CN101007838A - Nucleic acid molecule RTN4BSR22 and its application in preparing anticancer medicine - Google Patents
Nucleic acid molecule RTN4BSR22 and its application in preparing anticancer medicine Download PDFInfo
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- CN101007838A CN101007838A CN 200710036918 CN200710036918A CN101007838A CN 101007838 A CN101007838 A CN 101007838A CN 200710036918 CN200710036918 CN 200710036918 CN 200710036918 A CN200710036918 A CN 200710036918A CN 101007838 A CN101007838 A CN 101007838A
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Abstract
The invention belongs to the field of biological pharmacology, and relates to the use of RTN4BSR22 in preparing antitumor medicament, wherein a nucleotide molecule is provided which can be used for preparing antitumor medicaments, wherein the sequence includes 5'-AAACACCACTCCAGTCTTC-3' or 5'-AAACACCACUCCAGUCUUC-3', and is named RTN4BSR22 in the present invention. The nucleotide molecule RTN4BSR22 of the invention can be injected into tumors of the nude mice, compared with nude mice injected with other nucleotide molecule under the identical culture conditions, the tumor growth is inhibited appreciably, which is becoming more appreciable with lapses of time. The invention provides a novel approach for the treatment and alleviation of tumor.
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, its sequence comprises 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 ', is called as RTN4BSR22 in the present invention.
Wherein, A, U, T, G and C are respectively adenine nucleotide, uridylate, thymidylic acid, guanylic acid and cytidylic acid(CMP).
In the present invention, term " nucleic acid molecule RTN4BSR22 " refers to have inhibition RTN4B protein-active and contains and 5 '-AAACACCACUCCAGUCUUC-3 ' sequence height homologous nucleotide sequence.This term also comprises the homology at least 70% with 5 '-AAACACCACUCCAGUCUUC-3 ', preferably at least 80%, and at least 90% nucleotide sequence more preferably.
This term also comprises the nucleotide sequence variation form of 5 '-AAACACCACUCCAGUCUUC-3 '.These variant forms comprise (but being not limited to): 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 '-AAACACCACUCCAGUCUUC-3 ' back.
Nucleic acid molecule among the present invention, its sequence can comprise 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUCdTdT-3 '.
Nucleic acid molecule among the present invention, its sequence can comprise 5 '-AAACACCACUCCAGUCUUC-3 '.
Nucleic acid molecule among the present invention, its sequence can comprise 5 '-AAACACCACUCCAGUCUUCdTdT-3 '.
Nucleic acid molecule among the present invention, its sequence can be 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 '.
Nucleic acid molecule among the present invention, its sequence can be 5 '-AAACACCACUCCAGUCUUCdTdT-3 '.
The present invention also provides a kind of carrier, and it contains the nucleic acid molecule that sequence comprises 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 '.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, selecting commercially available carrier for use, the 5 '-AAACACCACUCCAGUCUUC-3 ' that will contain RTN4BSR22 nucleotide sequence of the present invention then operationally is connected in expression regulation sequence, can form recombinant vectors., for example, can adopt slow virus, adenovirus or fores encephalitis virus expression system (Semliki Forest 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.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear rna sequence can influence the activity of same other parts of linear rna sequence.For example, if signal peptide RNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) RNA operationally is connected in polypeptide RNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means adjacent and do not influence function for nucleic acid molecule of the present invention.
A kind of host cell also is provided among the present invention, and it contains the nucleic acid molecule that sequence comprises 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 '.
In the present invention, term " host cell " is an eukaryotic cell, as Chinese hamster ovary celI, COS-7,293 cells etc.
On the other hand, the present invention also provides the preparation method of above-mentioned nucleic acid molecule RTN4BSR22, and the sequence of promptly pressing RTN4BSR22 is with each ribonucleic acid molecule dehydrating condensation successively.
Nucleic acid molecule RTN4BSR22 of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule RTN4BSR22 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 RTN4BSR22 in the preparation antitumor drug.
Verify all that with in vitro results RTN4BSR22 of the present invention can obviously cut down the expression of RTN4B in the body.
Experiment showed, that RTN4B (the NCBI number of landing AF148538) can promote tumor growth, therefore, the expression of cutting down RTN4B just means the inhibition tumor growth.
The injection cell that will contain RTN4BSR22 is gone in the tumour of nude mice, compares with the nude mice of not injecting RTN4BSR22 under the same culture conditions, and the growth of knurl body obviously is suppressed, and this phenomenon is As time goes on obvious day by day.
Nucleic acid molecule RTN4BSR22 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 RTN4BSR22 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 RTN4BSR22 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, BRSK2 of the present invention also can use with the other treatment agent.
When nucleic acid molecule RTN4BSR22 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 RTN4BSR22 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.
Among the present invention, described medicine can be injection, pulvis or tablet.
Nucleic acid molecule RTN4BSR22 of the present invention is injected in the tumour of nude mice, compares with the nude mice of not injecting RTN4BSR22 under the same culture conditions, and 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
Embodiment 1 RTN4B makes nude mice growth tumor experiment
1) the cell strain enlarged culturing of the cell strain SMMC-7721 of stable transfection RTN4B and stable transfection empty carrier
2) use trypsin digestion cell, centrifugal 1000rpm * 5min
3) PBS washes twice, counts with cell counting count board
4) with PBS diluting cells suspension to 2 * 10
6Cell/200 microlitres
5) (medicine institute of Shanghai Chinese Academy of Sciences animal center, BALB/c) subcutaneous vaccination is carried out in the oxter, a shot 200 μ l cell suspensions nude mice.The stable cell line of oxter, same nude mice left side injection stable transfection empty carrier, the cell strain of the right injection stable expression of exogenous RTN4B.
6) after about 2 weeks, the nude mice of inoculation grows glucagonoma, takes out the knurl body after cervical vertebra is put to death, and measures the knurl body line of apsides and weight.
The result shows that obviously faster than the right in left side, this showed that expression RTN4B can promote tumor growth to the tumor growth on right side.
Embodiment 2 knurl body freezing microtome sections are analyzed
1) glucagonoma that grows of nude mice is cut into small pieces and is immersed in the embedding liquid, cuts into slices after quick freezing in the liquid nitrogen, and all slide glasss are handled through the anti-flake of poly-lysine
2) section is at (20 ℃) acetone internal fixing 20S of ice, then at 37 ℃ of baking sheets (but 12h).Slice, thin piece can be placed 1～2 year at-20 ℃
3) slice, thin piece is washed in PBS 2 times, 5min/time
4) sealing 15～30min (confining liquid: 5%BSA, 5%Goat serum)
5) PBS washes 3 times, 5min/time
6) 37 ℃ of 1.5h or 4 ℃ spend the night (background is few) among anti-(CD31) 0.5%BSA, volume 20～30 μ l
7) PBS washes 3 times, 5min/time
8) 37 ℃ of 30min of two anti-0.5%BSA (the available 1%BSA of the words that background is high, 1%Goat serum)
9) PBS washes 3 times, 5min/time
10) drip the 80% glycerine mounting that heat is crossed
11) 20 times of fluorescent microscopes detect down and take pictures
12) numeration blood vessel number, the blood vessel in the glucagonoma of the statistics left and right sides is counted difference
The result shows that in the section of the hepatoma that inoculation b6 (cell strain of stably express RTN4B) generates, obviously increase is many for the blood vessel number in the hepatoma section that blood vessel number ratio inoculation a6 (cell strain of stable transfection empty carrier) generates.
Embodiment 3 endogenous screening RTN4B inhibitor
According to the sequence of RTN4B, implementation sequence is that the nucleic acid molecule of 5 '-AAACACCACUCCAGUCUUCdTdT-3 ' is as the RTN4B candidate inhibitor.Be called RTN4BSR22.
1. plant the 293T cell in 24 orifice plates, plant plate density 2*10
4/ hole.
Behind kind of plate 24h (hour), 72h, 120h repeat transfection RTN4BSR22 fragment, (Opti-MEM I Reduced Serum Medium, invitrogen company 31985-070) is dissolution with solvents, makes final concentration reach 40nM with Opti-MEM.Transfection reagent uses lipofectamin2000 (11668-027, invitrogene company).
3. 24h, 72h, 120h peptic cell after the transfection first time are collected 72h and 120h cell 5*10
4As sample.Western detects, antibody Nogo (N-18) SC-11027 (santa cruz company) RTN4B intrinsic protein changes of expression level.
Conclusion: relatively, 72h is that visible RTN4B protein expression level is obviously reduced after the RTN4BSR22 transfection with the negative fragment NS of siRNA (5 '-UUCUCCGAACGUCACGUdTdT-3 ').
The cell levels screening experiment of the consumption of 4 pairs of RTN4B genetic expressions of embodiment
Experimental procedure:
1. plant the 293T cell in 96 orifice plates, plant plate density 0.8*10
4/ hole
2.24h after will carry RTN4BSR22 lentivirus (slow virus, Ji Kai company) transduction go into cell.The Lentivirus titre is 10
6TU/ul, getting MOI is 100, promptly every hole adds 80ul virus liquid.Concrete transduction method is as follows:
Change 50ul fresh culture (PH7.0) for every porocyte, add an amount of viral liquid, the 50ul substratum is added in every hole behind the 7-8h.Change liquid behind the 24h.48-72h observes GFP (green fluorescence) fluorescence, detects transduction efficiency.
3. with the cell enlarged culturing, utilize the flow cytometry pair cell to carry out the sorting of GFP green fluorescence, filter out virus transduction positive cell, and detect its positive rate.
4.Western detect the RTN4B intrinsic protein changes of expression level of positive cell and negative cells.
Conclusion: A.48h observe GFP fluorescence, positive rate is about 50%, illustrates that virus transduction experimental technique is correct.
B. flow cytometry sorting transducer cell, positive group and negative group the transduction rate all greater than 90% (FCM results show data).
C.Western detects positive cell RTN4B protein expression level to be reduced greatly than negative cells.
Embodiment 5 RTN4B inhibitor suppress the experiment of nude mice tumor growth
One, experiment material
1. nude mice: Shanghai Slac Experimental Animal Co., Ltd.
2.Lentivirus vector (lentiviral vectors): the Shanghai triumphant gene engineering of Ji company limited
Two, experimental procedure
1. nude mice is put into SPF level Animal House (no-special pathogen level experimental animal room), make it adapt to an about week of culture environment.
With DMEM (Dulbecco ' s Medified Eagle Medium, invitrogene company, 12800-82)+10%FBS cultivates SMMC-7721 cell (Chinese Academy of Sciences's cell bank).
The SMMC-7721 cell is centrifugal after with trysinization 3., remove supernatant, resuspended with the DMEM of serum-free, remove supernatant then, add an amount of PBS, make every milliliter about 4 * 10
7The suspension of individual cell.
4. the oxter injection 0.2ml to the nude mice in age in 4-6 week contains 8 * 10 approximately
6The PBS suspension of the cell of individual unit.
5. when treating that knurl body diameter reaches 3-5mm (millimeter), be classified as experimental group and control group respectively, and all cut the ear numbering for this every mouse of 2 groups, the major diameter minor axis and the body weight of every mouse tumor body of record this moment by identical 2 of the big young pathbreaker of knurl volume.
6. virus and the viral empty carrier of difference direct injection 0.1ml (milliliter) in the knurl body of 2 groups of mouse are contrast with the injecting virus empty carrier.Duplicate injection in per 4 days is once injected three times altogether.
7. since the day of the injecting virus first time, measured and write down the major diameter minor axis and the body weight of the knurl body of every mouse, the volume v=ab of knurl body in per 3 days
2/ 2 (a major diameter length, b minor axis length).Get knurl after about 4 week.
The result shows that compare with control group, the nude mice knurl bulk-growth of injecting virus (containing RTN4BSR22) obviously slows down.
Claims (8)
1. a nucleic acid molecule is characterized in that, its sequence comprises 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 '.
2. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence comprises 5 '-AAACACCACUCCAGUCUUC dTdT-3 '.
3. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence is 5 '-AAACACCACTCCAGTCTTC-3 ' or 5 '-AAACACCACUCCAGUCUUC-3 '.
4. a nucleic acid molecule as claimed in claim 1 is characterized in that, its sequence is 5 '-AAACACCACUCCAGUCUUC dTdT-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 dehydrating condensation successively.
6. as the application of any nucleic acid molecule among the claim 1-4 in the preparation antitumor drug.
7. application as claimed in claim 6 is characterized in that the pharmaceutical composition that described antitumor drug is made up of any nucleic acid molecule among the claim 1-4 that contains effective therapeutic dose and carrier pharmaceutically or vehicle.
8. application as claimed in claim 6 is characterized in that, described medicine is injection, pulvis or tablet.
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| CN 200710036918 CN101007838A (en) | 2007-01-29 | 2007-01-29 | Nucleic acid molecule RTN4BSR22 and its application in preparing anticancer medicine |
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|---|---|---|---|
| CN 200710036918 CN101007838A (en) | 2007-01-29 | 2007-01-29 | Nucleic acid molecule RTN4BSR22 and its application in preparing anticancer medicine |
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