CN105770858A - Human-derived obesity inhibitory peptide - Google Patents
Human-derived obesity inhibitory peptide Download PDFInfo
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Marine Sciences & Fisheries (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a novel human-derived polypeptide capable of promoting white adipose browning and brown adipose differentiation. The polypeptide derived from IRS-1 protein and containing 56 amino acids is of great significance to prevention and treatment of obesity and complications thereof. An animal model is based on that a mouse only with IRS-1 amino terminal 56aa being kept is identified; compared with wild-type and heterozygous mice, the mouse is characterized in that subcutaneous white adipose tissues decrease obviously and brown adipose tissues in interscapular regions increase obviously, and is resistant to obesity caused by high-fat diet; compared with mice with IRS-1 genes being knocked out completely, the mouse with more brown adipose is capable of resisting the obesity and the complications thereof caused by high-fat diet, so that effect of IRS-1 amino terminal 56aa is determined primarily, and the technical problem of large side effect of slimming drugs in the prior art can be solved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of humanized's obesity peptide for inhibiting.
Background technology
At present, the whole world has the population of nearly 1/5th to suffer from obesity, obesity can involve the multiple organ of whole body and
System, with diseases such as cardiovascular and cerebrovascular disease, hypertension, diabetes, blood fat disorder, OSAS even cancers
There is close association.Every year because of total more than the death toll because of war, the attack of terrorism and smoking of the fat and number of death
With；Additionally, obesity patient's especially child patient is because build obesity easily forms the individual character such as timidity, Social Withdrawal and behavior
Feature.Therefore, the focus that the most effectively obesity controlling, always scientists are explored.
Moving and going on a diet is safest method of weight-reducing, but obese patient is gone on a diet only with simple and being moved not
Obvious fat-reducing effect can be played, and people are extremely urgent to physically and mentally healthy and body shaping beauty treatment pursuit, so that newly
Type medicine becomes the only selection for the treatment of obesity.At present slimming drugs the most on the market probably can be divided into appetite inhibitor,
Pancreatic lipase inhibitor and thyroxine preparation etc..
But, slimming drugs are while quick weight-lossing is shaped, mostly with multiple side effect and bad reaction.Such as appetite
Inhibitor can cause nervous system, intestines and stomach and cardiovascular system bad reaction；Pancreatic lipase inhibitor can cause steatorrhea, makes
Become fat-soluble avitaminosis, may also result in hepatic disorder simultaneously；Thyroxine preparation makes fat body during loss of weight
Weight excessively reduces, and causes osteoporosis and cardiovascular bad reaction.
Polypeptide drugs have molecular structure transformation little, easy synthesis, purity is high, biologically active is high, pharmaceutical dosage is little, security
The plurality of advantages such as height, high specificity, industrialization development are with the obvious advantage, is widely used to tumour, hepatitis, AIDS, generation at present
The prevention of thanking property disease etc., diagnose and treat, there is wide Prospect of R & D.Polypeptide can be divided into two big classes: endogenous polypeptide, i.e.
Endogenous polypeptide (such as enkephalins, thymic peptide, pancreas polypeptide etc.) that human body is intrinsic and exogenous polypeptid are (such as snake venom, sialic acid, honeybee
Poison, frog poison, scorpion venom, hirudin, an ancient wind instrument spiral shell toxin derivant and the cecropin etc. of fly secretion).Wherein, endogenous polypeptide structure is clear
Chu, mechanism of action clearly, quality level close to traditional small-molecule chemical medicine, activity close to pharmaceutical grade protein, self
Immune response is inconspicuous, and above feature becomes the main source of polypeptide drugs.
Summary of the invention
The present invention provides a kind of humanized's obesity peptide for inhibiting, to solve the technology that in prior art, fat-reducing medicine side effect is big
Problem.
The present invention provides a kind of IRS-1 aminoterminal 56aa application in preparing slimming medicine.
Preferably, the described IRS-1 aminoterminal 56aa concentration that plays a role is 0.4-2 μ g/ml.
Preferably, the described IRS-1 aminoterminal 56aa concentration that plays a role is 1 μ g/ml.
The present invention provides a kind of IRS-1 aminoterminal 56aa application in preparing diet food.
Preferably, the described IRS-1 aminoterminal 56aa concentration that plays a role is 0.4-2 μ g/ml.
Preferably, the described IRS-1 aminoterminal 56aa concentration that plays a role is 1 μ g/ml.
The technical scheme that embodiments of the invention provide can include following beneficial effect:
The present invention be a kind of based on humanized, have promote white adipose brown and brown fat differentiation novel many
Peptide, this polypeptide derives from IRS-1 albumen, comprises 56 amino acid, has important function in the fat preventing and treating with its complication.
The animal model of the present invention is according to being: we identify a kind of mouse only retaining IRS-1 aminoterminal 56aa, compared with wild type and miscellaneous
Zygote is compared by mouse, and the subcutaneous white adipose tissue of this mouse significantly reduces, interscapular region BAT showed increased, and
IRS-1-/-Mouse can resist the obesity caused by high fat diet.Compared with the mouse that IRS-1 gene is rejected completely, this mouse has
There is more brown fat, and obesity and the complication thereof of high fat diet induction can be resisted, thus primarily determine that in vivo
The effect of IRS-1 aminoterminal 56aa, can solve the technical problem that in prior art, fat-reducing medicine side effect is big.
It should be appreciated that it is only exemplary and explanatory, not that above general description and details hereinafter describe
The present invention can be limited.
Accompanying drawing explanation
Figure 1A is the genotype mice figure figure provided in the embodiment of the present invention；
Figure 1B is each mouse genotypes white adipose tissue and BAT's body weight provided in the embodiment of the present invention
Ratio chart；
Fig. 1 C is the food effect experimental result picture provided in the embodiment of the present invention；
Fig. 1 D is the three kinds of subcutaneous white adipose tissue of mouse genotypes provided in the embodiment of the present invention and interscapular region palm fibre
The HE staining versus figure of look adipose tissue；
Fig. 1 E is the qPCR test knot of the three kinds of subcutaneous white adipose tissue of mouse genotypes provided in the embodiment of the present invention
Fruit figure；
Fig. 1 F is the WB test result of the three kinds of subcutaneous white adipose tissue of mouse genotypes provided in the embodiment of the present invention
Figure；
Fig. 1 G is that the qPCR of the three kinds of mouse genotypes interscapular region BATs provided in the embodiment of the present invention surveys
Test result figure；
Fig. 1 H is the WB test of the three kinds of mouse genotypes interscapular region BATs provided in the embodiment of the present invention
Result figure；
Fig. 2 A is the molecular secondary structural representation of the IRS-1 provided in the embodiment of the present invention；
Fig. 2 B is the three-dimensional structure schematic diagram of the IRS-1 molecule provided in the embodiment of the present invention；
Fig. 3 A is that the polypeptide analysis device provided in the embodiment of the present invention measures IRS-1 aminoterminal 56aa polypeptide physicochemical property
Test result figure；
Fig. 3 B is the mass spectrogram of the purifying IRS-1 aminoterminal 56aa polypeptide provided in the embodiment of the present invention；
Fig. 4 A is that the mtt assay provided in the embodiment of the present invention measures the variable concentrations polypeptide propagation to front white adipocyte
Effect figure；
Fig. 4 B is that the mtt assay provided in the embodiment of the present invention measures the variable concentrations polypeptide propagation to front brown fat cell
Effect figure；
Fig. 4 C is to provide in the embodiment of the present invention3It is thin to front white adipose that H-TdR incorporation methods measures variable concentrations polypeptide
The multiplication effect figure of born of the same parents；
Fig. 4 D is to provide in the embodiment of the present invention3It is thin to front brown fat that H-TdR incorporation methods measures variable concentrations polypeptide
The multiplication effect figure of born of the same parents；
Fig. 4 E is that in the embodiment of the present invention, before the polypeptide intervention of the variable concentrations gradient of offer, white adipocyte becomes fat to lure
Lead rear UCP1 and express tendency chart；
Fig. 4 F is that in the embodiment of the present invention, before the polypeptide intervention of the variable concentrations gradient of offer, brown fat cell becomes fat to lure
Lead rear UCP1 and express tendency chart；
Fig. 5 A is the 9th day oil red O stain pair after the front white adipocyte brownization induction provided in the embodiment of the present invention
Than figure；
Fig. 5 B is the 9th day oil red O stain comparison diagram after the front brown fat cell induction provided in the embodiment of the present invention；
Fig. 5 C is white adipocyte brownization induction UCP1 expression change before the WB detection provided in the embodiment of the present invention
Figure；
Fig. 5 D is UCP1 expression variation diagram after the front brown fat cell induction of WB detection provided in the embodiment of the present invention.
Detailed description of the invention
Here will illustrate exemplary embodiment in detail, its example represents in the accompanying drawings.Explained below relates to
During accompanying drawing, unless otherwise indicated, the same numbers in different accompanying drawings represents same or analogous key element.Following exemplary embodiment
Described in embodiment do not represent all embodiments consistent with the present invention.On the contrary, they are only with the most appended
The example of the device that some aspects that described in detail in claims, the present invention are consistent.
Each embodiment in this specification all uses the mode gone forward one by one to describe, identical similar portion between each embodiment
Dividing and see mutually, what each embodiment stressed is the difference with other embodiments.
The explanation of the abbreviation appeared in the present invention:
IBAT: interscapular region BAT
SWAT: subcutaneous white adipose tissue
HE: haematoxylin eosin staining procedures
WB: protein imprinted
QPCR: real time fluorescent quantitative nucleic acid amplification detects
MTT: colorimetric method
3H-TdR: tritiated thymidine
DMSO: dimethyl sulfoxide (DMSO)
PBS: phosphate buffer
TCA: trichloroacetic acid
RhBMP7: humanized's BMP-7
Insulin: insulin
T3: trilute
IBMX:3-isobutyl group-1-methyl xanthine
Dex: dexamethasone
The amino acid sequence of IRS-1 aminoterminal 56aa: MASPPESDGFSDVRKVGYLRKPKSMHK
RFFVLRAASEAGGPARLEYYENEKKWRHK, the nucleotides sequence encoding described humanized's obesity peptide for inhibiting is classified as:
atggcgagccctccggagagcgatggcttctcagacgtgcgcaaggtgggctacctgcgcaagcccaagagtatgca
taagcgctttttcgtgctgcgggcggccagcgaggccgggggcccagcgcgcctggagtattatgagaacgagaaga
agtggcggcacaagtcg.Spontaneous gene mutation can cause the sequence after IRS-1 coding aminoterminal 56 amino acids the most eventually
Only.
Embodiment 1
IRS-1-/-Mouse phenotype checking and sWAT and iBAT change:
Randomly select the 1 sub-IRS-1 of monthly age Male homozygous-/-, heterozygote IRS-1+/-And wild type IRS-1+/+Each 3 of mouse,
Take iBAT, sWAT respectively and weigh, relatively each mouse genotypes white and BAT's body weight ratio；HE dyes observation group
Knit morphological change；The differential expression of WB and qPCR detection brown fat mark UCP1.56aa regulation and control before internal confirmation IRS-1
BAT differentiation and sWAT brown.
Refer to Figure 1A, show in the embodiment of the present invention provide genotype mice figure figure, picture from a left side to
The right side is followed successively by the IRS-1 at 1 monthly age-/-、IRS-1+/-And IRS-1+/+Mouse.By Figure 1A it can be seen that IRS-1 protein function lacks
IRS-1-/-Mouse, with IRS-1+/-And IRS-1+/+Mouse is compared, and build is the most modest, loses weight.
Refer to Figure 1B, each mouse genotypes white adipose tissue and brown fat provided is provided in the embodiment of the present invention
Fat organizer anharmonic ratio illustration.From Figure 1B, IRS-1-/-Mouse subcutaneous white adipose tissue weight ratio significantly reduces, and iBAT
Mass body anharmonic ratio dramatically increases, and illustrates that (i.e. in IRS-1, amino acid sequence is the IRS-1 aminoterminal 56aa that the present invention provides
The polypeptide of MASPPESDGFSDVRKVGYLRKPKSMHK RFFVLRAASEAGGPARLEYYENEKKWRHK) there is promotion white
The function of fat brown.
Refer to Fig. 1 C, the food effect experimental result picture provided is provided in the embodiment of the present invention.The present embodiment is random
Choose the 1 sub-IRS-1 of monthly age Male homozygous-/-And wild type IRS-1+/+Mouse feeds common food and high lipid food 16 weeks respectively,
In figure, curve C-IRS-1-/-And C-IRS-1+/+Represent that homozygote and wild-type mice feed the body weight change of common food respectively
Change；Curve HF-IRS-1-/-And HF-IRS-1+/+Represent that homozygote and wild-type mice feed the body weight change of high lipid food respectively
Change.From Fig. 1 C, no matter IRS-1-/-The nursing food of mouse is common food or high lipid food, its body weight the most relatively IRS-1+/+Mouse is greatly reduced, and IRS-1-/-Mouse is being fed in the case of high lipid food, body weight relatively IRS-1+/+Mouse is fed common
Food is also light, thus illustrates, IRS-1-/-Mouse can resist the obesity caused by high fat diet.
Refer to Fig. 1 D, the HE dye of three kinds of mouse genotypes sWAT and the iBAT provided is provided in the embodiment of the present invention
Look.The visible IRS-1 of HE dyeing-/-Mouse sWAT volume is obviously reduced, and is dispersed with the more little fat in many rooms and drips；Meanwhile, IRS-
1-/-Mouse iBAT relatively wild type is compared with heterozygote, drips containing substantial amounts of little fat, illustrates that IRS-1 aminoterminal 56aa can promote
White adipose brown and the differentiation of brown fat.
Refer to Fig. 1 E, Fig. 1 F, Fig. 1 G and Fig. 1 H, the three kinds of genotype provided in the shown respectively embodiment of the present invention are little
The three kinds of mouse genotypes provided in qPCR and the WB test result figure of the subcutaneous white adipose tissue of mouse and the embodiment of the present invention
QPCR and the WB test result figure of interscapular region BAT.From Fig. 1 E and Fig. 1 F, the subcutaneous white adipose of homozygote
Show that brown fat mark UCP1, PGC1a and Cidea mrna expression relatively other two kinds of genotype substantially raise, at protein level
On, homozygote IRS-1, without expression, UCP1 up-regulated, these results suggest that the subcutaneous white adipose tissue of homozygote exists white
Fat brown phenomenon.From Fig. 1 G and Fig. 1 H, homozygote and heterozygote interscapular region BAT UCP1, PGC1a
And Cidea mrna expression relatively wild type substantially raises, on protein level, homozygote IRS-1 is without expression, UCP1, PGC1a table
Reach rise, these results suggest that homozygote interscapular region BAT exists the phenomenon of differentiation.
Embodiment 2
IRS-1 aminoterminal 56aa analyzes
Refer to Fig. 2 A, the molecular secondary structural representation of the IRS-1 provided is provided in the embodiment of the present invention.Fig. 2 A table
Bright IRS-1 gene comprises the N-terminal high conservative of PH and PTB domain.56 amino acid whose sequences of N end are entered by further
Row is analyzed and is found: 56aa is high conservative in people, mouse, chicken, pig even frog.Refer to Fig. 2 B, show the present invention
The three-dimensional structure schematic diagram of the IRS-1 molecule provided in embodiment.This section of polypeptide of 56aa is carried out three-dimensional structural analysis discovery: this
56aa can form the structure of 4 hairpin structures, hairpin structure herein and other parts of IRS-1 the most not at the N end of IRS-1
With, the 56aa further illustrating IRS-1N end has specific function.
Embodiment 3
According to IRS-1 aminoterminal 56aa sequent synthesis polypeptide and analysis of physical and chemical property thereof
56aa sequence MASPPESDGFSDVRKVGYLRKPKSMHKRFFVLRAASEAGGPAR is encoded according to people's IRS-1 aminoterminal
LEYYENEKKWRHK, specific as follows: (the 6th and 7 amino acids people ES, mouse DT, 57aa codon c-a are IRS-
1-/-The mutational site of mouse, is terminator codon by mutant serine):
Refer to Fig. 3 A, it is many that the polypeptide analysis device that showing in the embodiment of the present invention provides measures IRS-1 aminoterminal 56aa
The test result figure of peptide physicochemical property.From Fig. 3 A, application polypeptide analysis program also carries out physics and chemistry to IRS-1 aminoterminal 56aa
Character finds: this section of polypeptide molecular weight 6540.5Da, net charge during isoelectric point 10.5, PH=7, and average hydrophilicity 0.5 is hydrophilic
Residue ratio 45%, favorable solubility in water.Synthesize IRS-1 aminoterminal 56aa polypeptide fragment according to amino acid sequence and carry out
Functional study.
Refer to Fig. 3 B, the mass spectrogram of the purified polypeptide provided is provided in the embodiment of the present invention.From Fig. 3 B, this
The theoretical molecular of bright polypeptide is 6540.5Da (100%M+H), Mass Spectrometer Method condition: flow velocity, 1.0ml/min, and wavelength is
220nm, volume is 20 μ l, and flow phase system is 0.1% trifluoracetic acid/second cyanogen solution-0.1% trifluoracetic acid/aqueous solution, purifies
The mass spectrum of polypeptide shows, its molecular weight is consistent with calculated value, and after synthesis, its purity is 95.97%.
Embodiment 4
Polypeptide is to front white adipocyte and the proliferative effect of front brown fat cell
White adipocyte and front brown fat cell before the polypeptide of variable concentrations gradient is intervened be set, respectively row MTT and3H-TdR incorporation methods surveys cell proliferation, intervenes PECTORAL LIMB SKELETON with the polypeptide of variable concentrations simultaneously, then carries out into brown fat
Induction differentiation, detects brown fat label UCP1, finally determines that the intervention concentration intervening cells play effect is 0.4-2 μ g/
Ml, optimal peptide concentration is 1 μ g/ml.
1, before mouse primary, brown fat cell and front white adipocyte are cultivated: (following steps are the most in an aseptic environment
Carry out)
(1) front white adipocyte is cultivated:
S11: take mouse in 7-10 days ages 6, along ventrimeson incision of skin, scissors blunt separation skin and peritoneal tissues are also cut
Open skin at lower abdomen and groin, take iliac region and subcutaneous white adipose；
S12: after being cut into small pieces by above-mentioned subcutaneous white adipose, uses 1mg/ml clostridiopetidase A to digest 45 at a temperature of 37 DEG C
Minute, repeatedly dispel mixing；
S13: when digestive juice becomes liquid completely, terminates digestion with the F12/DMEM culture medium containing 10% hyclone, and
The adipocyte of maturation is fallen at 70 μm strainer filterings；
S14:1200rpm × 10min is centrifuged, and outwells supernatant, is centrifuged again, so repeats two after the resuspended precipitation of complete medium
Time, cell is precipitated resuspended after plant in the culture dish of 3.5cm, 37 DEG C, 5%CO2Cultivate under environment.
(2) front brown fat cell is cultivated:
S21: take mouse in 2-3 days ages 6, along dorsal midline incision of skin, scissors blunt separation skin and hypodermis, takes
Interscapular region BAT；
S22: after above-mentioned interscapular region BAT being cut into small pieces, uses 1mg/ml clostridiopetidase A the temperature of 37 DEG C
Lower digestion 45 minutes, dispels mixing repeatedly；
S23: when digestive juice becomes liquid completely, terminates digestion with the F12/DMEM culture medium containing 10% hyclone, and
The adipocyte of maturation is fallen at 70 μm strainer filterings；
S24:1200rpm × 10min is centrifuged, and outwells supernatant, is centrifuged again, so repeats two after the resuspended precipitation of complete medium
Time, cell is precipitated resuspended after plant in the culture dish of 3.5cm, 37 DEG C, 5%CO2Cultivate under environment.
2, brown fat cell and front white adipocyte propagation before MTT detection
S31: collect logarithmic phase cell, adjusts concentration of cell suspension, and every hole adds 100 μ l, and bed board makes front white adipose thin
Born of the same parents and front brown lipocyte density are 5 × 103/ hole；
S32:5%CO2, hatch under conditions of 37 DEG C, be paved with (96 hole flat underside) at the bottom of hole to cell monolayer, after 12 hours,
Add the polypeptide intervention (0,10 of variable concentrations gradient-3, 10-2,10-1, 1,2,5 μ g/ml), every hole 100 μ l, each concentration sets 8
Multiple hole, and stay 8 blank well；
S33:5%CO2, after hatching 24 hours under conditions of 37 DEG C, every hole add 10 μ l MTT solution (5mg/ml, i.e.
0.5%MTT), terminate cultivating after continuing to cultivate 4 hours, carefully suck nutrient solution in hole；
S34: every hole adds 100 μ l DMSO, puts low-speed oscillation 10 minutes on shaking table, makes crystal fully dissolve, and joins at enzyme
The light absorption value in each hole is measured at immune detector 490nm.
Refer to Fig. 4 A and Fig. 4 B, the shown mtt assay provided that is respectively in the embodiment of the present invention measures variable concentrations polypeptide
The mtt assay provided in the multiplication effect figure of front white adipocyte and the embodiment of the present invention is measured variable concentrations polypeptide to front palm fibre
The multiplication effect figure of look adipocyte.From Fig. 4 A and Fig. 4 B, front white adipocyte and front brown fat cell MTT curve
Figure shows that polypeptide promotes that the concentration range of cell proliferation is 0.4-2 μ g/ml, and both OD values all reach when concentration is 1 μ g/ml
Big value, illustrating that polypeptide intervenes the concentration of cells play effect is 0.4-2 μ g/ml, and the most most preferably intervening concentration is 1 μ g/ml.
3、3Brown fat cell and front white adipocyte propagation before the detection of H-TdR incorporation methods
Principle illustrates: thymidine (TdR) is the distinctive base of DNA, is also the required material of DNA synthesis, apparatus
Active isotope3H mark TdR is i.e.3The front physical efficiency that H-TdR synthesizes as DNA mixes DNA anabolic process, by surveying
Determine the radioactive intensity of cell, cell DNA metabolism and cell proliferative conditions can be reflected.Detailed process is as follows:
S41: front white adipocyte or front brown fat cell are with 5 × 103/ ml density is inoculated in 96 orifice plates, 37 DEG C,
5%CO2Middle cultivation 24 hours；
After S42:12 hour, add the polypeptide intervention (0,10 of variable concentrations gradient-3, 10-2,10-1, 1 μ g/ml), every hole
100 μ l, not add any medicine group as blank (each concentration group sets 8 groups of parallel holes)；
S43: after cultivating 24 hours respectively, every hole adds 50 μ l's3H-TdR working solution (0.5uCi) cultivates 24 hours；
S44: cultivate after terminating, be centrifuged and suck supernatant, wash 3 times with the 200 cold PBS of μ l, then add 200 μ l precoolings
5%TCA washes 1 time, then washes twice with 95% ethanol, and the NaOH adding 200 μ l 0.5M in every hole dissolves, and is cracked by cell with the volley of rifle fire
Liquid is transferred in special 96 orifice plates of scintiloscope configuration, is dried at 60 DEG C and processes 30 minutes, is then allowed to be cooled to room temperature；
S45: collect lysate with DYQ-II type bull cell collector, moves in flicker plate, adds scintillation solution 100 μ l, put
Liquid scintillation counter measures radiocounting (cpm)；
With SI (SI), S46: represent propagation degree with umber of pulse per minute (cpm), represents that experimental group relatively compares
The propagation degree that group is compared and propagation efficiency；SI=experimental port cpm/ control wells cpm；If the SI value of given the test agent group is significantly higher than
The SI value of control group, i.e. can determine that this experimental result positive.
Refer to Fig. 4 C and Fig. 4 D, the shown respectively embodiment of the present invention provides3H-TdR incorporation methods measures different dense
Degree polypeptide is to providing in the multiplication effect figure of front white adipocyte and the embodiment of the present invention3H-TdR incorporation methods measures difference
The concentration polypeptide multiplication effect figure to front brown fat cell.From Fig. 4 C and Fig. 4 D, front white adipocyte and front brown
Adipocyte all reach maximum when concentration is 1 μ g/ml, illustrate intervene cells play effect optimal peptide concentration be 1 μ g/
ml。
4, determine and most preferably intervene concentration
When current white adipocyte and front brown fat cell attachment 80%, respectively with the polypeptide of variable concentrations gradient
(0,10-3, 10-2,10-1, 1 μ g/ml) intervene 48 hours after, 3.3nM rhBMP7 intervene 72h (day 0), use 20nM insul
In, 1nM T3,0.5mM IBMX, 5 μMs of Dex with 0.125mM Indomethacins become brown fat induction differentiation, induction the 3rd day
(day 3) WB detection UCP1 expresses, and before further clear and definite IRS-1, the polypeptide of 56aa most preferably intervenes concentration is 1 μ g/ml.
Refer to Fig. 4 E and Fig. 4 F, the shown polypeptide intervention that the variable concentrations gradient provided is provided in the embodiment of the present invention
After front white adipocyte and front brown fat cell adipogenic induction, UCP1 expresses tendency chart.From Fig. 4 E and Fig. 4 F, UCP1
Expression effect along with intervene concentration (0,10-3, 10-2,10-1, 1) increase and raise, intervene concentration be 1 μ g/ml time,
The expression of UCP1 is optimal, illustrates that the optimal peptide concentration intervening cells play effect is 1 μ g/ml.
IRS-1 amino terminal 56aa effect experimental
Front white adipocyte and front brown fat cell are divided into the 5 groups: 1st group to be negative control group, and remaining 4 groups carry out palm fibre
It is further divided into ins+T after lookization induction differentiation3(positive control), polypeptide+T3, T3, ins+ polypeptide+T3Changing liquid, polypeptide is intervened concentration and is
1 μ g/ml, in the 9th day (day 9) oil red O stain observation of cell differentiation situation of induction, and utilizes Western blot to detect brown fat
Mark UCP1 expresses change.
Refer to Fig. 5 A and Fig. 5 B, the shown front white adipocyte brownization induction that embodiment of the present invention offer is provided
9th day oil red O stain figure after latter 9th day oil red O stain figure and front brown fat cell induction.From Fig. 5 A and Fig. 5 B,
In front white adipose and the induction differentiation of front brown fat, polypeptide+T3Organize relatively T3Group fat drips increased number, ins+ polypeptide+T3Group fat drips
Relatively ins+T3Group increases, and shows that IRS-1 aminoterminal 56aa polypeptide can promote white adipose brown and brown fat differentiation.
Separate the front white adipose of wild-type mice and front brown fat cell, when cell attachment 80% with 1 μ g/ml's
After the polypeptide of IRS-1 aminoterminal 56aa is intervened 48 hours, 3.3nM rhBMP7 intervene 72 hours after (day 0), use 20nM
insul in,1nM T3, 0.5mM IBMX, 5 μMs of Dex with 0.125mM Indomethacins become brown fat induction 3 days (day of differentiation
3) respectively with ins+T after,3(positive control), polypeptide+T3, T3, ins+ polypeptide+T3Change liquid 3 times, at the 9th day (day 9) of induction
Oil red O stain, observes polypeptide for becoming the effect of fat differentiation.Collect the 9th day (day 9) product, utilize the table of WB detection UCP1
Reach change, using β-actin as internal reference, compare the polypeptide impact on two groups of cellular fat differentiation marks.
Refer to Fig. 5 C and Fig. 5 D, white adipocyte brown before the shown WB detection that embodiment of the present invention offer is provided
Change UCP1 after induction UCP1 expresses variation diagram and the front brown fat cell induction of WB detection and express variation diagram.Shown in Fig. 5 C and Fig. 5 D
WB testing result show that polypeptide group UCP1 is expressed and increase, further checking IRS-1 aminoterminal 56aa polypeptide can promote white
The sizing differentiation of fat brown and front brown fat cell.
Invention described above embodiment, is not intended that limiting the scope of the present invention.Any in the present invention
Spirit and principle within amendment, equivalent and the improvement etc. made, should be included within the scope of the present invention.
The above is only the detailed description of the invention of the present invention, makes to skilled artisans appreciate that or realize this
Bright.Multiple amendment to these embodiments will be apparent to one skilled in the art, as defined herein
General Principle can realize without departing from the spirit or scope of the present invention in other embodiments.Therefore, the present invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and principles disclosed herein and features of novelty phase one
The widest scope caused.
Claims (6)
1.IRS-1 aminoterminal 56aa application in preparing slimming medicine.
The IRS-1 aminoterminal 56aa the most according to claim 1 application in preparing slimming medicine, it is characterised in that institute
Stating the IRS-1 aminoterminal 56aa concentration that plays a role is 0.4-2 μ g/ml.
The IRS-1 aminoterminal 56aa the most according to claim 2 application in preparing slimming medicine, it is characterised in that institute
Stating the IRS-1 aminoterminal 56aa concentration that plays a role is 1 μ g/ml.
4.IRS-1 aminoterminal 56aa application in preparing diet food.
The IRS-1 aminoterminal 56aa the most according to claim 4 application in preparing diet food, it is characterised in that institute
Stating the IRS-1 aminoterminal 56aa concentration that plays a role is 0.4-2 μ g/ml.
The IRS-1 aminoterminal 56aa the most according to claim 5 application in preparing diet food, it is characterised in that institute
Stating the IRS-1 aminoterminal 56aa concentration that plays a role is 1 μ g/ml.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102078315A (en) * | 2011-02-25 | 2011-06-01 | 昆明医学院 | Application of ampelopsin in preparing medicines for treating obesity |
| CN102716134A (en) * | 2012-06-07 | 2012-10-10 | 中国人民解放军第四军医大学 | Application of oleanolic acid to medicine for treating obesity |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102078315A (en) * | 2011-02-25 | 2011-06-01 | 昆明医学院 | Application of ampelopsin in preparing medicines for treating obesity |
| CN102716134A (en) * | 2012-06-07 | 2012-10-10 | 中国人民解放军第四军医大学 | Application of oleanolic acid to medicine for treating obesity |
Non-Patent Citations (2)
| Title |
|---|
| VINCENZO MARZOLLA等: "Mineralocorticoid Receptor Antagonism Promotes Brown-like Remodeling of Murine White Adipose Tissue Via Reduction of Autophagic Flux in Adipocytes", 《JOURNAL OF PHARMACOLOGICAL SCIENCES》 * |
| 洪靖等: "IRS-1基因与肥胖、2型糖尿病", 《中日友好医院学报》 * |
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