The specific embodiment
Below in conjunction with specific embodiment the present invention is carried out detailed description.
One, the new purposes of Nafamostat Mesilate treatment retinal neovascularization property disease
The Nafamostat Mesilate medicine is bought the company in Sigma-Aldrich; Mouse peritoneal injection consumption is 50mg/kg; Utilize C57BL/6J mice (purchasing Laboratory) to set up oxygen and induce retinopathy varying model OIR in Jackson; To place volume fraction be that 75% oxygen environment is raised and got back to air behind the 5d and continue to raise with cleaning the level mice (P7) age on the 7th, from 12 days (P12) beginning, and mouse peritoneal injection every day Nafamostat Mesilate medicine; At 17 days the most vigorous (P17) of retinal neovascularization growth, irritate 5 μ lFITC-conjugated isolectin-B4 (20mg/ml) through jugular vein.Experiment is drawn materials after finishing, and carries out retina shop sheet subsequently, observes the retinal neovascularization growing state.Show that through fluorescence microscope Nafamostat Mesilate can obviously suppress the growth of RNV, the growth of experiment mice optical fundus blood vessel is tending towards normal.
Yet, utilize the receptor C3aR of complement system endotoxin molecule C3a and C5a and C5aR to knock out Mus (C3aR-/-And C5aR-/-Mice) equally handle the growth change of retinal neovascularization and not obvious behind the injection Nafamostat Mesilate.In addition, through retina shop sheet, fluoroscopic visualization is observed, the statistics of blood vessel endothelium check figure, relatively C57BL/6J mice and C3aR-/-And C5aR-/-Mice, concrete operations are following:
(1) retina shop sheet: utilize C57BL/6J mice and C3aR-/-And C5aR-/-Mice is set up oxygen and induces retinopathy varying model OIR, and concrete grammar is with the above.According to the fluorescence microscope result, C3a and C5aR knock out the growth that has suppressed RNV.According to the standards of grading of RNV, the RNV growth, the formation of blood tube bundle, the tortuous degree of blood vessel that knock out Mus obviously reduce than the C57BL/6J mice, and its no perfusion area of central authorities is significantly greater than the normal group mice.
(2) fluoroscopic visualization art: fluorescein optical fundus blood vessel system shadow is the ophthalmologic examination handicraft of rising the sixties in 20th century.Utilize fluorescein sodium to inject fast as system shadow agent farmland forearm vein; When fluorescein sodium gets into optical fundus blood vessel with blood flow; Through tearing the Fundus photography machine of none group light filter open, receive the fluorescence form that excitation line is launched when continue taking the outer dyestuff samsara of optical fundus blood vessel, to watch the process of the dynamic samsara of retina; Thereby be able to know from experience the fine structure of optical fundus blood vessel and the variation of little samsara, for the pathogeny of much retinopathy, diagnosis, doctor's unrest and prognosis evaluation supply with according to.Utilize this means,, carry out Real Time Observation with the optical fundus contrast machine subsequently, the line item of going forward side by side mouse peritoneal perfusion 0.1ml10% fluorescein sodium.Presentation of results C3aR-/-And C5aR-/-The central authorities of mice do not have perfusion area obviously greater than the C57BL/6J mice.
(3) blood vessel endothelium check figure counting: set up the OIR model, get C57BL/6J mice and C3aR and C5aR respectively and knock out the rathole eyeball, and carry out liver histopathological analysis, method is following:
Fixing (10% formalin, BouinShi fixative etc.): the protein denaturation of tissue, cell is solidified, preventing cell self-dissolving after death or the decomposition of antibacterial, thus the original morphosis of maintenance cell.Carry out subsequently
It is transparent to dewater: generally use by low concentration and make dehydrant to alcohol in high concentration, slough the moisture content in the piece of tissue gradually.Again piece of tissue is placed both to be dissolved in ethanol, be dissolved in the clarifier xylene of paraffin transparently again, replace out the middle ethanol of piece of tissue with xylene, could the waxdip embedding.
Waxdip embedding: transparent piece of tissue is placed the paraffin that has dissolved, put into the wax-dissolving box insulation.Treat to carry out embedding after paraffin immerses piece of tissue fully: prepare container (like a folding small paper box) earlier, pour the paraffin that has dissolved into, the piece of tissue that paraffin has been soaked in rapid gripping is put into wherein.The cooled and solidified in bulk.The piece of tissue hardening that embedding is good could be cut into very thin section on microtome.
Section and paster: the wax stone that embedding is good is fixed on the microtome, thinly slices, and is generally 4 micron thick.The thin slice that downcuts is fold often, be put in the water of heating to plate, and is attached on the microscope slide again, puts in 45 ℃ of calorstats and dry.
Dewaxing dyeing (HE dyeing): before the dyeing, must slough the paraffin in the section, arrive low-concentration ethanol via high concentration again, go into distilled water at last, just can dye with xylene.Detailed process is following:
1. hematoxylin aqueous solution dyeing several minutes is put in the section that will go into behind the distilled water.
2. color separation in sour water and the ammonia, each several seconds.
3. the flowing water flushing is gone into distilled water for a moment after 1 hour.
4. go into to dewater in 70% and 90% ethanol each 10 minutes.
5. go into ethanol Yihong dyeing liquor dyeing 2-3 minutes.
It is transparent to dewater: the section after the dyeing is dewatered through absolute alcohol, makes section transparent through xylene again.
Sealing: canada balsam is dripped in transparent section, the covered sealing.After treating that natural gum is slightly done, stick the mark writing paper, section preparation just can use.
Through serial section, dyeing is observed, statistics blood vessel endothelium check figure, and the result shows C3aR-/-And C5aR-/-The blood vessel endothelium check figure of the breakthrough internal limiting membrane of mice is starkly lower than the growth that the C57BL/6J mice shows that endotoxin molecule C3a and C5a in the complement system can obviously promote retinal neovascularization.
Set up the OIR model, get C57BL/6J mice and C3aR and C5aR respectively and knock out Mus (PBS matched group and FUT-175 processed group) retina, extract total protein and carry out western blot analysis, the concrete operations step is following:
The extraction of total protein: add protein lysate and protease inhibitor, fully shred and grind well, place on ice, fully extract, centrifugal, get supernatant.
SDS-PAGE gel electrophoresis: glass cleaning plate assembling electrophoresis tank, encapsulating and last appearance, electrophoresis (be generally constant voltage 120V electrophoresis, constant voltage 160V electrophoresis is till bromophenol blue reaches the gel bottom behind sample entering lower floor glue).
Change film: cut glue, be equipped with film, the dress film changes film
Immunoreation, specific operation process is following:
1. sealing: with film with rinsing among the TBST 3 times, each 5min.After the rinsing nitrocellulose filter is put into the plate that is added with the BSA confining liquid, shaking table 2h under the room temperature, perhaps 4 ℃ of refrigerator overnight.
2. add one anti-: the nitrocellulose filter after will seal put into contain TBST wash on the cylinder shaking table rinsing 3 times, 5min at every turn.After washing TBST is drained as far as possible (but film must keep moistening), put into and be added with an anti-plate side-sway shaking table room temperature low speed and hatch 2h.
3. add two anti-: reclaim one anti-ly, nitrocellulose filter is washed in the cylinder rinsing of room temperature shaking table 3 times at TBST, each 5min puts into the nitrocellulose filter of rinsing then and is added with two anti-plates, lucifuge incubated at room 1h.After hatching nitrocellulose filter washed at TBST and wash each 5min in the cylinder 3 times.
Photographic fixing is developed in chemiluminescence
Gel images is analyzed: film is scanned or takes pictures, with the molecular weight of gel images processing system evaluating objects band and OD value only.
Through analyzing gel images; The protein expression that shows FUT-175 scalable VEGF-A; In knocking out Mus, not having the expression that detects VEGF-A changes; Preliminary explanation FUT-175 expresses to VEGF-A through complement system (C3a and C5a), and then regulates the growth of RNV, and more morning, more effective treatment retinal neovascularization property disease.Therefore, Nafamostat Mesilate can be used for preparing the medicine of treatment retinal neovascularization property disease.
Embodiment 1: Nafamostat Mesilate adds C3a antibody
C3a antibody is bought in Abcam company (ab48581).Including mass volume ratio g/ml in the medicine of preparation is that 2% methanesulfonic acid naphthalene is not taken charge of; Add mass volume ratio g/ml simultaneously and be 0.002% C3a antibody; The addition mass volume ratio g/ml of antiseptic phenethanol is 1.1%, is supplemented to final volume with phosphate buffer at last.
For the methanesulfonic acid naphthalene not the consumption of department and antibody, antiseptic be as the criterion to meet pharmacology dosage, those skilled in the art can confirm addition through the method for routine, and is not limited only to the scope put down in writing in the description of the present invention.
Concrete method for preparing is that the Nafamostat Mesilate pressed powder is dissolved in phosphate buffer (sodium chloride 9 grams; Sodium hydrogen phosphate 6 grams, sodium dihydrogen phosphate 0.4 gram adds distilled water to 1000ml, PH7.2 to 7.4) in; Adding concentration is the antibody of 200 μ g/ml, adds phenethanol then and accomplishes preparation.
Embodiment 2: Nafamostat Mesilate adds C3a and C5a antibody
C5a antibody is bought in Santa Cruz company (sc-21941).In the 90ml phosphate buffer, adding 1g methanesulfonic acid naphthalene does not take charge of; Add the C3a of 0.003g and the mixture of C5a antibody again; Wherein the adding proportion of C3a and C5a antibody is 1:1, adds 1g antiseptic phenethanol again as antiseptic, at last with the phosphate buffer polishing to 100ml.
Wherein C3a and C5a antibody can equal proportion add, the ratio that also can select for use arbitrary pharmacology to allow.
Intraperitoneal administration dosage 50mg/kg according to Nafamostat Mesilate; P12 days from the oxygen guidance model begin; Inject the medicine of these embodiment 2 preparations every day; Can observe at the most vigorous P17 of retinal neovascularization growth, the growth of injecting the mice retinal neovascularization of this embodiment medicament injects more merely that Nafamostat Mesilate is more effective must to be able to control, and angiogenic growth is more normal.