CN101914541B - Inhibitor for inhibiting expression of MBD2 and application thereof - Google Patents
Inhibitor for inhibiting expression of MBD2 and application thereof Download PDFInfo
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Abstract
The invention discloses an inhibitor for inhibiting the expression of MBD2 and application thereof, and relates to an inhibitor capable of inhibiting the expression of methylated CpG binding domain protein 2 in vivio and in vitro. The inhibitor comprises a nucleic acid molecule, a genetic structure, an siRNA molecule and a compound containing one or more of the nucleic acid molecule, the genetic structure and the siRNA molecule, and can be introduced into mammalian cells to inhibit the expression of the MBD2. The inhibitor for inhibiting the expression of the MBD2 is an siRNA, which has a nucleotide sequence in which the sense strand is 5'-GCAAGAGCGAUGUCUACUA-3' and the antisense strand is 5'-UAGUAGACAUCGCUCUUGC-3', or of which the 3' end is provided with two suspended basic groups tt, and has a nucleotide sequence in which the sense strand is 5'-GCAAGAGCGAUGUCUACUAtt-3' and the antisense strand is 5'-UAGUAGACAUCGCUCUUGCtt-3'. The application of the inhibitor in preventing and treating related diseases of vascular endothelial cell dysfunction provides a novel option for preparing medicaments or preparations for preventing and treating the related diseases of the vascular endothelial cell dysfunction by taking the MBD2 as a target.
Description
Technical field
The invention belongs to molecular biology and biological medicine technology field, be specially with elevation of methyl-CpG binding domain 2 (Methyl-CpG Binding Domain Protein 2, MBD2 is designated hereinafter simply as MBD2) be to suppress inhibitor and application thereof that MBD2 expresses by target spot.
Background technology
Vascular endothelial cell is mounted lining at the internal surface of whole cardiovascular systems, it not only regulates nutritive substance, the transportation of multiple bioactive molecules and blood cell, and be an important homeostasis organ, normal endotheliocyte can be replied multiple physiology and chemical signal generation, discharge a group autocrine and paracrine substance such as nitrogen protoxide (nitric oxide, NO), prostacyclin and the endothelium derived hyperpolarization factor etc., regulate the tension force of blood vessel, anticoagulant, cell adhesion, smooth muscle cell proliferation, in the vascular function regulation and control, playing the part of crucial role, in vascular disease and initial defence thereof, playing a significant role simultaneously.But, when endotheliocyte is exposed to the cardiovascular risk factor such as smoking, diabetes, hypertension, hyperlipemia, obesity and chronic system inflammation etc., it will be in a kind of active state------endothelial dysfunction, express the synthetic NO minimizing of a large amount of chemokineses, cytokine and adhesion molecule and eNOS, biological activity reduction etc., cause generation, the development of cardiovascular disorder, the people of often causing a disease is dead and disabled.Therefore blood vessel endothelium dysfunction is the major cause of cardiovascular disorder.Because the cause of disease of vascular disease comprises heredity and posteriori many factors, employed medicine spininess is to the different causes of disease, and various, and therefore establishing one is a huge challenge for Different types of etiopathogenises, therapeutic goal best, that side effect is minimum.
As a main epigenetics integral part, right and wrong are usually seen in animal and human's class vascular disease in the change of dna methylation pattern.Nearest studies show that, dna methylation can be used as one " footprint " reflection environmental exposure to the consequence of dissimilar cells.This dna methylation pattern can be by conservative elevation of methyl-CpG binding domain family (MBD) " reading ", comprising MBD1, and MBD2, MBD3, MBD4 and MeCP2.They and its chaperone are brought into play positive effect transcribing of dna methylation mediation in inhibition and/or the heterochromatic formation.Every kind of MBD albumen is different from the avidity of methylate DNA, and what avidity was the highest is MBD2 albumen, but MBD3 can not selectivity identification methylate DNA in the Mammals.Can set up MeCP2 at present, MBD1, the animal model of MBD2 and MBD4 defective, but the MBD3 defective causes embryonic death.Studies show that MeCP2 or MBD1 deficient mice are relevant with specific neuroscience defective; And MBD4 is found to suppress the CpG sudden change and tumour occurs.By contrast, the MBD2 deficient mice shows an almost normal phenotype, only observe small unusual maternal behavior (Hendrich B, Guy J, Ramsahoye B, Wilson VA, Bird A.Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development.Genes Dev 2001 March15; 15 (6): 710-23), the present expression that studies show that application RNA perturbation technique inhibition MBD2 can suppress the growth of tumour, and MBD2 has become the novel targets (WO/2004/001027) that people study the treatment kinds of tumors.This shows that MBD2 may become a suitable epigenetic therapy target, by suppressing the expression of MBD2, reaches the purpose of prevention and treatment Dysfunction of vascular endothlial cells relative disease.
RNA disturbs (RNA interference, RNAi) to refer to the gene silencing phenomenon of being brought out by double-stranded RNA on a kind of molecular biology, and its mechanism is by the translation that hinders specific gene or transcribes inhibition of gene expression.When the double-stranded RNA that imports in the cell with endogenous mRNA coding region homology, degraded occurs and causes reticent (the Fire A of genetic expression in this mRNA, Xu S, Montgomery M, Kostas S, Driver S, Mello C.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature, 1,998 391 (6669): 806-11).RNAi mechanism is prevalent in animals and plants, especially in the unicellular lower eukaryote.Therefore gene expression regulation relatively conservative on being considered to evolve is machine-processed.Studies show that RNAi mechanism may exist as the defense mechanism of resisting poisoning intrusion at rna level.
RNAi has high efficiency and simplicity aspect gene silencing, the double-stranded RNA of 19 Nucleotide is the fully expression of suppressor gene almost, but also has very high specificity, and the mispairing of a base all can significantly weaken the effect of gene silencing between the said target mrna, so be the important tool of gene functional research.Most drug belongs to the inhibitor of target gene (or disease gene), so RNAi simulated the effect of medicine, is the important tool that drug targets is confirmed in the pharmaceutical industries of today.Simultaneously, those proof effective siRNA/shRNA in the target experiment itself can also be developed further into the medicine into RNAi.
Gene knockout (knock-out) mouse is the disallowable mouse of one or more specific gene that utilizes gene knochout technique to make under a kind of gene engineering.This technology is one of instrument of observing and study in vivo gene performance.In addition, the variation that more can produce mouse after the gene knockout, the pathogenic machine of accurately understanding disease that human congenital gene defect causes turns, and also is one of main advantages of this technology.
We utilize RNA perturbation technique and knock out mice, understand the MBD2 gene silencing or knock out animal and human's class Dysfunction of vascular endothlial cells role, thereby determine whether MBD2 can become a kind of brand-new prevention and treatment Dysfunction of vascular endothlial cells relative disease target spot.For Different types of etiopathogenises, medicine or preparation best, that side effect is minimum extremely important meaning is arranged to inventing one.
Summary of the invention
The object of the present invention is to provide the novel targets MBD2 of prevention and treatment Dysfunction of vascular endothlial cells relative disease.Relate to the expression inhibitor that suppresses MBD2, the technical scheme that realizes above-mentioned purpose is as follows: it is a kind of nucleic acid molecule, genetic construction, siRNA molecule and comprises above one or more mixture, can be in vivo or/and the expression of vitro inhibition MBD2, and the application in preparation prevention and treatment Dysfunction of vascular endothlial cells relative disease medicine and preparation.
A kind of siRNA that the present invention relates to, complementary with the mRNA sequence homology of MBD2, feature is that its nucleotides sequence is classified positive-sense strand as: 5 '-GCAAGAGCGAUGUCUACUA-3 '; Antisense strand: 5 '-UAGUAGACAUCGCUCU UGC-3 '.It can import mammalian cell, suppresses the expression of MBD2.
3 ' end of above-mentioned double stranded rna molecule can add that two are hung base tt, and active to increase siRNA, its nucleotides sequence is classified positive-sense strand as: 5 '-GCAAGAGCGAUGUCUACUAtt-3 '; Antisense strand: 5 '-UAGUAGACAUCG CUCUUGCtt-3 '.It can import mammalian cell, suppresses the expression of MBD2.
A kind of siRNA that suppresses the MBD2 expression involved in the present invention, complementary with the mRNA sequence homology of MBD2, but its sequence is different from above sequence 1,2,3,4, can import mammalian cell, suppresses the expression of MBD2.
3 ' end of above-mentioned double stranded rna molecule can add that two are hung base tt, and is active to increase siRNA, can import mammalian cell, suppresses the expression of MBD2.
SiRNA molecule involved in the present invention is that above-mentioned siRNA molecule is through the double stranded rna molecule of chemically modified.
Described chemically modified is the phosphoric acid backbone modification, comprises the part of the phosphodiester bond that connects Nucleotide is modified or with other chemical bonds replacements, such as thio-modification.Thio-modification is widely used at first and improves oligonucleotide in the antisense technology to the resistance of nuclease.Thiophosphoric acid is the non-bridging oxygen atom that replaces phosphodiester bond with a sulphur atom, and namely the P-S key substitutes the P-O key, has improved the ability of siRNA nuclease-resistant, increases its stability.
Chemically modified is that ribose is modified, and comprises chemically modified and change that the OH on 2 of these siRNA molecular nucleic acid pentoses is done, as introduce some substituting group such as methyl, fluorine in 2 ' position of ribose, makes siRNA have the performance of stronger opposing nuclease hydrolysis.It is to introduce one or more alkyl in 2 ' position of siRNA pentose that the first ribose is modified, such as 2 '-the O-methyl; The second modifies 2 '-5-FU be with 2 of the upper pyrimidine nucleotide of siRNA '-OH with 2 '-F is alternative, 2 '-F makes RNA enzyme siRNA not easy to identify, thereby increased the stability of siRNA.
Chemically modified comprises the modification to siRNA molecule base, and siRNA and mRNA form hydrogen bond by base complementrity, and therefore the effect of performance RNAi can modify base.Introducing bromine or iodine in 5 sites of uridylic is the normal base modification method of using, and can strengthen the connection between VITAMIN B4-uridylic (A-U) such as 5-bromo-uridylic, 5-iodo-uridylic, improves the interaction of base.
Other modify as introduce lipophilic group, strengthen the lipotropy of siRNA, increase the ability of its permeate through cell membranes; With 2 ' deoxythymidine acid substitution 3 ' outstanding uridylate, the effect that its RNA disturbs is identical, the digestion that more can resist nuclease.These modifications can increase the stability of siRNA, strengthen simultaneously its biologic activity, can import mammalian cell, suppress the expression of goal gene.
A kind of nucleic acid molecule that the present invention relates to comprises 7 or continuous nucleosides above and Mammals MBD2mRNA homologous complementary, can enter mammalian cell, suppresses the expression of MBD2.
A kind of genetic construction that the present invention relates to comprises above nucleic acid molecule, such as RNA molecule, dna molecular, plasmid, carrier, virus etc., can enter mammalian cell, suppresses the expression of MBD2.
The mixture that the present invention relates to, comprise above one or more inhibitor can enter mammalian cell, suppresses the expression of MBD2.
The application of the above-mentioned MBD2 inhibitor that the present invention relates in preparation prevention and treatment Dysfunction of vascular endothlial cells relative disease medicine and preparation.
What the present invention relates to is that MBD2 is used as target spot, the application in prevention and treatment Dysfunction of vascular endothlial cells relative disease.As being used for prevention and the treatment of diabetic cardiovascular complications, atherosclerosis etc.
Superiority of the present invention is to use RNA perturbation technique and correlation technique to verify the MBD2 gene silencing or knocked out can treat and prevent the caused disease of animal and human's class Dysfunction of vascular endothlial cells, thereby determine that MBD2 can become a kind of brand-new prevention and treatment Dysfunction of vascular endothlial cells relative disease target spot, for Different types of etiopathogenises, medicine or preparation best, that side effect is minimum extremely important meaning is arranged to inventing one, provide new selection for preparing related drugs or preparation, had broad application prospects.
Description of drawings
Fig. 1: MBD2 siRNA suppresses human umbilical cord vein blood vessel endotheliocyte MBD2 protein expression.MBD2 siRNA dose-dependent inhibition endotheliocyte MBD2 protein expression.When MBD2 siRNA concentration was 100nM, the MBD2 protein expression had reduced by 70% (n=4).
The inhibition that Fig. 2: MBD2 expresses has promoted vasculogenesis, and the protection endotheliocyte avoids the endothelial cell apoptosis of hydrogen peroxide-induced.
Fig. 2 A, MBD2 gene knockdown promotes into pipe.The left side is into the presentation graphics of pipe, the mean length of right illustrated tubes (n=4 example).Fig. 2 B, the inhibition of MBD2 has promoted the hyperplasia of HUVEC.The result represents (n=3) with mean ± SEM.Fig. 2 C, MBD2 knockdown protection endotheliocyte avoids H
2O
2The apoptosis of inducing.Left: representational flow cytometer data.Right: the diagram result is from 4 independently experiments.Ctl，Control；**，p＜0.001；*，p＜0.01.
Fig. 3: the expression of MBD2 and mouse ischemic perfused hindquarter recover after the posterior-limb ischemia damage.
Fig. 3 A does not have the expression of MBD2 in MBD2 deficient mice blood vessel.Fig. 3 B, the change in time and space that MBD2 expresses after the posterior-limb ischemia damage.With Western blotting hind leg MBD2 protein expression has been carried out checking (left side), with the relative content than value representation MBD2 of MBD2 albumen and beta-actin (right side, n=5).Fig. 3 C, ischemia and non-ischemic hind leg section MBD2 and endotheliocyte be immunohistochemical staining altogether.Nucleus is with DAPI dyeing (a﹠amp; E), endotheliocyte is with anti--CD31 antibody staining (b﹠amp; F), MBD2 dyeing is green (c﹠amp; G).Image (d﹠amp after the merging; H) by DAPI, anti--CD31 and the MBD2 overlapping generation of dyeing.The high resolution image that inserts among figure d and the h shows the common location of MBD2 and endotheliocyte.Fig. 3 D, the recovery of hind leg blood flow after the posterior-limb ischemia damage.The laser-Doppler imaging system is used for measure blood flow (left panel), and average hind leg blood flow is with the ratio value representation (right panel, n=6) of ischemic side with non-ischemic side signal.Fig. 3 E, ischemia injury after 7 days MBD2 deficient mice capillary vessel form and to compare obvious increase with wild-type.Left cut into slices (X200) for hind leg, with the quantity of endotheliocyte (CD31 positive cell) the formation index as capillary vessel.The average microvessel density (n=5) of each group of bar graph representative on right side.Fig. 3 F, 7 days metarterioles of ischemia injury form.The quantity of smooth muscle cell (anti-α smooth muscle actin positive cell) is as arteriolar formation index in the hind leg section (X200).The average arteriole density (n=5) of each group of bar graph representative on right side.*，p＜0.01.
Fig. 4, the blood vessel endothelium dysfunction that MBD2 deficient mice opposing diabetes cause.
Fig. 4 A, the vasoconstriction that Repone K (KCl, 120mM) is induced.Fig. 4 B, the vasoconstriction that phyenlephrinium (PE) causes.The endothelium-dependent vasodilatation that Fig. 4 C, vagusstoff cause.Fig. 4 D, behind L-NAME (10-4M) the blocking-up endogenous eNOS, the endothelium-dependent vasodilatation that SNP induces.(A and B) the vasoconstriction activity under physiology and diabetic disease states of MBD2 defective and control mice does not have difference, but, wild-type diabetic mice arterial dilation is obviously impaired, and MBD2 defective diabetic mice is similar to non-diabetic control group (C), and shows as endothelium-dependent relaxation (D).Data represent with mean ± SEM, n=4.*，p＜0.01。
Fig. 5: the activation endotheliocyte of losing of MBD2 is survived and the Angiogensis signal.
Fig. 5 A, the inhibition of MBD2 has strengthened the activity of ERK1/2.Left: representational total ERK1/2 and p ERK1/2 immunoblotting result; Right: the quantitative analysis that p ERK1/2 expresses in the HUVEC of transfected MBD2siRNA (n=3).Fig. 5 B, the transfection of MBD2siRNA has increased the level of BCL-2 among the HUVEC, is about 2.2 times of control group.Fig. 5 C, the inhibition of MBD2 has increased the expression of eNOS and VEGF-R2 among the HUVEC, the increase relevant (left side) of the increase that eNOS expresses and the eNOS of phosphorylation, the quantitative analysis results (n=3) of the total eNOS of right diagram and VEGF-R2.Fig. 5 D, MBD2 deficient mice blood vessel also demonstrates the result similar to HUVEC (n=3).Fig. 5 E, the expression that suppresses HUVEC cell MBD2 has promoted the activation of p38, the p38 of activation is higher than 1.3 times of control groups.
Fig. 6, MBD2 are incorporated into eNOS non-translational region and VEGF-R2 promotor high GC content district.
Fig. 6 A, the high GC content district is positioned at the eNOS non-translational region.Fig. 6 B, the ChIP analytical results of eNOS, total input DNA, MBD2 antibody and the control antibodies gained immunoprecipitate of pcr amplification purifying, HUVEC genomic dna (positive control).Fig. 6 C, 2 CpG islands on the VEGF-R2 promotor.Fig. 6 D, the ChIP analytical results of VEGF-R2.
Fig. 7, the expression that MBD2 suppresses eNOS is defined to endotheliocyte.
Fig. 7 A, TSA have eliminated the impact that MBD2siRNA expresses eNOS.Fig. 7 B, the expression that the inhibition of MBD2 can not induced Hcla cell eNOS, the transfection of MBD2siRNA and control group do not detect the expression of eNOS, however TSA but can induce the expression of these two groups of cell eNOS.Fig. 7 C can not express eNOS from the splenocyte that the MBD2 deficient mice separates, but the splenocyte that TSA can induce the MBD2 deficient mice to separate with wild-type mice is expressed eNOS.
Embodiment
The contriver adopts the siRNA Design Rule, designs and filter out one section siRNA sequence that can suppress MBD2 genetic expression, positive-sense strand: 5 '-GCAAGAGCGAUGUCUACUAtt-3 '; Antisense strand: 5 '-UAGUAGACAUCGCUCUUGCtt-3 ', close siRNA by Santa cruz biotech company chemistry, become and the human umbilical cord vein blood vessel endotheliocyte of successful transfection; Utilize gene Knockout to obtain the MBD2 deficient mice and be used for invention.Following examples are not limited only to scope of the present invention, also related mechanism of the present invention are now expanded.
Embodiment one: MBD2siRNA suppresses human umbilical cord vein blood vessel endotheliocyte MBD2 protein expression
Human umbilical cord vein blood vessel endotheliocyte (Human Umbilical Vein Endothelial Cells, HUVECs) is incubated in the EBM-2 epithelial cell substratum, places 5%CO
2, cultivate in 37 ℃ of incubators.Front 24 hours of transfection is inoculated in 12 well culture plates with cell, and inoculum density is 4 * 10
4, second day carries out transfection according to Lipofectamine (Invitrogen, Carlsbad, CA) reagent operational guidance, establishes 50,100nmol/LMBD2siRNA and control group.24-48 hour cell after the collection transfection extracts the capable immunoblotting of albumen and checks.Albumen forwards on the pvdf membrane after separating in 8%SDS-PAGE glue.Seal in 5% skimmed milk TBS-T solution, film was hatched 16 hours in 4 ℃ of MBD2 antibody-solutions, more at room temperature with two anti-hatching 1 hour, after the TBS-T washing, add ECL reagent, film development.The result shows that MBD2siRNA suppresses the expression (Fig. 1) of MBD2 in dose-dependent mode.
Embodiment two: MBD2siRNA promotes vasculogenesis in experiment in vitro, and the protection endotheliocyte avoids H
2O
2The apoptosis of inducing
For being detected as pipe, matrigel is placed in 96 orifice plates (100 μ L/well), 37 ℃ at least 30 minutes, again will be 24 hours endotheliocyte of transfection siRNA (100nmol/L) be inoculated in (nutrient solution is the EBM-2 nutrient solution that contains 2% foetal calf serum) in 96 orifice plates, density is 2 * 10
4The cells/ hole is in 5%CO
2, hatched 24 hours in 37 ℃ of incubators.Under opticmicroscope, checked into the pipe situation in per 4 hours, and found that, namely can be observed typical one-tenth pipe in the MBD2siRNA transfection group in inoculation after 4 hours, and contrast siRNA group is not almost found into pipe; (10.4 ± 0.8mm) are significantly higher than control group (3.3 ± 0.5mm, Fig. 2 A) to the average one-tenth length of tube of MBD2siRNA transfection group after 24 hours.
Be to detect MBD2siRNA to the impact of HUVEC propagation, transfection siRNA24 hour endotheliocyte is inoculated in 96 orifice plates after by trysinization, and density is 5 * 10
3Cells/well is with mark
3The thymidine of H was hatched 16 hours, and harvested cell is counted on 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter.The result shows, compares with control group, and the application of MBD2siRNA has significantly promoted the hyperplasia 9 (Fig. 2 B) of HUVEC.
For confirming MBD2 role in apoptosis, transfection siRNA24 hour endotheliocyte is by 0.2%mmol/L H
2O
2Process guiding apoptosis in 24 hours, subsequently with annexin-V and propidium iodide (PI) dyeing, with Flow cytometry, the apoptosis cell of MBD2siRNA group is starkly lower than control group (Fig. 2 C).
Find out that from above result the generation of MBD2 negative regulator blood vessel promotes H
2O
2The apoptosis of inducing.
Embodiment three: detect the impact that the MBD2 gene knockout recovers the mouse hind leg perfusion with the posterior-limb ischemia experiment
The foundation of posterior-limb ischemia model: expose at left inguinal region place behind the mouse anesthesia, separate proximal femoral, make a call to two knots with the 7-0 silk thread in proximal femoral, and between two knots, cut off femoral artery with scissors, sew up the incision.
The expression of ishemic part MBD2: get different period left hind femoral artery, extract the capable immunoblotting of albumen and detect, observe the expression of MBD2.Ischemic tissue and the non-ischemic tissue of offside are embedded among the OCT, cut into slices (10 μ m) simultaneously, and the row immunohistochemical staining detects the location of MBD2 in blood vessel.
Immunoblot experiment shows, does not express (Fig. 3 A) at MBD2 deficient mice MBD2 albumen; And wild-type mice only has low-level MBD2 to be detected under physiological status, yet ischemic induces the expression of ischemic area MBD2 to increase gradually, peaked in the 4th day to postoperative, then reduce gradually, fortnight drops to a relatively low level (Fig. 3 B).Immunostaining is found, mainly is positioned at endotheliocyte (CD31 positive cell) at non-ischemic region MBD2, but at ischemic region, along with the formation of new vessel, a large amount of MBD2 are expressed in (Fig. 3 C) in the endotheliocyte that increases.
The assessment that the mouse hind leg perfusion recovers: checked the situation that the left hind perfusion recovers in 0,2,4,7 and 14 day after surgery with PIM 3scanning Laser Doppler imaging system (Perimed, Stockholm, Sweden).Average hind leg blood flow is with the ratio value representation of ischemic side with non-ischemic side signal.MBD2 defective and wild-type mice did not all have the blood flow signal after surgery the same day, but MBD2 deficient mice left hind after surgery second day can detect part blood flow and recover signal, the 7th day restoration of blood flow to 50%, blood flow recovered fully in the 14th day; And the wild-type mice left hind is observed restoration of blood flow the 4th talent, and blood flow only returned to 30% and 60% (Fig. 3 D) in the 7th and 14 day.。
The formation of ishemic part new vessel: the non-ischemic tissue of ischemic tissue and offside is embedded among the OCT, section (10 μ m), and the row immunohistochemical staining detects kapillary (CD31) and the arteriole (formational situation of unstriated muscle α-actin).(formation of unstriated muscle α-actin) (Fig. 3 F) is obviously more than wild-type mice at MBD2 deficient mice ischemic region kapillary (CD31) (Fig. 3 E) and arteriole.Anti-MBD2, CD31, unstriated muscle α-actin antibody come from respectively Millipore (Bedford, MA), BD pharmingen (San Diego, CA), Abcam (Cambridge, MA)
Find out from above experiment, the regeneration that the losing of MBD2 promoted the ischemic region blood vessel and the formation of artery, the perfusion that has improved left hind recovers.
Embodiment four: the MBD2 deficient mice can avoid the endothelial dysfunction of diabetes-induced fully
The foundation of diabetes model: adopt low dose repeatedly abdominal injection STZ method (STZ, every day 50mg/kg, continuous 5 days), induce MBD2 knock out mice and the wild-type mice of 8-10 size about week, make occurrence of diabetes.The control mice of age and gender matched then gives the abdominal injection citrate solution every day.Every other day mouse tail blood sampling detects glucose level with blood-glucose meter.The mouse that only has glucose level to surpass 250mg/dl will be used to next step research.
After the animal that suffers from 6 weeks of diabetes is condemned to death, take out rapidly thoracic aorta, and to be cut to two segment length be 3 millimeters arterial ring, to avoid damaging endothelial layer.Then the aorta fragment is hatched (NaCl 115mmol/l, KCl 4.6mmol/l, KH2PO4 1.2mmol/l in fresh Krebs solution, MgSO4 1.2mmol/l, CaCl2 2.5mmol/l, NaHCO3 25mmol/l, glucose 11.1mmol/l and EDTA 0.02mmol/l; And pass to 95%O2 and 5%CO2 pH7.4).Be fixed on moving system (Catamount, St.Albans, the Vermont) row of tracing of four-way flesh with the aorta posterior fragment and equidistantly shrink measurement.The response capacity of vasoconstrictor and diastole agent is assessed the function of endotheliocyte by them.Repone K (120mmol/L) and phyenlephrinium (10
-9To 10
-5Mol/L) be used as vasoconstrictor, vagusstoff (ACh) (10
-9To 10
-5Mol/L) and sodium Nitroprusside ion (SNP) (10-9 is to 10-5mol/L) be used as phyenlephrinium (10
-5Mol/L) pretreated arteries diastole agent.L-nitroarginine-methylester (L-NAME, 10
-4Mol/L), the rival of L-arginine is used to suppress the activity of endogenous eNOS.
Under physiology and diabetic disease states, the vasoconstriction that Repone K or phyenlephrinium cause is not showing difference (Fig. 4 A﹠amp; B); But the wild-type diabetic mice is compared with the non-diabetic mouse, and it is to vagusstoff (ACh, 1 * 10
-9To 1 * 10
-5The stretching reaction of mol/L) inducing is obviously impaired; Form therewith sharp contrast, MBD2 defective diabetic mice but demonstrates to greatest extent diastole (Fig. 4 C).But, after L-NAME blocking-up endogenous nitric oxide, to the stretching reaction that Sodium Nitroprusside is induced, MBD2 defective and wild-type diabetic mice and similar (Fig. 4 D) thereof, this decline that shows wild-type diabetic mice relaxation ability is caused by Endothelial dysfunction.Therefore, the disappearance of MBD2 can watch for animals fully and avoid the endothelial dysfunction of diabetes-induced.
Embodiment five: inhibition activation endotheliocyte existence and Angiogensis signal that MBD2 expresses
Whether activate endotheliocyte survival signaling path for detecting the inhibition that MBD2 expresses, transfection the HUVEC concentration of MBD2 and contrast siRNA be the H of 0.2mmol/L
2O
2Processed 24 hours, its split product is used to immunoblotting and detects two kinds of main endotheliocyte survival signaling extracellular signal-regulated kinase 1/2 (ERK1/2) and serine-threonine kinase Akt, and the expression of ERK1/2 associated protein BCL-2 (ERK1/2 phosphorylation BCL-2 and stop its degraded).The result shows, Akt reach total ERK1/2 and do not showing difference in the expression of transfection MBD2siRNA group and control group, but the expression of the ERK1/2 of phosphorylation (Fig. 5 A) and BCL-2 (Fig. 5 B) obviously raises in transfection MBD2siRNA group.Compare with the wild-type diabetic mice, MBD2 deficient mice blood vessel also demonstrates same trend.This shows that MBD2 regulates endothelial cell apoptosis and may realize by regulating ERK1/2 and BCL-2 signal.Be the mechanism that research MBD2 regulates vasculogenesis, transfection the HUVEC split product of MBD2 and contrast siRNA be used to immunoblotting and detect the Angiogensis signal eNOS of two kinds of keys and the expression of VEGF-R2 and p38 mitogen activated protein kinase.ENOS, VEGF-R2 express in transfection MBD2siRNA group and obviously raise (Fig. 5 C), compare with wild-type mice, and MBD2 deficient mice blood vessel also demonstrates same trend (Fig. 5 D).The p38 that has activated also obviously raises in transfection MBD2siRNA group, but the total amount between of p38 do not have difference (Fig. 5 E).
This shows, MBD2 lose may active cells existence and Angiogensis signal improve endotheliocyte existence and vasculogenesis ability.
Embodiment six, and MBD2 is incorporated into eNOS and the high GC of VEGF-R2 promotor district
For understanding fully whether MBD2 is incorporated into eNOS and the high GC of VEGF-R2 promotor district, chromatin immunoprecipitation (Chromatin immunoprecipitation, ChIP) be used for dragging lower MBD2/DNA mixture, carry out relating operation according to the explanation of chromatin immunoprecipitation analysis test kit.The result shows that MBD2 is incorporated into eNOS and the high GC of VEGF-R2 promotor district (Fig. 6 A, B, C, D), and then suppresses transcribing of eNOS and VEGF-R2.
Embodiment seven: MBD2 relates to Chromatin Remodeling to the inhibition that eNOS transcribes, and is defined to endotheliocyte.
For inquiring into MBD2 whether the inhibition that eNOS transcribes is related to Chromatin Remodeling, add TSA (hdac inhibitor) to transfection the HUVEC of MBD2 and contrast siRNA, add DMSO and be contrast, after 48 hours, detect the expression of eNOS.The inhibition of MBD2 has promoted the expression of eNOS, and (Fig. 7 A) eliminated in this effect fully by TSA.This shows that MBD2 relates to Chromatin Remodeling to the inhibition that eNOS transcribes.
For whether the inhibition of research MBD2 can promote the expression of non-endotheliocyte eNOS, the Hela cell is used to carry out similar experiment with splenocyte (separating with the MBD2 deficient mice from wild-type).In the Hela cell, the expression of eNOS is not induced in the inhibition of MBD2, and after TSA processes, transfection the Hela cell of MBD2 and contrast siRNA can both detect the expression (Fig. 7 B) of eNOS; On splenocyte, also obtained analog result (Fig. 7 C).This explanation MBD2 relates to Chromatin Remodeling to the inhibition that eNOS transcribes, and is defined to endotheliocyte.
Claims (2)
1. suppress the inhibitor that vascular endothelial cell MBD2 expresses, it is characterized in that: the inhibitor that described inhibition MBD2 expresses is siRNA, and its nucleotides sequence is classified positive-sense strand as: 5 '-GCAAGAGCGAUGUCUACUA-3 '; Antisense strand: 5 '-UAGUAGACA UCGCUCUUGC-3 '; Or its 3 ' end has two to hang base tt, and nucleotides sequence is classified positive-sense strand as: 5 '-GCAAGAGCGAUGUCUACUAtt-3 '; Antisense strand: 5 '-UAGUAGACA UCGCUC UUGCtt-3 ', can import the Mammals vascular endothelial cell, suppress the expression of MBD2.
2. the inhibition vascular endothelial cell MBD2 according to claim 1 inhibitor of expressing, it is characterized in that: described siRNA is the double-stranded siRNA molecule through the chemically modified gained, and this chemically modified comprises the non-bridging oxygen atom of the part of the phosphodiester bond that connects Nucleotide being modified or replaced with a sulphur atom phosphodiester bond; Chemically modified comprises chemically modified and the change that the OH on 2 of these siRNA molecular nucleic acid pentoses is done, and chemically modified comprises the modification of siRNA molecule base or other lipotropy genetic modifications.
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| CN102178958B (en) * | 2011-03-09 | 2012-09-12 | 中国人民解放军第三军医大学第一附属医院 | MBD2-mTEM fused DNA vaccine and preparation method and application thereof |
| CN110917351A (en) * | 2018-09-20 | 2020-03-27 | 华中科技大学同济医学院附属同济医院 | Use of MBD2 inhibitors for the prevention and treatment of fibrotic diseases |
| CN116790603B (en) * | 2023-08-18 | 2023-10-31 | 成都中科奥格生物科技有限公司 | sgRNA and CRISPR/Cas9 vector as well as construction method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1206714A (en) * | 1997-07-24 | 1999-02-03 | 中国科学院上海生物化学研究所 | Oligomeric deoxynucleotide of vascular endothelial cell growth factor |
| WO2004001027A1 (en) * | 2002-06-20 | 2003-12-31 | Mcgill University | Oligonucleotide inhibitors of mbd2/dna demethylase and uses thereof |
| CN1938035A (en) * | 2004-11-05 | 2007-03-28 | 金泰润 | Therapeutic use of CPG oligodeoxynucleotides for skin disorders |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1206714A (en) * | 1997-07-24 | 1999-02-03 | 中国科学院上海生物化学研究所 | Oligomeric deoxynucleotide of vascular endothelial cell growth factor |
| WO2004001027A1 (en) * | 2002-06-20 | 2003-12-31 | Mcgill University | Oligonucleotide inhibitors of mbd2/dna demethylase and uses thereof |
| CN1938035A (en) * | 2004-11-05 | 2007-03-28 | 金泰润 | Therapeutic use of CPG oligodeoxynucleotides for skin disorders |
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