CN102703447B

CN102703447B - Oligonucleotide with breast cancer treatment effect

Info

Publication number: CN102703447B
Application number: CN 201210206725
Authority: CN
Inventors: 王丽颖; 杨亮; 于永利
Original assignee: Changchun Huapu Biotechnology Co Ltd
Current assignee: Huapu Biotechnology Hebei Co ltd
Priority date: 2007-06-26
Filing date: 2007-06-26
Publication date: 2013-10-23
Anticipated expiration: 2027-06-26
Also published as: CN102703447A

Abstract

The invention provides oligonucleotide. The oligonucleotide has the function of stimulating the proliferation and activation of immune cells in vitro. Animal experiments show that the oligonucleotide can be used for preventing and curing the breast cancer. The oligonucleotide can be used for preparing a preparation for preventing and curing the breast cancer of people.

Description

A kind of oligonucleotide with the effect for the treatment of mammary cancer

Technical field:

The present invention relates to a kind of oligonucleotide with and be used for the treatment of the purposes of mammary cancer.

Background technology:

Mammary cancer (mammary carcinoma) is the malignant tumour that occurs in the breast epithelium tissue, is one of modal malignant tumour of global women, and sickness rate is soaring year by year, and is rejuvenation situation, is the first place of women's malignant tumour cause of the death.Mammary cancer is only second to cervical cancer at the sickness rate of China, and crowd's morbidity is 23,/10 ten thousand; Account for 7～10% of the various malignant tumours of whole body.

Methods for the treatment of and the measure of mammary cancer are more, comprise operation, radiotherapy, chemotherapy, endocrine therapy and biotherapy etc.Operative treatment is the Main Means of breast cancer treatment at present, and is better to the mammary cancer curative effect that there is no in early days axillary lymphatic metastasis; After radiotherapy was generally used for operation, to prevent local recurrence, for the local recurrence focus of isolatism, and the bone metastasis of mammary cancer all had certain Palliative curative effect; Chemotherapy is a kind of general assisting therapy of necessity, and common drug has endoxan, lomustine, Chlorambucil etc.; Endocrine therapy mammary cancer is non-healing, but can receive in various degree the curative effect of appeasing for the hormonal dependent breast cancer; Biotherapy is divided into immunotherapy, gene therapy, stem-cell therapy and vasculogenesis suppression therapy, a class treatment means (Wei YQ who rises along with molecular biology, immunology, cytobiology, Wu Y, Advances in Biotherapy for Breast Cancer, China J Bases Clin General Surg, 2004,11 (1): 3-5).Although mammary cancer has multiple therapeutic modality, application all is difficult to obtain satisfied curative effect separately.Therefore, it is main that the at present treatment of mammary cancer is mostly adopted to perform the operation, take various therapeutic modalities as auxiliary complex therapy.

Summary of the invention:

1, the invention provides a kind of oligonucleotide.This oligonucleotide is in external function with immune stimulatory cell proliferation, activation.Experimentation on animals shows that this kind oligonucleotide can be used for prevention and treatment mammary cancer.

2, oligonucleotide provided by the invention is when being used for the purposes of prevention and treatment mammary cancer, this oligonucleotide can be used with the carrying of Drug carrier, its administering mode can be through application such as enteron aisle, non-enteron aisles (injection in such as tumour direct injection, the other lymphoglandula injection of knurl, lymphoglandula, subcutaneous injection, intramuscular injection and intravenous injection etc.).

3, oligonucleotide provided by the invention can form various pharmaceutical compositions or formulation as main component, and this class oligonucleotide that comprises dose therapeutically effective alone or in combination other therapeutic modality is used for the treatment of the purposes of mammary cancer and other tumour.

4, oligonucleotide provided by the invention can pass through various chemically modifieds.

Term in the invention:

Unless lay special stress on, the term among the present invention have can be by the ordinary meaning that the technician understood in the field under the present invention.If the conflict on the implication occurs, should defer to explanation among the present invention, define or illustrate.

" oligonucleotide ": oligonucleotide is by sugar (such as ribodesose or ribose), phosphate group is connected the molecule that mononucleotide connects and form with based composition, wherein glycan molecule and base connect into nucleosides (nucleoside), nucleosides is connected to form Nucleotide (nucleotide) by phosphate group, the base that forms nucleosides has pyrimidine and purine, pyrimidine has thymus pyrimidine (thymine, be abbreviated as T or t) and cytosine(Cyt) (cytosine, be abbreviated as C or c), purine has VITAMIN B4 (adenine, be abbreviated as A or a) and guanine (guanine is abbreviated as G or g).Oligonucleotide can be strand also can be double-stranded.In the present invention, " oligonucleotide " (Oligodeoxynucleotide, ODN) can replace with its english abbreviation ODN.

" chemically modified ": compare with the DNA of nature, the oligonucleotide among the present invention can be through various chemically modifieds, and the position of modification can occur in the phosphodiester bond between the nucleosides, ribose units is or/and organic base (A, T, C, G, uridylic, uridine is abbreviated as U or u).Can modify between the synthesis phase of oligonucleotide or after synthetic.Chemically modified between synthesis phase can be modified at inside or the 5` end of oligonucleotide.Oligonucleotide after synthetic can but be not limited to carry out chemically modified at active group (such as phosphoric acid or the hydroxyl of 5` or 3` end).The professional can understand the concrete mode of these chemically modifieds.Chemically modified among the present invention comprises the backbone modification of oligonucleotide.Wherein, the non-bridge phosphoric acid Sauerstoffatom at least one internucleotide linkage is replaced by sulphur atom.Nonionic DNA analogue also can occur in the skeleton of oligonucleotide, modify such as alkyl, fragrance-phosphonate (charged phosphonate Sauerstoffatom is replaced by alkyl, aromatic base), the part of the Sauerstoffatom in phosphodiester and the alkyl phosphotriester is modified by alkylation for another example.Oligonucleotide can also be the mosaic of phosphorothioate and phosphodiester.Chemically modified comprises that also base substitutes, and substitutes such as C-5 propine pyrimidine and the alternative purine of 7-deaza-7.Chemically modified also comprises base modification.The base of base in chemically being different from typical nature of modifying, but have their basic chemical structures.Oligonucleotide among the present invention can also be with cytosine derivative or thymidine Derivatives Modified.Here cytosine derivative refers to cytosine(Cyt) sample nucleosides (except cytosine(Cyt)).The thymidine derivative refers to the phonetic sample nucleosides of thymus gland (not comprising thymus pyrimidine).In addition, the modification of the oligonucleotide among the present invention can be two or dibasic alcohol of a terminal connection at oligonucleotide, such as TEG or six ethylene glycol.

" tumour ": " tumour " among the present invention is the tumour of modern medicine definition, can be divided into the innocent tumour of non-carcinogenic and the malignant tumour of carinogenicity.

" individuality ": the individuality among the present invention refers to people or inhuman vertebrates.

" immune response ": immunne response has similar meaning with immune response.The immune response reaction that to be immunocyte such as B cell, T cell, NK cell, gamma delta T cells, NKT cell, dendritic cell, scavenger cell and granulocyte etc. make antigen or other stimulation.Immunne response comprises innate immunity and acquired (specificity) immunne response.Acquired immune response comprises cellullar immunologic response and humoral immunoresponse(HI).

" treatment tumour ": the treatment here refers to therapeutic measures that tumorigenic individuality is taked, be used for controlling the progress of disease, prolong the lifetime of Cancer individuality, improve the life quality of Cancer individuality, the symptom of the disease that alleviates makes cancerous swelling dwindle even eliminate.

" pharmacology acceptable carrier ": the pharmacology acceptable carrier refers to weighting agent, thinner or the encapsulating substance of one or more solids or liquid, and this carrier is fit to the oligonucleotide among the present invention is applied to individuality.This carrier can be organic, inorganic, natural or synthetic.This carrier comprise various solution, thinner, solvent, dispersion agent, liposome, emulsion, sugar-coat, antiseptic-germicide, anti-mycotic agent, etc. the carrier used of the oligonucleotide among that ooze and reagent delayed absorption and other suitable the present invention.The carrier that injectable is used comprises water, physiological saline, balanced salt solution, damping fluid, glucose solution, glycerine etc.For solid mixture (such as powder, ball, tablet, capsule form), non-toxic solid carriers commonly used comprises: N.F,USP MANNITOL, lactose, starch and Magnesium Stearate etc. with pharmacology purity.In addition, the carrier as biologically neutral can contain atoxic auxiliary substance in the pharmacology component of application, comprises humidifying or emulsifying agent, sanitas, PH buffer reagent, sodium-acetate and mono-laurate etc.

" treatment effective dose ": in order to treat tumour, giving individual applications dosage is the treatment effective dose.This treatment effective dose refers to give can produce the oligonucleotide of desirable prevention or result for the treatment of or the dosage of its functional analogue behind the individuality in the process for the treatment of tumour.This " dosage " how much be decided by the standard technique that those skilled in the art should be known, also to reference to other factors, comprise being not limited to individual size and the severity of health condition and disease.Oligonucleotide among the present invention can be as single therapy or repeatedly treatment.Oligonucleotide among the present invention or its functional analogue are applied to individual dosage range from 1 μ g to 1000mg at every turn.In order to reach desirable result for the treatment of, those skilled in the art can adjust the dosage of using, such as the doctor in charge on the basis of reliable medical judgment, the dose titration of making, its dosage range can be 10 times to 1000 times of aforementioned range.

" route of administration ": the oligonucleotide among the present invention is when using separately or being made into pharmaceutical composition and using by prescription, and its route of administration can be in the intestines, intestines are outer, external application or inhalation route.Wanted approach to comprise per os, stomach, colon or rectal administration in the intestines; Intestines external administration approach comprises in vein, peritonaeum, sheath, muscle, subcutaneous, intracutaneous, part, the other lymphoglandula of knurl, tumor tissues direct injection, transvaginal, external application, nasal mucosa and lung suction etc.The topical administration approach comprises through skin, oral cavity, eyes, ear and nose.

" pharmaceutical composition ": the mixture of acceptable carrier composition on the oligonucleotide that pharmaceutical composition refers among the present invention the treatment effective dose and the pharmacology.Pharmaceutical composition divides the oligonucleotide that can comprise among one or more the present invention.Injection medicine composition of the present invention comprises the pharmaceutically acceptable aqueous solution, non-aqueous solution, dispersion agent, clouding agent, emulsion or pulvis, dissolves with aseptic water for injection or dispersion agent before the injection.In some cases, in order to prolong oligonucleotide action effect among the present invention, pharmaceutical composition is used after can being processed over suitable sustained release system.Oligonucleotide in the invention or its functional analogue or pharmaceutical composition can be processed into the suspension of crystallization or the less amorphous substance of moisture, stably delay to discharge.Oligonucleotide or its functional analogue delays that discharge can be by realizing with hydrophobic substance (such as, acceptable oiliness carrier) dissolving among the present invention of injection.The pharmaceutical composition that is used for injection form can be oligonucleotide or the breast grain of liposome, or biodegradable semipermeable polymers, such as polylactide, polyorthoesters or polyanhydride.

" anti-tumor agent ": anti-tumor agent refers to be used for the treatment of at individuality the preparation of tumour, comprises chemotherapeutics, nonspecific immunity strengthening agent, EGFR (EGFR)-Tyrosylprotein kinase (TK) activation inhibitor, tumour cell mutain tyrosine kinase inhibitor, tumor vessel inhibitor, anti-tumour antibody and tumor vaccine, RNA interferences etc.

" chemotherapeutics "; Chemotherapeutics refers to the chemicals for oncotherapy.Chemotherapeutics includes but not limited to mustargen, endoxan, nitrocaphanum, mustine hydrochlcride, carmustine, ring second Nitrosourea, thiophene is for group, Myelosan, cis-platinum, nitrosourea, 5-fluor-uracil, cytosine arabinoside, Rheumatrex, purinethol, Ismipur, hydroxyurea, Zorubicin, pidorubicin, daunorubicin, mitoxantrone, mitomycin, widely collect mycin, zilimeisu, dactinomycin, vincristine(VCR), vincaleucoblastine, vindesine, tricuspid sugi gum alkali, camptothecine, Zuyeyidal, maytenin, Elemenum Emulsion, tamoxifen, adrenocortical hormone, procarbazine, hydroxymethyl benzyl hydrazine, L-asparaginase, platinum class (cis-platinum, carboplatin, RP-54780), dacarbazine, hexamethylmelamine, taxotere or pemetrexed (Alimta), nvelbine, xeloda, irinotecan, topotecan, RP-54780, Docetaxel (Docetaxel).The chemotherapeutics of combined utilization such as PTX (taxol)+cis-platinum, PTX+carboplatin, gemzar (gemcitabine hydrochloride)+cis-platinum, taxotere (Docetaxel)+cis-platinum, PTX D or gemzar+cis-platinum, cis-platinum (Cisplatin)+Vinorelbine (vinorelbine), gemcitabine (gemcitabine)+carboplatin (Carboplatin), taxol (paclitaxel)+carboplatin.

" nonspecific immunity strengthening agent " refers to the preparation of the individual anti tumor immune response of the non-specific enhancing of energy, and these preparations comprise list but are not limited to bacille Calmette-Guerin vaccine (BCG), alpha-interferon (IFN-α) and interleukin (IL-2).

" EGFR (EGFR)-Tyrosylprotein kinase (TK) activation inhibitor ": this type of preparation includes but not limited to Gefitinib or Iressa (Gefitinib, Iressa) and erlotinib (Erlotinib, Tarceva) (Bezjak, A., et al.J Clin Oncol 24:3831-38372006).

" tumour cell mutain tyrosine kinase inhibitor " refers to the preparation of inhibition tumor cell mutain tyrosine kinase activity.This type of preparation includes but not limited to imatinib mesylate (Gleevec or STI-571 or Imatinib mesylate).Imatinib mesylate is mutein (Bcr-Abl) Tyrosylprotein kinase phosphorylation inhibitor (Rubin BP, Duensing A.Mechanisms of resistance to small molecule kinase inhibition in the treatment of solid tumors.Lab Invest.2006Aug 21).

" tumor vessel inhibitor " refers to form the preparation of bringing into play antitumor action by suppressing tumor vessel.This class preparation includes but not limited to Avastin (Avastin or bevacizumab), rhEndostatin (ENDOSTAR) and pegaptinib.Avastin is in conjunction with the Humanized monoclonal antibodies of vascular endothelial growth factor (VEGF) (Gridelli C, et al.New antiangiogenetic agents and non-small cell lung cancer.Crit Rev Oncol Hematol.2006Jul 12).RhEndostatin is recombinant human vascular endothelial inhibin.Pegaptinib is a kind of medicine of the angiogenesis inhibitor take small molecules nucleic acid with antibody sample recognition function and avidity as the target vascular therapy endothelial cell growth factor (ECGF) that mainly will study minute, eye part disease (Gatto B, the Cavalli M.From proteins to nucleic acid-based drugs:the role of biotech in anti-VEGF therapy.Anticancer Agents Med Chem.2006Jul for the treatment of take blood vessel hyperplasia as feature; 6 (4): 287-301).

" anti-tumour antibody " refers to the medicative antibody class medicine of growth of tumour cell, such as Herceptin etc.

" tumor vaccine " refers to induce the individual preparation that produces the antineoplastic specificity immunne response.Tumour antigen in the tumor vaccine is the core substance that induces tumour-specific immune response, and these tumour antigens include but not limited to MUC1 and HER-2.

The small RNA molecular of (sequence-specific gene silencing) effect that the RNA interferences refers to have the sequence-specific gene silencing; it can be the small RNA molecular of synthetic, also can be to produce RNA interferences plasmid or virus vector such as adenovirus.Multiple RNA interferences shows antineoplastic action (Takeshita F, Ochiya T.Therapeutic potential of RNA interference against cancer.Cancer Sci.2006Aug in experimentation on animals; 97 (8): 689-96).

" delivery vector ": the oligonucleotide among the present invention or its functional analogue can be used the delivery vector delivery applications.Delivery vector includes but not limited to: steroid (such as cholesterol), complex compound, emulsification, immunostimulating complex (ISCOMs), lipid (such as cation lipid and negatively charged ion lipid), liposome, the bacteria carrier of living is (such as Salmonellas, intestinal bacteria, mycobacterium will is congratulated (family name) bacillus, lactobacillus) and virus vector (such as Smallpox Vaccine, adenovirus, hsv), virosomes, virus-like particle, microballoon, nucleic acid vaccine, macromolecular material is (such as carboxymethyl cellulose, chitosan), the ring-type polymer.Delivery vector also is the ligand molecular of targeting specific acceptor.

" biotherapy ": " biotherapy " of mammary cancer comprises immunotherapy (such as antibody class medicine and tumor vaccine), vasculogenesis suppression therapy, gene therapy and stem-cell therapy etc.

" endocrine therapy ": the endocrine therapy of mammary cancer is to use hormone to come assisting therapy hormonal dependent mammary cancer.

Description of drawings:

Fig. 1: oligonucleotide (ODN) is to the hormesis of human peripheral blood single nucleus cell hyperplasia

Abscissa is the dosage of synthetic ODN, and ordinate is 1 minute radioactivity reading (cpm) of sample.The result shows that YW101 is stimulation human peripheral blood mononuclearcell propagation effectively, and its hormesis strengthens along with the increase of the dosage of YW101.

Fig. 2: oligonucleotide (ODN) is to the hormesis of mouse boosting cell hyperplasia

The ODN that the X-coordinate representative is different, ordinate zou is 1 minute radioactivity reading (cpm) of sample.The result shows, YW101, YW102 can effectively stimulate the splenocyte propagation of mouse.

Fig. 3: oligonucleotide (ODN) is to human peripheral blood B cell, NK cell and monocytic activation

The scatter diagram transverse axis is forward scatter, and the longitudinal axis is respectively CD19, the CD56 of PE mark and the monoclonal antibody of CD14, and CD19, CD56 and CD14 are respectively the B cells, NK cell and monocytic characteristic surface sign; Histogrammic transverse axis is the average intensity of FITC, i.e. the Activation of corresponding CD69, and the longitudinal axis is the number of cells of respective strengths.

The result shows, YW101, YW102 can effectively raise B cell among the human PBMC (Fig. 3 A), the expression of CD69 in NK cell (Fig. 3 B) and the monocyte (Fig. 3 C).

Fig. 4: oligonucleotide (ODN) is to the therapeutic action (gross tumor volume) of mouse breast cancer

Abscissa is the fate after the injection tumour, and ordinate is the volume of tumour.

The result shows, PBS compares with injection, inguinal region injection YW101, YW102 can both effectively suppress the growth (P＜0.05) of tumour, wherein suppresses the effect the most obvious (P＜0.01) of tumor growth behind right side (being the homonymy of tumour) the lymphoglandula injection YW101.

Fig. 5: oligonucleotide (ODN) is to the therapeutic action (survival rate) of mouse breast cancer

Abscissa is the fate after the injection tumour among the figure, and ordinate is the survival rate of mouse.

The result shows, compare with the PBS group, can effectively suppress the growth (p＜0.05) of tumour at the inguinal region injection YW101 of tumour homonymy and offside, also can establishment tumor growth (p＜0.05) at the inguinal region of tumour homonymy injection YW102, wherein in tumour with the tumor killing effect of side injection YW101 the most obvious (p=0.001).

Fig. 6: oligonucleotide (ODN) is to the prophylactic effect (tumor weight) of mouse breast cancer

Abscissa represents group: PBS: right side inguinal region injection PBS; 25 μ g: the right side inguinal region is injected 25 μ g YW101,50 μ g: the right side inguinal region is injected 50 μ gYW101; Ordinate zou represents tumor weight.

The result shows that PBS compares with injection, and injecting 50 μ g YW101 can (P＜0.05) occur the establishment tumour, and injecting 25 μ g before the kind knurl also can establishment tumor growth (P=0.035).

Embodiment

Embodiment 1 oligonucleotide (ODN) is to the hormesis of human peripheral blood single nucleus cell hyperplasia

1. the separation of people's peripheral blood mononuclear cells

(1), plant and instrument, equipment, reagent and material

Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, the dropper of all size etc.

ODN: synthetic by Takara company, the OPC rank, its phosphoric acid skeleton is full thio-modification except special indicating.With PBS (prescription is with reference to " molecular cloning experiment guide (third edition) ") dissolving, ultraviolet spectrophotometer is quantitative.

The human peripheral leucocytes of anticoagulant heparin: Central Blood Ban, Changchun City.

Poly-glucose-urografic acid methylglucamine salt: proportion 1.077 ± 0.001, Beijing ancient cooking vessel state biotech company.

RPMI1640 nutrient solution: the RPMI1640 of L-glutaminate (GIBCOBRL) 10.4 gram, sodium bicarbonate 2.0 grams, gentamicin 100,000 units add ultrapure water to 1000 milliliter, 0.22 micron filter membrane suction filtration degerming, packing.

The RPMI1640 substratum that contains 10% foetal calf serum: 10 milliliters of foetal calf serums (Invitrogen), 90 milliliters of RPMI1640 substratum

(2) method:

Separate the mononuclearcell of human peripheral with poly-glucose-urografic acid methylglucamine salt lymphocyte separation medium.Parting liquid and anticoagulant heparin White Blood Cells Concentrate volume ratio are 2: 1, horizontal centrifugal (1,000 * g, 30 minutes), with the liquid band of dropper absorption mononuclearcell, insert in another centrifuge tube, add isopyknic serum free medium, centrifugal 10 minutes of 1,000g abandons supernatant.Repeated washing twice is with 5 milliliters substratum re-suspended cell, cell counting.

2.3H-Tdr mix experiment

(1) instrument, equipment, equipment, reagent and material

Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, β calculating instrument, sample injector, glass fiber filter paper etc.

3H-thymidine: Shanghai Atomic Nucleus Inst., Chinese Academy of Sciences.

Scintillation solution: take by weighing 2,5 diphenyl-oxazoles (PPO), 4 grams, 100 milligrams of two [5-Ben Ji oxazolyl-2] benzene of Isosorbide-5-Nitrae are dissolved in 1000 milliliters of dimethylbenzene.

RPMI1640 nutrient solution: the same.

The ODN that adopts is YW101,2216,2006 and C274.2216 refer to CpG2216 (5 ' gggggacgatcgtcgggggg3 ') (Dominique De Wit, et al.Blood plasmacytoid dendritic cell responses to CpG oligonucleotides are impaired in human newborns.Blood, 1Feb, 2004, Vol 103, Num 3:1030-103).2006 refer to CpG2006 (5 '-tcgtcgttttgtcgttttgtcgtt-3 ') (Dominique De Wit, et al.Blood plasmacytoid dendritic cell responses to CpG oligodeoxynucleotides are impaired in human newborns.Blood, 1Feb, 2004, Vol 103, Num 3:1030-103).C274 refers to CpG C274 (5 '-tcgtcgaacgttcgagatgat3 ') (Omar Duramad, et al.Inhibitors of TLR-9Act on Multiple Cell Subsets in Mouse and Man In Vitro and Prevent Death In Vivo from Systemic Inflammation.The Journal of Immunology, 2005,174:5193-5200).

The sequence of YW101 is: 5 '-tcgcgaacgttcgccgcgttcgaacgcgg-3 '.

2006 sequence is: 5 '-tcgtcgttttgtcgttttgtcgtt-3 '.

2216 sequence is: 5 '-GGgggacgatcgtcGGGGGg-3 ' (wherein the G of capitalization is full thio-modification).

The sequence of C274 is: 5 '-tcgtcgaacgttcgagatgat-3 '.

(2) method:

Adopt culture plate at the bottom of 96 hole circles, with the RPMI 1640 cultivator peripheral blood mononuclear cell that contain 10% foetal calf serum, 6 * 10 ⁵Individual cells/well.Set up experimental group and control group, experimental group adds ODN YW101, and control group adds respectively ODN2006,2216, C274.The final concentration of ODN is 3 μ g/ml in every group of culture hole.

After the ODN effect 48 hours, add 3H-Tdr, 0.5 μ Ci/ hole remake with 16 hours, and collecting cell was dried for 50 ℃ on glassine paper in 3 hours.Corresponding glass paper is joined in the scintillation solution, act on 12 hours, detect 1 minute radioactivity reading (cpm) with the β calculating instrument.

3. experimental result:

YW101 is stimulation human peripheral blood mononuclearcell propagation effectively, has dose-dependent linear relationship (Fig. 1).

Conclusion: YW101 is the activation and proliferation of stimulation human peripheral blood mononuclearcell effectively.

Embodiment 2 oligonucleotide (ODN) are to the hormesis of mouse boosting cell hyperplasia

1. the separation of mouse boosting cell

(1) instrument, equipment, equipment, reagent and material:

Cryogenic refrigerator, carbonic acid gas incubator, Bechtop, inverted microscope, liquid nitrogen container, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, 200 mesh filter screens, the dropper of all size etc.

Erythrocyte cracked liquid: take by weighing respectively KHCO ₃1g, EDTA.Na ₂37.2g, NH ₄Cl8.29g adds 800ml water, transfers pH value to 7.2～7.4, adds water to 1L, filtration sterilization again.

RPMI1640 nutrient solution: the same.

C57/BL, BALB/c and Kunming mouse: all available from Beijing dimension tonneau China company.

The ODN that adopts is YW101, YW102,1585,1826 and C274.1585,1826 refer to CpG 1585 (Heidi H.van Ojik, et al.CpG-A and B Oligodeoxynucleotides Enhance the Efficacy of Antibody Therapy by Activating Different Effector Cell Populations.Cancer Research, 1Sep, 2003, Vol 63,5595-5600).CpG 1826 refers to (Heidi H.van Ojik, et al.CpG-A and B Oligodeoxynucleotides Enhance the Efficacy of Antibody Therapy by Activating Different Effector Cell Populations.Cancer Research, 1 Sep, 2003, Vol 63,5595-5600).

The YW101 sequence is the same;

The sequence of YW102 is: 5 '-tcgcgacgttcgcccgacgttcggta-3 '.

1585 sequence is: 5 '-GGggtcaacgttgaGGGGGg-3 ' (wherein the G of capitalization is full thio-modification);

1826 sequence is: 5 '-tccatgacgttcctgacgtt-3 ';

C274: sequence is the same.

(2) method:

After mouse was put to death in dislocation, aseptic separating spleen was placed spleen on the obscure glass, grinds the pressure spleen, filtered through 200 mesh filter screens and obtained cell suspension.Horizontal centrifugal (1,000g, 5 minutes), abandon supernatant, behind the re-suspended cell, add the 1ml erythrocyte cracked liquid, room temperature is placed 1min, add the RPMI1640 to 5ml of 10% foetal calf serum, again horizontal centrifugal (1,000g, 5 minutes), after resuspended, 200 mesh filter screens filter to get mouse boosting cell suspension, cell counting.

2.3H-Tdr mix experiment

(1) instrument, equipment, equipment, reagent and material:

RPMI1640 nutrient solution: the same.

(2) method:

Adopt culture plate at the bottom of 96 hole circles, cultivate mouse boosting cells, 5 * 10 with the RPMI 1640 that contains 10% foetal calf serum ⁵Individual cells/well.Set up experimental group and control group, experimental group adds ODN YW101, YW102, and control group adds respectively 1585,1826, C274.The final concentration of ODN is 3 μ g/ml in every group of culture hole.

After the ODN effect 48 hours, add 3H-Tdr, 0.5 μ Ci/ hole, after 16 hours, collecting cell 50 ℃, was dried on glass fiber filter paper in 3 hours.The corresponding glass fiber filter paper is joined in the scintillation solution, after 12 hours, with 1 minute radioactivity reading (cpm) of β calculating instrument detection.

3. experimental result:

YW101, YW102 can effectively stimulate mouse boosting cell propagation (Fig. 2).

Conclusion: YW101, YW102 can effectively stimulate the activation and proliferation of mouse boosting cell.

Embodiment 3 oligonucleotide (ODN) are to human peripheral blood B cell, NK cell and monocytic activation

1, the separation of people's peripheral blood mononuclear cells: method is the same.

2, the activation experiment of ODN stimulation human peripheral blood mononuclearcell

Adopt 24 orifice plates, with the RPMI 1640 cultivator peripheral blood mononuclear cell that contain 10% foetal calf serum, 3 * 10 ⁶Individual cells/well.Set up experimental group and control group, experimental group adds ODN YW101, YW102, and control group adds respectively 2216,2006, C274.The final concentration of ODN is 3 μ g/ml in every group of culture hole, and its sequence is as implied above.

After the ODN effect 18 hours, use the CD19 of the PE mark of BD company (USA) production, the CD69 of CD56 and CD14 antibody and FITC mark advances dyeing to cell.Concrete grammar is: the antibody of cell and PE mark and the CD69 of FITC mark were hatched 30 minutes altogether 4 ℃ of lower lucifuges.Then collecting cell utilizes flow cytometer (BD, USA) to detect the intensity of FITC of the cell of corresponding PE mark, the i.e. intensity of CD69.CD19, CD56, CD14 are respectively B cells in the human peripheral, and NK cell and monocytic surface marker molecule, CD69 are mainly expressed at T as the significant antigen of lymphocyte early activation, the surface of B and NK cell.

3. experimental result:

YW101, YW102 can effectively raise B cell among the human PBMC (Fig. 3 A), CD69 in NK cell (Fig. 3 B) and the monocyte (Fig. 3 C).

Conclusion: YW101, YW102 can effectively activate B cell among the human PBMC, NK cell and monocyte.

Embodiment 4 oligonucleotide (ODN) are to the therapeutic action of mouse breast cancer

1. instrument, equipment, equipment, reagent and material:

The carbonic acid gas incubator, Bechtop, inverted microscope, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, vernier callipers, the dropper of all size etc.

RPMI1640 substratum: the same.

0.25% trypsin Trypsin).

2. experimental technique:

Cultivate EMT6 cell [ATCC with RPMI 1640 subculture in vitro separately that contain 10% foetal calf serum, the breast cancer cell line of BALB/c mouse (Miaden Korbelik, et al, The Role of Host Lymphoid Populations in the Response of Mouse EMT6 Tumor to Photodynamic Therapy, 15Dec, Vol 56,5647-5652)], when cell enters logarithmic phase, to contain 0.25% the trypsin digestion cell of 2mM EDTA, with serum-free RPMI 1640 centrifuge washing cells, regulating cell concn is 2.5 * 10 ⁶/ ml, this cell suspension of subcutaneous injection 200 μ l is to close root of the tail section about 0.7cm place, back of the body bottom, BALB/c mouse right side.Until tumour can touch (after about 3 * 3mm), injection ODN or PBS.

With 6～8 ages in week, 18～22g mouse is divided into 4 groups, 12 every group, per 48 hours injections 100 μ lODN or PBS once, the concentration of ODN is 250 μ g/ml, injects altogether 6 times.

The experiment grouping:

The YW101 group, the subcutaneous injection of right hind inguinal region;

The YW102 group, the subcutaneous injection of right hind inguinal region;

YW101 (to) group, the subcutaneous injection of left hind inguinal region;

The PBS group, the subcutaneous injection of right hind inguinal region.

3. experimental result

Behind injection ODN, length and the width of per two days in-vitro measurements tumours calculate the gross tumor volume (volume of tumour=1/2 * length * wide ²) (Fig. 4); The date of death (Fig. 5 .) of record mouse. with SPSS the result is carried out statistical analysis.

The result shows, compare with the PBS group, at the inguinal region injection YW101 of tumour homonymy and offside energy establishment tumor growth (p＜0.05), also can establishment tumor growth (p＜0.05) at the inguinal region of tumour homonymy injection YW102, wherein in tumour with the tumor killing effect of side injection YW101 the most obvious (p＜0.01) (Fig. 4).

Compare with the PBS group, can effectively prolong the lifetime (p＜0.05) of tumor-bearing mice at the inguinal region injection YW101 of tumour homonymy and offside, also can effectively prolong lifetime (p=0.027) of tumor-bearing mice at the inguinal region of tumour homonymy injection YW102, wherein in tumour with the effect the most obvious (p=0.001) of side injection YW101 (Fig. 5).

101,102 pairs of mouse breast cancers of conclusion: YW have therapeutic action.

Embodiment 5 oligonucleotide (ODN) are to the prophylactic effect of mouse breast cancer

1. instrument, equipment, equipment, reagent and material:

The carbonic acid gas incubator, Bechtop, inverted microscope, Tissue Culture Flask, Hematocyte Counter, horizontal centrifuge, sample injector, electronic balance, the dropper of all size etc.

RPMI1640 substratum: the same.

0.25% trypsin Trypsin).

2. experimental technique:

The method of setting up the mouse breast cancer model is the same.

With 6～8 ages in week, 18～22g BALB/c mouse mouse is divided into 3 groups, 10 every group, take the injection mouse tumor cell as the 0th day, respectively-4, respectively injects 100 μ l ODN or PBS in-2 and 0 days once.

The experiment grouping:

YW101 25 μ g group, regulating concentration is 250 μ g/ml, the subcutaneous injection of right hind inguinal region;

YW101 50 μ g group, regulating concentration is 500 μ g/ml, the subcutaneous injection of right hind inguinal region;

The PBS group, the subcutaneous injection of right hind inguinal region.

3. experimental result

Observe and record out ratio of outflow (table 1); Put to death tumor-bearing mice in 40 days after the kind knurl, take out lump, weigh, record tumor weight (Fig. 6).With SPSS the result is carried out statistical analysis.

Table 1 mouse goes out ratio of outflow

101,102 pairs of mouse breast cancers of conclusion: YW have prophylactic effect.

Claims

1. oligonucleotide, its sequence is the sequence of SEQ ID NO:1.

2. the application of oligonucleotide claimed in claim 1 in preparing the medicine that makes people or the enhancing of mouse immunologic function by stimulation people or mouse immune cell propagation.

3. oligonucleotide claimed in claim 1 is for the preparation of the application in the medicine of activation people immunocyte.

4. the application of claim 3, wherein said medicine is activating B cell by the B cell high expression level CD69 molecule in the stimulation human peripheral blood; Described medicine activates the NK cell by the NK cell high expression level CD69 molecule in the stimulation human peripheral blood; Or described medicine by the monocyte high expression level CD69 molecule in the stimulation human peripheral blood activated monocyte.

5. oligonucleotide claimed in claim 1 is for the preparation of the application in the medicine of prevention or treatment animal model mouse breast cancer.

Oligonucleotide claimed in claim 1 in preparation by strengthening immunologic function and prevent or treating application in the medicine of animal model mouse breast cancer.

7. the application of claim 6, wherein said immunologic function is anti tumor immune response.

8. oligonucleotide claimed in claim 1 is for the preparation of the purposes of the preparation that is used for the prevention human breast carcinoma.

9. oligonucleotide claimed in claim 1 is for the preparation of the purposes in the preparation that is used for the treatment of human breast carcinoma.

10. oligonucleotide claimed in claim 1 is for the preparation of the purposes that is used for preventing or treating the preparation of individual mammary cancer.

11. according to claim 9 or 10 arbitrary described purposes, oligonucleotide is wherein giving lotus mammary cancer when individual, described Drug combination is in the methods for the treatment of of following one or more mammary cancer: chemotherapy, radiotherapy, biotherapy, endocrine therapy.

12. according to the arbitrary described purposes of claim 9-10, oligonucleotide wherein is the form of using separately or the form of using with the pharmacology acceptable carrier at form of medication when giving people or other individuality.

13. according to the arbitrary described purposes of claim 9-10, oligonucleotide wherein when giving individuality as the effective constituent in the pharmaceutical composition.

14. according to the arbitrary described purposes of claim 9-10, oligonucleotide wherein is to be suitable in intestines or/and the form that intestines external administration approach is implemented when giving individuality, wherein intestines external administration approach comprise that injection in tumor tissues direct injection, swollen tumor-side injection, the lymphoglandula, lymphoglandula sidenote are penetrated, peritoneal injection, intradermal injection, subcutaneous injection, intramuscular injection.

15. according to the arbitrary described purposes of claim 9-10, oligonucleotide wherein when giving individuality for being suitable for independent application, or the form of self combined utilization.

16. according to the arbitrary described purposes of claim 9-10, oligonucleoside wherein when giving individuality for can with the form of delivery vector combined utilization.

17. according to the arbitrary described purposes of claim 9-10, the dosage of oligonucleotide wherein is the treatment effective dose.