CN105126122A - Application of micro RNA mimics in preparation of drug treating choroidal neovascularization - Google Patents
Application of micro RNA mimics in preparation of drug treating choroidal neovascularization Download PDFInfo
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Abstract
The invention provides application of micro RNA mimics in the preparation of a drug treating choroidal neovascularization (CNV). The inventor adjusts bone marrow derived cells MMP expression through micro RNA, utilizes the characteristic of chemotaxis of the bone marrow derived cells to CNV to realize high precision targeted drug delivery and sustained release drug delivery for a part of the CNV part, so as to inhibit the bone marrow derived cells from forming pathological new vessels together with in situ cells. The invention also provides application of micro RNA agomir in the preparation of a drug treating the choroidal neovascularization. The inventor simultaneously adjusts two types of MMP expressions of BMCS through the micro RNA agomir, so the invasion of the BMCS is inhibited to reduce CNV cells and factor sources, and the in situ cells are inhibited from migrating, and the CNV can be efficiently treated; besides, due to the characteristic of the BMCS only enriching at a local part of the CNV, a high precision targeted therapy can be realized at the part of the CNV.
Description
Technical field
The invention belongs to bio-medical technology field, be specifically related to the application of microRNAmimics for the preparation for the treatment of choroidal neovascularization medicine.
Background technology
Choroidal neovascularization (choroidalneovascularization, CNV) is more than the 40 total pathological changes of planting ocular disease, and sickness rate is high, serious threat visual function.The CNV cause of disease is complicated, and pathogenesis is still not clear.Though have multiple therapy methods for CNV clinically at present, there is unsatisfactory curative effect, relapse rate is high, long-term safety is imprecise and the problem such as expensive, brings white elephant to family and society, this area research has become the great Strategic Demand of country.Exploring CNV pathogenesis, actively find the generation of efficient target spot suppression new vessels, is the hope place fundamentally curing CNV relevant disease.Formation due to new vessels is various kinds of cell, the coefficient result of molecule, and cell, intermolecular interaction are intricate again, and the medical treatment effect for single cell/factor exploitation is often limited.The positive key node found in regulation and control new vessels generting machanism, the control for CNV is significant.
Summary of the invention
The invention provides the application of microRNAmimics for the preparation for the treatment of choroidal neovascularization medicine.
Described microRNAmimics is miR188-5p analogies.
Present invention also offers the application of microRNAagomir for the preparation for the treatment of choroidal neovascularization medicine.
Described microRNAagomir is through the miR188-5p analogies modified that methylate.
The invention has the beneficial effects as follows:
Neovascularization growth is a comparatively long-term process, adopts Angiogenesis Inhibitor treatment CNV to need to find more efficiently mode.In the generation evolution of CNV, bone marrow-derived cells can specificity chemotactic to CNV place, participate in Angiogenesis, be the important composition composition of CNV.Inventor regulates bone marrow-derived cells MMP to express by microRNA, bone marrow-derived cells is utilized to realize bone marrow-derived cells at the accurate target administration of the height at CNV position and sustained-release administration to the characteristic of CNV chemotactic by conventional vein input, pathology new vessels is suppressed to be formed, realize the object of effectively treatment CNV, and avoid the risk of intraocular injection administration repeatedly.
Inventor finds, when CNV generates, BMCS invades and the divide a word with a hyphen at the end of a line main source of necessary MMP2/13 of cells in situ is exactly BMCS, and in BMCS, miR-188-5p can regulate and control the expression of MMP2/13 simultaneously.Inventor utilizes the microRNA analogies agomir of synthetic to regulate miR-188-5p level in BMCS, the cascading of single medicine generation is realized to the intervention of multiple target spot by Conventional glass body cavity injection miR-188-5pagomir, namely reduce MMP2/13 secretion simultaneously, BMCs is suppressed to invade to CNV region on the one hand, thus reduce cell and the Factor Source of CNV, cells in situ is suppressed to be divided a word with a hyphen at the end of a line on the other hand, reduce vessel lumen to be formed, Mutiple Targets treatment CNV, pathology new vessels is suppressed to be formed, reach the object of efficient treatment CNV, can it be significantly suppressed to grow in early days at CNV, shorten treatment time, avoid the risk of prolonged and repeated administration.In addition, because BMCS is only in the characteristic of CNV local specific enrichment, intravitreal administration only produces effect at CNV place, reaches targeted therapy.
Accompanying drawing explanation
Fig. 1 detects different time points MMP-2/MMP-13 protein expression for organizing ELISA; The expression of MMP-2 after A, laser induced CNV; The expression of MMP-13 after B, laser induced CNV;
Fig. 2 is the expression that real-time quantitative PCR detects miR188-5p;
Fig. 3 observes under laser confocal microscope, and the BMCs of GFP+ is green, and MMP-2/MMP-13/miR188-5 is red, and both coexpression regions (arrow) are yellow; Within after A, laser induced CNV 3 days, locate the BMCs of MMP-2 and GFP+ altogether; Within after B, laser induced CNV 7 days, locate the BMCs of MMP-2 and GFP+ altogether; Within after C, laser induced CNV 7 days, locate the BMCs of MMP-13 and GFP+ altogether; Within after D, laser induced CNV 28 days, locate the BMCs of MMP-13 and GFP+ altogether; Within after E, laser induced CNV 1 day, locate the BMCs of miR188-5p and GFP+ altogether; Within after F, laser induced CNV 7 days, locate the BMCs of miR188-5p and GFP+ altogether;
Fig. 4 is the expression constituent ratio of MMP-2/MMP-13/miR188-5p in each time point BMCs of statistical analysis; Total expression of MMP-2 and the expression constituent ratio of wherein BMCs in each time point after A, laser induced CNV; Total expression of MMP-13 and the expression constituent ratio of wherein BMCs in each time point after B, laser induced CNV; Total expression of miR188-5p and the expression constituent ratio of wherein BMCs in each time point after C, laser induced CNV;
Fig. 5 is MMP-2, MMP-13 and miR188-5p immunofluorescence area statistics at each time point of BMCs expression in each time point after laser induced CNV.
Fig. 6 is miR188-5p and MMP-2/MMP-13 that after administration, in each time point in CNV region, BMCs expresses; Fig. 6 A, frozen section fluorescence in situ hybridization representative graph.Red: miR-188-5p; Green: GFP+BMCs; Upper: NC group; Under: agomir group; Fig. 6 B, frozen section immunofluorescence representative graph.Red: MMP-2/MMP-13; Green: GFP+BMCs.
Fig. 7 is that after administration, dynamically the observation CNV order of severity and BMCS raise situation.After Fig. 7 A, choroid paving sheet fluorescence staining, copolymerization Jiao takes 3D imaging representative graph and the CNV volume statistical analysis of CNV.Red: new vessels; Fluorescence microscope shooting CNV representative graph after Fig. 7 B, choroid paving sheet.Red: new vessels; Green: GFP+BMCs; Left: NC group; Right: agomir group.
Fig. 8 is the Complementary binding sites that there is miR188-5p 3 ' noncoding region of MMP-2 and MMP-13 messenger RNA.
Detailed description of the invention
Matrix metalloproteinase (matrixmetalloproteinases, MMPs) degradation of cell epimatrix and endogenous Anti-angiogenesis factors in CNV generative process, promote cell divide a word with a hyphen at the end of a line and new vessels sprout, segment dislocation, be conducive to bone marrow-derived cells (bonemarrowderivedcells simultaneously, BMCs) invade in CNV, participate in new vessels and formed.The MMPs that CNV cells in situ is expressed is limited, and BMCs participates in CNV and generates, and is the important sources of MMPs in CNV.Inventor studies discovery, and the expression of miR188-5p to MMP-2/-13 plays regulating and controlling effect.This research is by the CNV model of structure allophenic mice, and whether the BMCs observing discussion participation CNV generation is whether the important sources of MMPs and the MMP-2/-13 of BMCs expression regulate and control and then affect CNV to develop by miR188-5p.Further, utilize the microRNA analogies agomir of synthetic to regulate miR-188-5p level in BMCs, suppress MMP2/13 secretion, thus suppress the intrusion of BMCs and cells in situ to be divided a word with a hyphen at the end of a line, reach efficient, targeted therapy CNV effect.
Regulate and control BMCs about microRNA and express the experimentation that MMPs affects CNV generation development and corresponding treatment strategy
1 materials and methods
1.1 reagent and instrument
Rabbit anti-mouse MMP-2/MMP-13 polyclonal antibody (Abcam company of the U.S.); The goat anti-rabbit antibody (Earth company of the U.S.) of DyLight594 labelling; Biotin labeled goat anti-rabbit antibodies (Neomarkers company of the U.S.); MMP-2 organizes ELISA kit (Abcam company of the U.S.); MMP-13 organizes ELISA kit (Wuhan doctor's moral biological product company limited); Mir-188-5p in situ hybridization test kit (Wuhan doctor's moral biological product company limited); Real-time quantitative PCR test kit (Japanese TaKaRa company)
1.2 animal origin
Adopt green fluorescent protein (greenfluorescentprotein, GFP) female transgenic mice as the donor of bone marrow transplantation, in 6 ~ 8 week age, body weight 18 ~ 24g, is provided by neurobiology teaching and research room of The Fourth Military Medical University; Receptor Mus is wild females C57BL/6J mice, in 6 ~ 8 week age, body weight 18 ~ 24g, is provided by The Fourth Military Medical University's Experimental Animal Center, 50 the C57BL/6J.GFP allophenic mices accepting bone marrow transplantation are experimental group, and 48 the wild type C57BL/6J mices not doing to transplant are matched group.
1.3 bone marrow transplantations and chimeric degree analyzing
Bone marrow transplantation: receptor Mus accepts
60co irradiates, exposure dose 8.0Gy, 1.0Gy/min.The cell suspension prepared through tail vein injection in 12h after irradiating, 3.0 × 10
7/ 300ul/ is (HouHY only, WangYS, XuJF, etal.Thedynamicconductofbonemarrow-derivedcellsinthechor oidalneovascularizationmicroenviroment [J] .CurrEyeRes, 2006,31:1051-1061).
Peripheral blood flow cytometry analysis: transplant latter one month, bone marrow chimerism degree (Espinosa-HeidmannDG is judged with cell proportion in mononuclear cell of expressing GFP in flow cytometry analysis sample, CaicedoA, HernandezEP, etal.Bonemarrow-derivedprogenitorcellscontributetoexperi mentalchoroidalneovascularization [J] .InvestOphthalmolVisSci, 2003,44:4914-4919).
1.4 laser photocoagulations bring out CNV
Matched group 42 (mice do not transplanted) and experimental group 42 (the successful mice of bone marrow transplantation) all row 532nm laser photocoagulation (wavelength 532nm, time of exposure 0.1s, spot diameter 75um, power 100mw) every 6 ~ 8 laser spots, apart from optic disc 1 ~ 1.5 dish diameter (Yang Xiumei, Wang Yusheng. the animal model [J] of choroidal neovascularization. international ophthalmology is scanned, 2006,30:166-170).
1.5 organize ELISA to detect the expression of MMP-2/MMP-13
Experimental group and matched group respectively get 24 mices, each time point 3, immerses in the 100ulPBS containing 0.05% phenylmethylsulfonyl fluoride after quick-freezing by the retina of eyeball of mouse and tela chorioidea, at manual homogenization on ice, homogenate completes 3 freeze-thaw cycle from liquid nitrogen to ice, collects supernatant.According to organizing ELISA kit, the MMP-2/MMP-13 detecting supernatant is described.
1.6qRT-PCR detects the expression of miR188-5p
Matched group gets 24 mices, each time point 3.It is (TAGTTGCATGGTGGAGGGAAA) that miR188-5p is 21bp length (CAUCCCUUGCAUGGUGGAGGG) according to its design primer at humans and animals.MiR188-5p in the retina and tela chorioidea detecting eyeball of mouse is described according to qRT-PCR test kit.
1.7 immunofluorescence dyeings observe the expression of MMP-2/MMP-13
MMP-2/MMP-13 is Expression in CNV: experimental group gets 24 mices, each time point 3.Specimen is done perpendicular to amphiblestroid 8 ~ 10um frozen section at-20 DEG C.Section drying, closed rear and rabbit anti-mouse MMP-2/MMP-13 polyclonal antibody (1:200; 1:300) at room temperature overnight incubation; Drip two after washing to resist, the goat anti-rabbit IgG antibody (1:300) of red fluorescence labelling, incubated at room temperature 3h, observe under laser confocal microscope after washing, mounting and take pictures.Primary antibodie is replaced to make blank with PBS.To take pictures observation: the MMP-2/MMP-13 that in CNV, GFP positive cell (medullary cell namely transplanted) is expressed.The shown in green fluorescence of BMCs labelling under the microscope, the shown in red fluorescence of MMP-2/MMP-13, both are shown as yellow fluorescence by common focusing block.ImageJ is adopted to measure area (yellow) and CNV position the MMP-2/MMP-13 gross area (redness) (in units of the pixel) of the MMP-2/MMP-13 that BMCs expresses.Total expression of MMP-2/MMP-13 and the expression constituent ratio of wherein BMCs in statistical analysis CNV.
1.8 in situ hybridizations observe miR188-5p Expression in CNV
Frozen section process is the same.Illustrate according in situ hybridization test kit and detect miR188-5p.Its take pictures observation and statistical analysis the same.
1.9 microRNA target prediction
The microRNA target prediction software RNA22microRNAtargetdetection (http://cbcsrv.watson.ibm.com/rna22) of microRNA is utilized to detect the microRNA of regulation and control MMP-2 and MMP-13 expression.
1.10 prepared by medicine
MicroRNAagomir is through the microRNA agonist that special chemical is modified, and enters miRISC complex regulate the expression of said target mrna and play a role by simulation microRNA.Agomir can overcome the obstacles such as cells in vivo film, tissue and be enriched in target cell.Can carry out administration by methods such as whole body or local injection, suction, medicine feeds in zoopery, the action effect persistent period is 6 weeks.
Agomir preparation method: on mimics basis, is prepared as ripe microRNA double-stranded RNA after methylating and modifying.
The mimics of mir-188-5p and the sequence of negative control thereof are:
mir-188-5pmimics(sense):5’-CAUCCCUUGCAUGGUGGAGGG-3’
Mir-188-5pmimics (antisense): 5 '-CUCCACCAUGCAAGGGAUGUU-3 ' detection method: in vitro and in vivo respectively with the expression of mir-188-5p after real-time quantitative PCR and hybridization in situ technique detection agomir transfection.
1.11 mice intravitreal medicines
After laser modeling at once, first after the distance limbus of corneae of mice temporo side, 2mm place hole is pricked with more sharp-pointed 30G injection of insulin syringe needle under Stereo microscope, again with 33G round needle head link 5ul microsyringe, thrust in vitreous chamber with 40 ° of-60 ° of angles towards equatorial region along Zha Kongchu, slowly inject 1ulPBS (PBS group), unrelated sequences (NC group) and miR-188-5pagomir (agomir group) respectively, and the concentration of negative control and experimental group is 1nmol/ul, do not inject group as blank.After injecting, slowly extract syringe needle out, and clamp injection port with tweezers and complete intravitreal after 5 seconds.
The horizontal detection of miR-188-5p and MMP-2/13 in the BMCS of CNV region after 1.12 administrations
Fluorescence in situ hybridization technique detects the expression of miR-188-5p.Often organize and get 15 mices, each time point 3, after intravitreal 3,5,7,14 and 28 days, after mouse anesthesia, 4% paraformaldehyde (containing 1/1000DEPC) pours into.Extract bilateral eyeball, fix 2h after 4% paraformaldehyde at 4 DEG C, in 30% sucrose solution, antifreezing protection spends the night.Specimen is done perpendicular to amphiblestroid 8 ~ 10um frozen section at-20 DEG C.After distilled water fully washs, each sample drips 20ul prehybridization solution, and calorstat 38 DEG C ~ 42 DEG C, absorbs unnecessary liquid after 2h.Each sample drips 20ulmiR-188-5p hybridization solution, calorstat 38 DEG C ~ 42 DEG C hybridized overnight.Next day, taking-up was washed with the SSC of about 37 DEG C water temperatures.Drip confining liquid 37 DEG C 30 minutes, get rid of surplus liquid, do not wash.Drip biotinylation mouse-anti digoxin, 37 DEG C of 60 minutes or room temperatures 120 minutes.The special PBS of in situ hybridization washs 4 times.Add the antibody 1:200 of antibiotin red fluorescence labelling, 37 DEG C of 1h.Get rid of surplus liquid, PBS washs 2 times.Observe under laser confocal microscope after glycerol mounting and take pictures.To take pictures observation: the miR188-5p that in CNV, GFP positive cell (medullary cell namely transplanted) is expressed.The shown in green fluorescence of bone marrow-derived cells labelling under the microscope, the shown in red fluorescence of miR188-5p, both are shown as yellow fluorescence by common focusing block.Measure each site area and compare (in units of pixel).
Immunofluorescence dyeing detects the expression of MMP-2/13.The frozen section chosen lucifuge under room temperature is placed 2-3h, dries; PBS washs, jog 20min on shaking table, and period changes liquid 3-4 time; Close: 5% [NGS+BSA]+1%Triton+PBS, room temperature, lucifuge hatches 3h; Primary antibodie is hatched: 1% [NGS+BSA]+1%Triton+ primary antibodie (antibody dilution: MMP-131:200; MMP-21:300)+PBS, room temperature, hatches 24h (at least 20h); PBS washs 30min, changes liquid 5 times; Blank: PBS substitutes primary antibodie, parallel dyeing; Negative control: normal calf serum (dilution factor: 1:50) substitutes primary antibodie.Two anti-hatch: the anti-(antibody dilution: biotinylated goat anti-rabbit IgG antibody, 1:300, red fluorescence of 1% [NGS+BSA]+1%Triton+ two; Antibody dilution: biotinylated goat anti-rabbit IgG antibody, 1:300, green fluorescence)+PBS, lucifuge, incubated at room 3h (or 4 DEG C are spent the night); PBS washs 30min; Reagent D API redyes nucleus; Observe under laser confocal microscope after glycerol mounting and take pictures.To take pictures observation: the MMP-2/MMP-13 that in CNV, GFP positive cell (medullary cell namely transplanted) is expressed.The shown in green fluorescence of BMCs labelling under the microscope, the shown in red fluorescence of MMP-2/MMP-13, both are shown as yellow fluorescence by common focusing block.Measure each site area and compare (in units of pixel).
The 1.13 dynamic observation CNV orders of severity and BMCS raise situation
After laser modeling 7,14 and 28 days, row RPE-choroid-sclera paving sheet: often organize and get 15 mices, each time point 3, after intravitreal 7,14 and 28 days, carry out the excessive anesthesia of mice (ditto) respectively, after heart perfusion 20ml normal saline and 40ml4% paraformaldehyde, extract eyeball.Fix 30min in 4% paraformaldehyde after, be placed on PBS shaking table and embathe 2h.Microscope goes down and first carefully cuts off unnecessary muscle and connective tissue, then remove cornea, crystal, retina neural sensory layer and optic nerve.The RPE-of remainder choroid-sclera complex is put into 1%Triton 4 DEG C of 24h.The RCA (1:200) of rhodamine labelling is placed in 24 orifice plates, every hole 500 μ l, then paving sheet is placed in one is put in incubated at room temperature 24h on shaking table.Finally in PBS buffer, wash 24h.RPE-choroid-sclera complex after washing makes 5-6 radial incision to be made it to flatten.Paving sheet is flattened on microscope slide, detects under laser confocal microscope after the glycerol mounting of 50%.Choose paving sheet effect to organize preferably, often group gets 12 CNV points, adopts area and the volume of Image-proplus6.0 professional image software measurement redness and green fluorescence.
1.14 statistical analysis
Statistics software adopts SPSS17.0 to carry out data analysis.The data information of this research Testing index is normal distribution through W inspection, with
represent, mean through between Levene check groups, variance neat (equal P>0.05).Adopt completely random to divide into groups two horizontal experimental designs, matched group and experimental group comparison in difference adopt one factor analysis of variance inspection.Adopt two tail detection method, P<0.05 is that difference has statistical significance.Relation between two factors adopts dual factors correlation analysis.
2 results
Chimeric degree after 2.1 bone marrow transplantations
After bone marrow transplantation 4 weeks, chimeric degree > 85% totally 48 (transplanting succeed rates 96%) included subsequent experimental in, expresses GFP cell proportion average 90.67 ± 3.02%, meet later experiments requirement in peripheral blood lymphocytes.
2.2 organize ELISA to detect protein concentration:
Bone marrow transplanted mice and MMP-2/MMP-13 in wild-type mice totally express the consistent (one factor analysis of variance: MMP-2:F=0.060, p=0.810 of trend; MMP-13:F=0.012, p=0.915).Result display MMP-2 just starts quick increase in early days bringing out CNV, to expression the highest (5765.056 ± 73.952pg/ml during CNV the 3rd day; 5693.572 ± 88.330pg/ml), start subsequently to decline.MMP-13 slowly increases in early days at CNV, to expression the highest (1603.885 ± 77.781pg/ml during CNV the 7th day; 1535.267 ± 91.478pg/ml), start subsequently to decline.Fig. 1 organizes ELISA to detect different time points MMP-2/MMP-13 protein expression; A. the expression of MMP-2 after laser induced CNV; B. the expression of MMP-13 after laser induced CNV, bone marrow transplanted mice and MMP-2/MMP-13 in wild-type mice totally express trend consistent (MMP-2:F=0.060, p=0.810; MMP-13:F=0.012, p=0.915).
2.3qRT-PCR detects the expression of miR188-5p:
MiR188-5p is along with the increase of CNV natural law before laser induced and after laser induced CNV model, and the trend risen afterwards appears first declining in miR188-5p.MiR188-5p relative expression quantity when CNV the 7th day is minimum.Fig. 2 qRT-PCR detects the expression of miR188-5p, and along with the increase of CNV natural law, the trend risen afterwards appears first declining in miR188-5p.
Organize ELISA to detect the expression of MMPs and the expression of miR188-5p and do correlation analysis: the Expression of miR188-5p and the Expression of MMP-13 before laser induced be negative correlation (wild-type mice: r=-0.740, bone marrow transplanted mice: r=-0.738, p < 0.05) in 28 days after laser induced CNV; The Expression of miR188-5p and the Expression of MMP-2 are negative correlation (wild-type mice: r=-0.957, bone marrow transplanted mice: r=-0.960, p < 0.05) in first 5 days before laser induced and after laser induced CNV.
2.4 microRNA target prediction: there is the Complementary binding sites of miR188-5p 3 ' noncoding region (untranslatedregions, UTR) of MMP-2 and MMP-13 messenger RNA as shown in Figure 8.
MMP-2/MMP-13 and miR188-5p expressed in BMCs in 2.5CNV
Bone marrow transplanted mice immunofluorescence image shows, and after laser photocoagulation, the BMCs of the GFP positive is in the enrichment of CNV region; The BMCs in CNV region can express MMP-2/MMP-13 and miR188-5p (Fig. 3).ImageJ is adopted to analyze total expression of three in sxemiquantitative CNV and the expression constituent ratio (Fig. 4) of wherein BMCs.The MMP-2 that BMCs expresses just starts quick increase in early days bringing out CNV, peaks, account for 64.21% of total expression to expression during CNV the 3rd day.The MMP-13 that BMCs expresses just starts slow increase in early days bringing out CNV, peaks, account for 79.61% of total expression to expression during CNV the 7th day.The miR188-5p that BMCs expresses just starts quick decline in early days bringing out CNV, reaches minimum to expression during CNV the 7th day.Immunofluorescent localization sxemiquantitative statistical analysis show that the expression of the Expression of the miR188-5p in CNV in BMCs and the MMP-13 of BMCs is negative correlation (r=-0.868, p < 0.05); The Expression of miR188-5p after laser induced CNV in first 5 days in BMCs and the expression of the MMP-2 of BMCs are negative correlation (r=-0.997, p < 0.05) (Fig. 5).Have no green fluorescence in the immunofluorescence dyeing result of wild-type mice eyeball section, illustrate that green fluorescence is really sent by donor murine myeloid cells.
Immunofluorescent localization sxemiquantitative statistical analysis show that the expression of the Expression of the miR188-5p in CNV in BMCs and the MMP-13 of BMCs is negative correlation (r=-0.868, p < 0.05); The expression of the Expression of the miR188-5p after laser induced CNV in 5 days in BMCs and the MMP-2 of BMCs is negative correlation (r=-0.997, p < 0.05).
Result shows that BMCs is the important sources of MMP-2/-13, and miR188-5p regulates and controls BMCs to the expression of MMP-2/MMP-13, so affect that vascular cell sprouts, divides a word with a hyphen at the end of a line, apoptosis and CNV fibrosis.
The horizontal detection of miR-188-5p and MMP-2/13 in the BMCS of CNV region after 2.6 administrations
In NC group, light coagulates CNV region miR-188-5p expression decline in latter 3 days, express minimum, and agomir group miR-188-5p expresses apparently higher than other groups (P<0.05) (Fig. 6-A) when 7 days.The MMP-2/MMP-13 expression of BMCs secretion comparatively other group decline (P<0.05) (Fig. 6-B) in agomir group.
After 2.7 dynamic observation administrations, the CNV order of severity and BMCS raise situation
MiR-188-5p alleviates the mice CNV order of severity.Choroid paving sheet and lectin dyeing display, it is [(153.02+27.59) × 10 that light coagulates latter 7 days NC group CNV volumes
3] μm
3, agomir group CNV volume is [(98.0+16.11) × 10
3] μm
3; It is [(130.24+11.28) × 10 that light coagulates latter 14 days NC group CNV volumes
3] μm
3, agomir group CNV volume is [(86.68+5.00) × 10
3] μm
3; It is [(106.00+18.09) × 10 that light coagulates latter 28 days NC group CNV volumes
3] μm
3, agomir group CNV volume is [(69.72+7.38) × 103] μm 3; Light coagulates latter 7 days, 14 days and 28 days agomir group CNV volumes are significantly less than other groups (P<0.05) (Fig. 7 A).PBS group, blank group and NC group no difference of science of statistics (P>0.05).MiR-188-5p suppresses BMCs to participate in CNV generation.By the impact that the choroid paving picture understanding miR-188-5p analyzing NC group and agomir group generates BMCs chemotactic and participation CNV.Two groups of image displays, have GFP+ cell to participate in, and in the CNV of agomir group, GFP+ cell obviously reduce (Fig. 7 B) in the CNV that optic disc generates.
Claims (4)
1.microRNAmimics is for the preparation of the application for the treatment of choroidal neovascularization medicine.
2. apply as claimed in claim 1, it is characterized in that, described microRNAmimics is miR188-5p analogies.
3.microRNAagomir is for the preparation of the application for the treatment of choroidal neovascularization medicine.
4. apply as claimed in claim 3, it is characterized in that, described microRNAagomir is through the miR188-5p analogies modified that methylate.
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| CN117230118A (en) * | 2023-09-21 | 2023-12-15 | 武汉科技大学 | Method for constructing weak sperm mouse model by targeting spermatogenic cells |
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| CN113930428A (en) * | 2021-10-20 | 2022-01-14 | 中国人民解放军联勤保障部队第九〇四医院 | MiRNA for treating CNV and preparation method and application thereof |
| CN113930428B (en) * | 2021-10-20 | 2023-04-25 | 中国人民解放军联勤保障部队第九〇四医院 | miRNA for treating CNV and preparation method and application thereof |
| CN117230118A (en) * | 2023-09-21 | 2023-12-15 | 武汉科技大学 | Method for constructing weak sperm mouse model by targeting spermatogenic cells |
| CN117230118B (en) * | 2023-09-21 | 2024-05-03 | 武汉科技大学 | Method for constructing weak sperm mouse model by targeting spermatogenic cells |
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