CN103920144B - The new opplication of the deoxyribonuclease I of recombined human - Google Patents

The new opplication of the deoxyribonuclease I of recombined human Download PDF

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CN103920144B
CN103920144B CN201310014825.4A CN201310014825A CN103920144B CN 103920144 B CN103920144 B CN 103920144B CN 201310014825 A CN201310014825 A CN 201310014825A CN 103920144 B CN103920144 B CN 103920144B
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pq
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lung
deoxyribonuclease
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吴庄民
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吴庄民
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Abstract

The invention belongs to biomedicine field, disclose gene recombinant human deoxyribonuclease I with intravenously administrable, the application in the application of the lung preversation of the acute lung injury for the treatment of drug induccd, and the rheumatism for the treatment of acute interstitial pneumonia.Gene recombinant human deoxyribonuclease I has been excavated new medical application by the present invention, has opened up a new application.There is good prospect in medicine.And gene recombinant human deoxyribonuclease I abundance, preparation technology is simple, easy to use.

Description

The new opplication of the deoxyribonuclease I of recombined human

Technical field

The invention belongs to field of biological pharmacy, the deoxyribonuclease I (rhDNaseI) being specifically related to gene recombinant human exists Prepare the application in the medicine of curative source property acute lung injury.

Background technology

The deoxyribonuclease I one class endonuclease of people, acts on the phosphodiester bond of DNA, catalytic dna water Solve.Numbered EC 3.1.21.1.

I.e. Deoxyribonuclease, Chinese is deoxyribonuclease, is that one can digest strand or double-strand DNA produces the Cobra venom endonuclease of the oligodeoxynucleotide of monodeoxyribonucleotide or strand or double-strand.

DNase I, i.e. Deoxyribonuclease I, Chinese is deoxyribonuclease I, is that one can digest list Chain or double-stranded DNA produce the Cobra venom endonuclease of the oligodeoxynucleotide of monodeoxyribonucleotide or strand or double-strand.DNase I Product after hydrolysing single or double-stranded DNA, 5' end is phosphate group, and 3' end is hydroxyl.

DNase I activity depends on calcium ion, and can be activated by magnesium ion or divalent manganesetion.Under magnesium ion existence condition, DNase I can any site of random shearing double-stranded DNA;Under divalent manganesetion existence condition, DNase I can be in same position Point shears DNA double chain, forms flat end, or the viscous end that 1-2 nucleotide is prominent.

Deoxyribonuclease I is used for the treatment bronchiectasis relevant with mucosa effect, lung abscess, capsule the most clinically Property pulmonary fibrosis and oral cavity department disease etc..

The deoxyribonuclease I (rhDNaseI) of gene recombinant human contains 260 amino acid whose polypeptide chains, has total score Protonatomic mass is 37000 dalton, and molecular formula is C1321H1995O396S9

Injury of lung belongs to chest diseases, and penetrance damage (except projected at high velocity beyond the region of objective existence) is relatively easily tolerated, pulmonary parenchyma by lungs There is good repair ability, unless hilus pulumonis structural damage, the gas leakage of general lung tissue and hemorrhage quickly stopping, peripheral part Substantial damage seldom need excision;On the other hand, although passivity injury of lung causes lesser degree of local damage, but due to The gross area of multiple injuries strengthens and secondary reactions sexually revises, and it can cause complication more serious, the most life-threatening.

Acute lung injury (acute lung injury, ALI) is that the alveolar epithelium that various directly or indirectly causative factors of injury cause is thin Born of the same parents and capillary endothelial cell damage, cause diffusivity interstitial lung and intra-alveolar edema, the acute hypoxia respiratory function caused The most complete.Reduce with Pulmonary volume, lung compliance reduces, ventilation/blood flow is out of proportion as pathophysiological features, clinical signs For showing as the exudative process of heterogencity in Progressive symmetric erythrokeratodermia hypoxemia and respiratory distress, lung image, it is developed to sternly The weight stage (oxygenation index < 200) it is referred to as adult respiratory distress syndrome (acute respiratory distress Syndrome, ARDS).The reason causing ALI includes infection, chemical factors damage, autoimmune etc., the most clinical scarce Weary special treatment means, only to remove possible inducement, anti symptom treatment, if desired mechanical ventilation support.Existing Therapeutic Method focuses primarily on suppression inflammatory factor, reduce inflammation reaction, or improves oxygenate, corrects anoxia, or in promotion lung The absorption oozed out, including glucocorticoid, pentoxifylline, lisofylline, antioxidant, granulocyte colony stimulating factor, Surfactant, liquid ventilation, nitric oxide (NO), activated protein C etc., but all lack enough evidence-based medicals Support.

Rheumatism is one group of infringement joint, skeleton, muscle, blood vessel and is main disease about soft tissue or connective tissue, its Middle majority is autoimmune disease.Morbidity is much more hidden and slow, and the course of disease is longer, and mostly has genetic predisposition.Examine Disconnected and treatment all acquires a certain degree of difficulty;How blood can check different autoantibodys, may be relevant from different HLA hypotypes; To nonsteroidal anti-inflammatory agent (NSAID), glucocorticoid and immunosuppressant have preferable short-term or the reaction of long-term palliative.

Acute interstitial pneumonia (AIP) is a kind of rare fulminant injury of lung quickly grown.Acute traumatic for lung Pathological changes.Onset, drastically (in a few days to several weeks), shows as heating, coughs and out of breath, respiratory failure followed by occur, exactly like Agnogenic idiopathic ARDS.AIP case fatality rate high (> 60%), most dead in 1~2 months.Clinical non-infection The property representational example of AIP includes: AIP caused by paraquat poisoning, Diffuse Connective Tissue Disease and follow-up irreversible Pulmonary fibrosis.It there may be total injury of lung and causes a disease path, and wherein paraquat poisoning constitutes unique disease Model.

N,N'-dimethyl-.gamma..gamma.'-dipyridylium (paraquat, PQ) chemical name 1,1 '-dimethyl-4,4 ' bipyridinium dichloride, are that one extensively makes Organic heterocyclic class contact defoliant and herbicide.Since within 1962, being applied to agricultural, PQ acute poisoning and death Case report of common occurrence.PQ poisoning can be divided into two stages, is first with inflammation that ALI/AIP is main performance Sexual stage, pulmonary edema, cellular infiltration and alveolar bleeding occurred within a couple of days;The patient survived from this stage will be developed To the further pulmonary fibrosis multiplicative stage, this process continued for several weeks time, final patient is dead with respiratory failure, and it is dead Rate high (75%-100%).The toxicological mechanism of PQ poisoning is failed to understand, is badly in need of studying its definite pathogenesis, and basis at this The effective intervention means of upper development.Exploration to the ALI caused by PQ not only facilitates the Clinical toxicology difficulty of this lethal of solution Topic, also will provide important intervention clue to the ALI/AIP caused by other reasons.

And cystic fibrosis (cysticfibrosis, CF) is a kind of hereditary invading multi viscera.Mainly show as Eccrine dysfunction, mucus gland hyperplasia, juice thickness, perspiration sodium chloride content increases.Have clinically lungs, The glandular tube of air flue, pancreas, intestinal, biliary tract, deferent duct, cervix uteri etc. is blocked caused series of symptoms by thick secretions, And it is the most prominent with respiratory system damage.Research thinks that generation and the transmembrane regulator gene (CFTR) of CF suddenlys change and direct Cause charrin disease relevant.The direct result of charrin disease is to cause airway mucus to block and Progressive symmetric erythrokeratodermia Lung tissue is downright bad.Complication: repeatedly spitting blood as having when merging bronchiectasis, the later stage can have cyanosis and drumstick finger health to search Rope often merges the severe complication such as pulmonary heart disease and heart failure.

Cystic fibrosis is owing to being positioned at the autosomal recessive hereditary diseases that the 7th pair of chromosome CF gene mutation causes, patient Being homozygote, its parents are heterozygotes.In the compatriot of patient, half can be with recessive gene, and 1/4 can fall ill.General band The heterozygote having recessive gene accounts for birth neonatal 2%~5%, and about having one in 2000~2500 neonates can fall ill. The dysglandular pathogeny of cystic fibrosis external secretion is unclear.Point out the epithelial cell chloride of patient according to the study Passage regulation is defective;The water of respiratory mucosa epithelium, electrolyte transmembrane transport have obstacle;Acid sugar in mucus glands secrete thing Protein content increases, and changes the rheol characteristic of mucus, the reason that may become viscous for secretions.

Primary disease occurs mainly in white people, and Northern Europe, U.S.'s sickness rate are higher, and black race is less, and Aisan seldom sees.Various places District's prevalence is inconsistent, and patient and neonatal ratio are about 1: 500 to 1: 3500.Infant morbidity in period, mainly Betiding child, about 3% makes diagnosis after growing up, and mortality rate is high.In recent years owing to early diagnosis is with rationally, actively control Treating, patient's survival rate increases, and at least 25% patient can live to and grow up, and 9% age was more than 30 years old.

Acute lung injury and interstitial pneumonia are all different types of disease with capsule pulmonary fibrosis.

Deoxyribonuclease I (DNase I), as the biologics of a kind of high-efficiency low-toxicity, increasingly comes into one's own.

Summary of the invention

It is an object of the invention to provide the new application of the deoxyribonuclease I of recombined human, i.e. new opplication in pharmacy.

The technical scheme realizing objects of the present invention is as follows:

The present invention relates to the application in the medicine of preparation treatment injury of lung of the gene recombinant human deoxyribonuclease I.

The present invention relates to the application in rheumatismal medicine is treated in preparation of the gene recombinant human deoxyribonuclease I.

Described injury of lung be acute, described acute lung injury be drug induccd.

Described rheumatism is interstitial pneumonia.Described interstitial pneumonia is acute.

The production method of DNase I

One, natural extract

Past DNase I is mainly from vertebrate pancreas and the extraction of the parotid gland, because the two position is rich in DNase Ⅰ.But being limited by raw material sources, DNase I yield is the highest and expensive.It is presently mainly the urine from animal to carry Taking DNase I, obtaining DNase I from animals urine mainly has the advantage in terms of two: be first to ensure that DNase The homogeneity of I, because fresh urine can be obtained from single animal individual continuous several times, and measures general enough experiment and needs Want;Next avoids the injury to humans and animals.Extracting method sees reference document: Mark Lewis-Francis. Ma Kai.Use quick egg White liquid chromatograph carries out rapid purification .FEBS Letters.1984,167 (1) to DNase I: 155159

The isolated and purified DNase of chromatography I is the most conventional method.Conventional chromatography has IEC and GFC.Paudel Phenyl Sepharose filtering chromatogram is used to combine the method for concanavalin A, Con A agarose affinity chromatography respectively with Yasuda etc. From cattle, sheep, the pancreas of horse and Anguillar japonica, chicken pancreas extract DNase I, Yasuda use this method also from the urine of people In gathered in the crops hDNase I.2000, the affinity chromatograph filler of mouse-anti people DNase I monoclonal antibody was by Nakajima Exploitation is used for isolated and purified extraction hDNase I from human urine, and the method makes whole extraction engineering more fast and simple.Tool Body step and conditioned reference: Guo Xiongjun etc. recombined human DNase I expression in escherichia coli and isolated and purified [D]. Beijing University of chemical technology academic dissertation .2009.

Two, gene engineering research

Owing to the restriction of natural extract method raw material sources and the differences between batches of product quality are relatively big, current scientific research personnel will study Directional steering gene engineering research.Express hDNase I efficiently at engineering bacteria or engineering animal and plant cells and have the biggest excellent Gesture, because the problem that this method can solve natural extract method raw material sources and product quality.The nineties in 20th century DNase I in Chinese hamster cell (CHO) successful expression, subsequently scientists just use this achievement in research develop treatment lung fiber The aerosol that encapsulated is sick, and presently commercially available rhDNase I uses expressing cho cell.2006 Fujihara etc. use COS 7 cell successful expression rhDNase I.Additionally Guo Xiong army etc. used large intestine bar in 2008 Bacterium engineering bacteria successful expression rhDNase I.Refer to: Liu great He.Recombined human deoxyribonuclease _ renaturation research [D]. Beijing University of Chemical Technology's master thesis, 2012.

People's deoxyribonuclease I product that the most existing many companies produce commercially is sold, people's deoxyribonuclease I (DNase) product is the colourless solution that concentration is 1mg/ml of sterilizing;Wherein people's deoxyribonuclease I, two water chlorine Change calcium, the mass ratio of NaCl is: 1000:150:8.77.

People's deoxyribonuclease I (DNase) solution can preserve 3 weeks at 15 DEG C (pH 6.3);It is placed in 4 after packing DEG C stored refrigerated, 2 years shelf-lifves.Used for intravenous injection.Dosage is 100IU/kg

Recombined human deoxyribonuclease I used in the present invention is that ProSpec-Tany company of Israel produces, Amy Prompt Science and Technology Ltd. agency's: injection, 1000IU/1mg, every milligram containing 150 microgram calcium chloride dehydrations and 8.77 Milligram sodium chloride.

Accompanying drawing explanation

Fig. 1 is that PQ of the present invention causes II type alveolar epithelial cells (A549) mitochondrial injury and the release figure of mitochondrial DNA;

In figure, (A-B) PQ is the mitochondrial membrane potential (being become green from redness) of dose-dependent reduction A549 cell.(C)PQ Dosage and time dependence ground reduce the survival rate (red line) of A549 cell, and increase releasing of supernatant Mitochondria DNA Put (black line).(D) A549 cell is hatched 12 hours with the PQ of 600uM, continues training with fresh culture after eluting PQ Support and the cell conditioned medium that 12h, PQ induced exists lasting mitochondrial DNA release.

Fig. 2 is that the mtDNA of PQ of the present invention induction can promote PBMC chemotactic response figure;

The supernatant that in figure, (A) is enriched with through the mtDNA of PQ pretreatment increases becoming of PERIPHERAL BLOOD MONONUCLEAR CELL (rather than PMN) Change index.(B-C) endotheliocyte transwell fluorescence leakage experiment, or guinea pig detects, and does not all show PQ Or the mtDNA of PQ induction weakens endothelial barrier function.

Fig. 3 is that the mtDNA of PQ of the present invention induction promotes II type alveolar epithelial cells (A549) secretion TGF-β 1 figure;

(A) effect that the non-stimulated lung fibroblast of the mtDNA (HLF) of PQ or PQ induction breeds.(B) mtDNA of PQ induction The expression of A549 cell TGF-β 1 can be raised, but the biomarker of epithelial-mesenchymal is not made significant difference (C-D). Fig. 4 is PQ injury of lung Establishment of mouse model figure of the present invention;

In figure (A) C57 mice give PQ (25mg/kg, lumbar injection, the next day twice totally), typical acute lung injury occurs And interstitial pulmonary fibrosis, its pathological score is better than classical bleomycin model (through airway administration).(B) blood plasma and The dynamic change of the time point that BALFmtDNA is different after PQ modeling.

Fig. 5 is the survival rate figure that intravenous injection DNaseI of the present invention is obviously improved acute fatal amount PQ mice.

In figure, (A) C57 mice gives disposable PQ acute lethal dose (40mg/kg, IP, d1).Experimental mice at d0, 2,5 and 8 DNA enzymatic I giving tail vein injection various dose.Result display DNA enzymatic I can prevent the mice that PQ induces Injury of lung, significantly improves survival rate.(B) DNA enzymatic I suppresses, in dose-dependent mode, circulation and the BALF mtDNA that PQ induces Level.

Detailed description of the invention

Embodiment 1

DNaseI reverses PQ induction, the injury of lung of mitochondrial DNA mediation in vitro

1.1 PQ cause the release (as shown in Figure 1) of II type alveolar epithelial cells (A549) mitochondrial injury and mitochondrial DNA

1.1.1 take the logarithm trophophase A549 cell, with 20000 cells/well kinds in 96 orifice plates, insert 37 DEG C, 5%CO Overnight incubation in 2 incubators;Next day every hole add variable concentrations N,N'-dimethyl-.gamma..gamma.'-dipyridylium 100 μ l (concentration includes 100 μm ol/l, 300 μm ol/l, 600 μm ol/l, 800 μm ol/l, 1000 μm ol/l, prepare after being all filtered), matched group adds completely Culture fluid, puts into 37 DEG C, cultivates 24 hours in 5%CO2 incubator;600 μm ol/l concentration groups give different time Stimulating (1h, 4h, 8h, 24h, 48h, 72h), each experiment of each concentration is in triplicate;Corresponding time point, discards on each hole Clear liquid, PBS rinses one time, and every hole adds 100 μ l fresh mediums and 10 μ lCCK8, inserts 37 DEG C, 5%CO2 Incubator is cultivated 1 hour, and wherein blank well is not contain the CCK8 culture fluid of PQ and cell;96 are taken out after 1 hour Orifice plate, microplate reader reads OD value during 490nm wavelength;Calculate cells survival rate according to each hole OD value, calculate public affairs Formula is as follows: cell survival rate=[experimental port-blank well/control wells-blank well] × 100%

1.1.2 mitochondrial membrane potential detection: trophophase A549 cell of taking the logarithm, adds copolymerization with the density of 10000 cells/well In burnt special culture dish, put into 37 DEG C, 5%CO2 incubator overnight incubation;With variable concentrations, (concentration includes 100 μm Ol/l, 300 μm ol/l, 600 μm ol/l, 800 μm ol/l) N,N'-dimethyl-.gamma..gamma.'-dipyridylium solution stimulate A549 cell, then place into cultivation Case continues cultivate 24 hours;Removing the N,N'-dimethyl-.gamma..gamma.'-dipyridylium solution of variable concentrations, carefully rinse with PBS twice, then every hole adds Enter the JC-1 liquid 200 μ l prepared, be put in incubator and hatch 30min;Discard JC-1 solution, rinse twice with PBS, Then under laser confocal microscope, imaging is taken pictures;Number according to red green fluorescence calculates the ratio of red green fluorescence, and root The situation of change of alternative line mitochondrial membrane potential is observed according to its ratio.

1.1.3 the detection of mitochondrial DNA: the foundation of the standard curve of detection human mtdna content, designs mankind's line grain The primer of body specific sequence cytochrome B, primer sequence is Human cytochrome B-F:5-ATGACCC CAATACGCAAAAT-3, Human cytochrome B-R:5-CGAAGTTTCATCATGCGGAG-3.P CR expands mankind's CYTO 1 B gene;After 2% agarose gel electrophoresis, cut glue, DNA fast purifying/recovery test kit returns Receive product, couple PGM-T plasmid, errorless through DNA sequencing after converting amplification, plasmid extraction;Then as standard substance, After different dilutions, qPCR draws standard curve.In aforementioned cells culture supernatant, the extraction of DNA is according to QlAamp DNA Blood Mini Kit description is carried out, so as template row qPCR, by standard curve quantitative determination mtDNA copy Shellfish number.

Result: PQ is the damage A549 cell mitochondrial of dosage and time dependence, reduces mitochondrial membrane potential, causes supernatant The release of middle mtDNA;PQ Yu A549 co-culture of cells 12h, is further cultured for 12h, A549 cell and still deposits after eluting PQ Discharge at the mtDNA of persistence.

The mtDNA of 1.2 PQ inductions can promote PBMC chemotactic response (as shown in Figure 2)

1.2.1 extraction normal person's EDTA-anticoagulation 5 milliliters, dilutes by equivalent PBS;Density gradient centrifugation separates PBMC or use PolymorphprepTMSeparation of Neutral granulocyte;All add 1% hyclone re-suspended cell, inverted microscope with RPIM1640 Lower counting, according to 1 × 107The cell density diluting cells of/ml;The upper indoor addition 100 μ l cell suspension of Transwell (contains 1×106Individual cell), lower room adds 500 μ lRPIM1640 culture medium, wherein contains 20% hyclone, puts into cultivation Case cultivates 2 hours respectively, 4 hours, 6 hours.Drawing lower room culture fluid, 800rpm, 5min are centrifugal removes supernatant, with new Fresh culture fluid re-suspended cell, counts under inverted microscope, calculates chemotactic index.And carry out PBMC by flow cytometry Cell typing.

Chemotactic index=experimental group cell number/cellular control unit number

1.2.2 vascular permeability detection: the detection of endothelial cellular membrane resistance uses Roche Xcelligence detector, cell Detection plate (E-Plate) adds huve cell suspension, according to the density of 10000 cell/100 μ l, treats cell After adherent, change serum-free medium, starved cells 5-6 hour, make cell synchronization;Add containing 20% tire Sanguis Bovis seu Bubali Clear each group stimulus object stimulates cell 4 hours, observes each group of cell membrane resistance variations situation.Additionally use vascular permeability Tra Nswell test kit (vascular permeability Kit, Millipore Corporation) by specification operates, and detects fluorescence Leakage.

The mtDNA of result: PQ induction can promote PBMC, rather than the chemotactic effect of neutrophilic granulocyte;This chemotactic effect is to T Cell, B cell, NK cell, mononuclear cell non-selectivity;The chemotactic of its inflammatory effector cell be active process and also Non-is the seepage being increased by vascular endothelial cell permeability and causing.This effect can by ectogenic addition DNaseI (from And remove mtDNA) institute's antagonism.

The mtDNA of 1.3 PQ inductions can promote II type alveolar epithelial cells (A549) secretion TGF-b1 (as shown in Figure 3)

1.3.1 A549 cell is cultivated and is seen 1.1 parts, adds supernatant (and the nothing of PQ pretreatment (after eluting) enrichment mtDNA PQ), check TGF-b1 (ELISA method) in supernatant after cultivating 48h, or cultivate extracting cell RNA after 12h, The relevant label of quantitative PCR detection epithelial-mesenchymal after reverse transcription.

1.3.2 human fibroblasts (HLF) is cultivated, and adds different disposal condition, is divided by Roche Xcelligence system Analysis Proliferating antigen Ki67.

Result: PQ induction mtDNA to fibroblastic propagation without direct facilitation;It has promotion TG to A549 The effect of F-b1 secreting, expressing, thus participate in fibrotic processes.This effect can be exogenously added DNaseI institute antagonism.

Embodiment 2

DNaseI stops the injury of lung of PQ mouse model in vivo and significantly improves survival rate

The foundation (Fig. 4) of 2.1 PQ model of lung injury mices

2.1.1 8-10 week old male SPF level C57BL/6j mice, body weight 20-24g, PQ lumbar injection 25mg/kg, every 2 It once, totally 2 times.Blood mark is collected in modeling group mice 2h, 12h, 1,2,3,7,14,28 day after being administered This, after mouse weights, after 1% pentobarbital sodium (80mg/kg) intraperitoneal injection of anesthesia mice, extract eyeball and take blood; Fixing mice, first exposes thoracic cavity, then cuts off mice skin of neck, and before blunt separation trachea, muscular tissue exposes trachea, On trachea, cut an osculum with eye scissors, insert the puncture needle being connected with 1ml syringe, slower pumpback broncho-pulmonary Bubble irrigating solution (BALF), pumpback 5-8 time repeatedly, when pumpback amount reach 0.8 and above time extract puncture needle.Left lung is put into 4% paraformaldehyde fixes 48 hours, routine paraffin wax embedding, and 4 μm paraffin sections carry out HE dyeing respectively, and Masson contaminates Color, SABC.

2.1.2 the mtDNA detection of mouse peripheral blood and BALF: the primer sequence of mice CytoB: Mouse cytochrome B forward primer 5-CCTATCAGCCATCCCATAT-3, Mouse cytochrome B downstream primer 5-GGAAGA GGAGGTGAACGA-3.Embodiment 1.1 between the standard curve of quantitative PCR and Samples detection.

Result: set up PQ injury of lung mouse model, acute lung injury in early days and later stage pulmonary fibrosis pathological manifestations typical case, homogeneity Bleomycin mouse model due to classical airway administration;The mtDNA of PQ model peripheral blood and BALF increases, and two Person's pattern is different, and peripheral blood mtDNA is that persistence increases, and BALF the 7th day mtDNA of modeling reach peak it Rear decline, the bridge that prompting mtDNA possible composition acute lung injury to pulmonary fibrosis is excessive.

2.2 intravenous injection DNaseI are obviously improved the survival rate (as shown in Figure 5) of acute fatal amount PQ mice

8-10 week old male C57BL/6j mice, 1 time, PQ abdominal cavity property injection 40mg/kg (d1), some animals is opened at d0 Begin give DNaseI (0.3mg/kg, or 3mg/kg, or 30mg/kg) tail vein injection, the DNaseI used for Se Lie ProSpec-Tany company produces, the injection of Amy victory Science and Technology Ltd. agency, 1000IU/1mg, every milligram Containing 150 dehydration of microgram calcium chloride and 8.77 milligrams of sodium chloride, hereafter d2, d5, d8 are repeated 3 times administration.Observe existence Curve.Periodically (d3, d7, d14, d28) each group of draw materials mice detection peripheral blood, mtDNA level (method of BALF See 2.1).

Result: PQ acute fatal amount group mice is the most dead in 7 days, and gives DNaseI and there is significant protective effect, 2 8d survival rate reaches as high as 90%, in dose-dependence;Parallel with this is its be dose-dependent effectively inhibit outside The mtDNA release of all blood and BALF.

Embodiment 3

Intravenous injection DnaseI treats rat acute Radioactive synovectomy

1 material and method

1.1 material male Wistar rats 45, body weight (240 soil 20) g, random packet is matched group (0Gy), single list Pure full breast irradiation group (30Gy) and DnaseI prophylactic treatment group (the full breast of single irradiates 30Gy+DnaseI).

1.2 method

1.2.1 single irradiation group merely and DnaseI treatment group Wistar Mus lumbar injection 1.5 pentobarbital sodium 2ml/kg, With Zhou Ding and the screening arrangement row of designed, designed after Animal Anesthesia60Co gamma-rays irradiates, and field size is 4.5cm × 4.0 Cm, exposure dose 30Gy close rate is 6.02 × 10C/ (kg min), and irradiation distance is 2.5m, and irradiation time is 12min。

1.2.2DnaseI group accepts DnaseI intravenous injection, once a day, until put to death in batches, often in pre-irradiation 2 days Secondary dosage 500IU/kg, simple irradiation group the most in kind injects sterile saline.Standby group was 0.5 month, January 1.5 The preparation of 1.2.3 specimen is put to death in the moon dislocation of neck in batches and dyeing takes the sagittal half and half leaf lung group of each leaf in right lung upper, middle and lower Knit to put in 10 neutral formalin one PBS fixatives and fix 48h, hereafter row routine dehydration, leaching Scarabaeiform, embed, cut into slices. Row HE dyeing respectively, ammonia-alum one Masson dyeing and SABC Vimentin of cutting into slices dyes, and the latter uses SABC Method dyes, and after methyl green counterstain, dimethylbenzene is transparent, mounting.The DNaseI used is that Israel ProSpec-Tany is public Department produces, and the injection of Amy victory Science and Technology Ltd. agency, 1000IU/1mg, every milligram takes off containing 150 microgram calcium chloride Water and 8.77 milligrams of sodium chloride.

Under optical microscope, observed result is as follows.

Normal group (0Gy): normal lung tissue, end sees that exception, ammonia-alum-Masson dyeing end are shown in proliferation of fibrous tissue, Vi Mentin is negative staining.

Simple full breast irradiation group (30Gy)

Irradiating latter 0.5 month and be separated with hyperemia in various degree between lung, indivedual visible point stove shapes are hemorrhage, and alveolar wall slightly thickens, Capillary endothelial cell swelling, has a small amount of inflammatory cell infiltration based on neutrophilic granulocyte.Ammonia-alum-Masson dyes And Vimentin dyeing compared with Normal group end see exception

The slight hypertrophy of fibrous tissue under January lung tissue visceral pleura after irradiation, blood vessel wall basement membrane thickened, perivascular edema, Visible relatively multi-lymphocytes, plasma cell are the infiltration of oversleeve sample, and part thin vessels is inaccessible or sees thrombosis alveolar septum edema, hence it is evident that increase Width, has the homogeneous substance deposition that a small amount of cellulose sample powder contaminates, fibroblast hypertrophy in various degree, have more neutrophilic granulocyte, Lymphocyte, plasma cell, the macrophages infiltration alveolar wall I obvious hypertrophy of type epithelial cell, dash forward to alveolar space, accidental double-core, Multinuclear and mitosis figures alveolar space part diminish, partial rupture, form pulmonary belb, and intracavity has cellulose sample to ooze out and edema Liquid, has more inflammatory cell to ooze out in alveolar space, have more macrophage, multinucleated giant cell, also visible one-tenth in some alveolar space The foamy cell lung bronchia epithelial hyperplasia of heap, basement membrane is that lacery thickens.Plating Alumen-Masson dyeing, Under part pleura and the collagen fiber of very thin dark blue dyeing seen from alveolar wall, in minority alveolar space, hyaline membrane is dark blue dye, shows Collagen is had to be formed.Vimentin dyes display, and alveolar asks fibroblast, macrophage and the lung that matter has more pale brown dyeing The brown color macrophage of bubble intracavity.

Irradiate latter 1.5 months lung tissues, pathological changes compared with January after irradiating, lung tissue was even more serious, lung ask matter proliferation of fibrous tissue and Hyperplasia becomes apparent from, and part alveolar space disappears, and has the most all types of inflammatory cell infiltrations.

DnaseI treatment group (30Gy)

Irradiating latter 0.5 month DnaseI treatment group alveolar wall blood capillary mild hyperaemia, other has no compared with Normal group The most abnormal.

After irradiation, DnaseI treatment in January group lung tissue lung hears matter mild hyperaemia, and there is a small amount of lymphocyte indivedual thin vessels peripheries Infiltration alveolar wall blood capillary mild hyperaemia, part alveolar wall has a small amount of inflammatory cell infiltration.

Irradiate latter 1.5 months DnaseI treatment group lung tissues in addition to alveolar wall blood capillary has mild hyperaemia, show no obvious abnormalities.

Ammonia-alum-Masson dyeing and Vimentin immunohistochemical staining, 0.5 month, January and 1.5 months DnaseI treatment Group lung tissue is showed no obvious abnormalities.

Injection DnaseI has good therapeutic effect to rat acute Radioactive synovectomy as can be seen here.

Claims (2)

1. gene recombinant human deoxyribonuclease I application in the medicine of the acute lung injury that N,N'-dimethyl-.gamma..gamma.'-dipyridylium causes is treated in preparation.
Apply the most as claimed in claim 1, it is characterised in that the agent of the gene recombinant human deoxyribonuclease I applied Type is injection.
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