CN104288782A - Application of Beclin1 interacting protein and its gene - Google Patents

Abstract

The invention relates to an application of a Beclin1 interacting protein and its gene, and concretely relates to an application in the preparation or screening of drugs for regulating adipocyte differentiation and autophagy. BAD gene and YWHAQ gene can be used for preparing or screening drugs for regulating subcutaneous fat autophagy caused by hunger; AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3 or YWHAQ gene can be used for preparing or screening drugs for regulating subcutaneous fat autophagy caused by high fat diet; BAD, BIRC5 or TICAM2 gene can be used for regulating internal organ fat autophagy caused by high fat diet; and the TNFAIP3 gene can be used for preparing or screening drugs for regulating adipocyte differentiation The genes related with the fat autophagy and differentiation in the Bclin1 interacting protein are screened, and a new direction is provided for the screening of drugs for treating obesity.

Description

The interaction protein of Beclin1 and the application of gene thereof

Technical field

The present invention relates to biological medicine and molecular biological arts, be specially the interaction protein of Beclin1 and the application of gene thereof, the application especially in fatty autophagy.

Background technology

Along with the raising of socioeconomic development and living standards of the people, fatly become a current large health problem gradually.So-called fat, namely body fat is too much accumulated, and has influence on health, reduces the life-span, reduces quality of life.The harm that obesity is brought not only is confined to itself, and multiple Other diseases is more easily sent out, especially the tumor and osteoarthritis etc. of heart disease, diabetes, obstructive sleep apnea, some type.Fat modal pathogenic factor is the too much and momental deficiency of Energy intaking, toward contact along with genetic predisposition.But, also have the morbidity of small part obese patient to cause primarily of related gene, endocrine disturbance, medicine and psychotic disorder.Because the sickness rate of obesity and fatality rate worldwide constantly rise, and the age of morbidity present rejuvenation trend, fatly become one of 21st century the most serious public health problem already.Scientist all over the world constantly attempts setting about fat research from all angles, ites is desirable to find to some extent in various aspects such as the pathogenesis of obesity, fat prevention, the pathogenesis of obesity and the treatments of obesity, thus contributes to answering a fat difficult problem.

Autophagy, namely carries out " engulfing " autologous material, and it realizes the self renewal of cell by degrade long-lived albumen, damaged cell device etc. of lysosomal pathway, to resist the change of external environment, maintains homeostasis.Under the extremities such as hunger, injury, autophagy decomposes the recovery that cell component realizes utility, ensure the synthesis of important albumen, maintain the survival of cell and body, the regulating action in the pathogenesis such as tumor, nervous system disease, incretion metabolism disease.In inner-sphere reorganization energy discipline category, the change of autophagy under the high pathological state such as sugared, fat receives much concern.Document shows, autophagy likely take part in the generation of diabetes and diabetic complication.

Autophagy roughly can be divided into three types: the first, large autophagy.Mainly be used for digesting damaged cell device and protein, in large autophagy process, double membrane structure wraps up treats that digestion material forms autophagosome, and autophagosome is combined with lysosome and forms lyase autophagosome then, completes autodigestion process; The second, little autophagy.Be mainly used to digest the less material of granule and structure, in little autophagy process, lysosome membrane directly wraps up treats that degradation material completes digestion process; 3rd, the autophagy of molecular chaperones mediation.The autophagy process of molecular chaperones mediation is comparatively complicated, in the process, treats that degradation material combines with molecular chaperones in advance, is transported in lysosome subsequently, completes degradation process.Namely general said autophagy refers to the first type, large autophagy.Autophagy process involved in the present invention also refers to large autophagy.

LC3-II is unique protein labeling relevant to autophagy reliably, and the conversion using the method for WesternBlot to detect LC3 is a kind of method of monitoring autophagy of extensive use.The appearance of LC3-II indicates the generation of autophagy, and therefore the expression of LC3-II can react the height of autophagy.But the ratio of LC3-II and LC3-I more can describe the process that autophagy occurs, thus LC3-II/LC3-I becomes the index more objectively reflecting autophagy amount.Beclin1 (BECN1) is first mammal autophagy related gene that scientist clones, and the homologous genes in mammal for yeast Atg6 and nematicide Bec-1, plays vital effect in autophagy process.Meanwhile, Beclin1 also take part in the process such as apoptosis and cell differentiation, becomes the bridge connecting autophagy, apoptosis and differentiation.But, Beclin1-/-MEFs apoptosis and not obvious, and autophagy disappearance is obviously, and this illustrates relative apoptosis, the autophagy controlling gene that Beclin1 is important especially.Also there are some researches show that Beclin1 plays certain role in the generation of tumor and neurodegenerative diseases.Thought in the past all autophagy processes be Beclin1 rely on, although found the dependent autophagy process of non-Beclin1 in recent years, the importance of Beclin1 had had some idea of.

Research shows, autophagy also plays an important role in adipocyte lipolysis.2009, first Singh research group found that autophagy regulates and controls a New function of lipid metabolism in liver, and called after " macrolipophagy ", open the new knowledge of people to this field.When namely this function refers to hunger, born of the same parents' lactone drips and can enter lysosome after autophagy component is combined, fast hydrolyzing TG, and release FFA, for mitochondrial beta-oxidation is supplied raw materials.Macrolipophagy not only can occur in hepatocyte, all there is this approach as endotheliocyte, lymphoblast, neurocyte etc. in nearly all studied cell, the destruction of autophagy can cause a large amount of accumulations of born of the same parents' inner lipid to cause the infringement of body, shows that autophagy plays in lipid mobilization and acts on widely.

The most classical derivant of autophagy is hungry or nutritional deficiency.Initial research in yeast finds, hunger can induce autophagy to increase, and promotes the necessary protein of non-existence and amino acid whose degraded, for the synthesis of existence institute proteins necessary.At higher eucaryote, the histiocyte carrying out fasting process or In vitro culture to animal is deprived nutrition and autophagy also can be caused to raise.Except hungry and nutrient deprivation, also have factors can activate autophagy, such as anoxia, motion, high fat diet, x-ray, hyperinsulinemia etc.

Autophagy is different in the generation of different internal organs.Such as, at liver and pancreas, the autophagy of hungry induction generally continues several hours, and in skeletal muscle, the formation energy last from days of autophagosome.Meanwhile, different skeletal muscle fiber such as the level of autophagy in musculus soleus and gastrocnemius is also discrepant, fast muscle fiber for the sensitivity of the autophagy caused by high sugar far away higher than slow switch fibers.Fat, as an endocrine organ, can secrete the hormones such as leptin.Whether different fatty tissuees presents difference, whether there is different responses for different autophagy processes in this physiological processes of autophagy.

Beclin1 is a kind of novel BH3 domain protein, has the characteristic be combined with each other with other albumen, the role of " porter " that play the part of in autophagy, and many signal paths are by the autophagy of Beclin1 regulating cell.Anti-apoptotic proteins Bcl-2/Bcl-xL is in conjunction with Beclin1 autophagy approach capable of blocking.Beclin1 and III type Phosphatidyl inositol triphosphate kinases (Class III PI3K) combine the complex formed, significant in the formation of regulation and control autophagic vacuole.The object of the invention is to find out the Beclin1 mutual action protein gene relevant to autophagy, for new enlightenment served by the Mechanism Study band of fatty autophagy.

The Beclin1 interaction protein title that table 1 relates to and effect

Title	Main Function
		AMBRA1	Regulation and control autophagy and nervous system development
BAD	Promote cell death
		BCL2L1	T suppression cell is dead
BIRC5	Promote cell differentiation, stop apoptosis
		FEZ1	Regulate cellular morphology and axon growth
GOPC	Participate in intracellular protein transhipment and degraded
		SLAMF1	Signal lymphocyte (T cell/B cell) anakmetomeres
TICAM2	Participate in immunoreation
		TNFAIP3	Participate in immunoreation and inflammatory reaction
WAC	Participate in genetic transcription and histone ubiquitination, regulate autophagy
		YWHAQ	Wide participation signal transduction, apoptosis inhibit

Summary of the invention

The present invention is intended to the interaction protein and the gene thereof that screen the Beclin1 participated in different fatty tissue, different autophagy approach, Adipose Differentiation process and steatolysis process.

Autophagy, as a kind of special mass degradation approach, plays important role in the maintenance of the energy balance.One of core protein that Beclin1 occurs as autophagy, plays a very important role in the whole process of autophagy.By the expression of interaction protein in different fatty tissue, different autophagy approach, Adipose Differentiation process and steatolysis process of observation and comparison Beclin1, screening participates in the gene that autophagy occurs and maintains fatty function.

By observing Beclin1 and 11 interaction protein expression at interior fat and subcutaneous fat, compare its differential expression; Observe the expression of Beclin1 and 11 interaction protein under two kinds of autophagy state of activation (autophagy that hunger causes and the autophagy that high fat diet causes), analyze its difference; Observe the expression of Beclin1 and 11 interaction protein in 3T3-L1 Adipocyte Differentiation and steatolysis process; The acetylation modification of desk study Beclin1.

By larva starvation group mice fasting 48 hours, high fat diet group mice high lipid food is fed 12 weeks, extract histone or tissue mRNA after sacrifice, observe the expression in the autophagy process that gene causes in different fatty tissue and the different stimulated factor by the method for WesternBlot and Real-timePCR; In 3T3-L1 Adipocyte Differentiation process, collect cell in 0,2,4,6,8 day respectively and carry out cell protein or cell mRNA extraction, by the expression of methods analyst gene in Adipocyte Differentiation process of WesternBlot and Real-timePCR, and use fluorescence microscope Taking Pictures recording 3T3-L1 atomization; IBMX and ISO is used to stimulate ripe 3T3-L1 adipose cell generation steatolysis, be respectively 0,3,6,9,12,16 hour action time, extract cell mRNA also by the methods analyst gene expression of Real-timePCR, use fluorescence microscope adipose cell fat to drip morphologic change simultaneously.

Result shows, and Beclin1, BAD, FEZ1, GOPC, TNFAIP3, WAC, YWHAQ are significantly higher than the expression at subcutaneous fat at the expression of interior fat, SLAMF1 in the expression of subcutaneous fat apparently higher than the expression at interior fat; In the autophagy activation process that hunger causes, the expression of Beclin1, BAD, YWHAQ reduces, and in the autophagy activation process caused in high fat diet, the expression of Beclin1, AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3, YWHAQ all significantly raises; In the differentiation process of 3T3-L1 adipose cell, the expression of Beclin1 and TNFAIP3 progressively raises, and the expression of BIRC5 presents first high rear low trend, and the expression of FEZ1 then continues to be in reduced levels; In the ripe 3T3-L1 adipocyte lipolysis process of IBMX+ISO induction, Beclin1 and 11 interaction protein does not detect to have distinctive change.

The present invention can by the interaction protein of Beclin1 and gene thereof for the preparation of or the medicine of screening regulation and control Adipocyte Differentiation and autophagy.

Preferably, BAD gene and YWHAQ gene for the preparation of or the medicine of the hungry subcutaneous fat autophagy caused of screening regulation and control.AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3 or YWHAQ gene for the preparation of or the medicine of subcutaneous fat autophagy that causes of screening regulation and control high fat diet.

The interior fat autophagy that BAD, BIRC5 or TICAM2 gene causes for regulating and controlling high fat diet.

TNFAIP3 gene for the preparation of or screening regulation and control Adipocyte Differentiation medicine.

The present invention screened in the interaction protein of Beclin1 with fatty autophagy and break up relevant gene, for the medicine of screening treatment of obesity provides new direction.

Accompanying drawing explanation

Fig. 1 is Beclin1 at the expression figure (Sub: subcutaneous fat of mice subcutaneous fat and interior fat; Vis: interior fat)

Fig. 2 is that the genes such as BAD there are differences at mice subcutaneous fat and interior fat expression

Fig. 3 be under starvation Beclin1 in the expression conditions of mice subcutaneous fat and interior fat

Fig. 4 is under starvation, and BAD, YWHAQ are in the expression of mouse adipose tissue

Fig. 5 is that under starvation, the genes such as AMBRA1 are in the expression of mouse adipose tissue

Fig. 6 is under high fat diet state, and the genes such as AMBRA1 are in the expression (mRNA relative expression quantity) of mouse adipose tissue

Fig. 7 is under high fat diet state, and the genes such as FEZ1 are in the expression (mRNA relative expression quantity) of mouse adipose tissue

Fig. 8 is protein expression change in 3T3-L1 atomization

Fig. 9 is the mrna expression change of PPARr and Beclin1 in 3T3-L1 atomization

Figure 10 is the mRNA relative expression of Beclin1 interaction protein in 3T3-L1 atomization

Figure 11 is in ripe 3T3-L1 adipocyte lipolysis process, the change (B) of the change (A) of the expression of Beclin1 and the mRNA level in-site of BECN, UCP1

Figure 12 is the expression of Beclin1 interaction protein in ripe 3T3-L1 adipocyte lipolysis process

In figure, * p < 0.05, * * p < 0.01, * * * p < 0.001

Detailed description of the invention

Laboratory animal: C57BL/6 mice is purchased from Chinese Academy of Sciences's Experimental Animal Center (Shanghai Slac Experimental Animal Co., Ltd.).Mouse feeder is in the environment of room temperature constant (22 ~ 24 DEG C), and the light and shade cycle is 12h (8am ~ 8pm), can free intake water, food.Larva starvation group mice fasting 48 hours, put to death early morning; Group of being satiated with food mice non-fasting, and larva starvation group mice is put to death simultaneously.The nursing that high fat diet group mice adopts high lipid food to carry out 12 weeks, full diet group mice adopts normal diet to feed

Animal feed: normal diet and high lipid food are all purchased from Shanghai Slac Experimental Animal Co., Ltd..Nutritional labeling is as follows:

Table 2

Composition (100g)	Normal diet (%)	High lipid food (%)
			Carbohydrate	60	48.2
Protein	22	15.8
			Fat	10	29.2
Fiber and other	8	6.8

Embodiment 1

Fatty tissue can be divided into subcutaneous fat, interior fat etc. according to the difference at position, and subcutaneous fat and interior fat all there are differences in structure, function etc.Increasing evidence shows, interior fat more easily decomposes than subcutaneous fat, therefore also becomes the main cause causing free fatty to raise.The anti-lipolytic hormone such as insulin is also more weak in the effect of interior fat, and namely interior fat is more insensitive to insulin, and insulin resistant is more relevant.This may be fatty from two kinds receptor distribute different relevant.In a word, subcutaneous fat and interior fat are not only variant on Germ distribution, in the generation and pathogenesis of disease, also play different effects.

Because LC3-I albumen the generation of autophagy to the transition flag of LC3-II albumen, therefore WesternBlot is selected to detect the height of autophagy level, compare visible to the subcutaneous fat of mice and interior fat basis autophagy situation, although the autophagy level of fatty tissue is lower under base state, but the LC3-II level of single mice viscera fat is all higher than the LC3-II level of its subcutaneous fat, the LC3-II expression of internal organs histone is generally also higher than subcutaneous group of level.The index of LC3-II/LC3-I is adopted to carry out analyzing also leading to the same conclusion.It can thus be appreciated that subcutaneous fat and interior fat there are differences in autophagy process, and under basic condition, the autophagy of interior fat is higher than subcutaneous fat.

Detect the expression conditions of Beclin1 at mice subcutaneous fat and interior fat by REAL-TIMEPCR, result shows that the expression of Beclin1 at interior fat is apparently higher than the expression at subcutaneous fat.For getting rid of the impact of internal reference, in this part, REAL-TIMEPCR date processing adopts 36B4 and L27 two internal references to correct, and result such as Fig. 1, A figure is the result using 36B4 to analyze as internal reference, and B figure is the result using L27 to analyze as internal reference.

The expression conditions of 11 Beclin1 interaction proteins at mice subcutaneous fat and interior fat is detected further by REAL-TIMEPCR, result shows that BAD, FEZ1, GOPC, TNFAIP3, WAC, YWHAQ expression at interior fat is apparently higher than the expression at subcutaneous fat, SLAMF1 is then contrary, and the expression in subcutaneous fat is apparently higher than the expression at interior fat.

The gene expression dose of REAL-TIMEPCR result display AMBRA1, BCL2L1, BIRC5, TICAM2 in subcutaneous fat and interior fat does not present significant difference.Wherein, when adopting L27 to analyze, AMBRA1 shows the trend of interior fat higher than subcutaneous fat, but does not reach significant difference, to illustrate etc. that gene expression histological difference is not obvious.

Interior fat and the difference of subcutaneous fat in autophagy level, also embody in former research report to some extent.The research of Juliaetal is thought, in the interior fat (omental adipose) and subcutaneus adipose tissue of obese people, autophagy is all raise, and under fat condition the autophagy level of interior fat higher than subcutaneous fat.In type 2 diabetes mellitus patient, the autophagy level of interior fat is also high.

Experimental result display of the present invention, there is significant difference in the mark LC3-II/I of autophagy, many interaction proteins of autophagy related gene Beclin1 and Beclin1 gene expression amount in the subcutaneous fat and interior fat of base state, and the expression of most gene in interior fat is higher than the expression in subcutaneous fat, only have the expression of SLAMF1 in subcutaneous fat higher than the expression in interior fat, these results illustrate that autophagy process is not quite similar in different fatty tissuees, and in interior fat, the autophagy of foundation level is slightly high.

In above-mentioned Beclin1 interaction protein, BAD, FEZ1, GOPC, TNFAIP3, WAC, YWHAQ apparently higher than the expression at subcutaneous fat, raise multiple at the expression of interior fat between 5 to 25 times.BAD is the member of BCL2 family, equally with other BCL2 family member regulates and mediated cell programmed cell death, and it also comprises BH3 domain simultaneously, can form dimer suppress it to block apoptotic effect with anti-apoptotic proteins.FEZ1 be nematicide unc-76 gene at mammiferous homologous genes, relevant with the growth promoter of neural axon, participate in the transhipment of nerve.FEZ1 is mainly present in neuronal cell, and it helps neuronal cell to block the infection of HIV virus to have research to think.The difference of GOPC in subcutaneous fat and interior fat comparatively other gene is more obvious, and carrying out correcting with L27 internal reference is that expression in expression ratio subcutaneous fat in interior fat has exceeded 23 times more than.The protein of GOPC coding comprises PDZ domain, and this domain may be exactly the binding site of itself and Beclin1 albumen.The mice generation sterility and infertility of GOPC homologous protein disappearance, prompting GOPC may be relevant with reproductive system.The transhipment of GOPC and intracellular matter and degrade closely related, and autophagy is also correlated with.GOPC can work in coordination with Beclin1 and just regulate autophagy.TNFAIP3 expresses and acutely increases in the environment having TNF to stimulate, the protein of its coding comprises zinc fingers, the activation of NFkB path can be suppressed, stop the apoptosis that causes of TNF, stop and limitation inflammatory reaction process in play pivotal role.The protein of WAC gene code contains a WW structure, and this structure makes its easy protein binding with comprising linear small peptide or proline rich.YWHAQ is the member of 14-3-3 family, and this family is very conservative, all exists in plant and animal, and this homology in Mouse and rat of YWHAQ reaches 99%.This gene is up-regulated in amyotrophic patient.SLAMF1 is contrary with above gene, and the expression in interior fat is starkly lower than the expression at subcutaneous fat, reduces multiple about about 3 times.SLAMF1 has another name called signal lymphocyte activation molecule families member 1, and it is relevant with organism immune response as seen.Dysgammaglobulinemia, subacute sclerosing panencephalitis etc. is comprised with the related disease of SLAMF1.Except participation immunologic process, SLAMF1 also take part in the physiological process such as combination, transmembrane signal transduction of antigen-antibody.Visible, the most and apoptosis of these genes and immune relevant, also may participate in autophagy process more or less, but its rarer report of effect in fatty tissue autophagy.

From result, interior fat basis autophagy level is higher than subcutaneous fat, and Beclin1 and interaction protein thereof also present the differential expression of subcutaneous fat and interior fat.These researchs are that the diversity response of fatty tissue autophagy adds evidence, point out the autophagy of different fatty tissue likely to relate to different genes, have different generations and mechanism and enhancement mechanism.

Embodiment 2 hunger causes autophagy model

Larva starvation group mice starts to carry out fasting in execution for first 48 hours, group of being satiated with food mice non-fasting.WesternBlot is adopted to analyze the expression of LC3 after extracting fatty tissue albumen, result is shown, no matter be at subcutaneous fat or at interior fat, the expression of larva starvation group mice LC3-II and the ratio of LC3-II/LC3-I are significantly higher than group of being satiated with food, and prove the hungry mouse model modeling success causing autophagy.

Beclin1 is in the expression conditions of mice subcutaneous fat and interior fat under starvation to adopt REAL-TIMEPCR technology for detection, and under result is presented at starvation, no matter be at interior fat or at subcutaneous fat, its gene expression reduces.Result such as Fig. 3, A figure is that 36B4 corrects result, and B figure is that L27 corrects result.Note: FED: group of being satiated with food mice; STARVED: larva starvation group mice Sub: subcutaneous fat; Vis: interior fat * p < 0.05**p < 0.01***p < 0.001.

The result display of REAL-TIMEPCR, BAD and YWHAQ gene is down-regulated expression (Fig. 4) under fatty tissue (subcutaneous fat and interior fat) starvation.The expression of AMBRA1, BCL2L1, BIRC5, FEZ1, GOPC, SLAMF1, TICAM2, TNFAIP3, WAC gene under subcutaneous fat and interior fat starvation does not all present the difference (Fig. 5) with statistical significance.

Note: FED: group of being satiated with food mice; STARVED: larva starvation group mice; Sub: subcutaneous fat; Vis: interior fat.

Embodiment 3

High fat diet group mice uses high lipid food to feed 12 weeks, and control group mice uses normal diet to feed.Extracting fatty tissue albumen after putting to death mice adopts WesternBlot to analyze LC3 expression, result shows, at subcutaneous fat and interior fat, the expression of high fat diet group mice LC3-II and the ratio of LC3-II/LC3-I are significantly higher than group of being satiated with food, and demonstrate high fat diet and can activate autophagy.

Beclin1 is in the expression conditions of mice subcutaneous fat and interior fat under HFD state to adopt REAL-TIMEPCR technology for detection, and result is presented at subcutaneous fat Beclin1 under HFD state and expresses and raise, and interior fat has no significant change.

The display of Beclin1 interaction protein AMBRA1 isogenic REAL-TIMEPCR result, AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3, YWHAQ in the expression of high fat diet group higher than matched group.Wherein AMBRA1, BCL2L1, GOPC, TNFAIP3, YWHAQ are in subcutaneous fat change obviously, and not remarkable in the change of interior fat; It is more obvious that BIRC5 changes comparatively subcutaneous fat in the expression of interior fat; BAD, BIRC5, TICAM2 are all obvious in the change of subcutaneous fat and interior fat.Result is as Fig. 6.Beclin1 interaction protein FEZ1, SLAMF1, WAC compare in the expression of high fat diet group the change having no and have statistical significance with matched group.The result of REAL-TIMEPCR is as Fig. 7.Note: CON: control group mice; HFD: high fat diet group mice Sub: subcutaneous fat; Vis: interior fat

Hungry and high fat diet is two the different stimulated factors activating autophagy, is also important paathogenic factor.No matter be long-term hunger or long-term high fat diet, all can bring metabolic imbalance and the disorder of body sugar part, lipid, protein, make body be in stress state, resistance reduces, and energy regulatory machinery is impaired, finally causes disease and the infringement of body.Autophagy procedure activation under the condition of hungry and high fat diet, is resist these pessimal stimulation important channels, likely alleviates the infringement of extraneous pessimal stimulation to body in the process, thus play the effect of protectiveness.But, the autophagy that hungry and high fat diet causes respectively has feature, not identical.Hunger is the most classical activator of autophagy, and hungry model is one of the most frequently used model of research body autophagy.It is reported, some autophagy related gene such as ATG17, ATG29, ATG31 participate in the autophagy that hunger causes specifically, and effect in other autophagy processes is not obvious., show in the research of body, the autophagy that hunger causes is not quite similar in Different Organs meanwhile.High fat diet activates the sight that autophagy also attracts researcher in recent years always.At present, high fat diet is commonly used for structure obese model, under the fat state that high fat diet causes, autophagy level raise, and with inflammation, metabolism, multiple pathological and physiological condition stress be waited relevant.Due to the specificity of fatty tissue, research adipose cell autophagy is also not easy, and is not therefore a lot of to the research of its autophagy, mostly concentrates on the organ such as muscle, liver at present to the research of autophagy.But adipose cell plays a part very important in energy metabolism, occur in the pathological phenomenons such as the lipidosis of fatty tissue, disorders of lipid metabolism, endocrine function imbalance closely related with the disease of endocrine metabolism system.Therefore, various pathophysiological process and the mechanism of studying fatty tissue have great importance.

Under starvation, the expression of subcutaneous fat and interior fat Beclin1 decreases, and reduce multiple about 2 times, and under high fat diet state, the expression of subcutaneous fat Beclin1 obviously raises, and raises multiple and reaches 4-5 doubly.Beclin1 is vital autophagy related gene, and the mechanism of the autophagy that its expression intimate contrary under starvation and under high fat diet state prompting hunger causes and the autophagy that high fat diet causes may be different.

Under high fat diet state, Beclin1 is fairly obvious in the rising of subcutaneus adipose tissue, illustrate Beclin1 take part in high fat diet state under autophagy process.But surprisingly, in the autophagy process that high fat diet causes, although the autophagy of subcutaneous fat and interior fat raises all highly significants, the expression of Beclin1 is visibly different at subcutaneous fat and interior fat.Observe Beclin1 in subcutaneous fat under high fat diet state, express significantly rise, multiple is at 4-5 times, and the expression of Beclin1 under high fat diet state is almost have no change in interior fat.This illustrates that more than this physiological process of basic autophagy shows specificity in different fatty tissue, in autophagy process under the stimulation state produced under the effect of some stimulating factors, also there is the difference of subcutaneous fat and interior fat in the expression of autophagy related genes, there is the specificity of different fatty tissue.

Above-mentioned 11 Beclin1 interaction proteins, in hungry 48 hours and the high fat diet mouse model of 12 weeks, also present different expression.At the former, the expression of BAD and YWHAQ reduces; The latter, the expression of AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3, YWHAQ raises, and presents diversity at subcutaneous fat and interior fat.Most gene (AMBRA1, BCL2L1, GOPC, TNFAIP3, YWHAQ) is more obvious in the change of subcutaneous fat, and BIRC5 is more obvious in the change of interior fat, BAD, TICAM2 are all obvious in the change of subcutaneous fat and interior fat.If compare separately all vicissitudinous gene BAD and YWHAQ in hungry model and hyperlipidemia model, then these two kinds of genes are all reduce in hungry model, and are all raise in hyperlipidemia model, and the change that general trend and Beclin1 express is consistent.

Embodiment 4

Under malnourished state, triglyceride is hydrolyzed into free fatty to be increased, to be supplied to the energy needed for cell.When there is a serious shortage in the supply for energy, cell can provide institute's energy requirement by autophagy self component.Research shows, autophagy can regulate the steatolysis of fat, and the formation suppressing the autophagy of 3T3-L1 PECTORAL LIMB SKELETON can block fat to drip, reduces the accumulation of triglyceride.Meanwhile, autophagy has been considered to the important channel regulating cell differentiation in recent years.Autophagy process plays requisite effect in white adipose atomization.After knocking out important autophagy related gene, white adipocyte poorly differentiated, adipose cell structure changes, and is no longer to drip with less mitochondrion for principal character with huge fat, but presenting the mitochondrion that most less fat drips and increase, the latter is structurally close to the spy of brown fat.Therefore, autophagy plays vital effect in the atomization of adipose cell, and meanwhile, the disappearance of autophagy is relevant with white adipose brownization with suppression.White adipose and brown fat have different physiological propertys, brown fat is main relevant to heat production, significant for the existence of body under severe external environmental condition, and the accumulation of white adipose, especially the generation etc. of the accumulation of interior fat and diabetes, obesity, metabolism syndrome, cardiovascular disease is closely related.Therefore, white adipose brownization tool is of great significance.By the expression change of research Beclin1 and 11 interaction protein in 3T3-L1 atomization and in the mature fat cell steatolysis process of IBMX and ISO stimulation, determine whether differentiation and the steatolysis process that may take part in adipose cell.

3T3-L1 fibroblast uses GPS survey sugar 10%FBS-11965DMEM solution to cultivate, and culture bottle is placed in 37 DEG C, 5%CO2 constant-temperature incubation case.Namely can be observed cell attachment in general 24 hours, in fusiformis, change culture fluid and continue to cultivate, need go down to posterity when cell density is about 90%.First by culture fluid sucking-off when going down to posterity, 0.25% pancreatin adding about 4ml digests, after about 2-3 minute, under microscope, visible cell is shrunk to circle by irregular polygon or fusiformis, add normal culture fluid and stop pancreatin reaction, repeatedly blow and beat cell residual on bottle wall with pipet, make cell detachment culture bottle wall, suck and be equipped with in the centrifuge tube of 10ml culture fluid, the centrifugal 3min of 1000rpm, collecting cell precipitates.Supernatant discarded, then add normal culture fluid, with pipet piping and druming, cell dispersal is opened.Get the celliferous culture fluid of part according to inoculum density to be inoculated in 24 orifice plate/6, orifice plate/12 orifice plate/batch cultur bottles.

3T3-L1 PECTORAL LIMB SKELETON strain differentiation-inducing 48 hours after passage change normal culture fluid, continue to cultivate 48h.After cell fusion, add induced liquid A (volume fraction 10%FBS-DMEM, IBMX0.5mmol/L, dexamethasone 1 μm of ol/L, insulin 1.67 μm of ol/L) and induce, this is designated as the 0th day.After 48 hours, remove induced liquid A, change and induce with induced liquid B (volume fraction 10%FBS-DMEM, IBMX0.5mmol/L, insulin 1.67 μm of ol/L).After 48 hours, change and continue to cultivate with normal culture fluid, now visible a few cell form is rounded, and there have a small amount of fat to ooze to be existing, within every 48 hours afterwards, change a subnormal culture fluid, rounded and occur that a large amount of fat drips about the 8th day visible more than 90% cell, show that 3T3-L1 PECTORAL LIMB SKELETON is successfully induced as ripe adipose cell.Mature fat cell 0.2%BSA-11965DMEM is hatched 12-18 hour, can subsequent experimental be carried out.In the differentiation-inducing process of 3T3-L1 PECTORAL LIMB SKELETON, receive plate 1 time every 48 hours, namely obtain the cell of different divergaence time point.

Become in the process of mature fat cell in the induction of 3T3-L1 fibroblast, cell experienced by the metamorphosis from spindle shape to circle, and final a large amount of fat oozes existing, and cell induction is ripe.In the process, PPARr constantly rises as the marker expression amount of cell differentiation.Our result display, in the whole process of 3T3-L1 differentiation (0 day to 8 days), the expression of LC3-II is progressively risen, and the expression of Beclin1 is also progressively risen, namely, in the process of 3T3-L1 differentiation, autophagy level is (Fig. 8) of raising gradually.This part data still adopt two internal references (36B4 and L27) to carry out correction analysis respectively, straight line is depicted as 36B4 analysis result, dotted line is depicted as L27 analysis result (Fig. 9), transverse axis be 3T3-L1 divergaence time (my god), the longitudinal axis is mRNA relative expression quantity.

Observed the expression of Beclin1 interaction protein in 3T3-L1 Adipocyte Differentiation process by REAL-TIMEPCR, the expression of 11 genes is as Figure 10.The expression of visible BIRC5, FEZ1 and TNFAIP3 has characteristic.BIRC5 is at differentiation the 2nd day up-regulated, and along with the further differentiation of cell, it expresses the level be progressively reduced to again far below 0 day; The expression of FEZ1 starts i.e. violent nearly 10 times of reduction from differentiation, maintains low-level state subsequently in whole atomization always; The expression of TNFAIP3 constantly increases along with the propelling of differentiation process, and to differentiation 8 days adipocyte maturation, it expresses rising 5-9 doubly.Other gene has no obvious characteristic change in 3T3-L1 atomization.As Figure 10, wherein the longitudinal axis is mRNA relative expression quantity, transverse axis be 3T3-L1 divergaence time (my god).

Steatolysis is the critical function of mature fat cell, and we adopt IBMX and ISO process mature fat cell to promote its steatolysis, and record steatolysis process and observe.In steatolysis process, we observe adipose cell fat and drip and diminish gradually, and the expression that the expression of UCP1 acts on 3 hours at IBMX and ISO is the highest, but the expression of 6,9,12,16 hours still higher than control level.But, not there is obvious change in the expression of autophagy process and Beclin1 in the process, as Figure 11 (A) and (B), in Figure 11 (B), the longitudinal axis is mRNA relative expression quantity, and transverse axis is IBMX and ISO processing time (hour).

REAL-TIMEPCR result shows, and the gene expression of Beclin1 interaction protein in ripe 3T3-L1 adipocyte lipolysis process does not present the change of obvious characteristic.As Figure 12, the longitudinal axis is mRNA relative expression quantity, and transverse axis is IBMX and ISO processing time (hour).

Autophagy plays critical effect in the atomization of white adipocyte.Knock out in the mice of autophagy related gene Atg7 fatty tissue is special, fatty tissue autophagy is suppressed.The fatty tissue detecting mice on this basis finds, the content of white adipose tissue significantly reduces, and about the white adipose tissue cells structure of half changes, no longer that single large chamber fat drips in endochylema, but many bubbles fat little in a large number drips, and mitochondrial quantity also showed increased, and the latter is the construction features of brown adipose tissue.Thus, when autophagy lacks, white adipose dysplasia and there occurs brownization, this illustrates that autophagy is that white adipose normal differentiation development is necessary.In 3T3-L1 Adipocyte Differentiation process, the generation of autophagy strengthens gradually, and the expression of Beclin1 also progressively raises, and after 3T3-L1 Adipocyte Differentiation maturation, its rising multiple reaches 3-5 doubly.The expression of Beclin1 interaction protein is then not quite similar, and wherein the distinctive change of most has 3: BIRC5 at differentiation the 2nd day up-regulated, and it expresses the level be progressively reduced to again far below 0 day subsequently, presents the trend of falling after rising generally; The expression self-induction of FEZ1 starts i.e. violent nearly 10 times of reduction, maintains low-level state subsequently in whole atomization always; The expression of TNFAIP3 constantly increases along with the propelling of differentiation process, and to differentiation 8 days adipocyte maturation, it expresses rising 5-9 doubly, presents the trend progressively increased generally.And in steatolysis process, along with the prolongation of IBMX and ISO action time, the fat of mature fat cell drips size and changes, dripped by larger fat and gradually become tiny fat and drip, seem to there occurs " fat drips cracked ".But Beclin1 and interaction protein thereof do not detect extremely have distinctive change.

TNFAIP3 can increase sharply by expression under the induction of TNF, and the protein of its coding has the double effects of ubiquitination enzyme and deubiquitinating enzymes, in cytokine mediated immunoreation and inflammatory reaction, play important effect.This gene is considered to a kind of antioncogene, plays a role in the generation of tumor, and the disease relevant with it is important Lymphatic disease, severe infections, fibrosarcoma, breast carcinoma etc.The expression of TNFAIP3 in Adipocyte Differentiation process raises gradually, point out its maturation that may take part in adipose cell and growth, plays the effect of certain positivity in the atomization of adipose cell.

Claims (5)

1. The interaction protein of 1.Beclin1 and gene thereof for the preparation of or the medicine of screening regulation and control Adipocyte Differentiation and autophagy.
2. 2.BAD gene and YWHAQ gene for the preparation of or the medicine of the hungry subcutaneous fat autophagy caused of screening regulation and control.
3. 3.AMBRA1, BAD, BCL2L1, BIRC5, GOPC, TICAM2, TNFAIP3 or YWHAQ gene for the preparation of or the medicine of subcutaneous fat autophagy that causes of screening regulation and control high fat diet.
4. The interior fat autophagy that 4.BAD, BIRC5 or TICAM2 gene causes for regulating and controlling high fat diet.
5. 5.TNFAIP3 gene for the preparation of or screening regulation and control Adipocyte Differentiation medicine.