CN106822864A - A kind of method for suppressing the expression of proprotein convertases subtilisin 9 - Google Patents
A kind of method for suppressing the expression of proprotein convertases subtilisin 9 Download PDFInfo
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Abstract
The invention discloses a kind of method for suppressing the expression of proprotein convertases subtilisin 9, the generation of body proprotein convertases subtilisin 9 (PCSK9) is suppressed by using lunasine, reduce the serum levels of proprotein convertases subtilisin 9.The invention also discloses a kind of new application of lunasine, can be as the inhibitor of proprotein convertases subtilisin 9 relevant disease that convertase subtilisin 9 is mediated before the treatment, including hypercholesterolemia, obesity, diabetes, ephrosis etc..Present invention firstly discovers that inhibitory action of the lunasine to proprotein convertases subtilisin 9, opens the new application field of lunasine, with high market application value.
Description
Technical field
The invention belongs to biomedicine field, and in particular to one kind suppresses proprotein convertases subtilisin 9 (PCSK9)
The method of expression, can suppress the generation of body proprotein convertases subtilisin 9, drop by using lunasine (lunasin)
The serum levels of low proprotein convertases subtilisin 9.Can further using lunasine as proprotein convertases withered grass bacteriolyze
Plain 9 inhibitor are used for the medicine of the relevant disease for preparing the mediation for the treatment of proprotein convertases subtilisin 9.
Background technology
Proprotein convertases subtilisin 9 (PCSK9) (proprotein convertase subtilisin/kexin
9, PCSK9) it is a kind of glycoprotein being made up of 692 amino acid, category proprotein convertases (proprotein
Convertases, PCs) the 9th member in family is a kind of secreting type serine protease, mainly in liver and enteron aisle
Deng tissue in express, be then secreted into (J Biol Chem.2004 in blood；279(47):48865-48875；Trends
Biochem Sci.2007；32(2):71-77).PCSK9 can be with the low-density lipoprotein of surface of hepatocytes after entering blood circulation
The epidermal growth factor-like domain specific binding of acceptor (low density lipoprotein receptor, LDL-R),
Guide it to enter liver cell and reach lysosome, LDL-R is degraded in lysosome, so as to cause surface of hepatocytes LDL-R to reduce,
And then the ability that liver is combined and removes LDL-C is reduced, LDL-C levels raise (Proc Natl Acad in ultimately resulting in blood
Sci U S A.2008；105(35):13045-13050).Therefore, hypercholesterolemia (Annu can be treated by suppressing PCSK9
Rev Pharmacol Toxicol.2014；54:273-293).Additionally, current research shows, PCSK9 is raised and fat and 2 types
Diabetes (Pediatr Diabetes.2017.PMID:28093849,DOI:10.1111/pedi.12490；Diabetes
Metab Res Rev.2016；32(2):It is 193-199.) closely related, also with the chronic kidney disease such as nephrotic syndrome and albuminuria
(Int Urol Nephrol.2017.PMID:28084558, DOI:It is 10.1007/s11255-017-1505-2) closely related,
Therefore, the important means for preventing and treating these relative diseases can be turned into by suppressing PCSK9.
Existing various PCSK9 inhibitor are in development or granted listing at present turns into treatment hypercholesterolemia
Medicine, wherein mainly including：The Evolocumab (AMG145) of the 1st, anti-PCSK9 antibody inhibitions, such as U.S. Amgen companies and
The Alirocumab (REGN727/SAR236553) of Regeneron/Sanofi companies is in the granted listing (J of US and European
Clin Pharmacol.2017；57(1):7-32)；2nd, siRNA (SiRNA) inhibitor, such as ALN- of Alnylam companies
PCS can suppress the transcription of PCSK9, and Phase I clinical trial (Pharmacol Ther.2016 are had been enter at present；164:183-194)；
3rd, micromolecular compound, such as jamaicin can suppress transcription and translation (the J Biol Chem.2015 of PCSK9；290(7):
4047-4058；Atherosclerosis.2008；201(2):266-273).
Lunasine (lunasin) is a kind of initially isolated from soybean active peptide, by 43 amino acid residue groups
Into its sequence is:SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDD(SEQ ID NO:1), molecular weight is
5KDa(J Biol Chem.1987；262(22):10502-10505).There are some researches show polypeptide lunasine has antitumor
Activity, can suppress the propagation (Oncotarget.2015 of non-small cell lung cancer cell in vitro；6(7):4649-4662), suppress
The migration of breast cancer cell and invasion and attack (Oncol Rep.2016；36(1):253-262), can suppress in vivo mouse skin cancer and
Generation (the J Food Sci.2010 of breast cancer；75(9):H311-316).
The content of the invention
It is an object of the invention to provide a kind of method of suppression PCSK9, specifically by using polypeptide lunasine
(lunasin) method for suppressing proprotein convertases subtilisin 9 (PCSK9) expression in liver cell tissue.PCSK9 is a kind of
Secreting type serine protease, blood is secreted into after mainly being expressed in the tissue such as liver, small intestine, it has now been found that PCSK9 with
The chronic kidney diseases such as hypercholesterolemia, obesity, diabetes B, nephrotic syndrome and albuminuria are closely related, the invention provides
A kind of method that use polypeptide lunasine effectively suppresses PCSK9 generations, can be applied to prepare the above-mentioned disease related to PCSK9
Preparation or medicine.
Concrete technical scheme of the present invention is as follows：
A kind of method for suppressing the expression of proprotein convertases subtilisin 9, albumen turns before suppressing body using lunasine
Change the generation of enzyme subtilisin 9, reduce the serum levels of proprotein convertases subtilisin 9, the amino of the lunasine
Acid sequence such as SEQ ID No：Shown in 1.
The lunasine amino acid C-terminal can be non-amidated or amidated.
The side that the lunasine can be extracted by gene engineering method, chemical combination synthetic method or from natural plant material
Method is prepared.
The above method, lunasine can be used in combination with other inhibitor of proprotein convertases subtilisin 9, it is described before
The inhibitor of convertase subtilisin 9 includes that PCSK9 monoclonal antibodies, PCSK9 siRNAs, PCSK9 gene silencings are few
One or more in nucleotides, jamaicin and its derivative, curcumin, alga oligosaccharides.
The invention also discloses a kind of new application of lunasine, it is characterised in that lunasine is used as proprotein convertases withered grass
The inhibitor of bacteriolysin 9 is used for the medicine of the relevant disease for preparing the mediation for the treatment of proprotein convertases subtilisin 9, including courage high
Sterol mass formed by blood stasis, obesity, diabetes (diabetes B), ephrosis (chronic kidney disease such as nephrotic syndrome and albuminuria).
Such use, lunasine can be prepared into any preparation for pharmaceutically receiving, preferred tablet, capsule, drop
Agent, powder-injection or liquid drugs injection.
Said medicine is included as the lunasine and pharmaceutical acceptable carrier of active component.Preferably, medicine of the invention has
The lunasine as active component of 0.1-99.9% percentage by weights.
" pharmaceutical acceptable carrier " is included but is not limited to：Ion exchange material, aluminum oxide, aluminum stearate, lecithin, self-emulsifying medicine
Thing transmission system (SEDDS) such as d- TPGS 1000s, tween or other similar polymerisation medium medicines
Surfactant, the haemocyanin of preparation such as human serum albumins, buffer substance such as phosphate, amion acetic acid, sorbic acid, mountain
Potassium sorbate, the mixing of saturated vegetable fatty acid partial glyceride, water, salt, electrolyte such as sulfate protamine, disodium hydrogen phosphate, phosphorus
Potassium hydrogen phthalate, sodium chloride, zinc salt, silica gel, magnesium silicate etc..Polyvinyl pyrrolidone, cellulosic material, polyvinyl alcohol, carboxymethyl cellulose
Plain sodium, polyacrylate, ethene-polyoxyethylene-block polymer and lanolin, cyclodextrin such as α-, β-, gamma-cyclodextrin or its
The derivative such as hydroxyalkyl cyclodextrin such as 2- and 3- HP-β-CDs or other soluble derivatives etc. through chemical modification is equal
Can be used to promote the drug delivery of lunasine.
Other pharmaceutically acceptable auxiliaries such as filler (such as Lactis Anhydrous, starch, lactose bead and glucose), adhesive are (such as micro-
Crystalline cellulose), disintegrant (such as crosslinked carboxymethyl fecula sodium, Ac-Di-Sol, low-substituted hydroxypropyl cellulose and friendship
Connection PVP), lubricant (such as magnesium stearate), sorbefacient, flavouring agent, sweetener, diluent, excipient, wetting agent, solvent,
Solubilizer and colouring agent etc. can be also added in medicine of the invention.
The therapeutically effective amount of said medicine be 0.001-100mg/kg/d between, can be used for the single drug of relevant disease or
Drug combination treat, be skilled artisans appreciate that scope.
Brief description of the drawings
Fig. 1：Lunasin is in the mRNA expressions of PCSK9 in dosage and time-dependent inhibition HepG2 cells.Fig. 1-
1：The influence that various concentrations lunasin is expressed PCSK9mRNA；Fig. 1-2：Lunasin difference action times are to PCSK9mRNA tables
The influence for reaching.*,**,***And****Expression compares P with normal group<0.05, P<0.01, P<0.001 and P<0.0001 (n=3,
means±SEM)。
Fig. 2：Lunasin is in the expression of dosage and time-dependent inhibition HepG2 cell PCSK9 albumen.Fig. 2-1：It is different
The influence of concentration lunasin PCSK9 protein expressions intracellular to HepG2；Fig. 2-2：Various concentrations lunasin is to HepG2 cells
Secrete the influence of PCSK9 albumen；Fig. 2-3：The shadow of lunasin difference action time PCSK9 protein expressions intracellular to HepG2
Ring.*And**Expression compares P with normal group<0.05 and P<0.01 (n=3, means ± SEM).
Fig. 3：Western blot detect PCSK9 albumen in ApoE-/-Expression in mouse liver.####Represent right with normal
Compare according to group, P<0.0001；***Expression is compared with model group, P<0.001 (n=8, means ± SEM).
Fig. 4：Immunofluorescence test lunasin is to ApoE-/-Mouse liver secretes the influence of PCSK9 albumen.
Specific embodiment
Specific steps of the invention are illustrated by the following examples, but are not limited by the example.
Term used in the present invention, unless otherwise indicated, is typically generally understood that with those of ordinary skill in the art
Implication.
The present invention is described in further detail with reference to specific embodiment and with reference to data.It should be understood that the embodiment is
In order to demonstrate the invention, rather than by any way the scope of the present invention is limited.
In the examples below, the various processes and method not described in detail are conventional methods as known in the art.
Material used, reagent etc. in following examples, unless otherwise specified, commercially obtain.Below by way of
Embodiment further illustrates the present invention：
The lunasin of embodiment 1 effectively suppresses the expression of the intracellular PCSK9 genes of HepG2
The gene engineering method that polypeptide lunasine (lunasin) medicine is set up using laboratory of the present invention
(Setrerrahmane, S.Appl Biochem Biotechnol 174 (2014), 612-622) is prepared.Human liver cancer is thin
(the Cat containing 10%FBS is used in born of the same parents' strain HepG2 (being purchased from Chinese Academy of Sciences's Shanghai cell bank):10099141, Gibco) MEM culture mediums
After (41500034, Gibco) are resuspended, with 5 × 105Individual cells/well is inoculated in 6 orifice plates, is placed in 37 DEG C of 5%CO2Cultivated in incubator
When converging to 80%, former culture medium is abandoned, PBS is washed once, 1ml Opti-MEM culture mediums (Cat is added per hole:51985042,
Gibco) agent-feeding treatment after balance 1h.Dose-effect experimental administration method：Lunasin administration concentrations are 0,0.2,1 and 5 μM, during administration
Between be 24h；Ageing experimental administration method：Lunasin administration concentrations are 5 μM, and administration time is 1,2,4,8,16 and 24h.Medicine
It is intracellular to HepG2 using Quantitative Real Time-PCR (qRT-PCR) detections lunasin after thing treatment terminates
The influence of PCSK9 gene expression doses.
1. Total RNAs extraction
After cell is through drug-treated, 6 orifice plates add 1ml RNAiso Plus (Takara, 9108Q) per hole, blow and beat repeatedly
To without obvious sediment.In 5min is stored at room temperature, 200 μ l chloroforms are added, acutely vibrate 15s, treat that solution is fully emulsified, be stored at room temperature
After 5min, 12000rpm, 4 DEG C of centrifugation 15min.It is careful to draw 400 μ l supernatants, it is careful not to touch protein band and EP pipe pipes
Wall.Addition isopropanol (isometric) in supernatant, mixing of turning upside down, after being stored at room temperature 10min, 4 DEG C, 12000rpm, centrifugation
10min, obtains RNA precipitate.75% ethanol is slowly added to, cleaning 5-6 times of gently turning upside down, 7500rpm, 4 DEG C of centrifugations
After 5min, to the greatest extent except ethanol.Drying at room temperature precipitates about 10min, remaining to no liquid, after the appropriate RNase-free water dissolves of addition-
80 DEG C save backup.With the RNA concentration and purity of NanoDrop 2000/2000c spectrophotometric determination each sample.RNA tries one's best
In same day reverse transcription into cDNA-20 DEG C of storage.
2.cDNA synthesizes
Sequentially add each reagent to remove the genomic DNA in RNA by table 1.Then reverse transcription system is set up by table 2 to synthesize
CDNA, adds 1 μ g RNA in every 20 μ l reaction systems.Related reagent is purchased from Dalian treasured biology Takara companies.
Table 1 removes genomic DNA reaction system
After mixing, brief centrifugation, 42 DEG C of 2min (or room temperature 5min), 4 DEG C of hold.
The cDNA synthetic reaction systems of table 2
After mixing, brief centrifugation carries out the synthesis of cDNA.Reverse transcription condition is as follows：37 DEG C of 15min, 85 DEG C of 5s, 4 DEG C of guarantors
Deposit.
3.qRT-PCR is detected
QRT-PCR primer sequences are shown in Table 3, are synthesized by Shanghai Jierui Biology Engineering Co., Ltd.Reaction system is set up by table 4,
Reagent provides (Cat#RR820A) by Dalian treasured biology Takara companies, after mixing, brief centrifugation, and program is carried out as shown in table 5
Realtime PCR react.Each gene PCR amplification efficiency curve is drawn according to below equation：E (%)=(10-/slope-1)×100。
Sample 100ng cDNA Realtime PCR reactions executed as described above are taken, while NTC groups (the no-temple control) is set,
Experimental result is analyzed with Δ Δ Ct methods.Its formula is：2-ΔΔCT=2-[(CT gene of interest-CT internal control)sample A -(CT gene of interest–CT internal control)sample B]
The qRT-PCR primer tables of table 3
The μ l PCR reaction systems of table 4 20
The PCR response procedures of table 5
QRT-PCR methods detect the influence of lunasin PCSK9 gene expression doses intracellular to HepG2, as a result see Fig. 1.Amount
Effect experimental result shows：0.2nd, after 1,5 μ Μ lunasin act on HepG2 cells 24h, compare with normal group, lunasin is in agent
Amount dependence suppresses the expression of the intracellular PCSK9 genes of HepG2, wherein, 5 μ Μ lunasin effects most significantly (P<0.0001)
(Fig. 1-1).Timeliness experimental result shows：After 5 μ Μ lunasin treatment HepG2 cells 1,2,4,8,16,24h, lunasin is in agent
Amount dependence suppresses the expression of the intracellular PCSK9 genes of HepG2, wherein, 5 μ Μ lunasin effects 24h inhibitory action is most notable
(P<0.0001) (Fig. 1-2).
Thus prove, lunasin is in dosage and time-dependent inhibition HepG2 cell PCSK9 genes in transcriptional level
Expression, 5 μ Μ lunasin act on HepG2 cell 24h, and depression effect is the most notable.
The lunasin of embodiment 2 effectively suppresses HepG2 cell PCSK9 protein expressions
1. total protein of cell is extracted
The HepG2 cells in 6 orifice plates are incubated at after medicine packet transaction, 1ml cell culture supernatants is collected with 10kd
Super filter tube is concentrated and is settled to 100 μ l.HepG2 cells wash 3 times, every 1 × 10 with 4 DEG C of PBS of precooling simultaneously6In individual cell,
Add 100 μ l RIPA cell pyrolysis liquids (PMSF:RIPA=1:100), scraped with cell and gently scrape attached cell, split on ice
After solution 30min, (ultrasound mode is ultrasound 1s, interval 2s, total duration 1min) ultrasonically treated under condition of ice bath, 4 DEG C, 12000rpm
Centrifugation 10min, collects cell lysate supernatant, and determine each group protein content with BCA methods.According to each group protein content, dilution
Each sample, to ensure that albumen applied sample amount is consistent, adds 5 × Loading Buffer to same concentration, and 5min is boiled after mixing, makes
Albuminous degeneration, -20 DEG C save backup.
2.SDS-PAGE electrophoresis
12%SDS-PAGE glue is prepared by table 6, is 20~30 μ g albumen, 5 μ l pre-dyed in control wells per hole applied sample amount
Marker, is separated by electrophoresis to sample, and deposition condition is：80V electrophoresis about 30min, 120V continue electrophoresis to bromjophenol blue to red
Below line.
The SDS-PAGE glue of table 6 is with tabulation
3. transferring film
After electrophoresis terminates, according to the size of separation gel, 0.22 μm of clip pvdf membrane 1 and filter paper 6.Pvdf membrane is first used
Methyl alcohol soaks 5min, rinses 2min in distilled water, then after pvdf membrane and filter paper are soaked into 10min in transferring film buffer solution, directly
Making " sandwich " on the negative plate of device is connected on, from bearing to being just followed successively by：The metafiltration paper of porous pad-3-glue-pvdf membrane-3
Metafiltration paper-porous pad.Note the alignment of four sides and carefully drive bubble out of, ice-water bath is according to destination protein molecular weight 60V-100V
Constant pressure transferring film 100min.After transferring film terminates, after taking out pvdf membrane mark positive and negative, TBST cleaning 5min add Ponceaux dyeing
3-5min is to there is clear band, Taking Pictures recording.
4. close
TBST is rinsed 2-3 times, each 5min, after removal Ponceaux, in 10ml confining liquids (TBST containing 5% skimmed milk power)
Middle room temperature closes 1h.TBST washes 3 times, 5min/ times.
5. antibody incubation
According to destination protein molecular weight, film is cut into correspondingly sized, and cuts off the upper left corner and mark.With containing 5% defatted milk
The TBST of powder is with 1：The 1000 anti-PCSK9 antibody (Cat of dilution:Ab125251, abcam), add on corresponding pvdf membrane, 4 DEG C incubate
Educate overnight.TBST washes 3 times, 5min/ times.With secondary antibody dilution with 1:5000 dilution secondary antibodies, add on pvdf membrane, and room temperature is in level
Shaking table is incubated 2h.
6. colour developing identification
TBST cleanings pvdf membrane 3 times, 5min/ times.Plus ECL nitrite ions are on pvdf membrane, day energy Tanon gel imaging instruments into
Picture, analyzes experimental result.
Western blot methods detection HepG2 is intracellular and secretes to the PCSK9 protein expressions in cell culture supernatant
Level.As a result (Fig. 2) display, after 0.2,1,5 μM of lunasin act on cell 24h, HepG2 is thin for the detection of Western blot methods
Intracellular (Fig. 2-1) and secretion are to PCSK9 albumen (Fig. 2-2) in cell culture supernatant.Thus illustrate, compared with normal group,
Lunasin is intracellular in dose-dependent inhibition HepG2 and secretes to the PCSK9 protein levels in cell culture fluid, and 5 μM
Lunasin inhibitory action most significantly (P<0.01).Further detect lunasin difference action times PCSK9 intracellular to HepG2
The influence of protein expression level, as a result as Figure 2-3, after 5 μM of lunasin act on cell 1,2,4,8,16,24h,
Lunasin is in the intracellular PCSK9 protein expressions of time-dependent inhibition HepG2, and 24h depression effects most significantly (P<0.01).
Thus prove, lunasin is in dosage and time-dependent inhibition HepG2 cells expression PCSK9 albumen.
Therefore, the results show of example 1 and 2, lunasin can significantly inhibit HepG2 cells in transcription and translation level
The expression of middle PCSK9 genes.
The Lunasin intraperitoneal administrations of embodiment 3 can effectively suppress ApoE-/-The expression of mouse liver tissue PCSK9
1. animal packet and drug-treated
6 week old ApoE-/-It is limited that mouse and each 16 of C57BL/6 mouse are purchased from Beijing dimension tonneau China experimental animal technology
Company, credit number:SCXK (capital) 2012-0001.Animal feeding maintains 25 DEG C of room temperature, humidity 40%- in SPF grades of Animal House
70%, each 12h of light and shade, mouse ad lib drinking-water.After mouse gives the nursing of basal feed adaptability 1 week, ApoE-/-Mouse is given
High lipid food (Research Diets, D12108C) is given, C57BL/6 mouse give basal feed, after feeding 6 weeks, ApoE-/-It is small
Mouse is randomly divided into model group (physiological saline), administration group (0.5 μm of ol/kg lunasin), and C57BL/6 mouse are randomly divided into normally
Control group (physiological saline) and negative medicine group (0.5 μm of ol/kg lunasin), every group of each 8 animal.Using intraperitoneal injection side
Formula is administered, and dosage is 0.5 μm of ol/kg, and 1 times/day, successive administration is put to death after 4 weeks after mouse anesthesia, separates liver, is carried out
Subsequent experimental.
2.Western blot detect lunasin to ApoE-/-The influence of mouse liver PCSK9 protein expressions
After separating mouse hepatic tissue, eye scissors is shredded, 4 DEG C of brines of precooling 3 times, to remove residual blood, with egg
After (adding 600 μ l lysates per the 100mg livers) homogenate of white lysate (platinum aristogenesis thing Products), crack on ice 30min (every
10min vortex oscillations 1 time), 4 DEG C of centrifugation 15min of 12000r are taken in supernatant to centrifuge tube, and -20 DEG C save backup.BCA methods are surveyed
Mouse liver PCSK9 protein expression levels, the same embodiment of specific method are detected using Western blot methods after protein concentration
2。
Result as shown in figure 3, after 0.5 μm of C57BL/6 mouse peritoneal injections ol/kg lunasin 4 weeks, in its hepatic tissue
PCSK9 protein levels, without significant difference, illustrate that lunasin does not influence normal C57BL/6 Mouse Livers group compared with Normal group
Knit expression PCSK9 albumen.In addition, comparing with Normal group, ApoE-/-Model group murine liver tissue PCSK9 protein levels are notable
Raise, significant difference (P<0.0001), however ApoE-/-0.5 μm of mouse peritoneal injection ol/kg lunasin 4 weeks, then can be notable
Suppress PCSK9 protein expression levels in its hepatic tissue and raise (P<0.001).As can be seen here, lunasin does not influence Mice normal liver
Tissue secretion PCSK9 albumen, but ApoE can be significantly inhibited-/-Mouse PCSK9 protein levels are raised.
3. Immunofluorescence test lunasin is to ApoE-/-Mouse liver secretes the influence of PCSK9 protein levels
After murine liver tissue is separated, it is immediately placed in 4% paraformaldehyde after fixing 48h, conventional dehydration, transparent, waxdip, bag
Bury, cut into slices, 4 μm of slice thickness.After the conventional dewaxing rehydration of section, immunofluorescence detection is carried out.
(1) after histotomy, 60 DEG C of roasting 1h；
(2) dewax successively：The 15min of dimethylbenzene I, the 15min of dimethylbenzene II, absolute ethyl alcohol 5min, 95% ethanol 5min, 85%
Ethanol 5min, 75% ethanol 5min, PBS 5min；
(3) section after dewaxing is placed in citrate buffer solution, and 20min is boiled in heating, and after natural cooling, PBS is washed 3 times,
5min/ times；
(4) 3%H2O2Room temperature 10min, PBS wash 3 times, 5min/ times；
(5) 10% lowlenthal serums are closed, room temperature 30min；
(6) confining liquid is abandoned, PCSK9 primary antibodies (1 is added dropwise：200), 4 DEG C of overnight incubations；
(7) 37 DEG C of rewarmings 45min, PBS wash 3 times, 5min/ times；
(8) the goat-anti rabbit secondary antibody (1 of the marks of Alexa Fluor 488 is added dropwise：500), 37 DEG C of lucifuges are incubated 60min；
(9) PBS washes 3 times, 5min/ times；
(10) DAPI is added dropwise, 10min is dyeed, dries, glycerol adding mounting.Observation is taken pictures under inverted fluorescence microscope.
As a result (Fig. 4) display：Normal group C57BL/6 mouse and negative medicine group C57BL/6 murine liver tissues gap
It can be seen that a small amount of green fluorescence, represents there are a small amount of PCSK9 PEs in the murine liver tissue；Model group ApoE-/-Murine liver tissue
There are a large amount of bright green fluorescences in gap, represents that its hepatic tissue secretes PCSK9 albumen showed increaseds；Give lunasin abdominal cavities note
After penetrating 4 weeks, ApoE-/-Murine liver tissue gap green fluorescence is substantially reduced, and illustrates that lunasin intraperitoneal administrations can show after treating 4 weeks
Write and suppress ApoE-/-Murine liver tissue secretes PCSK9 albumen.
Above-mentioned the results show, ApoE can be significantly inhibited after polypeptide lunasin intraperitoneal administrations-/-Mouse liver tissue table
Up to and secretion PCSK9 albumen.
SEQUENCE LISTING
<110>China Medicine University
<120>A kind of method for suppressing the expression of proprotein convertases subtilisin 9
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 43
<212> PRT
<213> Soybean seeds
<400> 1
Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly Val Asn
Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly Arg Gly Asp Asp Asp
Asp Asp Asp Asp Asp Asp
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
AGGGGAGGACATCATTGGTG
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
CAGGTTGGGGGTCAGTACC
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
CTCTTCCAGCCTTCCTTCCT
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
CAGGGCAGTGATCTCCTTCT
Claims (9)
1. it is a kind of to suppress the method that proprotein convertases subtilisin 9 (PCSK9) are expressed, it is characterised in that to use lunasine
Suppress the generation of proprotein convertases subtilisin 9, reduce the serum levels of proprotein convertases subtilisin 9, it is described
The amino acid sequence of lunasine such as SEQ ID No：Shown in 1.
2. the method for claim 1, it is characterised in that the lunasine amino acid C-terminal is non-amidated or amidatioon
's.
3. the method for claim 1, it is characterised in that the lunasine passes through gene engineering method, chemical combination synthetic method
Or the method extracted from natural plant material is prepared.
4. the method as described in claim any one of 1-3, it is characterised in that by lunasine and other proprotein convertases withered grass
The inhibitor of bacteriolysin 9 is used in combination.
5. method as claimed in claim 4, it is characterised in that the inhibitor of proprotein convertases subtilisin 9 includes
PCSK9 monoclonal antibodies, PCSK9 siRNAs, PCSK9 gene silencings oligonucleotides, jamaicin and its derivative, curcumin,
One or more in alga oligosaccharides.
6. a kind of new application of lunasine, it is characterised in that lunasine is used as the inhibitor of proprotein convertases subtilisin 9
In the medicine of the relevant disease for preparing the mediation for the treatment of proprotein convertases subtilisin 9.
7. purposes as claimed in claim 6, it is characterised in that the related disease of the mediation of proprotein convertases subtilisin 9
Disease includes hypercholesterolemia, obesity, diabetes, ephrosis.
8. purposes as claimed in claim 6, it is characterised in that the lunasine can be prepared into any one for pharmaceutically receiving
Preparation.
9. purposes as claimed in claim 8, it is characterised in that the lunasine can prepare piece agent, capsule, drops, powder-injection
Or liquid drugs injection.
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| CN201710106759.1A CN106822864A (en) | 2017-02-27 | 2017-02-27 | A kind of method for suppressing the expression of proprotein convertases subtilisin 9 |
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| CN201710106759.1A CN106822864A (en) | 2017-02-27 | 2017-02-27 | A kind of method for suppressing the expression of proprotein convertases subtilisin 9 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110638856A (en) * | 2019-10-18 | 2020-01-03 | 宜昌市中心人民医院 | Medicine for preventing or reversing primary hypertension, type 2 diabetes or homologous diseases and animal model construction method |
| WO2022002160A1 (en) * | 2020-07-01 | 2022-01-06 | 陈敏 | Use of pcsk9 inhibitor in preparation of product for treating multiple diseases |
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| CN1741805A (en) * | 2001-07-18 | 2006-03-01 | 索莱有限责任公司 | High protein, bowman-birk inhibitor concentrate and process for its manufacture |
| CN104840943A (en) * | 2015-05-05 | 2015-08-19 | 中国农业科学院作物科学研究所 | Application of Lunasin polypeptide in aspect of preparing substance with weight-reducing activity |
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| CN1741805A (en) * | 2001-07-18 | 2006-03-01 | 索莱有限责任公司 | High protein, bowman-birk inhibitor concentrate and process for its manufacture |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110638856A (en) * | 2019-10-18 | 2020-01-03 | 宜昌市中心人民医院 | Medicine for preventing or reversing primary hypertension, type 2 diabetes or homologous diseases and animal model construction method |
| WO2022002160A1 (en) * | 2020-07-01 | 2022-01-06 | 陈敏 | Use of pcsk9 inhibitor in preparation of product for treating multiple diseases |
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Application publication date: 20170613 |