CN101503449A - Nucleic acid molecule CXSI32 and use thereof in anti-cancer medicine preparation - Google Patents
Nucleic acid molecule CXSI32 and use thereof in anti-cancer medicine preparation Download PDFInfo
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- CN101503449A CN101503449A CNA2008100335781A CN200810033578A CN101503449A CN 101503449 A CN101503449 A CN 101503449A CN A2008100335781 A CNA2008100335781 A CN A2008100335781A CN 200810033578 A CN200810033578 A CN 200810033578A CN 101503449 A CN101503449 A CN 101503449A
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Abstract
The invention belongs to the field of biological medicine and relates to a nucleotide molecule and the application thereof in the preparation of antineoplastic medicine. The invention provides a nucleotide molecule used for preparing the antineoplastic medicine, which has the activity of inhibiting tumor growth and the sequence including 5'-AACGAACCGGUAAAUCUGC-3' or 5'-AACGAACCGGTAAATCTGC-3'. The nucleotide molecule is called as CXSI32. The nucleotide molecule CXSI32 can stop the cell in the stage of G1 and can obviously inhibit the tumor growth when being applied to a nude mouse compared with a nude mouse under the same condition of culture.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of nucleic acid molecule and the application in the preparation antitumor drug thereof.
Background technology
Tumor disease has now risen to No. the 2nd, the world " killer ", and its death toll is only second to cardiovascular diseases.In the past few years, the foreign medical science bound pair has had new understanding again in the pathogeny of tumor disease on cell base.Based on the further understanding to tumor invasion mechanism, people utilize various approach to develop and develop can be special, effectively killing tumor cell and to the avirulent medicine of normal cell.At present, for treatment for cancer is first-selection with chemotherapy and radiotherapy still, though both have obtained suitable curative effect to tumor treatment, but since lack to the specificity of tumour cell thus have bigger toxic side effect and some tumour cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.In recent years, can to kill and wound cancer cells specifically and normal cell is not had the medicine of toxic side effect in order to develop, from cell, paid much attention to and huge investment by the research on the molecular level to the pathogenesis of cancer for people.
Summary of the invention
The purpose of this invention is to provide a kind of nucleic acid molecule that is used to prepare antitumor drug.
Another object of the present invention provides the application of above-mentioned nucleic acid molecule.
The invention provides a kind of nucleic acid molecule that is used to prepare antitumor drug, it has active and its sequence that suppresses tumor growth and comprises 5 '-AACGAACCGGUAAAUCUGC-3 ' or 5 '-AACGAACCGGTAAATCTGC-3 '.Be called as CXSI32 in the present invention.
Wherein, A, T, U, G and C are respectively adenine nucleotide, thymidylic acid, uridylate, guanylic acid and cytidylic acid(CMP).
In the present invention, this term of term " nucleic acid molecule CXSI32 " also comprises the nucleotide sequence variation form of 5 '-AACGAACCGGUAAAUCUGC-3 ' or 5 '-AACGAACCGGTAAATCTGC-3 '.These variant forms comprise: several (are generally 1-15, preferably 1-10,1-5 more preferably) disappearance, insertion and/or the replacement of Nucleotide, and add several (being generally in 10, preferably is in 5) Nucleotide at 5 ' and/or 3 ' end.For example, (3 ' end) add the sequence that some dT (deoxythymidine) back forms in 5 '-AACGAACCGGUAAAUCUGC-3 ' back.
Among the present invention, CXSI32 has the sequence that suppresses the active of tumor growth and comprise 5 '-AACGAACCGGUAAAUCUGC-3 ' dTdT.
Among the present invention, CXSI32 can be to have the activity that suppresses tumor growth and be the sequence of 5 '-AACGAACCGGUAAAUCUGC-3 ' dTdT.
Among the present invention, CXSI32 can be to have the activity that suppresses tumor growth and be the sequence of 5 '-AACGAACCGGTAAATCTGC-3 '.
In the present invention, can select various carrier known in the art for use,, be connected to become recombinant vectors with CXSI32 as commercially available carrier.For example, can adopt slow virus, adenovirus or fores encephalitis virus expression system (SemlikiForest Virus).Slow virus (Lentivirus) belongs to the retrovirus subgenus, with human immunodeficiency virus (human immunod efficiency virus, HIV) being representative. slow virus not only has the division of infection target cell and integrates in its genome, especially has the multiple Unseparated Cell ability that comprises neuronal cell, scavenger cell, liver cell, myocardial cell and stem cell etc. that infects, thereby, lentiviral vectors is widely used in the especially research of gene therapy of gene function as the effective tool of transgenosis.
On the other hand, the present invention also provides the preparation method of above-mentioned nucleic acid molecule CXSI32, and the sequence of promptly pressing CXSI32 is with each ribonucleic acid molecule dehydrating condensation successively.
Nucleic acid molecule CXSI32 of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule CXSI32 sequence of the present invention can obtain with the method for enzymolysis process or synthetic usually.
The present invention also provides the above-mentioned application of nucleic acid molecule CXSI32 in the preparation antitumor drug.
Experiment shows, changes CXSI32 of the present invention over to the H1299 cell, and the result shows, the H1299 cell presents the tangible G1 phase and blocks.
CXSI32 of the present invention also can suppress tumor cell proliferation.CXSI32 or the cell that contains CXSI32 are applied to nude mice, compare with the nude mice of not using CXSI32 under the same culture conditions, the former obviously is suppressed in the growth of knurl body, and this phenomenon is As time goes on obvious day by day.
Nucleic acid molecule CXSI32 of the present invention and analogue thereof when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.
With nucleic acid molecule CXSI32 of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains compound and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Nucleic acid molecule CXSI32 of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, CXSI32 of the present invention also can use with the other treatment agent.
When nucleic acid molecule CXSI32 of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Among the present invention, the pharmaceutical composition that described antitumor drug is made up of the nucleic acid molecule CXSI32 that contains effective therapeutic dose and carrier pharmaceutically or vehicle.Described pharmaceutical composition can be injection or tablet.Its effective therapeutic dose can for every day 1 microgram/kilogram to 10 mg/kg body weight.Described medicine can be injection, pulvis or tablet.
Nucleic acid molecule CXSI32 of the present invention adds H1299, and cell presents the tangible G1 phase and blocks as a result; When being applied to nude mice, compare with the contrast nude mice under the same culture conditions, the growth of knurl body obviously is suppressed, and this phenomenon is As time goes on obvious day by day.The present invention is for tumor treatment and a kind of new approach and the means of providing are provided.
Embodiment
The influence of embodiment one CXSI32 cell cycle
Adopt the GeneChem Easy-siRNA synthetic CXSI32 of the Shanghai triumphant gene engineering of Ji company limited.
Experimental implementation step with Lipofectamine 2000 transfection siRNA:
(1) cell is inoculated in 24 orifice plates in transfection the day before yesterday, and it is 500ul that institute adds culture volume.Cell confluency should be 40%-50%. during transfection
(2) preparation work mother liquor: add 125ul 1 * UniversalBuffer among the double-stranded siRNA of 2.5nmol (1.0 OD), obtaining concentration is the siRNA mother liquor of 20uM.
(3) can not contain the substratum of serum with other with the Opti-MEM that does not contain serum (Invitrogen) in the following dilution step (4 and 5) yet.
(4), use 0.84ug siRNA two strands for each hole of 24 orifice plates.The siRNA of 3ul 20uM two strands is mixed with 50ul Opti-MEM.
(5) Lipofectamine 2000 test kits are softly shaken up, get 1ul Lipofectamine 2000 reagent and mix, hatched under the room temperature 5 minutes at another Guan Zhongyu 50ul Opti-MEM.
(6) the siRNA after the dilution with dilute after Lipofectamine 2000 mix mixing gently.Incubation is 20 minutes under the room temperature.
(7) mixed solution of siRNA and Lipofectamine 2000 is added culturing cell, mixing is put into incubator with cell then gently.
(8) before detecting the RNAi effect, generally need not to change liquid.If but transfection reagent has obvious cytotoxicity, can change liquid in the time of full 3 hours in transfection.
As a result, cell presents the tangible G1 phase and blocks.
Embodiment two suppresses the nude mice tumor growth
2.1 nude mice (Shanghai Slac Experimental Animal Co., Ltd.) becomes knurl: a large amount of cultivation detected the SK-Hep1 cell, trysinization, centrifugal results institute cultured cells with PBS washing back counting, is resuspended in PBS, and cell carried out subcutaneous injection with disposable syringe to nude mice, inject 〉=5, (female, 30-40 days) for every group, the injection cell amount of every nude mice is 200 μ l, and cell count is about 10
6, tumour grows after the week.
2.2 virus injection: when gross tumor volume grows to 10-30mm
3During volume, 20 μ l are contained 3 * 10
7The PBS of TU titre virus enters in the tumour with the syringe direct injection.Experimental group injection LV-CXSI32 virus, control group injection LV-non-silencing virus.Two days later, use the same method again and dosage injection virus once.
2.3 tumor monitoring and knurl piece cut: detect the state of mouse every day, tumor size of measurement in per three days.Each five of experimental group and control group mice are measured, and from infecting the 29th day, significant difference appears in experimental group tumour and control group gross tumor volume.After 32 days, put to death mouse, and carry out weighing from subcutaneous taking-up knurl piece.
The result shows, compares with control group, has injected the nude mice knurl bulk-growth that contains CXSI32 and has obviously slowed down.This explanation, CXSI32 of the present invention can obviously suppress tumor growth.
Claims (6)
1. a nucleic acid molecule is characterized in that, it has active and its sequence that suppresses tumor growth and comprises 5 '-AACGAACCGGUAAAUCUGC-3 ' or 5 '-AACGAACCGGTAAATCTGC-3 '.
2. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, it has active and its sequence that suppresses tumor growth and comprises 5 '-AACGAACCGGUAAAUCUGC-3 ' dTdT.
3. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, it has active and its sequence that suppresses tumor growth is 5 '-AACGAACCGGUAAAUCUGC-3 ' dTdT.
4. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, it has active and its sequence that suppresses tumor growth is 5 '-AACGAACCGGTAAATCTGC-3 '.
5. the preparation method as any nucleic acid molecule among the claim 1-4 is characterized in that, by the sequence of any nucleic acid molecule among the claim 1-4, with each Yeast Nucleic Acid group or thymus nucleic acid group dehydrating condensation successively.
6. as the application of any nucleic acid molecule among the claim 1-4 in the preparation antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100335781A CN101503449A (en) | 2008-02-05 | 2008-02-05 | Nucleic acid molecule CXSI32 and use thereof in anti-cancer medicine preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100335781A CN101503449A (en) | 2008-02-05 | 2008-02-05 | Nucleic acid molecule CXSI32 and use thereof in anti-cancer medicine preparation |
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| Publication Number | Publication Date |
|---|---|
| CN101503449A true CN101503449A (en) | 2009-08-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2008100335781A Pending CN101503449A (en) | 2008-02-05 | 2008-02-05 | Nucleic acid molecule CXSI32 and use thereof in anti-cancer medicine preparation |
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| CN (1) | CN101503449A (en) |
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Open date: 20090812 |