CN101225098A - Nucleic acid molecule CRYL1SI2 and application for preparation of anti-cancer drugs - Google Patents

Abstract

The invention relates to a nucleotide molecule and the application in preparing antitumor drug, belonging to biological medicine field. The nucleotide molecule and the application in preparing antitumor drug has the advantages that: the nucleotide molecule used for preparing antitumor drug has the activity of promoting tumor cell apoptosis; the sequence comprises 5'-CCAUGCAUCUCAAUGCAGA-3' or 5'-CCATGCATCTCAATGCAGA-3'; the nucleotide molecule is labeled as CRYL1SI2; the nucleotide molecule CRYL1SI2 and daunorubicin can be combined, thereby greatly lowering drug resistance of tumor cells.

Description

Nucleic acid molecule CRYL1SI2 and the application in the preparation cancer therapy drug thereof

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.

Drug resistance (drug resistance) is a difficult problem in the tumor therapeutic procedure.Tumour cell not only makes cancer therapy drug not reach the purpose of treatment to the tolerance of medicine, also can have side effects simultaneously, allows the easier damage that is subjected to medicine of normal cell, directly jeopardizes patient's life.Therefore, can to reduce drug-fast compound a kind of be one of the focus in tumor research field in searching.

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, active and its sequence that it has the promotion apoptosis of tumor cells comprises 5 '-CCAUGCAUCUCAAUGCAGA-3 ' or 5 '-CCATGCATCTCAATGCAGA-3 '.Be called as CRYL1SI2 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 CRYL1SI2 " also comprises the nucleotide sequence variation form of 5 '-CCAUGCAUCUCAAUGCAGA-3 ' or 5 '-CCATGCATCTCAATGCAGA-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 '-CCAUGCAUCUCAAUGCAGA-3 ' back.

Among the present invention, CRYL1SI2 has the sequence that promotes the active of apoptosis of tumor cells and comprise 5 '-CCAUGCAUCUCAAUGCAGA-3 ' dTdT.

Among the present invention, CRYL1SI2 can be to have the activity that promotes apoptosis of tumor cells and be the sequence of 5 '-CCAUGCAUCUCAAUGCAGA-3 ' dTdT.

Among the present invention, CRYL1SI2 can be to have the activity that promotes apoptosis of tumor cells and be the sequence of 5 '-CCATGCATCTCAATGCAGA-3 '.

In the present invention, can select various carrier known in the art for use,, be connected to become recombinant vectors with CRYL1SI2 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 CRYL1SI2, and the sequence of promptly pressing CRYL1SI2 is with each ribonucleic acid molecule dehydrating condensation successively.

Nucleic acid molecule CRYL1SI2 of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule CRYL1SI2 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 CRYL1SI2 in the preparation antitumor drug.

Above-mentioned nucleic acid molecule CRYL1SI2 can with the daunorubicin coupling, be applied to prepare antitumor drug.

CRYL1SI2 of the present invention has significantly promoted the generation of Hep3B cell apoptosis under the daunorubicin action condition, with CRYL1SI2 and daunorubicin coupling, can reduce the resistance of tumour cell to daunorubicin greatly.

Nucleic acid molecule CRYL1SI2 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 CRYL1SI2 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 CRYL1SI2 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, CRYL1SI2 of the present invention also can use with the other treatment agent.

When nucleic acid molecule CRYL1SI2 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 CRYL1SI2 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.

Daunorubicin (daunorubicin) extensively and effectively has been used for leukemia since being developed always clinically, mammary cancer, and malignant lymphoma is in the middle of the treatment of tumor diseases such as soft-tissue tumor.But resistance is than higher.When the present invention gave the tumour cell dosing, the daunorubicin final concentration was 0.4uM.

CRYL1SI2 of the present invention has significantly promoted the generation of Hep3B cell apoptosis under the daunorubicin action condition, with CRYL1SI2 and daunorubicin coupling, can reduce the resistance of tumour cell to daunorubicin greatly, therefore, the present invention provides a kind of new approaches and methods for tumor treatment.

Embodiment

The influence of CRYL1SI2 cell cycle

Adopt the GeneChem Easy-siRNA synthetic CRYL1SI2 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 (1.0OD), 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 3ul20uM two strands is mixed with 50ulOpti-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) transfection after 24 hours cell or do not carry out cells transfected and use trypsin digestion cell after the specified time with the daunorubicin irritation cell, harvested cell is in the flow cytometer sample hose.

(9) PBS cleans 1-2 time, and room temperature is centrifugal, and (1000rpm 5min), abandons supernatant.

(10) add PI (final concentration is 50ug/ml), behind the room temperature dark place reaction 3min, flow cytometer detects.

The result shows that CRYL1SI2 has significantly promoted the Hep3B cell under the daunorubicin action condition apoptosis to take place.

Claims (7)

1. a nucleic acid molecule is characterized in that, active and its sequence that it has the promotion apoptosis of tumor cells comprises 5 '-CCAUGCAUCUCAAUGCAGA-3 ' or 5 '-CCATGCATCTCAATGCAGA-3 '.

2. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, active and its sequence that it has the promotion apoptosis of tumor cells comprises 5 '-CCAUGCAUCUCAAUGCAGA-3 ' dTdT.

3. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, active and its sequence that it has the promotion apoptosis of tumor cells is 5 '-CCAUGCAUCUCAAUGCAGA-3 ' dTdT.

4. a kind of nucleic acid molecule as claimed in claim 1 is characterized in that, active and its sequence that it has the promotion apoptosis of tumor cells is 5 '-CCATGCATCTCAATGCAGA-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.

7. as the application of any nucleic acid molecule among the claim 1-4, it is characterized in that,, be applied to prepare antitumor drug this nucleic acid molecule and daunorubicin coupling.