CN101886076A - Oligonucleotide for inhibiting blood-mediated inflammatory reaction in pancreatic islet transplantation - Google Patents

Abstract

The invention discloses an oligonucleotide for inhibiting a blood-mediated inflammatory reaction in pancreatic islet transplantation. The oligonucleotide comprises 7 to 75 nucleotides, at least comprises 3 three continuous achiral 5' to 3' nucleoside-triphosphate bonds, comprises a 5'-end untranslated region, a translation initiation region, a 3'-end untranslated region and/or a translation termination region which are complemented for a TF gene, and has the melting temperature of between 75 DEG C and 115 DEG C under the concentration of 1mmol. The oligonucleotide can be a ribose oligonucleotide or a deoxyribose oligonucleotide, can be modified chemically and can be used for preparing medicinal compositions for resisting the blood-mediated inflammatory reaction in the islet transplantation.

Description

A kind of oligonucleotide that suppresses in the pancreatic islets transplantation through the inflammatory reaction of blood mediation

Technical field

The present invention relates to a class and in pancreatic islets transplantation, suppress oligonucleotide, be specifically related to a class and suppress the application of antisense oligonucleotide in the pancreatic islets transplantation process that tissue factor TF expresses through the inflammatory reaction of blood mediation.

Background technology

Diabetes are global, that sickness rate increases gradually serious metabolic diseases, and the exogenous insulin treatment can not be stablized controlling blood sugar, can not prevent the generation and the progress of diabetic complication.Setting up the endogenous insulin excretory system is the focus that people are studying always, pancreatic islets transplantation brings hope [the Ridgway DM of healing for the type 1 diabetes patient, White SA, Nicholson ML.Pancreatic islet cell transplantation:progress in the clinical setting treatments in endocrinology.2003,2 (3): 173-189; White SA, James RF, Swift SM.Human islet cell transplantation-future prospects Diabetic Medicine.2001,18 (2): 78-103].

A large amount of studies show that, initial stage after the pancreatic islets transplantation is often with [the Ricordi C.Human islet cell transplantation:new perspectives for an old challenge.Diabetes Review that loses of a large amount of pancreas islet, 1996,4:356-369.], the reaction that the moment that wherein neo-implanted pancreas islet contacts with blood flow causes is called as inflammatory reaction (the Instant blood-mediated inflammatory reaction that mediates through blood immediately, hereinafter to be referred as IBMIR) [Ozmen L, Ekdahl KN, Elgue G, et al.Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets:possible application of the thrombin inhibitor melagatran in clinical islet transplantation.Diabetes, 2002,51:1779-1784.], it is the damage the earliest after pancreas islet is implanted, the feature of IBMIR is the activation that graft causes acceptor blood coagulation system and complement system, and these waterfall type cascade reactions cause in 70% the several minutes of graft after transplanting dead.The research of IBMIR external model is verified, and when the pancreatic islets transplantation thing contacted with blood, the key factor that the IBMIR reaction starts was a coagulation system activation.Existing report shows: the pancreatic islets transplantation thing contacts with acceptor blood in the several minutes of back, the tissue factor (TF) that discharges has just activated the VII factor of acceptor blood, activation [the Walsh PN that has caused exogenous cruor pathway, Sinha D, Koshy A, et al.Functional characterization of platelet-bound factor XI a:retention of factor XIa activity on the platelet surface.Blood, 1986,68:225-230.], this ectogenic activation plays a significant role in the IBMIR reaction, the collagen of graft exposure simultaneously etc. are incitant XI again, thereby activate intrinsic coagulation approach [Walsh PN, Sinha D, Koshy A, et al.Functional characterization of platelet-bound factorXIa:retention of factor XIa activity on the platelet surface.Blood, 1986,68:225-230.].In vivo, the physiological hemostasis nearly all starts by tissue factor (TF) and FVIIa formation mixture with thrombotic coagulation process.Therefore, the how expression of inhibition of transplant TF to prevent the damage of IBMIR to the pancreatic islets transplantation thing, has become the focus of transplantation medicine research.

Antisense technology is according to the antisense oligonucleotide of nucleic acid hybridization principle design at the particular target sequence, through synthetic or manually express the vector expression antisense oligonucleotide, combine with target sequence (RNA or DNA) by the base complementrity principle, the expression of sealing or inhibition specific gene, thus the function of pair cell exerts an influence.Antisense oligonucleotide comprises sense-rna, antisense DNA and ribozyme (Ribozyme) or DNAzyme (DNAzyme).

Antisense technology has tangible advantage: (1) antisense oligonucleotide is synthetic at the sequences Design of specific said target mrna (or DNA), has high specificity; (2) antisense oligonucleotide is the target gene design synthetic at known array, because target-gene sequence is known, antisense oligonucleotide only has 15-30 base usually, and is simple in structure, easily design and external a large amount of synthetic; (3) antisense oligonucleotide enters in the cell and has nothing to do with the cell cycle, not only can enter propagation phase cell but also can enter non-propagation phase cell; (4) antisense oligonucleotide does not contain virus sequence, can not produce immune response, can not be integrated in the host chromosome yet.Particularly Antisense RNA Technique has important in theory and realistic meaning, can be used as a kind of effective means of gene therapy and play a significant role, application [Chang-Mei Liu is all arranged in multi-field research at present, Zhuo Yang, Chao-Wu Liu, RongWang, Po Tien, Roderick Dale and L-Q Sun (2007) .Effect of RNA oligonucleotide targeting Foxo-1 on muscle growth in normal and cancer cachexia mice.Cancer Gene Therapy, 1-8; Chang-Mei Liu, Zhuo Yang, Chao-Wu Liu, Rong Wang, Po Tien, Rod Dale, L-Q Sun (2007) Myostatin Antisense RNA-mediated Muscle Growth in Normal and Cancer Cachexia Mice.GeneTherapy, 1-6.].

Summary of the invention

The antisense oligonucleotide that the purpose of this invention is to provide the effective selectively targeted TF gene of a class suppresses the generation of IBMIR in the pancreatic islets transplantation.

IBMIR be pancreas islet implant the back by tissue factor (TF) mediation damage the earliest, cause in the several minutes of graft after transplanting dead.The present invention reaches the purpose of control pancreatic islets transplantation thing damage by the adjusting to TF genetic expression.

The oligonucleotide of IBMIR reaction in the inhibition pancreatic islets transplantation provided by the present invention has following feature:

(1) contains 7～75 Nucleotide;

(2) contain 3 successive achiralitys 5 ' at least to key between 3 ' phosphoric acid nucleoside;

(3) be complementary to 5 of TF gene ' end non-translational region, translation initiation district, 3 ' end non-translational region and/or translation termination district;

(4) under 1 millimolar concentration, has 75 ℃～115 ℃ melting temperature (Tm) (Tm).

When oligonucleotide of the present invention when suppressing in the pancreatic islets transplantation IBMIR reaction, can use a kind ofly, also can use two kinds or more kinds of.When using multiple oligonucleotide, these several oligonucleotide will be separately and the different zones complementation in 5 of target gene ' end non-translational region, translation initiation district, 3 ' end non-translational region and/or translation termination district.Described target gene is the TF gene.

Preferably, above-mentioned oligonucleotide is made up of 10～26 Nucleotide, has 40 ℃～85 ℃ melting temperature (Tm) under 1 pmol concentration.

In order to use oligonucleotide effectively in vivo, it is carried out chemically modified can increase the stability that oligonucleotide is antiacid and antienzyme is degraded significantly.To oligonucleotide provided by the present invention can be to carry out a place or many places chemically modified on the basis of natural acid molecular structure, obtains the modification oligonucleotide with specific structure and physics-chem characteristic.Described chemically modified comprises the modification to the phosphoric acid ester bond between base, glycosyl and the Nucleotide, terminal protection, protonated processing etc.

Oligonucleotide can be by to its 5 ' end and/or 3 ' terminal modified characteristic that obtains the degraded of anti-exonuclease.These end modified comprising: the terminal base that oppositely connects; Terminal nucleotide is a dideoxy nucleotide; Have blocking group at 3 ' end and/or 5 ' end; 3 ' end closure and/or 5 ' end closure.

Connecting key between ribosyl and the mononucleotide is called the trunk structure of nucleic acid.The modification of trunk structure is comprised that connecting key and other can enhanced stability and the modification of affinity, for example the glycosyl structure is modified, concrete example has: base with respect to natural dextrorotatory isomer sugar when reverse, can use left-handed (L-) isomer ribodesose; In 2 ' position or 3 ' the Yeast Nucleic Acid or the thymus nucleic acid that replace.Common as have the modification oligonucleotide of substituted radical on glycosyl 2 ' position, described substituted radical includes but not limited to oxygen, methoxyl group, propoxy-, methoxy-oxyethyl group, fluorine, chlorine, bromine, iodine.

Connecting key between the Nucleotide of the preferred oligonucleotide of the present invention (key between phosphoric acid nucleoside) is (achiral) of achirality, contain at least 3 successive achirality connecting keys, preferably contain 7～12 successive achirality connecting keys, most preferably whole connecting keys is achiral.In some cases, achirality connecting key and chirality connectivity can alternately exist, for example, chirality connecting key of three successive achirality keyed jointings, or four successive achirality connecting keys are followed two chirality connecting keys etc.The achirality connecting key includes but not limited to: phosphodiester bond, thiophosphoric acid diester linkage.

Between the phosphoric acid nucleoside in the oligonucleotide of the present invention key can be 5 ' to 3 ', 5 ' to 2 ' or 5 ' to 5 ' or with the combination of upper type.The modification of this connecting key can be intramolecular (single or repetition), also can be the end at RNA or DNA.

Oligonucleotide of the present invention comprises ribose oligonucleotide and deoxyribose oligonucleotide.Described oligonucleotide sequence is preferably from SEQ ID NO:1～3.

Oligonucleotide of the present invention can be an antisense oligonucleotide, comprises the Antisensedigonucleotsequence sequence of target human or animal (for example pig) TF gene.The sequence of representational antisense oligonucleotide sees Table 1.

Can prepare the pharmaceutical composition of multiple anti-IBMIR with oligonucleotide of the present invention as active substance, be applicable to the animal and human.Described animal comprises pig, ox, sheep, monkey etc.Can contain various pharmaceutically acceptable vehicle, assistant agent in the described pharmaceutical composition.In the described pharmaceutical composition, can contain one or more described oligonucleotide, such as, two kinds, three kinds, four kinds, the modification oligonucleotide more than five kinds or five kinds can be contained.

Oligonucleotide provided by the present invention can carry out extracorporeal treatment to graft by ex vivo (in vitro) mode before islet cell transplantation.

Oligonucleotide provided by the invention for animal or human's dosage is: per kilogram of body weight 0.01～100mg.

The present invention introduces antisense oligonucleotide in the IBMIR gene therapy first, is target with TF, uses the antisense technology means, and the expression by downward modulation islet cells TF suppresses the generation of IBMIR.

Description of drawings

Fig. 1 is that each the sense-rna transfection with target TF enters islet cells, extracts total RNA, suppresses the result that TF expresses with RT-PCR from mRNA level detection TF sense-rna.

Fig. 2 is the result that different time RT-PCR detects the TF expression behind the sense-rna TF1006 transfection islet cells.

Fig. 3 is that the sense-rna transfection of target TF enters behind the islet cells the 4th day and extracts total protein, detects the result that TF expresses with Western blotting from protein level.

Fig. 4 has shown the blood clot that the external IBMIR reaction of the sense-rna transfection islet cells of target TF back forms.

Embodiment

The present invention is described in further detail by the following examples, but the content to invention is limited never in any form, as Antisensedigonucleotsequence sequence listed in the table 1 is representational molecule, any other can suppress the antisense oligonucleotide that TF expresses, and all can be used in the pharmaceutical composition of the present invention.

One, the design of TF antisense oligonucleotide, synthetic and modification

The method of design antisense oligonucleotide comprises: (1) selects contiguous with target region or an overlap oligonucleotide, (2) measure and target gene bonded Gibbs free energy level, (3) analyze hybridization melting temperature (Tm) (Tm), (4) carry out the sequence library analysis, to determine its target site zone of living in, for example, 5 ' end non-translational region, the translation initiation district, 3 ' end non-translational region and translation termination district.Preferred antisense oligonucleotide generally is adjacent to the translation initiation district or has a Nucleotide at least and the translation starting point overlapping.This overlapping is no less than three Nucleotide in some example.

Secondary and higher structure feature based on RNA are carried out the design of sense-rna or DNA, generally can be selected in 5 ' end regions.In some cases, can be selected in 3 ' end regions, or around the RNA splice point.

Antisense oligonucleotide of the present invention can analyze to determine the bonding strength of itself and target RNA by the Gibbs free energy.This analysis can be undertaken by computer program, program commonly used can Ftp: //rna.chem.rochester.eduThe website is found, and can be with reference to people's such as Matthews article (J.Mol.Biol, 1999,288,911-940; RNA, 1999,5,1458-1469).The Gibbs free energy of oligonucleotide and target RNA hybrid molecule is general≤-20kcal (37 ℃).Be preferably≤-25kcal, for the oligonucleotide of a 12-14 unit, its free energy is≤-15kcal; The hybridization free energy of the oligonucleotide of 15-17 unit is≤-20kcal; The hybridization free energy of the oligonucleotide of 18-20 unit is≤-25kcal; The hybridization free energy of the oligonucleotide of 21-23 unit is≤-30kcal; The hybridization free energy of the oligonucleotide of 24-26 unit is≤-35kcal.

The sense-rna sequence of the TF gene of GeneID 396677 is as shown in table 1 among the target Genbank that screens.

The representative target TF of table 1. gene the sense-rna sequence

Code	The sense-rna sequence	Sequence number
			??TF114	??5′-GUC?UCC?AUG?UCU?ACC?AGU-3′	??1
??TF1006	??5′-ACA?GUG?CUU?CCU?UUA?UGA-3′	??2
			??TF1009	??5′-CCA?ACA?GUG?CUU?CCU?UUA-3′	??3

Chemically modified: adopt ribosyl 2 '-O-methyl to modify and 3 ' terminal phosphate-O-propyl group modification to above-mentioned sense-rna in the present embodiment.

Two, select suitable transfection reagent and transfection conditions

Chosen process has adopted TMP (tetra (4-methyridyl) porphyrine) and two kinds of transfection reagents of Oligofectamine.Divide TMP transfection group and Oligofectamine transfection group with islet cells, every component three holes, every hole reagent total amount is 1ml, adding islet cells 1000IEQ (pancreas islet equivalent) and concentration is the FITC-Antisense RNA 8 μ l of 1000mM;

The TMP transfection group is divided into low concentration group, middle concentration group and high density group again:

Low concentration group adds TMP 19.2 μ l;

Middle concentration group adds TMP 38.4 μ l;

The high density group adds TMP 76.8 μ l.

The Oligofectamine transfection group also is divided into low concentration group, middle concentration group and high density group:

Low concentration group adds Oligofectamine 4 μ l;

Middle concentration group adds Oligofectamine 8 μ l;

The high density group adds Oligofectamine 16 μ l.

4 hours adding calf serum to end levels are 10% (volumn concentration) after the transfection, place CO ₂Incubator is cultivated 12 hours in fluorescent microscope observation down.More than test repeats 3 times, and fluorescent microscope is observed down, determines best transfection reagent and concentration.Under the fluorescent microscope, with wavelength 488nm ultraviolet excitation, as seen: the islet cells with the low middle high group of TMP transfection has only on a small quantity by fluorescent mark; High group had the some amount islet cells by fluorescent mark during the Oligofectamine transfection was low, with maximum by fluorescently-labeled cell quantity in the high density group.The result shows that the transfection reagent Oligofectamine of high density more helps the islet cells transfection.

Three, the sense-rna of the effective target TF of screening

Is transfection reagent with the sense-rna of three target TF of design synthetic with Oligofectamine according to the top condition of finding out, the sense-rna of TF is transfected in the islet cells, extract total RNA, detect the expression of TF by RT-PCR, detect the inhibition activity of the sense-rna of target TF at rna level.

Design of primers is as follows:

TF gene primer sequence:

Forward primer: 5 '-GGAGCCTCTGTATGAGAACT-3 '

Reverse primer: 5 '-CGGAGGCTTAGGAAAGTGTT-3 '

Internal control gene (18S RNA) primer sequence:

Forward primer: 5 '-CCGAGAAGTTTCAGCACATCC-3 '

Reverse primer: 5 '-TGGCAGTGATAGCGAAGGCT-3 '

The RT-PCR amplification program:

Three sense-rna transfections are entered behind the islet cells the 2nd day extract total RNA, the detected result of quantitative RT-PCR as shown in Figure 1, wherein TF1006, TF1009 and TF114 are the sense-rnas of target TF, do not carry out any treatment group in contrast, other is provided with the Control RNA meaningless RNA of any gene (not at) transfection group as parallel control, the inhibition effect of TF1006 is best as can be seen, and TF1009 and TF114 also can partly suppress the expression of TF.Fig. 2 is the inhibition situation of in eight days TF being expressed after the TF1006 transfection, and as can be seen from Figure 2, second day TF1006 can obviously suppress the TF expression after the transfection, suppresses effect and lasts till the 8th day, suppresses the most obvious at the 4th day.

The sense-rna transfection enters behind the islet cells the 4th day extracts total protein, detects result that TF expresses as shown in Figure 3 with Western blotting from protein level, and the sense-rna of target TF suppresses TF at protein level expresses.

Four, set up external IBMIR model

(Medtronic.Inc USA) makes in-vitro simulated blood vessel inner blood round-robin pipeline (Tubing Loops), length 50cm, internal diameter 6.3mm by silicone tube.Totally 3 groups of silicone tubes, the heparin saline 0.7ml that in every group of silicone tube, adds different concns respectively, the every interior healthy people's fresh whole blood dosage that adds non-anti-freezing of pipe is 7ml, make the ultimate density of heparin in the interior fresh blood of pipe be respectively 0.001mg/ml, 0.01mg/ml, 0.02mg/ml, the detection of before experiment, taking a sample of each group pipe inner blood, each group Guan Junyong is waved the detection of taking a sample after simulate blood flowed 60 minutes, the detection index is routine blood test, coagulation indexes and complement system experimental index, to determine best heparinization dosage in managing.

Silicone tube is fixed in 37 ℃ of shaking tables, and row arc pendulum model is waved, and regulating shaking table speed is 100 times/minute, and rocking tendency is about 30 °, makes in the pipe blood reach maximum and waves amplitude, simulates quick mobile blood, more than respectively organize repeated experiments 6 times.

The research sample packet:

Group before the experiment: healthy people's fresh whole blood is not put into the extracorporeal circulation pipeline after extracting out, does correlation detection immediately;

Control group: healthy people's fresh whole blood 7ml adds 100 μ l nutrient solutions, puts into the extracorporeal circulation pipeline and waves 60 minutes;

Experimental group: healthy people's fresh whole blood 7ml adds the 100 μ l nutrient solutions that comprise different quantities (being respectively 500IEQ, 1000IEQ) pancreas islet, specific as follows: 500IEQ (pancreas islet equivalent) experimental group, 1000IEQ experimental group, each experimental group is put into the extracorporeal circulation pipeline, is further divided into 5,10,60 minutes 3 groups according to the time of waving.

Below respectively organize repeated experiments 6 times, determine that the IBMRI external model adds pancreas islet quantity.

In the experiment to extracorporeal circulation pipeline internal surface heparinization, the optimal dose heparinization of determining according to previous experiments.

Test item:

(USA) filter, with the liquid portion of donor thrombus and non-thrombus in the blood separately by BD company through 70 μ m filtering nets for each group's blood.The capable HE dyeing of solid thrombus pathology detect [Xu B Y, Yu Y, Al-Abdullah I H, et al.Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice.[J] .Transplant Proc, 2008,40 (2): 584-586.].The non-thrombus of blood partly detects routine blood test (comprising thrombocyte, white corpuscle etc.), goes out the clotting time (PT, APTT); The ELISA method detects TAT (thrombin-antithrombine complex: the index of coagulation system activation) [Berman D M, Cabrera O, Kenyon N M, et al.Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.[J] .Transplantation, 2007,84 (3): 308-315; Cabric S, Sanchez J, Lundgren T, et al.Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation.[J] .Diabetes, 2007,56 (8): 2008-2015.]; The ELISA method detects complement C 3 (people's complement fragment), C5b-9 (robot end complement mixture) content [Rood P P, Bottino R, Balamurugan A N, et al.Reduction of early graft loss after intraportal porcine islet transplantation in monkeys.[J] .Transplantation, 2007,83 (2): 202-210.]; Pork insulin content [Goldman H C E.Human pancreatic islets in culture:effects of supplementing the medium with homologous and heterologous serum[J] .Science in the chemiluminescence determination blood, 1976 (192): 1014-1016; Tjernberg J, Ekdahl K N, Lambris J D, et al.Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may causetissue loss in clinical islet transplantation.[J] .Transplantation, 2008,85 (8): 1193-1199.].

The pipeline adding fresh blood that contains three kinds of different concns heparin waves after 60 minutes with before the experiment organizes (sample that is fresh extraction detects immediately) contrast, when heparin concentration is 0.001mg/ml, to blood coagulation function effect minimum, analogue body inner blood and to keep its normal physiological function constant for a long time; When concentration is 0.01mg/ml, 0.02mg/ml, though also can well keep the flowability of blood, keep the stable of various hemocyte quantity in the blood, go out the clotting time index and all obviously prolong, what can not keep blood normally goes out coagulation function.Therefore, in the follow-up external IBMIR experiment of carrying out, heparinization dosage is 0.001mg/ml.

The fresh whole blood of table 2. different concns heparin is biochemical and go out the clotting time detected result

Annotate: * compares with group before the experiment, and difference has statistical significance;

/ blood coagulation takes place, index of correlation fails to record;

*/and the APTT overlong time, exceed the apparatus measures scope, fail to measure.

From table 3,4,5 as can be seen, wave the experimental group that contained different islet cells quantity in 60 minutes, thrombocyte (PLT), neutrophil leucocyte (Neutrophils), monocyte (Monocytes), lymphocyte (Lymphocytes) obviously consume, and blood coagulation system, complement system significantly activate simultaneously; And control group does not have thrombosis, its significantly change be after 60 minutes platelet consumption 15%.And when adding the 1000IEQ islet cells, can form apparent in view thrombus, and prompting blood coagulation system and each index of complement system activated have significant difference than 500IEQ islet cells group, therefore in the external IBMIR experiment of proceeding in the back, we will adopt the 1000IEQ islet cells to experimentize.

Table 3. waves the blood hemocyte detected result of each group after 60 minutes

Annotate: * compares with control group, and difference has statistical significance;

#500IEQ compares with the 1000IEQ group, and difference has statistical significance.

Table 4. waves the blood complement and the Regular Insulin result of each group after 60 minutes

Annotate: * compares with control group, and difference has statistical significance;

#500IEQ compares with the 1000IEQ group, and difference has statistical significance.

Table 5. is respectively organized the thrombus weight (g) of different time points

Five, the external IBMIR test of TF sense-rna transfection porcine islet

Determine external IBMIR experiment condition by above-mentioned experimental result, select the higher TF1006 of transfection efficiency for use, the porcine islet of TF1009 and TF114 group carries out external IBMIR experiment.

The research grouping

Blank group: untreated porcine islet;

Blank transfection control group: add the porcine islet of cultivating behind the transfection reagent oligofetctmine;

TF sense-rna-Oligofectamine transfection experiment group: Tubing Loops model is divided into TF1006 group, TF1009 group and TF114 group, with the porcine islet after the TF1006 transfection, the porcine islet after porcine islet after the TH1009 transfection and the TH114 transfection is inserted other Tubing Loops model of respective sets respectively respectively.

More than each group add that 37 ℃ of constant temperature waved repeated experiments 6 times 30 minutes in the external IBMIR model.

Test item

(USA) filter, and the solid thrombus is weighed by BD company through 70 μ m filtering nets for each group's blood.The non-thrombus of blood partly detects routine blood test (comprising thrombocyte, white corpuscle etc.); The ELISA method detects TAT (thrombin-antithrombine complex: the index of coagulation system activation); The ELISA method detects complement C 3 (people's complement fragment), C5b-9 (robot end complement mixture) content.

The result is as shown in table 6, and the hemocyte numerical value of TF1006, TF1009, TF114 group is learned processing data by statistics than the control group height, represents that promptly lymphocyte, monocyte consumption are less, and it is little that lymphocyte system and non-specific immunity system activate degree; Hematoblastic numerical value is than the control group height, and prompting platelet activation degree is low than control group, and the coagulation system activation degree is also low than control group.

Complement C 3, the C5a-9 content of TF1006, TF1009, TF114 and control group are compared, be worth lower, C3a, C5b-9 are few than control group in the prompting blood plasma, C3a, C5a further activate granulocyte so, amount of mononuclear cells will reduce, white corpuscle is assembled this level of response to the pancreatic islets transplantation thing and will be alleviated, and forms the thrombus degree and alleviates.

Estimate the occurrence degree of IBMIR according to the big I of blood clot, to filter out gravimetric value and the control group of blood clot lighter for each group of sense-rna silence in the experiment, and statistical significance is arranged, and the intensity of prompting IBMIR generation is lower.

Show that according to above all analyses the expression of TF1006, TF1009, the reticent islet cells TF of TF114 energy is effectively lowered the activation of coagulation system degree, thereby alleviated the occurrence degree of IBMIR, in these three sense-rnas, the reticent TF best results of TF1006.

Respectively organize routine blood test, thrombus and complement detected result before and after the table 6.IBMRI reaction

Annotate: * represents to compare with blank transfection control group, and P＜0.05 difference has statistical significance

The external IBMIR reaction of target TF sense-rna transfection islet cells back forms blood clot as shown in Figure 3, from left to right be TF1006 transfection group, TF1009 transfection group, TF114 transfection group, blank transfection control group and blank group successively, the gravimetric value and the control group of the blood clot that each sense-rna transfection group filters out are lighter.

The weight evaluation that table 7.TF sense-rna suppresses thrombosis

Annotate: * represents to compare with blank transfection control group, and P＜0.05 difference has statistical significance

Claims (10)

1. oligonucleotide that suppresses in the pancreatic islets transplantation through the inflammatory reaction of blood mediation has following feature:

1) contains 7～75 Nucleotide;

2) contain 3 successive achiralitys 5 ' at least to key between 3 ' phosphoric acid nucleoside;

3) be complementary to 5 of TF gene ' end non-translational region, translation initiation district, 3 ' end non-translational region and/or translation termination district;

4) under 1 millimolar concentration, has 75 ℃～115 ℃ melting temperature (Tm).

2. oligonucleotide as claimed in claim 1 is characterized in that, this oligonucleotide is made up of 10～26 Nucleotide, has 40 ℃～85 ℃ melting temperature (Tm) under 1 pmol concentration.

3. oligonucleotide as claimed in claim 1 is characterized in that, this oligonucleotide is the modification oligonucleotide through chemically modified.

4. oligonucleotide as claimed in claim 3 is characterized in that, described modification oligonucleotide has blocking group at 3 ' end and/or 5 ' end.

5. oligonucleotide as claimed in claim 3 is characterized in that, described modification oligonucleotide is 3 ' end closure and/or 5 ' end closure.

6. oligonucleotide as claimed in claim 3 is characterized in that, described modification oligonucleotide is to have substituted radical on glycosyl 2 ' position, and described substituted radical is oxygen, methoxyl group, propoxy-, methoxy-oxyethyl group, fluorine, chlorine, bromine or iodine.

7. oligonucleotide as claimed in claim 1 is characterized in that, this oligonucleotide is the antisense oligonucleotide of target human or animal TF gene.

8. oligonucleotide as claimed in claim 7 is characterized in that, the sequence of described antisense oligonucleotide is selected from SEQ ID NO:1～SEQ ID NO:3.

9. arbitrary described oligonucleotide is used for preparing the purposes of anti-pancreatic islets transplantation through the pharmaceutical composition of the inflammatory reaction of blood mediation among the claim 1-8.

10. purposes as claimed in claim 9, it is characterized in that, contain one or more described oligonucleotide in the described pharmaceutical composition, when containing multiple oligonucleotide, this multiple oligonucleotide respectively with the different zones complementation in 5 of TF gene ' end non-translational region, translation initiation district, 3 ' end non-translational region and/or translation termination district.

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