CN1206242C - Anti-angiogenesis and anti-cancer function of angiogenin combined with nucleotide - Google Patents

Abstract

The present invention provides a nucleotide-angiogenin combined element (ABE) which is used for inhibiting angiogenin induction and is synthesized by r RNA. The length of oligonucleotide is from 10 to 150 bp, the C+T content of the oligonucleotide accounts for 80% to 100% of the total oligonucleotide, and the oligonucleotide contains 5 to 30 continuous CT repeating cells. ABE and angiogenin are specially combined so as to inhibit the r RNA synthesis by being proved through experiment, and the ABE can inhibit the growth of a human body tumour on a nude mouse by being proved through animal experiment.

Description

The angiogenesis inhibitor of angiogenin binding nucleotide and antitumous effect

Technical field

The present invention relates to field of medicaments.Particularly, the present invention relates to a kind of and the single-minded bonded oligonucleotide of angiogenin.Thereby this oligonucleotide can suppress the combination of the transcription regulatory region of human angiogenin and ribosomal rna gene and suppress transcribing and ribosomal synthetic, protein translation and cell growth of rRNA.The invention still further relates to the pharmaceutical composition that contains this angiogenin oligonucleotide binding.

Background technology

Tumor growth and transfer are accepted extensively by scientific circles the dependency of angiogenesis.Unless set up its oneself blood vessel network, otherwise tumor tissues can only be grown the size of diameter 1-2 millimeter.Size tumor does not constitute harm to human body like this.The previous theory of extensively being paid close attention to and accepting of order is the medicine that life-time service suppresses vasculogenesis, can make tumour cell be in latent period always.Like this, cancer can the control of long-term prescription thing as hypertension and diabetes.This is an important directions (the Harris AL.Antiangiogenesis for cancer therapy.Lancet 1997 of present cancer therapy research; 349 Suppl 2:SII13-5.).

The substantial connection of noumenal tumour and vasculogenesis is realized the earliest.Known that various malignant tumours such as sarcoma, fibroma, myxoma, osteoma, epithelioma or the like all secrete angiogenesis factor.Classify from tumorigenic organ, various organ cancerations such as lung cancer, liver cancer, breast cancer, osteocarcinoma, prostate cancer, ovarian cancer and head and neck cancer or the like all closely related (Hui YF, Ignoffo RJ.Angiogenesis inhibitors.A promising role in cancer therapy.CancerPractice 1998 in the known human body with blood vessel hyperplasia; 6 (1): 60-2.).

Nearest experimental result also proves, also relevant (the Schneider P of various body fluid cancers such as each quasi-leukemia with blood vessel hyperplasia with lymphatic cancer, Jerome MV, Paysant J, Soria PC, Vannier JP.The roleof angiogenesis in leukemia proliferation.American Journal of Pathology1999; 155 (3): 1007-8.).Like this, almost all pernicious and benign tumours are all relevant with blood vessel hyperplasia in the human body.The transfer of blood vessel hyperplasia and tumour also has confidential relation.This relation embodies both ways, and the one, original tumour must rely on vasculogenesis just can enter blood circulation, arrives transfer destination ground.The 2nd, when entering sanguimotor tumour cell after the implantation of point of destination, the formation that must impel new vessel is to reach growth, the purpose of expansion.

Blood vessel hyperplasia is meant that deriving capillary blood vessel network of capillary blood vessel formation from a blood vessel that has existed forms a microcirculatory process (Yancopoulos GD, Klagsbrun M, Folkman J.Vasculogenesis, angiogenesis, and growth factors:ephrins enter the frayat the border.Cell 1998; 93 (5): 661-4.).Blood vessel hyperplasia is a whole set of process of being made up of several steps.It is that normal physiological processes such as fertility, growth, wound healing, menstrual cycle are necessary, simultaneously, in multiple pathologic process, as tumor growth and transfer, rheumatic arthritis, diabetes, retinopathy plays keying action in the psoriasis etc.On cell and biochemical level, vasculogenesis divides four steps: (1) normal blood vessels endotheliocyte is subjected to the stimulation of angiogenesis factor and activates; (2) activated vascular endothelial cell intrusion surrounding tissue moves towards the direction at angiogenesis factor place; (3) following thereafter vascular endothelial cell division fills up migratory cell and interspaces; (4) the new microvascular anastomosis that forms, maturation forms the recycle system with function.So blood vessel hyperplasia is a lasting continual complex process.The mechanism and the regulatory factor of each concrete steps are illustrated substantially.

Vasculogenesis is a complex process that multistep is rapid.So the regulation and control of vasculogenesis also are complicated.This is a process dynamic, that control is very tight, is determined by angiogenesis factor and angiogenesis inhibitor.Present known angiogenesis factor has about 25 kinds (Iruela-Arispe ML, DvorakHF.Angiogenesis:a dynamic balance of stimulators and inhibitors.Thrombosis﹠amp; Haemostasis 1997; 78 (1): 672-7.).Their physico-chemical property has nothing in common with each other.But a common character is all arranged, and that is exactly the generation that promotes blood vessel.They all have been purified order-checking, and its gene is all cloned.More common angiogenesis factor has angiogenin (Angiogenin), vascular endothelial growth factor (VEGF), acidity and Basic Fibroblast Growth Factor (aFGF and bFGF), Urogastron (EGF), PDGF (PDGF), platelet activating factor (PAF), thrombocyte endothelial cell growth factor (ECGF) (PD-ECGF), placenta growth factor (PIGF), pHGF (HGF), rhIGF-1 (IGF) and interleukin 8 (IL-8) or the like.Under many circumstances, concentration and the tumor growth of these somatomedins in blood is proportionate.

Angiogenin is the protein with intense stimulus vasculogenesis vigor, and human angiogenin is made up of 123 amino acid, and molecular weight is 14400, and iso-electric point is 9.2.Angiogenin was found early than 1985, be that first also is up to the present unique one and only depends on the vasculogenesis vigor in the body of measuring and the isolating angiogenesis factor of purifying (Fett JW, Strydom DJ, Lobb RR, Alderman EM, BethuneJL, Riordan JF, Vallee BL.Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells.Biochemistry 1985; 24 (20): 5480-6.).In numerous angiogenesis factors, angiogenin induction of vascular new life's vigor is the highest.In the vigor system of the mensuration vasculogenesis of fertilized eggs chorion (Chorioallantoic Membrane), the pure angiogenin of 1 nanogram (1ng) just has the vigor that significantly impels vasculogenesis.So angiogenin has its special meaning in the vasculogenesis field.

Recent findings, angiogenin directly enters nucleus, concentrate in the kernel, directly act on (the Hu G of genetic transcription system of cell with the DNA combination, Xu C, Riordan JF.Human angiogenin israpidly translocated to the nucleus of human umbilical vein endothelialcells and binds to DNA.J Cell Biochem 2000; 76 (3): 452-62.).Angiogenin induce rRNA (rRNA) thus transcribe and impel ribosomal biosynthesizing.Because rrna is that protein translation is necessary, there is not the cell growth without protein translation, so, suppress the synthetic growth that can suppress vascular endothelial cell effectively of the angiogenin inductive rRNA of institute.

Yet, still do not have effectively to suppress the technology that angiogenin inductive rRNA transcribes so far.Therefore, this area presses for the Compounds and methods for that the new effective inhibition of exploitation is transcribed by angiogenin inductive rRNA.

Summary of the invention

Purpose of the present invention just provides a kind of new angiogenin oligonucleotide binding, and it can be efficiently, low toxicity or suppress the synthetic of angiogenin inductive rRNA innocuously, and then treatment and the excessive diseases associated of vasculogenesis.

In a first aspect of the present invention, a kind of oligonucleotide is provided, the single-minded combination of described oligonucleotide and human angiogenin, and the length of described oligonucleotide is 10-150bp, its C+T content accounts for the 80%-100% of whole oligonucleotide, and contains 5-30 successive CT repeating unit.

Preferably, the length of described oligonucleotide is 12-50bp.

In another preference, described oligonucleotide comprises 6-10 (as 6,7,8,9,10) successive CT tumor-necrosis factor glycoproteins.

In another preference, 3 ' terminal sequence is made of C and T basically, as CT or CCCTC.

In another preference, described oligonucleotide has to be selected from down organizes sequence: SEQ ID NO:1-5,7-12.

In another preference, the sequence of described oligonucleotide is SEQ ID NO:1,8,9,10,11 or 12.

In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the oligonucleotide of the present invention and the pharmaceutically acceptable carrier for the treatment of significant quantity.

In a third aspect of the present invention, the purposes of oligonucleotide of the present invention is provided, be used to prepare the medicine for the treatment of following disease:

(a) growth of treatment human tumor and transfer;

(b) treatment human body and the excessive diseases associated of blood vessel hyperplasia.

Description of drawings

Fig. 1 has shown the variation of the gel electrophoresis mobility that causes after angiogenin and the combination of rRNA transcription regulatory region dna fragmentation.Angiogenin causes that 3.0kb fragment mobility changes.

Fig. 2 has shown angiogenin and the segmental binding specificity of rRNA transcription regulatory region 3.0kb.Angiogenin causes that 3.0kb fragment mobility changes, yet N,O-Diacetylmuramidase (lysozyme) and ribonuclease A (RNase A) are to the not influence of 3.0 segmental mobilities.

Fig. 3 has shown 5 nucleotide sequences of cloning with the angiogenin bonded.

Fig. 4 has shown the sequence of 4 synthetic oligonucleotide.

Fig. 5 has shown the combination of human angiogenin and 4 synthetic oligonucleotide.In competitive assay, unlabelled nucleotide concentration is 10 times of isotope-labeled nucleotide concentration.

Fig. 6 has shown the normal chain of angiogenin and 4 synthetic oligonucleotide, anti-chain, or double-stranded combination.The affinity data on first hurdle is recorded by the affinity chromatography method.The binding constant on second hurdle is measured by the diaphragm method.

Fig. 7 has shown in order to measure angiogenin binding member (ABE) and has transcribed the collection of illustrative plates of 4 used luciferase expression plasmids of vigor.PGL3B is basic plasmid, does not contain exogenous promoter and enhanser.PGL3E contains the SV40 enhanser.PGL3P contains the SV40 promotor.PGL3C is that height is transcribed control plasmid, contains SV40 promotor and enhanser.

Fig. 8 has shown the transcribe vigor of angiogenin binding member (ABE) in 4 luciferase expression plasmids.ABE is inserted in the Hind III site of 4 expression plasmids.The result shows that ABE has the very strong vigor of transcribing in containing the plasmid pGL3E of SV40 enhanser.

Fig. 9 has shown angiogenin binding member forward and the vigor of transcribing that oppositely inserts.The result shows that ABE has only forward to be inserted in the HindIII site and just transcribes vigor, and expression ABE is a transcripting promoter.Fig. 9 A is the result in the HeLa cell; Fig. 9 B is the result in smooth muscle cell.

Figure 10 has shown the transcribe vigor of ABE in different cells.ABE is inserted in the Hind III site of pGL3E.The result shows that the vigor of transcribing of ABE and the content of cell angiogenin are proportionate: the highest in the HeLa cell, smooth muscle cell takes second place, and the U-937 cell is minimum.

Figure 11 has shown artificial raising or has reduced cell angiogenin level is transcribed vigor to ABE influence.Smooth muscle cell transforms to improve or to reduce the vasculogenesis cellulose content with expression plasmid pRM-Ang (+) or its antisense plasmid pRM-Ang (-) of angiogenin, measures ABE then at the intracellular vigor of transcribing of this kind.The result shows the vigor of transcribing of the cotransformation rising ABE of pRM-Ang (+), and the cotransformation of pRM-Ang (-) reduces the vigor of transcribing of ABE.

Figure 12 has shown that angiogenin antisense oligonucleotide CT-1 transcribes the influence of vigor to ABE.The result shows that CT-1 handles the vigor of transcribing that reduces ABE, and control oligonucleotide CT-2 is to the not influence of vigor of ABE.Adding external source angiogenin can offset the restraining effect of CT-1.

Figure 13 has shown that angiogenin antisense oligonucleotide CT-1 suppresses the synthetic of the endogenous angiogenin of people.Figure below is the pcr amplification result of beta-actin, and the homogeneous band in four samples represents that applied sample amount is identical.Last figure is that angiogenin pcr amplification result develops the color with ethidium bromide.The result represents that CT-1 handles and reduces the vasculogenesis cellulose content.And at blank, conversion reagent Effectene, or control oligonucleotide CT-2 processing back vasculogenesis cellulose content is constant.Expression CT-1 handles the specificity to the synthetic influence of angiogenin.

Figure 14 has shown that the angiogenin oligonucleotide binding suppresses human colon adenocarcinoma cell HT-29 in nude mice growth on one's body.The right side shoulder of each nude mice is gone in 10,000 HT-29 human colon adenocarcinoma cells subcutaneous injection.Every group of 8 nude mices, totally 3 groups.Handle with physiological saline for the 1st group, handle with 1 milligram of ABE for the 2nd group, the 3rd group with the processing of 1 milligram of contrast Nucleotide.Treatment process is subcutaneous injection every day.Injection site is near tumor cell inoculation point.Observational technique is to observe every day to touch, and measures gross tumor volume with vernier callipers.

Embodiment

The inventor is by extensive and deep research, by elaboration to the human angiogenin mechanism of action, find that Nucleotide that a class has a CT repeating unit is the oligonucleotide binding of single-minded human angiogenin, thus can be in order to suppress the inhibition that the nuclear vigor angiogenesis inhibiting of angiogenin in vascular endothelial cell finally cause tumor growth.On this basis, finished the present invention.

The length of angiogenin oligonucleotide binding of the present invention is not particularly limited.In general, for the bonding strength that acquires a certain degree, length is 10-150bp, preferably is 12-50bp, more preferably is 12-30bp.Wherein C+T content accounts for the 80%-100% of whole oligonucleotide, preferably 90-100%.And oligonucleotide of the present invention needs at least 5 CT repeating units, preferably at least 5 CT repeating units.

A kind of preferred angiogenin oligonucleotide binding is the sequence that can comprise angiogenin binding member (ABE) shown in the SEQ ID NO:1 in sequence.Sequence 5 '-CTCTCTCTCTCTCTCTCCCTC-3 ' (SEQ IDNO:1) is identical with clone 12 sequence among Fig. 3, is the transcription regulatory region clone gained from ribosomal rna gene.The binding ability of this oligonucleotide and angiogenin is the highest, and can high efficiency drive genetic expression.

The present invention discloses the vital role of angiogenin in vascular endothelial cell rRNA (rRNA) being transcribed first.The inventor finds, an important ring of the mechanism of action that the angiogenin induction of vascular generates is an intravasation endotheliocyte nuclear, be combined in the transcription regulatory region of ribosomal rna gene (rDNA), thereby impel transcribing of rRNA to strengthen ribosomal biosynthesizing, and then the growth of irritation cell and division.The present invention sets about from the mechanism of action of angiogenin, and utilization and rDNA transcription regulatory region angiogenin combining site have the angiogenin binding nucleotide of similar sequences, suppresses the synthetic of rRNA.

Because rRNA is a key component of forming cytoribosome, rrna is an intracellular protein synthetic factory, and its quantity what directly determine the speed of protein translation, thus the speed of decision cell growth.Therefore, the vigor that suppresses angiogenin with angiogenin binding nucleotide of the present invention can reach the purpose that suppresses vascular endothelial cell division, the necessary source mill of angiogenesis (rrna), and the effect of getting twice the result with half the effort is arranged.

Range of application of the present invention comprises all and the excessive diseases associated of blood vessel hyperplasia.

At first, human angiogenin oligonucleotide binding provided by the present invention can be used for treating human body and the excessive diseases associated of blood vessel hyperplasia, comprising but be not limited to: rheumatic arthritis, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, psoriasis, acne erythematosa, kaposi's sarcoma, specific reaction keratitis, epidemic keratoconjunctivitis, neovascular glaucoma, bacterial canker, mycotic ulcer, herpes-ness progenitalis infection, zoster infects, protozoal infections, mycobacterium infects, polyarteritis, sarcoid, scleritis, flush, the dry sacroiliitis syndromes of dry eye, systemic lupus erythematosus, AIDS-related complex, syphilis.

Secondly, in view of the substantial connection between blood vessel hyperplasia and tumor growth and the transfer, human angiogenin oligonucleotide binding provided by the present invention can not only suppress original growth of tumor and expansion, also can suppress the generation and the expansion of metastases simultaneously.The tumour example that is suitable for includes, but are not limited to: various noumenal tumours and leukemia, as lung cancer, small cell lung cancer, liver cancer, carcinoma of the pancreas, cancer of the stomach, osteocarcinoma, esophagus cancer, mastocarcinoma, prostate cancer, carcinoma of testis, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, adenocarcinoma cystic, cystocarcinoma, soft cancer, bronchogenic carcinoma, renal cell carcinoma, epithelial cancer, cholangiocarcinoma, choriocarcinoma, the embryo cancer, the spermatogonium cancer, wilms' tumor, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, the vocal cords neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, inochondroma, inocystoma, fibromyxoma, fibro-osteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondroma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, leukomyoma, lipomyxoma, chondrosarcoma, chondroma, chondromyoma, chordoma, chorioadenoma, the fine hair vascular tumor, the chorioepithelium knurl, chorioblastoma, osteosarcoma, osteoblastoma, osteoclastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, osteofibroma, the fibrosarcoma of bone, angiosarcoma, vascular tumor, angiolipoma, angiochondroma, hemangioblastoma, angiokeratoma, angioglioma, hemangioendothelioma, hemangiofibroma, angiomyoma, angiolipoma, hematolymphangioma, angiolipoleiomyoma, angiomyoliopma, angiomyoneuroma, angiomyxoma, angioreticuloendothelioma, lymphangiosarcoma, lymph meat odontoma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphepithelioma, lymphoblastoma, endothelioma, endothelioblastoma, lymphangioendothelial sarcoma, synovioma, synovial sarcoma, mesothelioma, mesocytoma, ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyoma, rhabdosarcoma, rhabdomyomyxoma, kemia, acute myelogenous leukemia, chronic leukemia, polycyth(a)emia, lymphoma, multiple myeloma.

Tumour needs blood vessel hyperplasia to set up the tumor vessel recycle system provides its required nutriment and eliminating waste material of growth fast.The present invention reaches the purpose that suppresses tumor growth and transfer by suppressing blood vessel hyperplasia.Clinical experiment is verified, and angiogenesis inhibitors can suppress the growth and the transfer of various human tumors effectively, and does not have resistance.

The present invention also provides a kind of pharmaceutical composition, and it contains angiogenin oligonucleotide binding of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.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.Pharmaceutical composition 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 50 mg/kg body weight.

When making pharmaceutical composition, be that angiogenin oligonucleotide binding with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 50 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 10 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.

When pharmaceutical compositions, usually, these oligonucleotide of the present invention can be formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, preferably pH is about 6-8, although the pH value can change to some extent with being prepared 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, intravenously, subcutaneous, intracutaneous or topical.

Oligonucleotide binding of the present invention can be directly used in disease treatment separately, also can with other treatment agent coupling, as angiogenin antisense oligonucleotide CT-1, neomycinsulphate A, B, C, the 2-methoxyestradiol, TNF-α, TGF-β, IFN-α, angiostatin (Angiostatin), endostatin (Endostatin), Glyfosfin, haematoporphyrin, lycobetaine, the kosam seeds breast, etoposide (being etoposide), the dehydration galactitol, Zorubicin, tamoxifen, methyl ECNU, 5 FU 5 fluorouracil, remove first spot chela element, Tegadifur, cucurbitacin, harringtonine, rubescensine B, Irisquinone A, polysaccharide-peptide, cytosine arabinoside, NSC-241240, taxol, lentinan, flutamide, ifosfamide, ubenimex, leuprorelin acetate, doxifluridine, Glass platinum, Yi Linnuoteken, bend azoles and Vumon etc.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

Cell cultures

People's placenta vein endothelial cell (HUVE) is incubated in the vascular endothelial cell special culture media (HE-SFM+10%FCS+10ng/ml bFGF) that contains 10% foetal calf serum and every milliliter 10 nanogram Prostatropin.Culture condition is 37 ℃ of fixed temperature and humidity, 5% carbonic acid gas gas phase.Changed a subculture every two days, with the cultivation of going down to posterity of 1: 3 ratio.HUVE cell with 15 generations of the third generation to the is done experiment.

People's placenta vein blood vessel smooth muscle cell (HuASM), human cervical carcinoma cell HeLa and human colon adenocarcinoma cell HT-29 are cultivated in the DMEM that contains 10% foetal calf serum, with the cultivation of going down to posterity of 1: 4 ratio.

The U-937 cell is cultivated in the RPMI that contains 10% foetal calf serum, with the cultivation of going down to posterity of 1: 4 ratio.

Embodiment 2

The separation of angiogenin binding nucleotide

The nucleus of angiogenin intravasation endotheliocyte is also induced synthetic (the Xu ZP of total RNA, TsujiT, Riordan JF, and Hu GF.The nuclear function of angiogenin inendothelial cells is related to rRNA production, Biochem.Biophys.Res.Commun.2002,294,287-292).Because showing angiogenin, Northern hybridization stimulates the synthetic of rRNA (rRNA), so carried out the combination that a series of experiment proves angiogenin and ribosomal rna gene (rDNA) transcription regulatory region.

The rDNA transcription regulatory region fragment that at first to obtain a length be 4.6kb also obtains 5 littler fragments with restriction enzyme DpnI processing.The experiment of gel electrophoresis mobility shows the fragment combination of angiogenin and 3.0kb, but to the not influence (Fig. 1) of other 4 fragments.

Fig. 2 shows that angiogenin is single-minded to the combination of 3.0kb dna fragmentation.Under same experiment condition, the fragment combination of N,O-Diacetylmuramidase (Lysozyme) and ribonuclease A (RNase A) discord 3.0kb.Because N,O-Diacetylmuramidase and ribonuclease A all are basic protein, its molecular weight is similar to angiogenin with iso-electric point, and the fragment combination of this explanation angiogenin and 3.0kb is not because the interaction of electric charge causes, but a species specific combination.

Embodiment 3

The clone of angiogenin binding nucleotide and sequential analysis

In the present embodiment, for the character of further Analysis and Identification angiogenin binding nucleotide, measure its sequence.

The binding substances of 3.0kb dna fragmentation and angiogenin is handled with deoxyribonuclease (DNase I).Like this, have only with the Nucleotide of the direct bound fraction of angiogenin can be by enzymolysis.Behind the separated clone of these fragments, measured nucleotide sequence.Fig. 3 has listed 5 sequences of clone like this.The general character of these 5 sequences is all to be rich in C and T, and comprises the CT tumor-necrosis factor glycoproteins.No. 12 clone's sequence (CTCTCTCTCTCTCTCTCCCTC, SEQ ID NO:1) is the shortest, is named as angiogenin binding member (ABE).

Embodiment 4

Angiogenin combines with the angiogenin binding nucleotide

Nucleotide described in the embodiment 3 obtains from the separation of ribosomal rna gene transcription regulatory region.For prove angiogenin really and the Nucleotide with CT tumor-necrosis factor glycoproteins in conjunction with and measure in conjunction with required minimum tumor-necrosis factor glycoproteins number, synthetic 4 kinds of Nucleotide as shown in Figure 4.They are measured with 3 kinds of diverse ways with the character that combines of angiogenin.

(1) method of gel electrophoresis mobility shift assay

At first form double-stranded respectively and usefulness 4 kinds of Nucleotide with its antisense strand ³²The P isotopic labeling.They with angiogenin in conjunction with after electrophoretic mobility different with unconjugated mobility.

The result as shown in Figure 5, synthetic length be 14 aggressiveness (14-mer) and 21 aggressiveness (21-mer) have the CT tumor-necrosis factor glycoproteins Nucleotide can and the angiogenin combination.Length is that the Nucleotide of 10 aggressiveness (10-mer) has slight combination (Fig. 5), the then debond of Nucleotide of 6 aggressiveness (6-mer).The combination that shows in the gel electrophoresis is single-minded, because do not compete with the Nucleotide of isotope-labeled corresponding nucleotide energy and mark.

(2) affinity chromatography

In order to confirm that angiogenin can and have the Nucleotide combination of CT tumor-necrosis factor glycoproteins, has also adopted the method for affinity chromatography.Will with sample on the Nucleotide of synthetic shown in Figure 4 to the angiogenin affinity column, use the salt gradient wash-out, the Nucleotide under the wash-out is measured photoabsorption at 260nm.The required salt concn of wash-out is represented bonded intensity.

Fig. 6 has shown usefulness angiogenin affinity column in conjunction with two strands, the result of normal chain and anti-chain Nucleotide.Length is that the two strands and the normal chain of the Nucleotide of 21 aggressiveness can combine with angiogenin is effective.Length is less than the Nucleotide debond of 10 aggressiveness.The angiogenin combination of all getting along well of all anti-chain Nucleotide.These results are with shown in Figure 5 consistent.

(3) diaphragm method

The third is used for measuring the bonded method is the diaphragm method.Press Stockley PG, Filter-bindingassay, in Methods in Molecular Biology, 1994, Vol.30:DNA-ProteinInteractions:Principles and Protocols (Kneale, G.G., Ed.), pp.251-261, Humana Press Inc., Totowa, the method described in the NJ. is carried out.The advantage of this method is the binding constant that can measure between the two.

The result as shown in Figure 6.Angiogenin and length are that the double chain nucleotide binding constant (Keq) of 21 and 14 aggressiveness is respectively 150 and 220nM.When length was reduced to 10 aggressiveness, binding constant was elevated to greater than 1000nM, and length was reduced to 6 o'clock and the avidity of angiogenin is vanished from sight (Fig. 6).

Embodiment 5

The angiogenin binding member is transcribed the mensuration of vigor

Obtain because angiogenin binding member (ABE) is separated by the ribosomal rna gene transcription regulatory region, it may have the vigor that drives genetic transcription.In the present embodiment, use this vigor of luciferase reporter gene systems measurement ABE.

4 luciferase reporting plasmids (available from Promega) have been used altogether.As shown in Figure 7, pGL3B is basic plasmid, does not contain exogenous promoter and enhanser, and total vigor is lower, can be used to measure the vigor of promotor or enhanser.PGL3E contains the SV40 enhanser, is fit to be used for measuring the vigor of promotor.PGL3P contains the SV40 promotor, is fit to be used for measuring the vigor of enhanser.PGL3C is that height is transcribed control plasmid, contains SV40 promotor and enhanser.

The data of Fig. 8 have showed the ability that angiogenin binding member (ABE) (SEQ ID NO:1) is inserted into the driving luciferase expression that Hind III is risen in the site in 4 luciferase reporting plasmids.Vigor was the highest when ABE was inserted into Hind III site in the pGL3E reporter plasmid, proved that ABE has the vigor of promotor.

If ABE is a transcripting promoter, its vigor will depend on it and present insertion site among the plasmid pGL3E at luciferase.Fig. 9 is presented among cervical cancer cell HeLa and the smooth muscle cell HuASM, and ABE only is inserted in Hind III site just the vigor that drives luciferase expression.These results have proved that further ABE has the function of promotor.In addition, if ABE oppositely inserts,, show that the vigor of ABE driving genetic transcription is single-minded no matter all to be not activated at what site the vigor of son in.

Embodiment 6

The driving vigor of transcribing with Nucleotide of different lengths CT tumor-necrosis factor glycoproteins

The driving vigor of transcribing for the Nucleotide of determining to have different lengths CT tumor-necrosis factor glycoproteins, synthetic double chain nucleotide as shown in table 1, they are inserted in the Hind III site of pGL3E luciferase reporting system, are converted into and measure the vigor of transcribing in the HeLa cell.

Table 1: the driving vigor of transcribing of the Nucleotide of different lengths CT tumor-necrosis factor glycoproteins

Nucleotide SEQ ID NO: direction of insertion vigor (the rising multiple in pGL3E)

Poly (G) ₂₁13 forwards 0.4 ± 0.1

ABE 1 forward 72 ± 4

Reverse 0.8 ± 0.2

(CT) ₅7 forwards 1.9 ± 0.3

Reverse 0.4 ± 0.1

(CT) ₆9 forwards 16 ± 1

Reverse 0.6 ± 0.1

(CT) ₇8 forwards 35 ± 3

Reverse 0.8 ± 0.1

(CT) ₈10 forwards 53 ± 4

Reverse 1.1 ± 0.1

(CT) ₉11 forwards 56 ± 10

Reverse 0.7 ± 0.2

(CT) ₁₀12 forwards 58 ± 8

Reverse 0.7 ± 0.1

The result shows need just have tangible driving transcriptional capability by at least 6 CT tumor-necrosis factor glycoproteinss.The binding ability consistent (Fig. 4-6) of these results and angiogenin described in the embodiment 1-4 and different lengths Nucleotide.Promptly have 5 CT at least, preferably at least 6 CT.

Embodiment 7

The transcripting promoter vigor of angiogenin binding member depends on the content of angiogenin

A series of in the present embodiment experimental results show that the angiogenin binding member must and angiogenin in conjunction with after the vigor that impels genetic transcription is just arranged.

At first, ABE is inserted into the Hind III site of pGL3E, and is transformed into the vigor of measuring luciferase in 3 kinds of different cells.In 3 kinds of cells that use, the HeLa cell contains the highest angiogenin, every day per 1,000,000 emiocytosis 1.5ng; Smooth muscle cell takes second place, every day per 1,000,000 emiocytosis 0.7ng; And in the U-937 cell, detection is less than secretion (the Moenner M. of angiogenin, Gusse M., HatziE., and Badet J.The widespread expression of angiogenin in differenthuman cells suggests a biological function not only related toangiogenesis, Eur.J.Biochem.1994,226,483-490).Result such as Figure 10 represent.The vigor that the driving luciferase of ABE is transcribed is the highest in the HeLa cell, takes second place in the smooth muscle cell, and is minimum in the U-937 cell.These results show that the transcripting promoter vigor of angiogenin binding member depends on the content of angiogenin.

Then, made up the expression plasmid of two angiogenins.PRM-Ang (+) contains vasculogenesis plain gene forward and inserts, and pRM-Ang (-) contains the vasculogenesis plain gene of reverse insertion, and mode that can antisense suppresses the expression of angiogenin in the born of the same parents.The vasculogenesis plain gene of pcr amplification is inserted the multiple clone site of pRMHa-3 (Promega company) forward or backwards, determine direction of insertion with the sequencing method.After they are transformed in the human smooth muscular cells, because external source angiogenin expression of gene, will be for raising respectively and reducing the content of the endogenous angiogenin of cell.Figure 11 is illustrated in pRM-Ang (+) cell transformed, and the vigor of ABE further raises.And in pRM-Ang (-) cell transformed, the vigor of ABE is vanished from sight substantially.The vigor that these results show the angiogenin binding member really and the content relation of being proportionate of the interior angiogenin of cell.

The 3rd is used for proving that the driving vigor of transcribing of angiogenin binding member depends on that the method for vasculogenesis cellulose content is the antisense nucleoside acid system.CT-1 is the single-minded antisense nucleotide of angiogenin, and sequence is CAACAAAACGCCCAGGCC (SEQ ID NO:14), with the 122-139 position complementation of human angiogenin mRNA.Intracellular vasculogenesis cellulose content can be reduced by CT-1.

The result as shown in figure 12.The promotor vigor of ABE is handled the back at CT-1 and is obviously reduced, and does not change and handle the back at contrast Nucleotide CT-2 (length is the random oligonucleotide of 18bp, and sequence is CCGGACCCGCAAAACAAC, SEQ IDNO:15).After antisense nucleotide CT-1 handles, add the external source angiogenin vigor of ABE is recovered substantially.This has confirmed that further angiogenin plays an important role transcribing in the vigor of ABE.

The content of angiogenin has reduced after CT-1 handles really in Figure 13 showed cell.Intracellular total mRNA separates with Trizol, and with the ethidium bromide colour developing, band shown in the last figure is the cDNA of human angiogenin after the RT-PCR amplification.Figure below is depicted as the pcr amplification result of beta-actin, the homogeneous of expression applied sample amount.The result shows that after CT-1 handled, the level of intracellular angiogenin reduced.Yet, under similarity condition, handle or only handle, not influence of vasculogenesis cellulose content in the pair cell through Lipofectin through CT-2.

Embodiment 8

The angiogenin binding nucleotide suppresses human colon adenocarcinoma cell HT-29 in nude mice growth on one's body

In order to prove the antitumor action of angiogenin binding nucleotide, carried out following experimentation on animals.Athymic mouse (nude mice) is because of immune deficiency, and the external source tumour cell can be set up and growth fast on one's body at it.Under the processing of tumor growth inhibitor, tumor growth rate descends, and stops or not taking place.

Concrete grammar is: 10,000 HT-29 go into the right side shoulder that each nude mice is gone in the colon adenocarcinoma cell subcutaneous injection.Every group of 8 nude mices, totally 3 groups.Handle with physiological saline for the 1st group.Use 1 milligram of angiogenin binding nucleotide of per kilogram of body weight (SEQ ID NO:1) subcutaneous injection for the 2nd group, injection site is near tumor cell inoculation point.The subcutaneous injection of 1 milligram of contrast of the 3rd group of per kilogram of body weight Nucleotide.Observational technique is to observe every day to touch, and measures gross tumor volume with vernier callipers.

As shown in figure 14, nude mice is behind human colon adenocarcinoma cell HT-29 knot kind, and all animals are bearing tumor and cause final death all.Under subcutaneous injection every day angiogenin binding nucleotide is handled, the not long tumour that goes up of 80% animal is arranged approximately.The result shows that the angiogenin binding nucleotide has good anticancer vigor.

Repeat above-mentioned experiment, use the nucleotide sequence shown in the SEQ ID NO:8,10,12 to replace the sequence shown in the SEQ IDNO:1 respectively, the result shows that these angiogenin binding nucleotides have significant anticancer vigor equally.

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.

＜110〉Fuyuan Biochemical Pharmacy R ﹠ D Co., Ltd., Shanghai

＜120〉angiogenesis inhibitor of angiogenin binding nucleotide and antitumous effect

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Claims (9)

1. an oligonucleotide is characterized in that, the single-minded combination of described oligonucleotide and human angiogenin, and the length of described oligonucleotide is 12-50bp, its C+T content accounts for the 80%-100% of whole oligonucleotide, and contains 6-12 successive CT repeating unit.

2. oligonucleotide as claimed in claim 1 is characterized in that, the length of oligonucleotide is 12-30bp.

3. oligonucleotide as claimed in claim 1 is characterized in that, described oligonucleotide comprises 7,8 or 9 successive CT tumor-necrosis factor glycoproteinss.

4. oligonucleotide as claimed in claim 1 is characterized in that, described oligonucleotide has the sequence of one of group of being selected from down: SEQ ID NO:1,8-12.

5. oligonucleotide as claimed in claim 4 is characterized in that, the sequence of described oligonucleotide is SEQ ID NO:1,10,11 or 12.

6. a pharmaceutical composition is characterized in that, contains described oligonucleotide of the claim 1 for the treatment of significant quantity and pharmaceutically acceptable carrier.

7. the purposes of oligonucleotide as claimed in claim 1 is characterized in that, is used to prepare the medicine that treatment is selected from down one of the group disease:

(a) human body and tumor growth and transfer diseases associated;

(b) the excessive diseases associated of human body and blood vessel hyperplasia.

8. purposes as claimed in claim 7, it is characterized in that, described tumour is selected from: lung cancer, small cell lung cancer, liver cancer, carcinoma of the pancreas, cancer of the stomach, osteocarcinoma, esophagus cancer, mastocarcinoma, prostate cancer, carcinoma of testis, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, adenocarcinoma cystic, cystocarcinoma, soft cancer, bronchogenic carcinoma, renal cell carcinoma, epithelial cancer, cholangiocarcinoma, choriocarcinoma, the embryo cancer, the spermatogonium cancer, wilms' tumor, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, the vocal cords neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, inochondroma, inocystoma, fibromyxoma, fibro-osteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondroma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, leukomyoma, lipomyxoma, chondrosarcoma, chondroma, chondromyoma, chordoma, chorioadenoma, the fine hair vascular tumor, the chorioepithelium knurl, chorioblastoma, osteosarcoma, osteoblastoma, osteoclastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, osteofibroma, the fibrosarcoma of bone, angiosarcoma, vascular tumor, angiolipoma, angiochondroma, hemangioblastoma, angiokeratoma, angioglioma, hemangioendothelioma, hemangiofibroma, angiomyoma, angiolipoma, hematolymphangioma, angiolipoleiomyoma, angiomyoliopma, angiomyoneuroma, angiomyxoma, angioreticuloendothelioma, lymphangiosarcoma, lymph meat odontoma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphepithelioma, lymphoblastoma, endothelioma, endothelioblastoma, lymphangioendothelial sarcoma, synovioma, synovial sarcoma, mesothelioma, mesocytoma, ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyoma, rhabdosarcoma, rhabdomyomyxoma, kemia, acute myelogenous leukemia, chronic leukemia, polycyth(a)emia, lymphoma, or multiple myeloma.

9. purposes as claimed in claim 7, the excessive diseases associated of described human body and blood vessel hyperplasia is selected from: rheumatic arthritis, diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, psoriasis, acne erythematosa, kaposi's sarcoma, specific reaction keratitis, epidemic keratoconjunctivitis, neovascular glaucoma, bacterial canker, mycotic ulcer, herpes-ness progenitalis infection, zoster infects, protozoal infections, mycobacterium infects, polyarteritis, sarcoid, scleritis, flush, the dry sacroiliitis syndromes of dry eye, systemic lupus erythematosus, AIDS-related complex, or syphilis.

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