CN105327335A - Medical application of CREG protein - Google Patents
Medical application of CREG protein Download PDFInfo
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- CN105327335A CN105327335A CN201410345638.9A CN201410345638A CN105327335A CN 105327335 A CN105327335 A CN 105327335A CN 201410345638 A CN201410345638 A CN 201410345638A CN 105327335 A CN105327335 A CN 105327335A
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Abstract
The invention relates to medical application of CREG protein, and in particular relates to application of the CREG protein in preparing a medicine or a medical instrument for preventing and/or treating atherosclerosis or atherosclerosis related diseases (such as coronary heart disease, cerebral apoplexy, abdominal aortic aneurysm and peripheral arterial disease). The invention also relates to application of a recombinant vector for expressing the CREG protein, a recombinant cell containing the recombinant vector, and a preparation capable of inhibiting down-regulation of the expression of the CREG protein inside blood vessel and capable of up-regulating the expression level of the CREG protein side the blood vessel in preparing a medicine or a medical instrument for preventing and/or treating atherosclerosis or atherosclerosis related diseases (such as coronary heart disease, cerebral apoplexy, abdominal aortic aneurysm and peripheral arterial disease).
Description
Technical field
The present invention relates to the medical usage of CREG albumen, be specifically related to CREG albumen for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy) or medical equipment.The invention still further relates to express CREG albumen recombinant vector, reconstitution cell containing this recombinant vector, CREG protein expression in blood vessel wall can be suppressed to lower or can raise CREG protein expression level in blood vessel wall preparation for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy) or medical equipment.
Background technology
Atherosclerosis (Atherosclerosis, AS) is modal type in hardening angiopathy.Along with the raising of living standards of the people, living-pattern preservation and life-time dilatation, the sickness rate of AS rises year by year.According to the difference of involved vessels, AS can show as the various ways such as myocardial infarction, apoplexy, is one of lethal principal disease disabled.Research shows, AS is the chronic inflammation processes of complicated, a multifactor participation.It comprises lipid aggregation, the intrusion of monocytes/macrophages, the hypertrophy of vascular smooth muscle cell (VSMC), the formation of foam cell, proliferation of fibrous tissue, inflammation and antigen antibody reaction etc.Up to the present, the mechanism of AS is illustrated not yet completely.Find affect AS occur development crucial endogenous molecule and with this molecule for target treatment AS is the focus studied at present.
People E1A activated gene repressor (humancellularrepressorofE1A-stimulatedgene, hCREG) gene is the cell differentiation controlling gene (VealE that first Harvard University medical college Gill professor in 1998 clones, MolCellBiol, 1998; 18 (9): 5032-5041).Early-stage Study confirms, at VSMC by shrinking phenotype in the process of synthesis Phenotypic Change, the expression of CREG albumen and mRNA significantly reduces (HanYa-Ling, ZhonghuaYiXueZaZhi, 2005; 85 (1): 49-53; HanYa-Ling, JMolCellCardiol, 2011; 50 (4): 723-30; HanYa-Ling, JMolCellCardiol, 2010; 48 (6): 1225-35), show that it participates in VSMC phenotype regulation process.In vitro study finds, process LAN CREG albumen can obviously suppress the propagation of VSMC and promote its differentiation (HanYa-Ling, ChineseJournalofCellularandMolecularImmunology, 2005; 21 (5): 570-574; HanYa-Ling, ProgBiochemBiophys, 2004; 31 (12): 1099-1105; HanYa-Ling, ProgBiochemBiophys, 2005; 32 (6): 517-522).Find in body research, in rat carotid artery blood vessel after balloon injured, the expression of the CREG gene of tunica media obviously declines, and process LAN CREG can suppress the propagation of VSMC and the formation (HanYa-Ling of new intima, CardiovascRes, 2008; 78 (3): 597-604; HanYa-Ling, JVascSurg, 2008; 48 (1): 201-9), simultaneously, the pLNCX2-hCREG retrovirus imported through membrane bound effectively inhibits generation (HanYa-Ling, ChineseJournalofPathophysiology, 2007 of mouse carotid stricture of artery after balloon injury; 23 (10): 1873-1877).Except VSMC, CREG also have regulating and controlling effect to vascular endothelial cell (EC).Process LAN CREG can promote that EC breeds (TaoJie, IntJMolSci, 2013; 14 (9): 18437-18456) the EC apoptosis (WangNa, Astherosclerosis, 2011 that, and to anti-inflammatory cause; 218 (2): 543-551) and barrier dysfunction (DuanYan, MolBiolRep, 2013; 40 (6): 3891-3900).Above-mentioned research prompting, CREG maintains the important endogenous regulation factor of vessel homeostasis.But still not there are some researches show, CREG albumen can as the target spot of AS treatment.
Summary of the invention
The present inventor is through great many of experiments and repeatedly grope, and finds, by improving CREG protein expression level in blood vessel wall, can reduce atheromatous plaque area, alleviate the inflammatory reaction in atherosclerosis, this completes the present invention.
First aspect present invention relates to CREG albumen for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment.
Second aspect present invention relates to the recombinant vector of expressing CREG albumen or the reconstitution cell containing this recombinant vector for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment; Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
The purposes of any one according to a second aspect of the present invention, wherein said recombinant vector is recombinant adenoviral vector.
In embodiments of the invention, wherein said recombinant adenoviral vector behaviour Adenovirus Type 5 Ad5-CREG, it is deposited in China typical culture collection center (CCTCC, Wuhan on January 2nd, 2008, Wuhan University), preserving number is CCTCC-V200801.This preservation information is open in Chinese patent CN101475961A.
Third aspect present invention relates to the preparation that Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise Ink vessel transfusing CREG protein expression level for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment.
Fourth aspect present invention relates to compositions, it comprises CREG albumen, express the recombinant vector of CREG albumen, reconstitution cell containing this recombinant vector, Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise the preparation of Ink vessel transfusing CREG protein expression level, and optional pharmaceutically acceptable carrier or excipient, described compositions is used for preventing and/or treating atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.); Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
Fifth aspect present invention relates to medical equipment, it comprises CREG albumen, express the recombinant vector of CREG albumen, reconstitution cell containing this recombinant vector, Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise the preparation of Ink vessel transfusing CREG protein expression level, and described medical equipment is used for preventing and/or treating atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.); Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
The invention still further relates to the method preventing and/or treating atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.), described method comprises to the CREG albumen of experimenter's effective dose in need, the recombinant vector of expressing CREG albumen, reconstitution cell containing this recombinant vector, Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise the step of preparation of Ink vessel transfusing CREG protein expression level.
The invention still further relates to the compositions of any one of fourth aspect present invention or the medical equipment of the 5th any one of aspect for the preparation of the purposes prevented and/or treated in the product of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.).
In the present invention, described CREG albumen (i.e. E1A activated gene repressor, cellularrepressorofE1A-stimulatedgene) both comprise the CREG albumen with complete sequence, also comprise the CREG albumen only containing partial sequence with CREG protein function.
In embodiments of the invention, the aminoacid sequence number of described CREG albumen is NP_003842.1.
In embodiments of the invention, No. Genbank of described CREG gene is NM003851.
In embodiments of the invention, described CREG albumen is human CREG (hCREG) albumen.
In the present invention, described atherosclerosis comprises each stage of atherosclerotic blood vessel narrow phase, but do not comprise atherosclerosis serious time the blood vessel obstruction phase.
In the present invention, described atherosclerosis comprises coronary atherosclerosis, atherosclerosis of aorta and Peripheral atherosclerosis.
In the present invention, described Atherosclerosis relevant disease comprises coronary heart disease, apoplexy, abdominal aortic aneurysm or peripheral arterial disease (such as arterial thrombus).
In the present invention, described carrier is prokaryotic expression carrier, carrier for expression of eukaryon or viral vector.In embodiments of the invention, described carrier is viral vector, such as, be adenovirus vector or gland relevant viral vector.In specific embodiment of the invention scheme, described carrier is adenovirus vector.
In the present invention, described cell is prokaryotic cell or eukaryotic cell, and described prokaryotic cell is such as escherichia coli, Salmonella, and described eukaryotic cell is such as yeast cells or mammalian cell.
In the present invention, described medical equipment is for being used for the treatment of atherosclerotic apparatus, and being generally the apparatus getting involved blood vessel, such as, is intravascular stent etc.
In the present invention, described CREG protein expression is lowered or is raised and refers to that the expression of CREG albumen is compared with undressed cell or tissue, improve or reduce at least 25%, such as at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or raising is greater than 100%.
In embodiments of the invention, by raising the expression of Ink vessel transfusing CREG albumen, atheromatous plaque area can be reduced, and alleviate the local inflammation reaction that atherosclerosis causes.
In specific embodiment of the invention scheme, by infecting Ad293 incasing cells, amplification obtains the recombined human CREG adenovirus of high titre;
In specific embodiment of the invention scheme, feed ApoE knock out mice by high fat and prepare atherosclerosis mouse model;
In specific embodiment of the invention scheme, by tail vein injection recombined human CREG adenovirus, infect atherosclerosis mice;
In specific embodiment of the invention scheme, evaluate recombined human CREG adenovirus by HE dyeing, oil red dyeing, inflammation index detection etc. and suppress atherosclerotic effect.
Accompanying drawing explanation
Fig. 1 .AdCREG infects Ad293 cell 3 days (200 ×).
(A) Ad293 form under light microscopic.
(B) under fluorescence microscope, the same visual field green fluorescence is expressed.
Fig. 2. the aorta HE that normal mouse blood vessel and apoE-/-knock-out mice height fat feed 8 weeks blood vessels dyes (100 ×).
Fig. 3. tail vein injection AdCREG infects high fat and feeds the aorta efficiency of infection of ApoE-/-mice and exogenous CREG expression rule.A) aorta GFP and the display of CREG immunofluorescence dyeing result are infected successfully, and have exogenous CREG to express (100 ×); B) after infecting, different time points GFP and CREG expresses, and bar diagram display WesternBlot band gray value is analyzed.Take normal blood vessels as reference, * P<0.05, n=5.
Fig. 4 .AdCREG treats the atherosis effect of rat aorta and judges.A aorta HE dyes and observes Mottling formation, and bar diagram display atherosclerotic plaque accounts for the percentage ratio of media area; 8w blood vessel is fed for contrast, * P<0.05, n=5 with high fat; The dyeing of B aorta oil red is observed atherosclerotic plaque and is formed, and bar diagram display atherosclerotic plaque accounts for the percentage ratio of vessel area; 8w blood vessel is fed for contrast, * P<0.05, n=5 with high fat; CELISA detects aorta TNF alpha expression level; 8w blood vessel is fed for contrast, * P<0.05, n=5 with high fat; DWesternBlot detects NF-κ B, and bar diagram display WesternBlot band gray value is analyzed; 8w blood vessel is fed for contrast, * P<0.05, n=5 with high fat; EWesternBlot detects I κ B α, and bar diagram display WesternBlot band gray value is analyzed, and feeds 8w blood vessel for contrast, * P<0.05, n=5 with high fat.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
The comparison application X 2 test of two sample rates, statistical procedures all applies the process of SPSS17.0 software kit.P<0.05 is for there being significant difference.
The amplification of embodiment 1. recombined human CREG adenovirus (AdCREG) and titer determination
AdCREG is deposited in China typical culture collection center (CCTCC on January 2nd, 2008, Wuhan, Wuhan University), title behaviour Adenovirus Type 5 Ad5-CREG, preserving number is CCTCC-V200801 (see Chinese patent CN101475961A, denomination of invention: express recombinant adenovirus of human CREG and uses thereof).5 × 10 are cultivated with the DMEM containing 5% hyclone
6ad293 cell (HEKC, purchased from American Stratagene company, article No. #240085).Get 0.5 μ lAdCREG virus conserving liquid, add the DMEM to 1ml containing 5% hyclone, mixing, obtains viral mixed liquor.Remove the culture fluid of Ad293 cell, carefully add viral mixed liquor, cross slowly rocks 3 times.Be placed in 37 DEG C of CO
2hatching 120min in incubator, add the DMEM culture fluid of 9ml containing 5% hyclone, is that the culture fluid of 10ml continues cultured cell with cumulative volume.Become after round coming off until most cells, collect whole cell, the centrifugal 5min sedimentation cell of 600g.With phosphate buffer (PBS) re-suspended cell of ice ,-80 DEG C to 37 DEG C multigelations 3 times.4 DEG C of centrifugal 10min of 16000g, remove cell debris, collect supernatant.Be placed in-80 DEG C of refrigerators frozen for subsequent use.
Before using, measure virus titer by 50% TCID (TCID50) method.Concrete grammar is collection one bottle of Ad293 cell, counting.With the DMEM re-suspended cell containing 2% hyclone, adjustment density is 10
5individual cell/ml, preparing cumulative volume is the cell suspension of 20ml.0.1ml cell suspension is added with the 12 road volley of rifle fires every hole in 2 piece of 96 orifice plate.Prepare virus dilution liquid: add 0.9mlDMEM culture fluid in the 1st pipe, all the other add 1.8ml, add 0.1ml virus conserving liquid in the 1st pipe again, inhale up and down and play 5 mixings.Use new rifle head instead, from the 1st pipe, draw 0.2ml add in the 2nd pipe, be repeatedly diluted to most high dilution.Carry out the 2nd with same pipe virus conserving liquid and take turns dilution.Last 8 diluents add 96 orifice plates, every hole 0.1ml, and each dilution factor 10 hole, 2 holes are negative control.Put 37 DEG C of CO
2cultivate 10 days in incubator.Observe under inverted microscope after 10th day, calculate the hole count occurring CPE (CPE) in each row, with Karbers formulae discovery virus titer.Virus titer T=10
1+d (S-0.5), wherein d=Log
10virus liquid dilution factor, sum/10, S=CPE hole.
Experimental result shows, and after adenovirus infection Ad293 cell, egfp expression in visible cell under fluorescence microscope, cell infection rate can reach more than 90% (Fig. 1).Virus is after amplification, and TCID50 method measures virus titer and reaches 4.75 × 10
10it is 1.85 × 10 that pfu/ml, HPLC measure granule number
12, purity is 81%.
Employing same method amplification restructuring green fluorescent protein adenovirus in contrast (AdGFP, purchased from AppliedBiologicalMaterialsInc., article No. 000541A) and preserve.
The preparation of embodiment 2. atherosclerosis mouse model
Adopt ApoE gene knockout (ApoE in 8 week age
-/-) mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), give high fat after wean and feed.High lipid food formula: 15% fat, 0.25% cholesterol, 84.75% normal diet.Continue nursing and after 8 weeks, complete the preparation of atherosclerosis mouse model.Experimental result shows, compare with normal mouse blood vessel, apoE-/-mice visible obviously atheromatous plaque after high fat feeds 8 weeks charges into tube chamber, tube wall is uneven, inner membrance obviously thickens, visible lipid cavity in speckle is cholesterol crystal, massive inflammatory cells infiltrated (Fig. 2).Above result shows that the atherosis model of rat aorta is successfully prepared.
Embodiment 3. tail vein injection Adenoviral Therapy atherosclerosis
High fat will be continued feed 8 weeks, and form atherosclerotic ApoE-/-mice and be divided into AdCREG group (n=20), AdGFP group (n=20) and saline control group (n=20) at random.The number of mice measuring often kind of index is 3-5.AdCREG group is 4.75 × 10 through tail vein injection titre
10individual virus plaque forms the normal saline 1ml of the AdCREG of unit (PFU).AdGFP group is 4.75 × 10 through tail vein injection titre
10the normal saline 1ml of the AdGFP of PFU.Saline control group is through tail vein injection hemopoietic reason saline 1ml.Continue nursing after 8 weeks, de-neck puts to death mice, gets aorta vessel.
To the blood vessel of having drawn materials, carry out the qualification of recombinant adenovirus efficiency of infection.In the present invention, recombinant adenovirus AdGFP is with green fluorescent protein (GFP) gene, restructuring AdCREG is also containing GFP gene, through the capable immunofluorescence dyeing of GFP antibody, the green fluorescence can observed under fluorescence microscopy in blood vessel wall is expressed, express ratio by graphical analysis green fluorescence, can calculate in body situation, the efficiency of infection of tail vein injection adenovirus.Concrete grammar is as follows: after animal blood vessels specimen is taken out, PBS cleans up.30min is fixed with 4% paraformaldehyde.Add 7.5% sucrose dehydration, 4 DEG C are spent the night.Embed with freezing embedding medium OTC, be placed in freezing microtome quick-freezing.Ultramicrotome is interrupted uniformly slicing, slice thickness 5 μm.With the penetrating 30min of 0.2%TritonX-100.1h is closed by the confining liquid room temperature containing 5% lowlenthal serum.Add GFP, CREG primary antibodie (purchased from American Abcam company, 1:100 dilutes) 4 DEG C of overnight incubation.PBS washes 3 times, each 5min.Add the fluorescence two anti-(1:100 dilution) of sheep anti-Mouse, incubated at room 2h.PBS cleans 3 times, each 5min.The expression of fluorescence microscopy Microscopic observation GFP, gathers image, and with Imageproplus software analysis image, calculates efficiency of infection.Experimental result shows, and infects after adenovirus, at the inner membrance of blood vessel wall, middle level, all visible a large amount of GFP positive cell of adventitia, accounts for 70% of all cells number.The CREG positive cell (Fig. 3 A) of red fluorescence is sent out as seen in the position of GFP positive cell.Successful expression CREG albumen in the successful AdCREG adenovirus cell of infection is described.
After understanding AdCREG tail vein injection further, the expression of metainfective aorta CREG, adopts WesternBlot to detect to infect the expression of different time points (1 week, 2 weeks, 3 weeks, 4 weeks) aorta CREG and GFP.Concrete grammar is as follows: get each time point mouse aorta blood vessel respectively and be placed in ice bath test tube, add ice bath lysate 300 ~ 500 μ l (10mmol/LHEPES, pH7.9,0.5mmol/LKCl, 0.5mmol/LEDTA, 1mmol/LDTT, 0.5mmol/LPMSF and 5% glycerol), DMSF, press down bright peptide and each 2 μ l of aprotinin, mixing, ice bath place 15min.Adopt BCA colorimetric kit to measure the concentration of protein in lysate, adjust protein concentration to 1 μ g/ μ l, with Eppendorf pipe subpackage, often pipe 35 μ l is stored in-80 DEG C.Sample through the capable SDS-PAGE electrophoresis of 12% separation gel, every swimming lane loading 30 μ l.With 300mA electric current, sample is gone on nitrocellulose filter in the circulator bath of 4 DEG C, time 2h.1h is closed by the TBS-T buffer room temperature containing 5% defatted milk powder.TBS-T washes film 3 times, each 15min.Add the primary antibodie (CREG primary antibodie and GFP primary antibodie) of 1:2000 dilution, 4 DEG C of overnight incubation.TBS-T washes film 3 times, each 15min.Add two anti-(1:2500 dilution) incubated at room 2h of 4 μ LHRP labellings.TBS-T washes film 3 times, each 15min.ECL chemiluminescence develops the color, the imaging of Kodak black and white film.
Experimental result shows, and after tail vein injection AdCREG, at 2 weeks and 3 time-of-weeks point, aorta has a large amount of GFP to express, and within 4 weeks, still has expression, and later exogenous GFP expresses and fades away; And namely CREG has great expression for 1 week in infection, exceed the expression of normal blood vessels, reach peak when 2 weeks, still express high than normal blood vessels when infecting 3 weeks, during to infection 4 weeks, expression weakens gradually to disappearance.These results show, adenovirus mediated CREG infects and effectively by CREG channel genes atheromatous plaque tissue, can recover CREG expression and meet or exceed normal level (Fig. 3 B).
Embodiment 4.AdCREG treats atherosclerosis effect assessment
Employing HE dyes, oil red dyes, ELISA and WesternBlot detects inflammation index and evaluate high fat nursing 8 weeks, and AdCREG treats the effect of latter 4 weeks.
To 3 groups of capable routine paraffin wax sections of mouse aorta and HE dyeing.Divide according to histology, with inside and outside elastic membrane for boundary, being tunica intima within interior elastic membrane, is tunica media between inside and outside elastic membrane, is tunica adventitia beyond outer elastic membrane.Under 4 times of object lens, complete vessel cross-sections HE is dyeed in image input Computer digital image analysis, Imageproplus5.0 Computer digital image analysis is utilized first to carry out picture editting, the profile of outer elastic membrane, interior elastic membrane and tube chamber is accurately ticked with mouse, progressively calculate lumen of vessels area, inner membrance and media area, and calculate inner membrance/media area ratio (I/M).Every animal random selecting 5 tangent planes carry out graphical analysis, average.
Experimental result shows, and AdCREG treatment group atheromatous plaque area is significantly less than saline control group and AdGFP matched group.Visible lipid core, cholesterol crystal in saline control group and AdGFP group blood vessel, and Ad-GFP group lacks cholesterol crystal.Statistical analysis, the ratio that AdCREG group atheromatous plaque area accounts for media area is significantly less than saline control group and AdGFP group (Fig. 4 A).
Atheromatous plaque formational situation is observed to 3 groups of capable oil red dyeing of mouse aorta.Blood vessel is fixed in 3% paraformaldehyde solution, 30min.After PBS washing, immerse PBS and spend the night.Reject extravascular tissue, rip cutting, expose tunica intima.With 60% oil red dye liquor (stock solution 60ml adds 40ml distilled water), dye 10-15min.Unnecessary dye liquor is washed away with 60% isopropyl alcohol.Distilled water cleans.Being put in by tremulous pulse scribbles on the microscope slide of poly-D-lysine, covered, and basis of microscopic observation is also taken pictures.Being dyed red position by oil red is atherosclerosis position.By Imageproplus software analysis image, calculate the ratio that red atherosclerosis area accounts for whole vessel area.
Experimental result shows, and red colored part is Lipid Plaque.AdCREG group vascular atherosclerosis plaque area is obviously less than saline control group and AdGFP group.The ratio that plaque area accounts for whole blood vessel is obviously less than other two groups (Fig. 4 B).
TNF-alpha content in vascular tissue is detected to 3 groups of capable ELISA of mouse aorta.Adopt mice TNF-α detection kit (RD company), operate by test kit description.Specific as follows: after standard substance doubling dilution, every hole adds 50 μ l.The concentration of dilution standard product is respectively 900 μ g/L, 600 μ g/L, 300 μ g/L, 150 μ g/L and 75 μ g/L.Establish blank well and testing sample hole respectively.Blank control wells does not add sample and enzyme marking reagent.All the other add the supernatant of the VSMCs of different group, negative control, 100 μ l/ holes.With the rearmounted 37 DEG C of incubation 30min of shrouding film shrouding.Carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, and static 30min discards, and 5 times repeatedly, pats dry.Every hole adds enzyme labelled antibody 50 μ l, except blank well.Incubation is the same.After the same washing 5 times, every hole first adds nitrite ion A50 μ l, then adds nitrite ion B50 μ l, shakes gently, 37 DEG C of lucifuge colour developing 15min.Every hole adds stop buffer 50 μ l, cessation reaction.Microplate reader surveys 450nm place OD value, and with OD value for abscissa, standard concentration is vertical coordinate drawing standard curve.Sample TNF-α concentration is calculated with sample OD value.
Experimental result shows, and in AdCREG group blood vessel, TNF-alpha expression amount is starkly lower than other two groups (Fig. 4 C), and prompting AdCREG treats the inflammatory reaction that can alleviate in atherosclerosis.
Inflammatory transcription factor NF-KB and I κ B α are detected to 3 groups of capable WesternBlot of mouse aorta.WesternBlot detection method is with described in embodiment 3.Experimental result shows, and matched group and AdGFP infect NF-κ B in matched group arteries and, all in high expressed, show that NF-κ B is the state of activation in atherosclerotic process, and the inflammatory reaction of prompting cells of vascular wall is in active state.And AdCREG group NF-κ B expresses significantly decline (Fig. 4 D), illustrate that CREG process LAN inhibits part inflammatory reaction in atherosclerotic blood vessel.The expression trend of I κ B is consistent with NF-κ B (Fig. 4 E).
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (8)
1.CREG albumen is for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment.
2. express the recombinant vector of CREG albumen or the reconstitution cell containing this recombinant vector for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment; Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
3. the purposes of claim 2, wherein said recombinant vector is recombinant adenoviral vector.
4. the purposes of claim 3, wherein said recombinant adenoviral vector behaviour Adenovirus Type 5 Ad5-CREG, it is deposited in China typical culture collection center (CCTCC, Wuhan on January 2nd, 2008, Wuhan University), preserving number is CCTCC-V200801.
5. the preparation that Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise Ink vessel transfusing CREG protein expression level is for the preparation of the purposes prevented and/or treated in the medicine of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.) or medical equipment.
6. compositions, it comprises CREG albumen, express the recombinant vector of CREG albumen, reconstitution cell containing this recombinant vector, Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise the preparation of Ink vessel transfusing CREG protein expression level, and optional pharmaceutically acceptable carrier or excipient, described compositions is used for preventing and/or treating atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.); Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
7. medical equipment, it comprises CREG albumen, express the recombinant vector of CREG albumen, reconstitution cell containing this recombinant vector, Ink vessel transfusing CREG protein expression can be suppressed to lower or can raise the preparation of Ink vessel transfusing CREG protein expression level, and described medical equipment is used for preventing and/or treating atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.); Wherein said recombinant vector contains the nucleotide sequence of coding CREG albumen.
8. the compositions of claim 6 or the medical equipment of claim 7 are for the preparation of the purposes prevented and/or treated in the product of atherosclerosis or atherosclerosis relevant disease (such as coronary heart disease, apoplexy, abdominal aortic aneurysm, peripheral arterial disease etc.).
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| WO2018028433A1 (en) * | 2016-08-08 | 2018-02-15 | 中国人民解放军沈阳军区总医院 | Medical use of creg protein for prevention or treatment of overweight, obesity and related diseases thereof |
| CN108261540A (en) * | 2016-12-30 | 2018-07-10 | 中国人民解放军沈阳军区总医院 | Applications of the CREG in treatment nonalcoholic fatty liver and diabetes B |
| CN117624348A (en) * | 2023-09-04 | 2024-03-01 | 中国人民解放军北部战区总医院 | anti-CREG monoclonal antibody 5E2 and application thereof |
| CN117624348B (en) * | 2023-09-04 | 2024-07-05 | 中国人民解放军北部战区总医院 | Anti-CREG monoclonal antibody 5E2 and application thereof |
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