretreated group relative cell viability under Etoposide 10uM concentration is reduced to about 40%, and corresponding non-treatment group still is about 80%.
Therefore as seen, antisense nucleotide of the present invention is except killing tumor cell, and its major function also is to improve the susceptibility of tumour to all kinds of chemotherapeutics, thereby improves result of treatment, reduces side effect.
In view of the huge injury that the traditional chemotherapeutics of heavy dose produces tumour patient, the good effect of enhanced sensitivity of the antisense drug that the present invention relates to can provide a possible solution for it, and produce comparatively far-reaching influence.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Fujian Jinshan Biomedicine Co., Ltd.
＜120〉antisense nucleotide of inhibition tumour
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ctcccagcat gtgccat 17
Claims (9)
1. an antisense nucleotide is characterized in that, described antisense nucleotide has the structure shown in the general formula (I):
5’-CTC CCA GCG TGC GCC AT Z-3’ (I)
Wherein, Z is selected from: C or CC.
2. antisense nucleotide as claimed in claim 1 is characterized in that at least 2 phosphate bonds are by thio-modification in the sequence of described antisense nucleotide.
3. antisense nucleotide as claimed in claim 2 is characterized in that all phosphate bonds are all by thio-modification in the sequence of described antisense nucleotide.
4. the purposes of the described antisense nucleotide of claim 1 is characterized in that, is used to prepare the medicine of prevention or treatment Bcl-2 relative disease.
5. purposes as claimed in claim 4 is characterized in that, described Bcl-2 relative disease is selected from: tumour.
6. purposes as claimed in claim 5 is characterized in that, described tumour is selected from: lung cancer, liver cancer, mammary cancer, cervical cancer, colorectal carcinoma, hemopoietic system cancer, endocrine system cancer, or lymphoglandula cancer.
7. a pharmaceutical composition is characterized in that, described pharmaceutical composition contains the described antisense nucleotide of claim 1 of significant quantity, and pharmaceutically acceptable carrier.
8. pharmaceutical composition as claimed in claim 7, it is characterized in that, wherein also can contain: taxol, fludarabine, cytosine arabinoside, daunorubicin, cis-platinum, 5 FU 5 fluorouracil, Dx, Etoposide, asparaginase, prednisone or methotrexate.
9. pharmaceutical composition as claimed in claim 8 is characterized in that, wherein contains Etoposide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100292024A CN100368424C (en) | 2006-07-21 | 2006-07-21 | Tumor inhibiting antisense nucleotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100292024A CN100368424C (en) | 2006-07-21 | 2006-07-21 | Tumor inhibiting antisense nucleotide |
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| Publication Number | Publication Date |
|---|---|
| CN1887896A CN1887896A (en) | 2007-01-03 |
| CN100368424C true CN100368424C (en) | 2008-02-13 |
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| CNB2006100292024A Active CN100368424C (en) | 2006-07-21 | 2006-07-21 | Tumor inhibiting antisense nucleotide |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5831066A (en) * | 1988-12-22 | 1998-11-03 | The Trustees Of The University Of Pennsylvania | Regulation of bcl-2 gene expression |
| CN1374088A (en) * | 2001-03-09 | 2002-10-16 | 暨南大学 | Bcl-2 gene antisense nucleic acid medicine for resisting leukemia and tumor drug-fast |
| CN1471408A (en) * | 2000-08-25 | 2004-01-28 | Methods of treatment of a BCL-2 disorder using BCL-2 antisense oligomers | |
| CN1500803A (en) * | 2002-11-19 | 2004-06-02 | 吕联煌 | Bc-2 antisense thiophosphoric acid oligodeoxynucleotides anticancer drugs, preparing method and uses thereof |
-
2006
- 2006-07-21 CN CNB2006100292024A patent/CN100368424C/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5831066A (en) * | 1988-12-22 | 1998-11-03 | The Trustees Of The University Of Pennsylvania | Regulation of bcl-2 gene expression |
| CN1471408A (en) * | 2000-08-25 | 2004-01-28 | Methods of treatment of a BCL-2 disorder using BCL-2 antisense oligomers | |
| CN1374088A (en) * | 2001-03-09 | 2002-10-16 | 暨南大学 | Bcl-2 gene antisense nucleic acid medicine for resisting leukemia and tumor drug-fast |
| CN1500803A (en) * | 2002-11-19 | 2004-06-02 | 吕联煌 | Bc-2 antisense thiophosphoric acid oligodeoxynucleotides anticancer drugs, preparing method and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| bcl-2反义寡核苷酸逆转人卵巢上皮性癌细胞耐药的体外研究. 李敏等.中华妇产科杂志，第41卷第2期. 2006 * |
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| CN1887896A (en) | 2007-01-03 |
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