CN103627709A - Deoxyribozyme capable of suppressing genetic expression of vascular endothelial growth factor - Google Patents

Abstract

The invention discloses a deoxyribozyme capable of suppressing genetic expression of a vascular endothelial growth factor. The deoxyribozyme comprises a catalytic functional sequence GGCTAGCTACAACGA, and 5'-terminated and 3'-terminated binding sequences which are connected with the two terminals of the catalytic functional sequence, wherein the catalytic functional sequence incises a connecting key of any purine nucleotide and pyrimidine nucleotide of a target sequence; the 5'-terminated and 3'-terminated binding sequences are complemented with the sequences on two sides of the target sequence cleavage site. The deoxyribozyme takes an epidermal growth factor receptor 1 (VEGFR-1) as a target gene, reduces expression of the VEGFR-1 gene through specific cleavage to mRNA or pre-mRNA of VEGFR-1, and suppresses tumor angiogenesis growth and tumor growth.

Description

The DNAzyme that suppresses expression of vascular endothelial growth factor receptor genes

Technical field

The DNAzyme that the present invention relates to a class target tumor angiogenesis blood vessel gene, relates in particular to a class and can suppress vascular endothelial growth factor receptor VEGFR1 genetic expression, suppresses the DNAzyme of tumor angiogenesis and tumor growth.

Background technology

New vessel generates the vascular system process multi-step referring to from both having deposited, the new vascular system forming under multifactorial effect, is the result that short angiogenesis factor and Angiostatin regulate and control mutually.The two,, in equilibrium state, will activate vascular system once this balance is broken under normal circumstances, promotes vasculogenesis or suppresses vascular system.Vasculogenesis only occurs in some specific physiological process normally, and as fetal development, wound healing, menstrual cycle etc., abnormal vasculogenesis is one of pathological manifestations of the diseases such as tumour, disease of immune system, atherosclerosis.

Tumor vessel is different from the blood vessel [1] of healthy tissues, grow and do not have a controllability, textural anomaly, distribute disorderly, lack normal microcirculatory function, but still the vigorous ability that nutrition and oxygen, excretion refuse are provided of tool is the pathologic basis of growth and metastasis of tumours, has critical role in tumor development.At present, tumor vessel has become most important target spot in tumour molecular targeted therapy.From Folkman propose first target vascular therapy generate can suppress tumor growth to become a kind of therapeutic modality since [2], a large amount of experimental studies and clinical experiment confirm that the vasculogenesis of inhibition tumour mediation can suppress growth and the transfer [3-5] of tumour effectively, the cancer therapy drug of existing dozens of target vascular therapy enters clinical, and the combined utilization of they and chemicotherapy demonstrates good application prospect.For tumor vascular targeted therapy, be mainly divided into two classes [6], the one, anti-angiogenic rebirth preparation (anti-angiogenic inhibitors, AI), suppress tumor vascular generation, Main Function is in early treatment stage of tumour or be used for prevention and have the oncotherapy of metastatic potential; The 2nd, tumor vessel blocking-up preparation (vascular-disrupting agents, VDA), destroy the blood vessel having generated, utilize the blood vessel correlation factor that the tumor vascular part of said preparation specific binding or selective destruction are abnormal, be mainly used to the larger tumour of volume that treatment has formed.Not same-action feature based on AI and VDA, both couplings can effectively improve curative effect [7].

In numerous angiogenic factors, vascular endothelial growth factor (VEGF) is the strongest angiogenic growth and the factor that increases vascular permeability of effect of finding the earliest, more and more studies show that [8], vascular endothelial growth factor family (vascular endothelial growth factor family, VEGFs) path plays an important role in the growth of tumour with in shifting.VEGF is by making VEGFR Dimerized with the combination of vascular endothelial growth factor receptor (VEGFR), form homodimer or heterodimer, the tyrosine residues of phosphorylation can regulate and control kinase whose vigor, participate in differentiation, propagation and the migration of endotheliocyte, also the signaling molecule can be in endochylema provides binding domains, participates in the cell signaling [9] of next stage.

One of VEGFR-l Shu Sange receptor tyrosine kinases family [10], molecular weight is about 180kDa, its protein structure is divided into intracellular region, cross-film district and three of extracellular regions part, wherein be responsible for and ligand binding extracellular region, have 7 immunoglobulin-like loops, ligand binding domain is positioned at 3 loop internal orifices of aminoterminal.The mankind, the gene of coding VEGFR-l is positioned at karyomit(e) 13q12-q13, full length gene 7680bp, 1338 amino acid of encoding.VEGFR-1 is one of three tyrosine kinase receptors of VEGF, is the tyrosine kinase receptor in conjunction with the cytolemma grappling of VEGF-A, is also the unique tyrosine kinase receptor [11] in conjunction with VEGF-B and P1GF.The signal path of VEGFR-1 mediation, is especially combined the formation of the main mediated pathology blood vessel of signal that causes with part PIGF.Large quantity research finds that this kinases territory regulates the perviousness of blood vessel and the angiogenesis of PIGF induction to be absolutely necessary for mononuclear macrophage, and the micromolecular inhibitor of target VEGFR-1 all can effectively suppress the angiogenesis [12] of tumour.

The Bone Morrow Hematopoietic Progenitor Cells of the VEGFR-1 positive can regulate the recruitment of the positive endothelial progenitor cells of VEGFR-2 and exosmose, and promotes VEGFR-2 intravasation to participate in the formation of new vessel, for the propagation of tumour cell, jointly sets up and shifts microenvironment [13].Selectively inhibiting VEGF R-1 has eliminated the formation of the small transfer tabernacle of tumour, thereby has reduced the micrometastasis of tumour.In sum, VEGFR-1 signal path is having vital effect aspect inflammation and transfer, adjusting and promotion tumor-blood-vessel growth, and the antineoplaston that the VEGFR-l of take is target spot is a field that application prospect is considerable.

DNAzyme is a kind of short-movie section single stranded DNA with catalysis that utilizes external molecular evolution technique to obtain, and has efficient catalytic activity and structure recognition capability.DNAzyme is combined efficient catalyzed degradation ability with the target recognition capability of antisense, can for target, from mRNA level, close target gene specifically, thereby goal of regulation and control protein expression is a kind of New Policy [14] of efficient special target gene therapy.The chemical nature of DNAzyme uniqueness is deoxy-oligonucleotide, and character is relatively stable; Molecular weight is little, and structure is relatively simple, good to the accessibility of substrate; Catalytic efficiency and specificity are high, and side effect is low; The restriction that target site is selected still less; Be easy to synthesize, cheap.Utilize DNAzyme target VEGFR-1 a kind of new antitumoral vasculogenesis to be provided and to suppress the preparation of tumor growth.

Reference

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Summary of the invention

The object of this invention is to provide a class and lower vascular endothelial growth factor receptor 1(VEGFR-1) express, by suppressing the DNAzyme targeted drug of tumor-blood-vessel growth treatment tumour.

DNAzyme provided by the present invention is by lowering the expression of vascular endothelial growth factor receptor 1, thereby suppresses the growth of tumour.DNAzyme is a kind of novel gene inhibitor, and it has the chemical stability of antisense oligonucleotide, have again the function of the catalyze cleavage RNA of ribozyme simultaneously, and its synthetic expense is very low.Based on these unique advantages, this technology has been widely used in body and external gene regulating.

Described DNAzyme is a kind of DNA molecular, can identify specifically and cut said target mrna or pre-mRNA, and its structure as shown in Figure 1.N=G wherein, U, C, A; R*Y is cleavage site; R=A or G; Y=U or C.DNAzyme comprises:

(1) catalysis sequence GGCTAGCTACAACGA, cuts arbitrary purine nucleotides of target gene mRNA or pre-mRNA and the connecting key of pyrimidine nucleotide with this;

(2) 5 ' ends and 3 ' end binding sequence, be respectively 5～12 Nucleotide, complementary with the sequence of target sequence cleavage site both sides respectively; 5 ' end is connected by catalysis sequence with 3 ' end binding sequence.

5 ' end of DNAzyme and 3 ' end binding sequence are called again its two arm, preferred, when selecting target cleavage site, and the G °≤-10kcal/mol that the length of adjustment DNAzyme two arms makes every arm be combined with target sequence.

The target gene of DNAzyme of the present invention is human VEGFR-3-1 gene.VEGFR-1 is high expression level in numerous tumours, to the inhibition of the genetic expression of VEGFR-1, can effectively suppress tumor growth.

DNAzyme is comprised of 27～39 nucleotide residues, from 5 ' to 3 ' direction, by phosphoric acid ester bond, connected.For strengthening the stability of DNAzyme, can carry out chemically modified to improve the ability of the antiacid and resistance to enzymic degradation of DNAzyme to its phosphoric acid ester bond, for example, with phosphorothioate bond, replace phosphoric acid ester bond, form lock nucleic acid (Locked nucleic acids, LNA) or peptide nucleic acid(PNA) (peptide nucleic acids, PNA).Preferably phosphorothioate bond is modified, and in the 5 ' end and/or 3 ' of described DNAzyme is held binding sequence, can contain 1-6 phosphorothioate bond.

The enhancing of stability can also realize by other chemically modifieds, comprising:

1) modification to base, glycosyl and trunk structure.Modification to base, for example, methylated base, methylolated base etc.Modification to glycosyl, for example 2 ' 3 ' replaces thymus nucleic acid.Connecting key between ribosyl and mononucleotide is called the trunk structure of nucleic acid.The modification of trunk structure is comprised to connecting key and other can enhanced stability and the modification of affinity, and increase some substituting groups, for example, diamines (diamine), cholesterol or other lipophilic group, 2 '-O-methyl acid phosphate diester linkage, 2 '-O-(CH2) n-OCH3,2 '-fluorine, heterozygosis connecting key, the modification of other trunk structure.Concrete example has, and while being reverse in base with respect to natural dextrorotatory isomer sugar, can use left-handed (L-) isomer ribodesose; Or by 2 ' of glycosyl-position with 2 '-halogens, 2 '-O-alkyl, 2 '-O-(alkylidene group) n-O-alkyl replaces; Or use following connecting key: 2 '-O-methyl acid phosphate diester linkage.DNAzyme of the present invention can have part modifies, or all modifies, or the combination of different modifying.Preferably glycosyl 2 '-O-methyl is modified.

2) end protection: the end of molecule add protecting group because of, to prevent the degraded of molecule.This protection can be 5 ' end, can be also 3 ' end, or two ends is all protected.For example, Opposite direction connection base, dideoxy nucleotide, METHAPHOSPHORIC ACID base, alkyl, aryl, cordycepin, cytosine arabanoside, 2 '-methoxyl group, oxyethyl group Nucleotide, phosphorothioate connecting key, 3 '-O-methyl base, fluorescein, peptide bond connects, two pyridyls, cholesterol, vitamin H, acridine, rhodaminepsoralen, glyceryl, butanols base, butyl, alcohol radical and 3 '-O-alkyl.Preferably 3 ' hold Opposite direction connection base.

In one embodiment of the invention, the design of the DNAzyme of target VEGFR-1 is by analyzing the mRNA sequence of target gene, filter out AU and GU site, further analyze free energy level, selecting target site.For these sites, design corresponding DNAzyme.Application DNA synthesizer device, synthetic designed DNAzyme, and carry out external cutting experiment, obtain the DNAzyme with high nicking activity.

The biologic activity of the angiogenesis inhibitor of target VEGFR-1 DNAzyme can utilize inside and outside model to verify; Anti-tumour effect utilizes tumor models and mice-transplanted tumor model to carry out experimental verification.

The biological safety of target VEGFR-1 DNAzyme is evaluated by conventional pharmacology and toxicologic method.

Experiment showed, that the DNAzyme of target VEGFR-1 of the present invention is by cutting specifically the mRNA of VEGFR-1, reduce the expression of VEGFR-1 gene, suppress tumor angiogenesis and tumor growth.

Accompanying drawing explanation

Fig. 1 is DNAzyme sequence and structural representation.

Fig. 2 has shown the external nicking activity analytical results of DNAzyme of target VEGFR-1 DNAzyme.

Fig. 3 has shown the impact that target VEGFR-1 DNAzyme forms microtubule DNAzyme.

Fig. 4 has shown target VEGFR1 DNAzyme DT18 and has contrasted external cutting power.

Fig. 5 has shown target VEGFR1 DNAzyme DT18 and has contrasted external cutting quantitative result.

Fig. 6 has shown that DNAzyme DT18 suppresses the expression of VEGFR-1 in cell.

Fig. 7 has shown that DNAzyme DT18 is on the angiopoietic impact of body interior opposite angle film.

Fig. 8 is the quantitative analysis results that DNAzyme suppresses corneal neovascularization.

Fig. 9 has shown the restraining effect (n=8) of DNAzyme DT18 to newborn melanoma growth.

Figure 10 has shown that DNAzyme DT18 suppresses nude mice Transplanted Human nasopharyngeal carcinoma growth (n=5).

Figure 11 has shown that DNAzyme DT18 suppresses the expression of VEGFR-1 in tumor tissues.

Figure 12 has shown the impact of DNAzyme DT18 on vascular permeability in transplanted tumor.

Figure 13 has shown the impact of DNAzyme DT18 on main organs techtology.

Embodiment

The invention will be further described by the following examples, but this is not limitation of the present invention, those skilled in the art, according to basic thought of the present invention, can make various modifications or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.

Embodiment 1: analysis and the design of target VEGFR-1mRNA DNAzyme cleavage site

VEGFR-1 is high expression level in many tumor tissues, and target suppresses VEGFR-1 and expresses the new vessel generation that can suppress tumour, thereby promotes death of neoplastic cells.

In the present embodiment, will utilize people HUVEC cell screening DNAzyme, and in mouse model, be analyzed and checking.All effective in mouse and people for guaranteeing the DNAzyme of target VEGFR-1, must design the DNAzyme for mouse/rat and people's VEGFR-1mRNA conservative region.In height homologous region, choose potential target site, the free energy (Δ G °) that uses nearest neighbor Equation for Calculating candidate DNAzyme to be combined with target sequence, the Δ G °≤-10kcal/mol that slightly length of inching ribozyme two arms makes every arm be combined with target sequence.In addition because mouse/rat and people's sequence homology there are differences, so two arm sequences of DNAzyme may be symmetrical or asymmetric chain.According to above principle, screen altogether 11 potential target sites, and for 11 target sites, built the DNAzyme (table 1) of target VEGFR-1.Meanwhile, the substrate recognition sequence of DNAzyme is constant, and the base sequence of catalytic center is changed into 5 ' AGCAACATCGATCGG3 ' (SEQ ID No:12), is built into the contrast INV-Ctrl of non-enzymatic activity.In order to improve the stability of DNAzyme, each 2 bases of two ends of DNAzyme and contrast molecule are carried out to thiophosphorylation modification; In order to detect the transfection efficiency of DNAzyme and in intracellular distribution situation, DNAzyme 5 ' to be held and used FITC mark.Carry out the DNAzyme of extracellular experiment all through PAGE purifying, carry out experiment in cell all through HPLC purifying.

Table 1: target site and the nucleotide sequence of target VEGFR-1 DNAzyme

# calculates according to homologous sequence length and free energy, and DNAzyme comprises respectively symmetrical or asymmetric combination arm.The following underlining of catalytic activity sequence.

Embodiment 2: the external nicking activity of target VEGFR-1 DNAzyme

DNAzyme has autocatalysis and efficient, the sequence-specific function of target, therefore, and its activity in vitro of external cleavage reaction direct reaction.With t7 rna polymerase, carry out in-vitro transcription and translate to prepare the needed template of external cutting experiment.T7Ampliscribe in-vitro transcription test kit (Epicentre, Wisconsin) is used in in-vitro transcription reaction, and operation is undertaken by test kit specification sheets.The linearization plasmid (pT7-VEGFR1) of take with the soluble fractions of T7 promotor coding VEGFR-1 is template, and t7 rna polymerase is transcribed out VEGFR-1RNA fragment, as the substrate of DNAzyme cleavage reaction.Cleavage reaction system is containing MgCl2(10mM), Tris-Cl (50mM, pH=7.5), the substrate RNA 1 μ l (1pmo1) of in-vitro transcription, DNAzyme 1 μ l (1pmo1), 0.1%DEPC water complements to 10 μ l.Reaction system is positioned over 85 ℃, 30 seconds; 37 ℃, 5 minutes; Incubation is 60 minutes again, then adds isopyknic stop buffer termination reaction (98% methane amide, the EDTA of 10mM and 0.1% loading dyestuff).Product is by 6% denaturing polyacrylamide gel electrophoresis 1 hour, 75W.Gel is shielded at PhosphorImager(phosphorus) exposure, band is used ImageQuant software to analyze.

As seen in Figure 2, except DT20, all there is the obvious cleaved products of band in DNAzyme DT10, DT11, DT12, DT13, DT14, DT15, DT16, DT17, DT18, DT19, and cleaved products size concentrates between 0.30-2.32kb.

Embodiment 3: the vascularization of target VEGFR-1 DNAzyme vitro inhibition

Endotheliocyte can be arranged in voluntarily capillary blood vessel spline structure after long-term cultivation, can observe by phase contrast microscope and transmission electron microscope.Micro-tube formation assay is the method that is usually used at present a kind of experiment in vitro of tumor-blood-vessel growth, often utilizes this phenomenon to observe and assess medicine and somatomedin for the impact of vasculogenesis in research.The method is to add extracellular matrix in original two-dimentional culture systems.Make endothelial cell growth in matrix, form dimensional culture, so that carry out Histological section and the structure of observing microtubule.

In order to screen the DNAzyme of target VEGFR-1mRNA best results, by micro-tube formation assay, analyze respectively and designed the ability that 11 kinds of synthetic DNAzyme suppress tubule formation.Transfection the day before yesterday, HUVEC is with 1-1.4*10 ⁴the even bed board in/hole, six orifice plates are placed in 37 ℃ and spend the night, and next day, cell density approached 80-90%.DNAzyme is carried out transfection by transfection reagent TMP, DNAzyme: TMP mixes 15min with the ratio of 1:3 before transfection, serum free medium cultivation changes complete training base into after 3 hours and spends the night, after transfection 48 hours, trysinization, complete training base stops the rear centrifugal 6min of 2000rpm of digestion, complete medium is washed recentrifuge one time, approximately with 30000 cells/well, plant in 96 orifice plates, be covered with the Matrigel of 100 μ l below, 2 as a child trained base afterwards changes low serum training base into, and 37 ℃ of overnight incubation, take pictures to next day for 4-6 hour.

By Fig. 3 and table 2, can find out, the endotheliocyte that target VEGFR-1mRNA DNAzyme DT18 processes does not almost see that complete microtubule forms, and the visible microtubule forming section of endotheliocyte that DT10, DT12, DT14, DT15, DT17, DT19 process is blocked; DT11, DT13, DT16, DT20 group all visible relatively complete microtubule form, but with respect to untreated fish group, the form of microtubule is slightly poor.Therefore, DT18 is chosen as guide's molecule (Lead molecule), is further studied.

Table 2: DNAzyme affects microtubule formation result and gathers *

* microtubule is formed to do not make significant difference (-); Part suppresses (+); Suppress completely (++).

Embodiment 4: target VEGFR1 DNAzyme vitro enzyme dynamics research

In cutting experiment, in fact the radioactive intensity of reaction system has reflected the process of DNAzyme cutting RNA substrate over time.Therefore, the radioactive intensity of system in the same time not in assaying reaction process, can obtain the information of cleavage reaction process, thereby obtain the kinetic parameter of reaction.

External dynamics index and the activity in vivo of DNAzyme have very strong dependency.Utilize following formula to calculate the external cutting efficiency (kobs) of DNAzyme, as the standard of selecting highly active DNAzyme:

[P]=[P∞](1-e ^-kobs·t)

Wherein [P] is the cutting rate of corresponding time point, and [P ∞] is final cutting rate, and t is the reaction times, and kobs is cutting efficiency constant.

Apply above principle of dynamics, the cutting power process of DT18 is analyzed.Under single-cycle condition, DT18 and contrast DNAzyme INV-Ctrl are hatched with together with the DNAzyme oligonucleotide substrate of 10 times of amounts respectively, to detect the nicking activity of DNAzyme.As shown in Figure 4 and Figure 5, DT18 can cut DNAzyme oligonucleotide substrate to result, and along with the prolongation of cleavage reaction time, visible substrate reduces gradually, and cleaved products increases gradually.Above result shows that the cutter of DT18 has sequence specific and time-dependent manner.

Embodiment 5: target VEGFR-1 DNAzyme suppresses VEGFR-1 and expresses in cell

With transfection reagent TMP transfection DNAzyme DT18 to HUVEC, by the protein level of VEGFR-1 in immunoblotting analysis of cells, result as shown in Figure 6.Near relative molecular mass 180kD band, HUVEC cell, transfection reagent group (TMP group), contrast transfection group (INV-Ctrl) all present positive band, and transfection DNAzyme DT18 group has no obvious band.Compare VEGFR-1 protein level obviously decline (P<0.05) after DT18 transfection with empty carrier transfection group.INV-Ctrl group is compared with TMP group, VEGFR-1 protein level no significant difference (P>0.05).With respect to blank group, INV-Ctrl group slightly declines with the VEGFR-1 protein level of TMP group, difference no difference of science of statistics (P>0.05).After this explanation DNAzyme DT18 transfectional cell, can obviously suppress the expression of VEGFR-1 albumen.

Embodiment 6: the impact of DNAzyme DT18 corneal new vessel

Cornea microencapsulation structure cornea rebirth blood vessel model is different from external dimensional culture cell formation tubule and evaluates vascularization ability, and it is a kind of in vivo model.In cornea micro-capsule, except endotheliocyte, also having the multiple factor such as various kinds of cell, extracellular matrix, body fluid to participate in, be equivalent to a microenvironment, and can observe directly the generation of blood vessel, is current widely used a kind of vasculogenesis analytical method.Utilize the method to detect the anti-angiogenesis effect of DT18, the DNAzyme DT18 of angiogenesis factor VEGF and target VEGFR-1mRNA is positioned in cornea of rats micro-capsule simultaneously, after 5 days, in VEGF stimulating growth region, by light microscopic, calculate angiogenic growth situation, after the area that new vessel is maximum is determined, 200 * the visual field under count (* 20 object lens and * 10 eyepieces, the visual field is 0.739mm2).

Found that, contrast molecule (INV-Ctrl) group is compared with physiological saline (Saline) group, both neovascularization growth no significant differences (P>0.05).With respect to physiological saline group and control group, DT18 has obviously suppressed the formation (Fig. 7 and Fig. 8) of blood vessel.In experimentation, there is not significant difference (P > 0.05) in the body weight change of three groups of rats.

Embodiment 7: target VEGFR1 DNAzyme DT18 suppresses mouse melanoma vasculogenesis

Melanin tumour b16 cell strain is the spontaneous tumor that is grown in C57BLP6 mouse ear root skin, is rich in abundant plexus vasculosus, a lot of blood vessel correlation factors are as high expression level all such as VEGF, bFGF, TGF β 1, easily shift, the B16 transplanted tumor of C57 mouse and metastatic tumor model are one of common experimental models of metastases invasion and attack research.Together with the DNAzyme DT18 of B16 cell and target VEGFR-1, inject subcutaneously, can see that experimental group tumour obviously dwindles, with respect to control group and physiological saline group, the reducing of tumour has statistical significance (P<0.05); INV-Ctrl group is compared with Saline group, INV-Ctrl group transplanted tumor volume slightly reduces, (Fig. 9), the Mouse Weight between group and group changes not statistically significant (P>0.05) to difference not statistically significant (P>0.05).We adopt MTT experiment to detect the effect of DT18 to B16 Growth of Cells.B16 cell seeding is in 6 orifice plates, with Fugene transfection DT18(2 μ g/ hole), peptic cell kind plate to 96 orifice plate after 48h, experiment is divided into 3 groups, every group 5 multiple holes, row MTT experiment after 48h, experimental result is found the propagation no significant difference (P>0.05) of three groups of cells, show DNAzyme on the propagation of B16 tumour cell without impact, further prove that DT18 is to form by affecting tumor vessel on melanomatous growth-inhibiting.

Embodiment 8: the impact of DNAzyme DT18 on the growth of subcutaneous transplantation knurl

Tumor inhibitory effect for checking DT18, is studied with nude mice Transplanted Human rhinopharyngocele cancer model.Found that: the DNAzyme DT18 of target VEGFR-1 acts on CNE1-LMPl(nasopharyngeal carcinoma cell) after subcutaneous transplantation knurl, gross tumor volume after one week DNAzyme group with respect to control group and physiological saline group, significantly dwindle, difference has statistical significance (P<0.05); Oligonucleotide group is with respect to physiological saline group, and gross tumor volume slightly reduces, and difference not statistically significant (P>0.05) (Figure 10).This result shows can the slow down rate of growth of nasopharyngeal carcinoma subcutaneous transplantation knurl of DNAzyme.

Active deoxidizing ribozyme DT18 is through subcutaneous being injected directly into after transplanted tumor tissue repeatedly, the capable immunohistochemical methods of 15 routine nasopharyngeal carcinoma transplanted tumor tissue sample, and result is not analyzed in the situation that knowing grouping by two refined hospital pathology department chief physicians in Hunan.In experimental group (DT18), the strong positive expression rate of VEGFR-1 is 0/5, positive expression 1/5, mainly be expressed in cytolemma and endochylema, become brown granular, the expression of physiological saline group (Saline) and control group (INV-Ctrl) VEGFR-1 is apparently higher than experimental group, and difference has statistical significance (P<0.01) (Figure 11).

Embodiment 9:DCE-MRI MR evaluation target VEGFR-1mRNA DNAzyme affects nasopharyngeal carcinoma transplanted tumor blood vessel

For inquiring into the impact of active deoxidizing ribozyme DT18 on mice-transplanted tumor angiogenesis, further clear and definite DT18 is affected angiogenesis and then is suppressed tumor growth by target VEGFR-1, and we adopt the inspection method DCE-MRI without wound, reaction tumor vessel state to assess the curative effect of DNAzyme.

Experiment divides three groups, and totally 15 mouse complete the parallel MRI of experiment and DCE-MRI smoothly, and measure Ktrans value.Ktrans refers to the volume transhipment constant of contrast medium from shifting to EES in blood vessel, main relevant with blood perfusion and vascular permeability etc., its iconography mark as tumor blood flow has obtained generally acknowledged, has been widely used in evaluating at present the effect of antitumor drug.In transplanted tumor, repeatedly inject after target VEGFR-1mRNA DNAzyme DT18, by DCE-MRI image is analyzed, as shown in figure 12, the Ktrans value of nasopharyngeal carcinoma transplanted tumor obviously declines, experimental group is compared with physiological saline group with control group, and Ktrans value has notable difference (P=0.028/P=0.026).Binding immunoassay group result, this experimental data again clear and definite DT18 has caused the change of tumour circulation and vascular permeability by suppressing the expression of VEGFR-1, reduced tumor tissues volume of blood flow.

Embodiment 10: the safety evaluation of target VEGFR1 DNAzyme

1. the impact of active deoxidizing ribozyme DT18 on Balb/C mouse organ index

Organ index refers to the ratio of internal organs weight in wet base and body weight, can react in a certain sense the impact of medicine on internal organs, can carry out preliminary toxicity judgement.Spleen is the immune organ of human body maximum, and index and spleen index can reflect the impact of medicine on immune organ indirectly.As can be seen from Table 3, by statistical analysis, with control group and the comparison of physiological saline group, experimental mice index and spleen index, liver index, lungs index and renal index are without significant difference, between three groups, compare mutually, difference does not all have statistical significance (P>0.05).Illustrate that active deoxidizing ribozyme DT18 all has no significant effect the non-specific immunity of mouse and vitals.

The impact of table 3:DT18 on organ index

2. the impact of active deoxidizing ribozyme DT18 on Balb/C mouse blood picture

As can be seen from Table 4, by statistical analysis, experimental mice and control group and the comparison of physiological saline group, the red corpuscle in blood picture, white corpuscle, oxyphorase, thrombocyte and neutrophil leucocyte all do not have notable difference (P>0.05); The data of three groups of mouse are compared equal no significant difference (P>0.05) between two, illustrate that active deoxidizing ribozyme DT18 has no significant effect the blood picture of mouse.

Table 4: hematological indices

3. active deoxidizing ribozyme DT18 is on the impact on Balb/C mouse hepatic and renal function

Any medicine enters in body all to be needed through liver metabolism, and gpt (ALT) and glutamic-oxal(o)acetic transaminase (AST) are requisite catalyzer in metabolism, is the important indicator of reflection liver function and healthy state.Medicine enters human body through renal excretion, and creatinine Scr and blood urea nitrogen Bun react the filtering function of renal glomerulus to a certain extent.As can be seen from Table 5, experimental group gpt and physiological saline group no significant difference, relative comparison group numerical value is higher, difference not statistically significant (P=0.292).The creatinine of DNAzyme experimental group and oligonucleotide group wants high compared with physiological saline group, but numerical value is in normal range, difference not statistically significant (P=0.478), careless transaminase, blood urea nitrogen and the control group of experimental mice and the comparison of physiological saline group, all do not have significant difference (P>0.05).This experimental result explanation active deoxidizing ribozyme DT18 has no significant effect the hepatic and renal function of mouse, to the vitals of mouse, is non-toxic and safe.

The impact of table 5:DT18 on biochemical indicator

ALT, gpt; AST, glutamic-oxal(o)acetic transaminase; Bun, blood urea nitrogen; Scr, serum creatinine.

4. active deoxidizing ribozyme DT18 is on the immune impact of Balb/C mouse

T lymphocyte is a kind of of peripheral blood lymphocyte, is the important indicator of evaluating cellular immunization, and therefore, on T, lymphocytic impact is to evaluate the important indicator of medicine on the impact of body's immunity situation to medicine.As can be seen from Table 6, each organizes CD3, the CD4 of mouse, the ratio of CD8 is very nearly the same, Epidemiological Analysis is found by statistics, the CD3 of experimental mice, CD4, CD8 and control group and life and the comparison of reason salt solution group, difference not statistically significant (P>0.05).This experimental data explanation active deoxidizing ribozyme DT18 has no significant effect the percentage of lymphocyte of mouse, and DNAzyme acts on the nonimmune mediation of biological effect possibility of tumour cell.

The impact of table 6:DT18 on lymphocyte subgroup ratio

5. the impact of active deoxidizing ribozyme DT18 on Balb/C mouse important organ

Hematoxylin-eosin staining method (HE dyeing) is that the difference of having a liking for acid-basicity according to endonuclear chromatin and tenuigenin is dyed respectively blueness and redness, comes evaluation of tissue to learn the most basic, most widely used a kind of technological method of difference.In order to assess the impact of DNAzyme DT18 on mouse important organ, we are by the capable HE dyeing of each organs and tissues.As seen from Figure 13, by each HE stained of microscopic examination, experimental mice and control group and the comparison of physiological saline group, liver, kidney, lungs and spleen be the normal configuration of visible internal organs all, have no obvious inflammatory cell infiltration and textural difference, each is organized does not all have notable difference between important organ histopathology.This coloration result explanation active deoxidizing ribozyme DT18 has no significant effect the morphology of the important organ of mouse, and intravenously medication is safe.

6. active deoxidizing ribozyme DT18 pharmacokinetic profiles in vivo

The distribution, elimination, the absorbing rule that enter drug disposition for further clear and definite DNAzyme DT18, our plans (10mg/kg) from mouse tail vein injection DT18 are inquired into medicine and at target organ, are reached required time and the action time of effective drug effect concentration, after disposable tail vein injection medicine, different time points is measured drug level, Plasma Concentration-the time data of matching is processed through 3P87 software analysis, meets the open two-compartment model of intravenously administrable.The pharmacokinetics process of DT18 in Mice Body shows as first order kinetics feature, and pharmacokinetic parameter is in Table 7.T _1/2α distribution half-life is 6min, illustrates that its distribution speed is in vivo very fast, and the elimination transformation period is 46.7h, illustrates that the medicine transformation period is in vivo longer, can within the longer time, bring into play drug effect concentration.Maximum plasma concentration is 84.4mg/ml, area 97mg/mlhr under pharmaceutical concentration-time curve, and apparent volume of distribution is 890ml/kg.

Pharmacokinetic analysis in table 7:DT18 body

T _1/2α: distribution half-life (distribution half-life); T1/2 β: eliminate the transformation period (elimination half-life); Cmax maximum plasma concentration (maximum concentration); Area under AUC pharmaceutical concentration-time curve (area under the curve); The apparent volume of distribution of Vd (aparent volume of distribution).

Claims (9)

1. a species specificity suppresses the DNAzyme of expression of vascular endothelial growth factor receptor genes, it is target sequence that this DNAzyme be take mRNA or the pre-mRNA of vascular endothelial growth factor receptor VEGFR-1 gene, comprise: a catalysis sequence GGCTAGCTACAACGA, and be connected to its two ends 5 ' end binding sequence and 3 ' end binding sequence; Wherein, described catalysis sequence is for cutting arbitrary purine nucleotides of target sequence and the connecting key of pyrimidine nucleotide; 5 ' end binding sequence and 3 ' end binding sequence are respectively 5～12 Nucleotide, complementary with the sequence of target sequence cleavage site both sides respectively.

2. DNAzyme according to claim 1, is characterized in that, described DNAzyme is comprised of 27～39 nucleotide residues.

3. DNAzyme according to claim 1, is characterized in that, the Δ G °≤-10kcal/mol that 5 ' end binding sequence of described DNAzyme and 3 ' end binding sequence are combined with target sequence separately.

4. DNAzyme according to claim 1, is characterized in that, the cleavage site of described target sequence is the connecting key of AU or GU.

5. DNAzyme according to claim 1, is characterized in that, described DNAzyme is selected from one of SEQ ID No:1 to SEQ ID No:11 in sequence table.

6. DNAzyme according to claim 1, is characterized in that, in 5 ' end binding sequence of described DNAzyme and/or 3 ' end binding sequence, contains 1-6 phosphorothioate bond.

7. DNAzyme according to claim 1, is characterized in that, described DNAzyme has following one or more chemically modifieds: 3 '-3 ' Opposite direction connection base, lock nucleic acid, peptide nucleic acid(PNA) and glycosyl 2 '-methoxyl group are modified.

8. DNAzyme according to claim 1, is characterized in that, the VEGFR-1 gene of described DNAzyme target is the VEGFR-1 gene of people and/or mouse.

9. the application of the arbitrary described DNAzyme of claim 1～8 in the medicine of preparation inhibition tumor-blood-vessel growth and tumor growth.

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