CN104174032A - siRNA molecular composition and application thereof in treatment on pathological scars - Google Patents
siRNA molecular composition and application thereof in treatment on pathological scars Download PDFInfo
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- CN104174032A CN104174032A CN201410381766.9A CN201410381766A CN104174032A CN 104174032 A CN104174032 A CN 104174032A CN 201410381766 A CN201410381766 A CN 201410381766A CN 104174032 A CN104174032 A CN 104174032A
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Abstract
The invention relates to a siRNA molecular composition and application thereof in treatment on hypertrophic scars and keloids. The siRNA molecular composition contains siRNA molecules with the sequence of 5'-CCCAAGGGCUACCAUGCCAACUUCU-3' and siRNA molecules with the sequence of 5'-GGUCUGGUGCCUGGUCUGAUGAUGU-3'. The siRNA molecular composition is proved to be capable of promoting in-vitro fibroblast apoptosis of the hypertrophic scars and keloids and have a good treatment effect on the hypertrophic scars in an animal model.
Description
Technical field
The present invention relates to molecular biology and gene therapy technology field, specifically, is the purposes of siRNA molecule in treatment hypertrophic cicatrix, keloid.
Background technology
Skin is human body largest organ, possesses the functions such as protection, immunity, excretion, and wherein of paramount importance function is to protect, maintain the stable state of internal milieu.When the integrity of skin is subject to compared with havoc, the stable state in body occurs uneven, and the extraneous invasion and attack factor easily enters in body, directly threatens individual existence.In the process of spore, there is the scar healing pattern of wound, this mode of healing can full out be recovered the integrity of skin, has greatly improved the existence probability of species.Therefore, cicatrix is the indispensable product of human body skin wound healing.
But in the process of wound healing, usually there is undue growth, the abnormality proliferation of cicatrix, form pathologic scar, comprise hypertrophic cicatrix and keloid.These cicatrixs, conventionally with sufferings symptom, affect patient's function and outward appearance.According to the literature, after the surgical operation postoperative patient of 40%-70% and 91% fire victim heal, form hypertrophic cicatrix, Plastic Surgery Clinic conventionally have over the patient of half because of cicatrix medical.Keloid clinical manifestation is " Eriocheir sinensis foot sample " infiltrative growth, exceeds former damage scope.Asia ethnic group, especially Chinese population are the group of people at high risks of keloid.Therapeutic Method for hypertrophic cicatrix, keloid comprises operation method and non-operative treatment at present, but all has some limitations.How to improve, treat pathologic scar, be the problem that plastic surgeon is paid close attention to always, is also the popular problem of being concerned about simultaneously.
The hypertrophic cicatrix cause of disease mainly comprises wound overtension, infection etc.The mechanism that hypertrophic cicatrix forms comprise fibroblast to the conversion of myofibroblast, fibroblastic propagation, excessively vascularization, cell collagen secretion increases and extracellular matrix is excessively piled up.In the Mechanism Study forming at hypertrophic cicatrix, the most study to TGF-β (transforming growth factor-beta).In three hypotypes of TGF-β, TGF-β 1 is the closest with the formation relation of hypertrophic cicatrix, and TGF-β 1 has participated in fibroblast and transformed to myofibroblast, and promotes emiocytosis collagen and tissue blood vessel.Existing many experiment reports, the expression that suppresses TGF-β 1 can alleviate hypertrophic cicatrix.COX-2(COX-2) be PGE(prostaglandin E) preemzyme, PGE is the important factor of inflammatory reaction, and angiogenesis is had to facilitation.TGF-β 1 and COX-2 have certain facilitation for the Epithelial and stromal conversion in the middle of wound healing process simultaneously.About COX-2, the research in treatment hypertrophic cicatrix is less at present, about the effect between TGF-β 1 and COX-2, also rarely has report.
The main pathological manifestations of keloid is fibroblast abnormality proliferation in tissue, a kind of fibrotic skin diseases of a large amount of depositions of cellular matrix.Keloid has aggressive behavior, is therefore also regarded as a kind of benign tumor of skin.In the formation Mechanism Study of keloid, TGF-β 1-smad path is the path of topmost promotion extrtacellular matrix deposition.The Fibrotic formation mechanism of keloid tissue is complicated, and existing many research, wherein has bibliographical information, has EMT (Epithelial and stromal conversion) phenomenon in keloid tissue.TGF-β 1 and COX-2 are the important path of EMT.In pulmonary fibrosis, renal fibrosis disease research, there is EMT in COX-2 induction epithelial cell, is considered to promote the important factor of tissue fibering.In addition, in the research of breast carcinoma, find, COX-2 induction EMT phenomenon, causes neoplasm metastasis.Thus, the formation of TGF-β 1 and COX-2 and keloid has certain dependency.Have not yet to see and suppress TGF-β 1 and COX-2 treatment keloid report.
Chinese patent literature CN201010260249.8, applying date 2010.08.24, denomination of invention is " promoting siRNA and the application of scarless wound healing of skin ", the siRNA molecule that promotes scarless wound healing of skin is disclosed, the cocktail combination of a plurality of siRNA molecules of a plurality of scarless wound healing related genes of targeting, using the pharmaceutical composition of siRNA molecule or its cocktail combination as effective ingredient, by cell experiment and mice and porcine skin trauma model, proved that this pharmaceutical composition can promote because of wound, the skin wound that surgical operation or diabetic skin ulcer etc. cause reaches without cicatrix and heals, wherein siRNA molecule can targeting those cause the gene of the reparation of wound pathologic or untoward reaction, siRNA two strands has different length and different ends, can targeted human, the homologous sequence of mice and pig cell same gene, a plurality of siRNA in cocktail combination can suppress the various related genes of wound inflammation and revascularization simultaneously, drug effect is more remarkable, HKP in pharmaceutical composition, the pharmaceutical carrier such as dendritic or liposome can strengthen siRNA and import skin histology with the form of nano-particle.But the document is disclosed, be siRNA to be applied to wound in agglutination, promote heal and reduce cicatrization, wound treatment and established scar treatment are diverse two concepts, relate to different physiological process, and at present about treating established hypertrophic cicatrix with siRNA, keloid have not been reported.
Summary of the invention
The object of the invention is for deficiency of the prior art, a kind of siRNA molecular composition is provided.
Another object of the present invention is that purposes and the route of administration of above-mentioned siRNA molecular composition is provided.
For achieving the above object, the technical scheme that the present invention takes is:
SiRNA molecular composition, described siRNA molecular composition comprises: sequence is that siRNA molecule and the sequence of 5 '-CCCAAGGGCUACCAUGCCAACUUCU-3 ' is the siRNA molecule of 5 '-GGUCUGGUGCCUGGUCUGAUGAUGU-3 '.
Preferably, described siRNA molecular composition also comprises pharmaceutically acceptable carrier, diluent or excipient.
Preferably, described pharmaceutically acceptable carrier is selected from the polymer of histidine and lysine.
Described siRNA molecular composition treatment hypertrophic cicatrix, the route of administration of keloid comprise that local organization injection and partial smearing puncture in conjunction with micropin.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
The purposes of siRNA molecular composition as above in the medicine of preparation treatment hypertrophic cicatrix, keloid.
Described treatment hypertrophic cicatrix refers to the established hypertrophic cicatrix for the treatment of, and preferably, described treatment hypertrophic cicatrix, keloid refers to and disappear established hypertrophic cicatrix or reduce the volume of established hypertrophic cicatrix, keloid.
SiRNA molecular composition as above is adjusted the purposes in the medicine of dying at preparation induction fibroblasts from hyperplastic scar or fibroblasts in keloid.
Herein, described " hypertrophic cicatrix " and " keloid " refer in the process of wound healing, in course of infection or the undue growth of the cicatrix occurring in other pathological processes, its mechanism comprise fibroblast to the conversion of myofibroblast, fibroblastic propagation, excessively vascularization, cell collagen secretion increases and extracellular matrix is excessively piled up.Need only the pathological process identical with above-mentioned mechanism, all include the category of " hypertrophic cicatrix " of the present invention, " keloid " in.
The invention has the advantages that:
The present invention's application TGF-β 1 and COX-2 associating siRNA method, striking that external hypertrophic cicatrix, fibroblasts in keloid are carried out to TGF-β 1 and COX-2 express is low, can reach the effect that promotes apoptosis of fibroblasts; In addition,, in animal model, confirm that TGF-β 1 and COX-2 associating siRNA have good therapeutic effect to disappearing of hypertrophic cicatrix; Local organization injection and partial smearing puncture in conjunction with micropin, are that reliable TGF-β 1 and COX-2 associating siRNA are in body route of administration.
Accompanying drawing explanation
Fig. 1 is form under the mirror of fibroblasts from hypertrophic scars in experiment in vitro.
Fig. 2 is genes of interest TGF-β 1 during experiment in vitro gene silencing effect detects, COX-2mRMA expression.
Fig. 3 is relevant genes of interest α-SMA, Col1a1, Col3a1mRMA expression during experiment in vitro gene silencing effect detects.
Fig. 4 is experiment in vitro α-SMA immunofluorescence dyeing result.
Fig. 5 is experiment in vitro Annexin V/PI fluidic cell apoptosis testing result.
Fig. 6 experiment in vitro cell conditioned medium hydroxyproline content testing result.
Fig. 7 is siRNA molecular fluorescence testing result after the local tissue injection administration of the interior experiment of body.
Fig. 8 is that inside and outside is with smearing in conjunction with siRNA molecular fluorescence testing result after micropin puncture administration.
Fig. 9 is the scar tissue piece general form result of experiment cicatrix animal model in body.
Figure 10 is the long-pending measurement result of scar tissue block of experiment cicatrix animal model in body.
Figure 11 is the expression of genes of interest TGF-β 1 in the scar tissue of experiment people hypertrophic scar tissue piece planting model in body, COX-2, α-SMA, Col1a1.
Figure 12 is scar tissue HE, the Masson coloration result of experiment cicatrix animal model in body, α-SMA, CD31, VEGF immunohistochemical staining result.
Figure 13 is the interior hydroxyproline content testing result of scar tissue of experiment cicatrix animal model in body.
Figure 14 is the testing result of the interior fibroblast generation apoptosis of scar tissue of experiment cicatrix animal model in body.
Figure 15 is the interior fibroblast generation apoptosis phenomenon of scar tissue of experiment cicatrix animal model in body and the statistical result of matched group comparison.
The specific embodiment
Below in conjunction with accompanying drawing, the specific embodiment provided by the invention is elaborated.
embodiment 1
1 experiment material
1.1 siRNA effective ingredient
TGF-β 1 siRNA molecule: 5 '-CCCAAGGGCUACCAUGCCAACUUCU-3 ' (SEQ ID NO.1);
COX-2 siRNA molecule: 5 '-GGUCUGGUGCCUGGUCUGAUGAUGU-3 ' (SEQ ID NO.2);
Pharmaceutical carrier: peptide (HKP), the polymer of histidine and lysine;
STP705(siRNA cocktail): above-mentioned TGF-β 1 siRNA molecule, COX-2 siRNA molecule and HKP are formed to nano-particle, wherein TGF-β 1 siRNA molecule, COX-2 siRNA molecule copy number ratio are 1:1, and the weight ratio of two kinds of siRNA molecules and HKP is 1:4.Above medicine is provided by the Suzhou biological company limited of holy promise.
antibody
The anti-human α-actin of rabbit (α-SMA, alpha smooth muscle Actin) monoclonal antibody (ab5694, Abcam, Iowa, USA), immunohistochemical staining primary antibodie dilution ratio 1:200, immunofluorescence dyeing primary antibodie dilution ratio 1:100;
The anti-human CD31 monoclonal antibody of rabbit (ab38264, Abcam, Iowa, USA), immunohistochemical staining primary antibodie dilution ratio 1:50;
Rabbit human vessel endothelium growth factor resisting (VEGF, vascular endothelial growth factor) monoclonal antibody (cat. #1909-1, Epitomics, California, USA), immunohistochemical staining primary antibodie dilution ratio 1:50.
use primer
2 experimental techniques
2.1 experiment in vitro
2.1.1 tissue specimen is originated
Hypertrophic scar tissue specimen and keloid tissue specimen all derive from the attached Jiu plastic surgery of the People's Hospital of Shanghai Communications University and are in hospital or out-patient.
the separation of people's fibroblasts from hypertrophic scars and cultivation
The hypertrophic scar tissue under operating room aseptic condition, operation being cut, keloid tissue piece are removed epidermis and subcutaneus adipose tissue, then on superclean bench, with eye scissors, tissue are cut into big or small about 0.5-1mm
3fritter, with 0.25% type i collagen enzymic digestion 2-4 hour, until piece of tissue be digested to cotton-shaped after strainer filtering, obtain the separated cell of digestion.Add again the appropriate DMEM culture fluid containing 20% calf serum, 100U/ml penicillin, 100 μ g/ml streptomycins to continue to cultivate, be inoculated in culture dish, at 95% air, 5%CO
2, cultivate under the saturated humidity condition of 37 ℃.Within three days, change liquid once.When cell fusion reaches 90%, go down to posterity.
the mensuration of cell proliferation curve
Cell proliferation experiment selects P3 that vegetative state is good for cell, cell counting.The 96 every holes of orifice plate add 2000 cells of 100 microlitre.Cell attachment after 6 hours, changes culture fluid, adds respectively STP705, HKP, TGF-β 1 siRNA, COX-2 siRNA in culture fluid, and drug level is respectively 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml.Intervene after 48 hours, exhaust culture fluid, PBS rinses 3 times, and every hole adds 100 μ l culture fluid and 10 μ l CCK-8 solution.Add respective amount cell culture fluid and CCK-8 solution but do not add the hole of cell as blank.In cell culture incubator, continue to hatch after 3 hours, by microplate reader (Varioskan flash, Thermo Electron Co.), detect.At wavelength 450nm, measure absorbance, each hole count value deducts blank value, obtains absolute light absorption value.Draw cell proliferation curve.
vitro Drug intervention experiment
Fibroblasts from hyperplastic scar reaches the third generation, with 2 * 10
6be inoculated in 6 orifice plates.After cell attachment, change liquid, add different intervention factors.Arrange 2 groups, matched group adds simple culture fluid, and treatment group adds STP705 in culture fluid, and concentration is 10 μ g/ml.Intervene and detect after 48 hours, observation of cell form.
reticent effect detection
Q-PCR detects the variation that matched group and genes of interest TGF-β 1, the COX-2 of each experimental group after intervening 48 hours express, and the variation expressed of relevant genes of interest α-SMA, Col1a1, Col3a1.
α-SMA immunofluorescence dyeing
Cell was intervened after 48 hours, exhausted culture fluid in culture dish.Fixing: 4% paraformaldehyde is 10min fixedly, and then PBS washes 3 times, each 5 minutes, dry rear cell dyeing region, the oil pen's labelling selected.Antigen retrieval: drip antigen retrieval liquid, effect 5min.Permeable membrane: add 0.1% Triton-X100 permeable membrane 10min, PBS washes three times.Sealing: 10% sheep blood serum sealing 1h.Primary antibodie is hatched: at cell surface, drip 1:100 α-SMA antibody, be placed in wet box, 4 ℃ are spent the night.Primary antibodie is hatched complete, PBS washing three times.Two anti-hatching: lucifuge operation, drips 1:200 goat-anti rabbit two at cell surface anti-, effect 30min.Negative control group is set, does not add primary antibodie and only add two anti-groups.Two anti-hatch complete, PBS washing three times.1:1000 DAPI dyeing 1 minute, PBS washes three times.Fluorescence microscopy Microscopic observation, takes pictures.
apoptosis detects
Intervene after 48 hours, trypsin digestion and cell, stops digestion, centrifugal 1500rpm, 5min, and adjusting cell concentration to be detected is 10
6individual/ml, gets 200 μ l, goes to flow cytometer detection pipe, 1000rpm * 5min(4 ℃).The PBS 1ml washed twice of pre-cooling, 1000rpm * 5min(4 ℃).Cell is resuspended in to 60 μ l binding buffer, adds 4 μ l Annexin V-FITC(20 μ g/ml), mix gently, lucifuge is placed 15 minutes on ice.Each sample adds 4 μ l PI(50 μ g/ml before facing machine), add Buffer and supply 200 μ l, after 2 minutes, detect rapidly.
hydroxyproline assay
Experimental principle: hydroxyproline accounts for 13.4% in collagen protein accounts for minute quantity in elastin laminin, in other albumen, does not all exist.Measure the hydroxyproline content in tissue, body fluid, culture fluid, can extrapolate the content of collagen protein.
T chloramines detection method, Hydroxyproline Colorimetric Assay Kit(Catalog #K555-100, BioVision, USA).Preparation T chloramines reagent, every hole 6 μ l T chloramines, 94 μ l oxidation reaction buffer.Preparation DMAB(methylamino benzaldehyde) reagent: every hole dimethylamino benzaldehyde 50 μ l, perchloric acid/isopropyl alcohol 50 μ l, keep in Dark Place.
The drafting of standard curve: configuration 0.1mg/ml hydroxyproline solution, 1mg/ml hydroxyproline standard substance 10 μ l add 90 μ l hydrogen peroxide, mix.Get respectively 0,2,4,6,8,10 μ l 1mg/ml hydroxyproline solution dilution to 100 μ l, make standard substance sample.Get 10 μ l master samples and add in 96 orifice plates, dry sample under vacuum.Every hole adds 100 μ l T chloramines reagent, and incubated at room 5 minutes adds 100 μ l DMAB reagent, hatches 90 minutes for 60 ℃.Under microplate reader 560nm, measure absorbance.The hole that adds 0 standard substance is blank, and the reading in every hole deducts blank value.Drawing standard curve.
Supernatant sample 10 μ l add 90 μ l H
2o.In 100 μ l sample liquid, add the dense HCl of 100 μ l, the politef EP seal of tube, 120 ℃ are hydrolyzed 3 hours.Get 10 μ l sample hydrolysis and also add in 96 orifice plates, dry sample under vacuum.Every hole adds 100 μ l T chloramines reagent, and incubated at room 5 minutes adds 100 μ l DMAB reagent, hatches 90 minutes for 60 ℃.Under microplate reader 560nm, measure absorbance.The light absorption value in every hole is converted into hydroxyproline content (Sa) (the μ g of unit) in standard curve.Adding the sample volume in 96 orifice plates is Sv(μ l), the hydroxyproline concentration in sample is calculated C=Sa/Sv μ g/ μ l.
experiment in body
2.2.1 the foundation of hypertrophic cicatrix animal model
(1) laboratory animal
Laboratory animal is provided by experimental animal center, Shanghai, and the use of laboratory animal meets laboratory animal ethics and Laboratory Animal Welfare clause (ethics is examined moving Cologne [the 2012]-7(HDKL[2012 in Shanghai]-7).
(2) tissue specimen source
Tissue specimen all derives from my plastic surgery of institute inpatient, and huge breast dwindles Operated Specimens in art.The use of specimen obtains patient's informed consent.
(3) method for building up
Full thickness skin graft is transplanted in the foundation of nude mice back people's hypertrophic cicatrix animal model.The skin histology of acquisition is pruned and made full thickness skin graft, big or small 2cm * 1.6cm.10% chloral hydrate 0.1ml intraperitoneal injection of anesthesia nude mice, adhesive tape is nude mice extremity fixedly, and excision nude mice back 2cm * 1.6cm skin, transplants people's full thickness skin graft in nude mice back.When skin grafting, skin graft and Mus deep fascia of back are fixed, make skin graft and rat tissue form stable contact surface, improve the survival rate of skin graft.Skin graft is played the bale of cotton and is fixed, and within postoperative 2 weeks, takes out stitches.Within postoperative one month, nude mice back forms hypertrophic scar tissue.Postoperative about one month, laboratory animal back formed scar model.
in body, intervene the reticent effect detection of genes of interest
Use cicatrix animal model to carry out STP705 intervention, intervene concentration 20 μ g/ml.Compound concentration is the STP705 of 80 μ g/ml.After anesthetized animal, 3D scanner is measured the volume of nude mice back piece of tissue, the cm of unit
3, by final concentration, be 20 μ g/ml, converse the injection volume of 80 μ g/ml STP705.
Injecting method: for medicine is injected in piece of tissue as far as possible equably, adopt 5 method injections.Each injection of 4 quadrants a bit, is divided equally a bit by piece of tissue central authorities.Drug injection is in scar tissue piece.
The collection of specimen: collect after single injection each 3 of the specimen of 4 time points 1 day, 3 days, 5 days, 7 days.
The detection of genes of interest: the tissue specimen of collection is carried out to Q-PCR detection, and frozen piece of tissue, pulverizes with bistrique, adds Trizol cracking in sample, detects TGF-β 1, COX-2 expression.
Testing result is analyzed, drawn the effective time limit of single injection.
The therapeutic scheme of formulating intervention experiment in cicatrix animal model is: after intervening, STP705 final concentration is 20 μ g/ml, administration 3 times, every minor tick 4 days.
vivo medicine-feeding mode
2.2.3.1 drug administration by injection mode
Set up after cicatrix animal model, measure scar tissue volume, by 20 μ g/ml administration concentration, calculate required drug dose.By the NC SiRNA molecule of FAM labelling, the polymer of HKP, subregion is evenly expelled in scar tissue, gathers latter 1 hour, 24 hours, 48 hours scar tissues of injection, after frozen section, and fluoroscopic examination SiRNA molecule amount retained.FAM-NC You Ji agate company is synthetic, NC sequence:5 ' TTCTCCGAACGTGTCACGT 3 '.
micropin puncture administering mode
Set up after cicatrix animal model, smear the FAM-HKP-NC of 80 μ g/ml concentration at scar tissue surface uniform, using dosage is the complete submergence scar tissue of medicine surface.Experimental group application 1.5mm micropin is in scar cuticle surface repeated localised puncture; Matched group does not puncture and only smears medicine.Gather and respectively to organize 1 hour scar tissue specimen after administration, after frozen section, fluoroscopic examination SiRNA molecule amount retained.
intervention experiment in body
Cicatrix animal model when implanting 2 weeks, piece of tissue is carried out to intervention experiment in body.
grouping:matched group and experimental group are set, every group of 4 laboratory animals.
processing method:it is 20 μ g/ml STP705 drug administration by injection interventions that experimental group animal scar tissue gives concentration, administration 3 times, every minor tick 4 days.The same experimental group of control animals scar tissue piece processing method, injection 20 μ g/ml NC.
the record of experimental result:taking Pictures recording tissue is data substantially.Measure the long-pending size variation of scar tissue block.After intervening there is significant difference in 4 weeks scar tissue pieces between two groups.
histology:collect the tissue specimen that each group is intervened latter 4 weeks, carry out HE dyeing, Masson trichrome stain.
dyeing:4% paraformaldehyde is fixed, dewaters transparent, and waxdip embedding, cooled and solidified in bulk, section and paster.Before dyeing, must slough the paraffin in section with dimethylbenzene, then arrive low-concentration ethanol via high concentration, finally enter distilled water, just can dye.HE dyeing: put into hematoxylin aqueous solution and dye several minutes entering section after distilled water.Color separation in sour water and ammonia, each several seconds.Flowing water rinses after 1 hour and enters distilled water for a moment.Enter in 70% and 90% ethanol and dewater each 10 minutes.Enter ethanol Yihong dyeing liquor dyeing 2-3 minute.Dewater transparent: the section after dyeing is dewatered through absolute alcohol, then make to cut into slices transparent through dimethylbenzene.Natural gum mounting.
trichrome stain:roasting sheet, dewaxing and aquation dye with HE.Celestine blue liquid dyes 15 minutes, and tap water rinses 2 minutes.Haematoxylin core dyes 15 minutes, and tap water rinses 2 minutes.1% hydrochloride alcohol differentiation, tap water rinses to mirror and sees karyon change indigo plant.Complex liquid (Ponceaux and acid fuchsin: 0.2% glacial acetic acid 1:9) dye 20 minutes (amount is 50 μ l/ specimen).0.2% acetate solution rinses 2 times.Phosphotungstic acid differentiation 15 minutes, 0.2% glacial acetic acid rinses 2 times.Viride nitens liquid dyes 15 minutes, and 0.2% acetic acid rinses 2 times, immerses fast 95% ethanol, dehydrated alcohol dehydration.Dimethylbenzene is transparent, resinene mounting.
sABC detects:collect the tissue specimen that each group is intervened latter 4 weeks, carry out α-SMA, VEGF, CD31 immunohistochemical staining.
The embedding of piece of tissue, section are dyeed with HE.Paraffin sheet dewaxes to water, and PBS rinses 3 times, each 5 minutes.3% hydrogen peroxide 5-10 minute, reduces the non-specific background dyeing/RT cell punching that endogenous peroxydase causes, and PBS rinses 3 times, each 5 minutes.10% sheep blood serum sealing 30 minutes.4 ℃ of overnight incubation of primary antibodie.PBS rinses 2 times, each 3 minutes.Two of peroxidase labelling resists 37 ℃ hatches 30 minutes, and PBS rinses 2 times, each 3 minutes.DAB(1ml ddH
2in O+ DAB colour reagent box, reagent A.B.C. is each 1) colour developing, DAB effect 10-30 minute, adds after nitrite ion and observes at any time, after finding to develop the color, puts into immediately water color development stopping.Redye Lignum Sappan uniformly dyeing core, dehydration, mounting.
genes of interest detects:collect the tissue specimen that each group is intervened latter 4 weeks, carry out Q-PCR detection.Testing goal gene TGF-β 1, COX-2 express, and the expression of relevant genes of interest α-SMA, Col1a1, Col3a1.Between organizing, compare.
hydroxyproline content detects:collect the tissue specimen that each group is intervened latter 4 weeks, organize hydroxyproline content to detect.Adopt T chloramine method.
apoptosis detects: collect the tissue specimen that each group is intervened latter 4 weeks, 4% paraformaldehyde is fixed, paraffin embedding.The breach end-labelling of TUNEL(deoxyribonucleotide terminal transferase mediation) dyeing is carried out the apoptotic cell in tissue, and concrete operation step is undertaken by explanation:
Pretreated: paraffin-embedded tissue slice pretreatment, conventional dewaxing, processed.E.C. 3.4.21.64 working solution, incubated at room 20 minutes, PBS rinses 3 times, each 5 minutes.Deoxyribonucleotide terminal transferase (10 *) 50 μ l and the reactant liquor reagent 450 μ l mixed preparing TUNEL reaction mixtures that contain nucleotide mixed liquor.The reactant liquor reagent that reserve part contains nucleotide mixed liquor is as negative control.Every section drips TUNEL reaction mixture 50 μ l, and 370C is hatched 60 minutes, and PBS rinses 3 times, each 5 minutes.Drip transforming agent-POD(enzyme labelling anti-fluorescein antibody) 50 μ l, 37
0c is hatched 30 minutes, and PBS rinses 3 times, each 5 minutes.Drip mono-of DAB, light Microscopic observation colour developing result, in time color development stopping.PBS rinses 3 times, each 5 minutes.Haematoxylin dyeing 3 seconds, rinses neutral gum mounting.Light Microscopic observation.
experimental result
3.1 experiment in vitro
After primitive cell culture, reached for 3 generations, carry out external intervention experiment.
By CCK8, detect and draw TGF-β 1 siRNA of variable concentrations, the impact of COX-2 siRNA on cell proliferation, result shows that TGF-β 1 siRNA, COX-2 siRNA have obvious inhibitory action to the scar fibroblast proliferation of In vitro culture, and intervention effect is better than TGF-β 1 siRNA or COX-2 siRNA intervention effect.Suitable pharmaceutical intervention concentration is 10 μ g/ml, and intervention time is 48 hours.
As shown in Figure 1, vitro Drug intervention experiment result is: Microscopic observation blank group proliferation of hypertrophic scar fibroblasts, propagation are well; There is little balloon-shaped structure in experimental group fibroblasts from hypertrophic scars periphery, cell density is lower than matched group.
The reticent effect of intervening as shown in Figure 2, rear genes of interest: TGF-β 1, COX-2 mrna expression obviously reduce, and experimental group and matched group have notable statistics difference.
As shown in Figure 3, after TGF-β 1, COX-2 siRNA intervene, the expression of the relevant genes of interest of cell changes: Q-PCR testing result shows that experimental group cell α-SMA, Col1a1, Col3a1 mrna expression level are starkly lower than matched group.
As shown in Figure 4, two groups of cells of experimental group and matched group carry out a-SMA immunofluorescence dyeing, and result shows that TGF-β 1 siRNA, COX-2 siRNA intervene rear α-SMA expression and be starkly lower than cellular control unit.
As shown in Figure 5, experimental group and cellular control unit application flow cytometer carry out apoptosis detection, and experimental group cell exists apoptosis phenomenon, and blank group has no apoptosis.
As shown in Figure 6, after TGF-β 1 siRNA, COX-2 siRNA intervene, the ability of experimental group emiocytosis collagen is starkly lower than cellular control unit.
administering mode
As shown in Figure 7, drug administration by injection mode: inject latter 1 hour SiRNA molecule and cause in scar tissue along injection needle diffusion; Within 24 hours, visible SiRNA molecular distribution is around nucleus; In 48 hours scar tissues, still have SiRNA molecule not degrade.
As shown in Figure 8, micropin puncture administering mode: the visible SiRNA molecule of experimental group, by micropore, enters scar tissue skin corium; Matched group SiRNA molecule cannot pass through epidermal area.
experiment in body
As shown in Figure 9, matched group, experimental group compare substantially: the piece of tissue volume of substantially seeing in experimental group cicatrix animal model is little compared with matched group, and piece of tissue surface is without congested phenomenon.As shown in figure 10, through measuring the long-pending size variation of scar tissue block, after show intervening, between 4 weeks matched groups and experimental group there is significant difference in scar tissue piece, and experimental group is remarkable with the rear scar tissue piece volume difference of injection before injection, shows can significantly reduce hypertrophic cicatrix after TGF-β 1 siRNA, COX-2 siRNA intervene.
As shown in figure 11, surrounding after intervening, in matched group and experimental group scar tissue, TGF-β 1, COX-2 mrna expression amount detect through Q-PCR, show that experimental group genes of interest TGF-β 1, COX-2 obviously reduce compared with matched group tissue expression.Other relevant genes of interest α-SMA, Cola1 mrna expression amount decline.
As shown in figure 12, HE dyeing, the visible treatment group scar tissue of Masson trichrome stain inner cell composition obviously reduce, fibrous bands fracture, dissolved form.In the visible treatment group scar tissue of VEGF immunohistochemical staining, VEGF secretion reduces.In the visible treatment group scar tissue of CD31 immunohistochemical staining, blood vessel, new vessels reduce.
As shown in figure 13, collagen content balance in matched group and experimental group scar tissue piece.In experimental group tissue, collagen content is starkly lower than matched group, and difference has statistical significance.
As shown in figure 14, pharmaceutical intervention is after 4 weeks, and in experimental group scar tissue, apoptosis of fibroblasts phenomenon increases.As shown in figure 15, experimental group is compared with matched group, and fibroblast generation apoptosis ratio exists significant difference.
embodiment 2
SiRNA molecular composition (siRNA molecular composition in siRNA molecular composition more of the present invention and Chinese patent literature CN 201310577630.0, described siRNA molecular composition comprises: nucleotide sequence contains a) 5 '-ccccggaggugauuuccaucuacaa-3 ', the siRNA molecule of 5 '-uuguagauggaaaucaccuccgggg-3 ', contain b with nucleotide sequence) 5 '-gucuuuggucuggugccuggucuga-3 ', the siRNA molecule of 5 '-ucagaccaggcaccagaccaaagac-3 ') effect in disappear hypertrophic cicatrix or keloid.
According to the method for embodiment 1, prepare siRNA cocktail, preparation cicatrix animal model then carries out intervention experiment in body when piece of tissue is implanted 2 weeks to cicatrix animal model.
grouping:201310577630.0 groups, of the present invention group of matched group, CN is set, and every group of 20 laboratory animals, respectively organize cicatrix animal model hypertrophic cicatrix size and have comparability before intervention.
processing method:it is 20 μ g/ml STP705 drug administration by injection interventions that treated animal scar tissue of the present invention gives concentration, administration 3 times, every minor tick 4 days.201310577630.0 groups of CN give concentration 20 μ g/ml siRNA cocktail drug administration by injection interventions, and processing method is with of the present invention group.The same experimental group of control animals scar tissue piece processing method, injection 20 μ g/ml NC.
experimental result record:taking Pictures recording tissue is data substantially.Measure the long-pending size variation of scar tissue block.Scar tissue piece difference between 4 weeks each groups after relatively intervening.
result:intervene 4 weeks front and back and respectively organize scar tissue piece change in volume in Table 1.Through significance analysis, 201310577630.0 groups of of the present invention groups, CN are compared with matched group, after intervening, scar tissue block amasss and all has significant difference (P<0.05), compare with 201310577630.0 groups of CN for of the present invention group, after intervening, scar tissue block amasss and also has significant difference (P<0.05).
Table 1 is intervened 4 weeks front and back and is respectively organized scar tissue piece change in volume
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University Sch
<120> siRNA molecular composition and the purposes in treatment pathologic scar thereof
<130> /
<160> 12
<170> PatentIn version 3.3
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Claims (7)
1.siRNA molecular composition, it is characterized in that, described siRNA molecular composition comprises: sequence is that siRNA molecule and the sequence of 5 '-CCCAAGGGCUACCAUGCCAACUUCU-3 ' is the siRNA molecule of 5 '-GGUCUGGUGCCUGGUCUGAUGAUGU-3 '.
2. siRNA molecular composition according to claim 1, is characterized in that, described siRNA molecular composition also comprises pharmaceutically acceptable carrier, diluent or excipient.
3. siRNA molecular composition according to claim 2, is characterized in that, described pharmaceutically acceptable carrier is selected from the polymer of histidine and lysine.
4. according to the siRNA molecular composition described in claim 1-3, it is characterized in that, described siRNA molecular composition treatment hypertrophic cicatrix, the route of administration of keloid comprise that local organization injection and partial smearing puncture in conjunction with micropin.
5. the purposes of the siRNA molecular composition described in claim 1-3 in the medicine of preparation treatment hypertrophic cicatrix, keloid.
6. purposes according to claim 5, is characterized in that, described treatment hypertrophic cicatrix, keloid refers to and disappear established hypertrophic cicatrix, keloid or reduce the volume of established hypertrophic cicatrix, keloid.
7. the siRNA molecular composition described in claim 1-3 is adjusted the purposes in the medicine of dying at preparation induction fibroblasts from hyperplastic scar or fibroblasts in keloid.
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| CN113425844A (en) * | 2021-07-09 | 2021-09-24 | 中国医学科学院北京协和医院 | Drug intervention target of keloid and application thereof |
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| CN113425844A (en) * | 2021-07-09 | 2021-09-24 | 中国医学科学院北京协和医院 | Drug intervention target of keloid and application thereof |
| CN115851710A (en) * | 2022-08-02 | 2023-03-28 | 中国医学科学院北京协和医院 | siRNA molecule composition and application thereof |
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