CN101884794B - Application of S100A16 gene in preparing medicaments for treating insulin resistance - Google Patents
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to an S100A16 gene and application of relevant accesses in the research and development of medicaments for treating metabolic diseases. The S100A16 gene is a new gene relevant to insulin resistance and provides the basis for the research on molecular mechanisms of the insulin resistance. The invention discloses the important function of the S100A16 in the insulin resistance for the first time through cells and the structures of animal models and provides the theoretical basis and the feasible basis for the research and development of new medicaments for insulin resistance, and the S100A16 gene can be used as a new medicament target.
Description
Technical field
The present invention relates to the application of S100A16 gene in preparation treatment insulin resistant medicine.
Background technology
It is conjugated protein that nervous tissue's Protein S 100 families are that a class is distributed widely in the acid solubility calcium of EF-hand type of different tissues, wherein S is soluble, the dissolubility of 100 these albumen of expression in saturated ammonium sulphate is 100%, S100 albumen contains 2 EF helical structures different with the calcium affinity, and the terminal EF helical structure of C-is formed typical C a by 12 aminoacid
2+Coupling collar, the terminal EF helical structure of N-comprises 14 aminoacid with S100 protein-specific.
The proteic gene of people S100 is in chromosome 1q21 district, tumor is in the normal frequent reorganization of the gene in this district, cause that easily S100 gene expression is out of control, therefore, the S100 unconventionality expression is arranged when advanced tumor and generation neoplasm metastasis, particularly in the tumor tissues of neuroectodermal origin unconventionality expression is arranged, and dependency is arranged with neoplasm staging and prognosis.S100 albumen is distributed with specificity mammal: S100A1 is mainly at neurocyte, Skeletal Muscle Cell, the endochylema of myocardial cell and nephrocyte; S100A2 is in the endochylema and nuclear of lung and nephrocyte; The distribution of S100A3 and S100A5 is still indeterminate; S100A4 is fibroblast, in myoepithelial cell and tumor cell endochylema and the extracellular fluid; S100A12 distributes and hippocampal cell; S100A13 is the abundantest at thyroid cell, and particularly nuclear week is distinguished; S100A16 is widely distributed, sees the kinds of tumors tissue.
Studies show that S100 albumen can participate in proliferation of cells, differentiation, migration apoptosis and cognition by regulating calcium ion and glycosylation receptor RAGE
[1]As whole S100 family the growth of neural axon is played regulating action
[2], and S100A7, S100A 13, and differential expression in tumor such as S100A 14 grades has been used as the marker gene of diagnosing tumor and prognosis
[3,4]
The S100A16 gene is that we use full genome micro-array chip technology, the new gene of differential expression that obtains in the research of examination metabolic disease related gene, be the newcomer of calmodulin, CaM S100 family, except having the basic structural feature of S100 family, as: be the less Ca of a class
2+, Zn
2+Conjugated protein, contain 2 EF helical structures different with the calcium affinity, the terminal EF helical structure of C-is formed typical C a by 12 aminoacid
2+Coupling collar still has following specificity:
1, be different from the proteic EF end of traditional S100 and be made up of 14 aminoacid, the proteic EF end of S100A16 is formed the loop ring by 15 aminoacid;
2, lack the glutamate, Glu residue in EF end rearmost position, this is very important for the site of coordinating calcium; So there is more complicated adjusting mechanism in prompting S100A16 gene.
S100 family is all bringing into play important role at proliferation of cells, differentiation, migration and apoptosis in the growth of neural axon and cognitive formation and the tumor development.About the research of its newcomer S100A16 gene and disease development,, still there is not clearly final conclusion at present because research is still shallow.Our research group was that target is carried out series of studies with it promptly in 2005, tentatively illustrated the effect of its performance in the developing of metabolic disease.
The S100A16 mRNA that studies show that the applicant is previous expresses in normal esophagus, fat and colon excessively, there are some researches show that S100A16 also is expressed in bladder, lung, thyroid, pancreas and the ovarian tumor excessively
[5], point out the multiformity of its biological function.Current research shows
[1], Ca in the cell
2+Concentration can regulate effectively that S100A16 is proteic to go into to examine out nuclear signal, Ca in the cell
2+Concentration increases, and S100A16 albumen mainly is distributed in endochylema, and with Ca in the cell
2+Concentration lowers, and the proteic nuclear translocation of S100A16 strengthens and accumulation in a large number in nuclear, and is closely related with kernel, and prompting S100A16 albumen may work in ribonucleoprotein complex complex process, gene silencing or cell division.The calcium dependent dystopy of S100A16 may be subjected to nearest description
[6,7]The netted adjusting of caryoplasm, nuclear near the termination, responsible free calcium is discharged into the subnucleus position of localized immobilization.The same with other members of S100 family, the S100A16 shortage is significantly gone into the nuclear signal peptide, and it goes into to endorse can be similar with S100A11, need react with transport protein
[6]Perhaps as regulating, calcium copper may take place by optionally promoting propagation path
[7]These results suggest S100A16 gene under the regulation and control of calcium signal path or other still unidentified path regulation and control, is being brought into play important biological function in some specific tissues.But S100A16 is at the nuclear effect and the S100A16-Ca in week
2+Whether the reason and the functional meaning that rely on the adipose cell differentiation and insulin secretion is relevant need further illustrate, do not appear in the newspapers at present both at home and abroad.
(Insulin Resistance IR) is meant the sensitivity decline of the target organ of insulin action to insulin action to insulin resistant, and promptly the insulin of normal dose produces a kind of state that is lower than normal biological effect.In adipose cell, insulin resistance causes the hydrolysis of the triglyceride that stores, and then improves the content of free fatty acid in the blood plasma.In muscle cell, insulin resistance reduces the absorption of glucose; And in hepatocyte, reducing the deposit of glucose, both cause the raising of blood sugar content jointly.Hyperinsulinism and high sugared content often cause metabolism syndrome and type 2 diabetes mellitus in the blood plasma that insulin resistance causes.Insulin resistant is the coefficient result of gene and external environment, and intracellular calcium plays an important role in the relevant insulin resistant of metabolism disorder
[8]Recently, the relation of calcium and insulin resistant becomes a research focus gradually, for the Therapeutic Method of seeking control insulin resistant and relevant disease thereof has been opened up the brand-new visual field of a slice.Studies show that intracellular calcium concentration plays an important role in the energy metabolism relevant with insulin resistant is unbalance
[9]The increase of intracellular calcium concentration cause lipogenesis expression of gene and lipogenesis increase, suppress steatolysis, finally increase fat generation and cause insulin resistant.Low calcium diet can cause 1, and the generation of 25-dihydroxyvitamin D3 increases, and promotes the generation of insulin resistant thereby stimulate calcium ion to flow in cell
[10,11]In this process, stream is an important step in the calcium ion cell of calcium channel mediation.Therefore carrying out further research round calcium channel and regulation and control factor thereof will have great importance.
Present patent application proves that first S100A16 has therapeutical effect to insulin resistant.
List of references
1.Emmanuel?Sturchler，Jos?A.Cox，Isabelle?Durussel，Mirjam?Weibel，Claus?W.Heizmann.S100A16，a?Novel?Calcium-binding?Protein?of?the?EF-hand?Superfamily.J?Biol?Chem.2006；281(50)：38905-38917.
2.Huttunen，H.J.，Kuja-Panula，J.，Sorci，G.，Agneletti，A.L.，Donato，R.，and?Rauvala，H.Coregulation?of?neurite?outgrowth?and?cell?survival?by?amphoterin?and?S100proteins?through?receptor?for?advanced?glycation?end?products(RAGE)activation.J?Biol?Chem.2000；275(51)：40096-400105.
3.Marenholz?I，Heizmann?CW.S100A16，a?ubiquitously?expressed?EF-hand?protein?which?is?up-regulated?in?tumors.Biochem?Biophys?Res?Commun.2004；313：237-244.
4.Denis?A.Smirnov，Daniel?R.Zweitzig，Bradley?W.Foulk，M.Craig?Miller，Gerald?V.Doyle，Kenneth?J.Pienta，Neal?J.Meropol，Louis?M.Weiner，Steven?J.Cohen，Jose?G.Moreno，Mark?C.Connelly，Leon?W.M.M.Terstappen，and?S.Mark?O’Hara.Global?Gene?Expression?Profiling?of?Circulating?Tumor?Cells.Cancer?Res.2005；65(12)：4993-4997.
5.Yao?R，Lopez-Beltran?A，Maclennan?GT，Montironi?R，Eble?JN，Cheng?L.Expression?of?S100?protein?family?members?in?the?pathogenesis?of?bladder?tumors.Anticancer?Res.2007；27(5A)：3051-3058.
6.Sakaguchi，M.，Miyazaki，M.，Takaishi，M.，Sakaguchi，Y.，Makino，E.，Kataoka，N.，Yamada，H.，Namba，M.，and?Huh，N.H.S100C/A11?is?a?key?mediator?of?Ca2+-induced?growth?inhibition?of?human?epidermal?keratinocytes.J?Cell?Biol.2003；163(4)：825-835.
7.Thorogate，R.，and?Torok，K.Ca2+-dependent?and?-independent?mechanisms?of?calmodulin?nuclear?translocation.J.Cell?Sci.2004；117：5923-5936.
8.Draznin，B；Sussman，K.E；Eckel，R.H；Kao，M；Yost，T.and?Sherman，N.A. Possible?role?of?cytosolic?free?calcium?concentrations?in?mediating?insulin?resistance?of?obesit
9.Kim，J.H，Mynatt，R.L，Moore，J.W，Woychik，R.P，Moustaid，N，and?Zemel，M.B.Theeffects?of?calcium?channel?blocade?on?agouti?induced?obesity.FASEB.J.1996，(10)：1646-1652.
10.Zemel?MB，Miller?SL.Dietary?calcium?and?dairy?modulation?of?adiposity?and?obesity?risk.Nutr?Rev.2004，62(4)：125-131.
11.Zemel?MB.Nutritional?and?endocrine?modulation?of?intracellular?calcium：implications?in?obesity，insulin?resistance?and?hypertention.Mol?Cell?Biochem.1998，188(1-2)：129-136.
Summary of the invention
Technical purpose
An object of the present invention is to provide the application in the S100A16 gene preparation treatment insulin resistant medicine.
Technical scheme
The application of S100A16 gene in preparation treatment insulin resistant medicine is confirmed by following technical solution:
One, adopt the preceding adipose cell 3T3-L1 cell model of mice, the glucose C14 that uses isotope tag confirms that by the glucose uptake test that detects insulin stimulating S100A16 crosses the inhibitory action of expression to glucose uptake.
Two, adopt the preceding adipose cell 3T3-L1 cell model of mice, use the influence that Western blot method detects Akt phosphorylation in the S100A16 pair cell, disclose the mechanism that S100A16 influences insulin action.
Three, adopt the fat Mus of diet induced and transgenic to confirm the effect of S100A16 in fat insulin resistant in the animal level.
Generally speaking S100A16 gene expression and insulin resistant have direct correlation, and promptly S100A16 crosses and expresses the aggravation insulin resistant, and low expression of S100A16 weakened insulin resistant, thereby a kind of target spot of selection is provided for exploitation glucagon opposing medicine.
Wherein the nucleotides sequence of S100A16 gene is classified Seq NO.1 as
Preceding adipose cell 3T3-L1 cell model is the model of the detection insulin resistant of classics.
Beneficial effect
1, a large amount of studies show that, obesity especially visceral adiposity are to produce insulin resistant (Insulin Resistance, key factor IR).(Insulin Resistance IR) is meant the sensitivity decline of the target organ of insulin action to insulin action to insulin resistant, and promptly the insulin of normal dose produces a kind of state that is lower than normal biological effect.In adipose cell, insulin resistant causes the hydrolysis of the triglyceride that stores, and then improves the content of free fatty acid in the blood plasma.In muscle cell, insulin resistant reduces the absorption of glucose; And in hepatocyte, reducing the deposit of glucose, both cause the raising of blood sugar content jointly.Hyperinsulinism and high sugared content often cause metabolism syndrome and type 2 diabetes mellitus in the blood plasma that insulin resistant causes.Our experimental result prompting S100A16 crosses expression in fat Mus that high fat diet is fed and ob/ob Adips Mus fat tissue, prompting is relevant with obesity and insulin resistant.
2, insulin resistant is the coefficient result of gene and external environment, and intracellular calcium plays an important role in the relevant insulin resistant of metabolism disorder.Recently, the relation of calcium and insulin resistant becomes a research focus gradually, for the Therapeutic Method of seeking control insulin resistant and relevant disease thereof has been opened up the brand-new visual field of a slice.Studies show that intracellular calcium concentration plays an important role in the energy metabolism relevant with insulin resistant is unbalance.The increase of intracellular calcium concentration cause lipogenesis expression of gene and lipogenesis increase, suppress steatolysis, finally increase fat generation and cause insulin resistant.The albumen that our result of study discovery high calcium can promote S100A16 gene expression is to cytoplasmic output, and low calcium can promote the albumen of S100A16 gene expression to change over to nuclear.Prompting S100A16 may be relevant with insulin resistant by stream in the calcium ion cell of calcium channel mediation.
3, the phosphorylation approach plays an important role in insulin resistant, Insulin receptor INSR has intrinsic protein tyrosine kinase activity, by making himself and IRS-1 (IRS-1) phosphorylation and mediated cell to the reaction of insulin, phosphorylation IRS-1 be equivalent to the second message,second messenger, it be rich in SH
2Protein phosphatidylinositol 3-kinase (PI-3k) combination, make it to activate, promote the synthetic of glucose transporter-4 (GLUT-4) and migrate to cell membrane by pond in the cell, start the picked-up of cell to glucose.Our result of study finds that S100A16 crosses expression for the phosphorylation approach in the adipose cell atomization, can suppress the phosphorylation of 3T3-L1 cell differentiation later stage Akt, thereby suppresses the glucose uptake of 3T3-L1 cell differentiation later stage insulin stimulating.Therefore, S100A16 brings into play biological function by suppressing phosphorylation in insulin resistant, promptly cross expression or S100A16 down-regulated expression by suppressing S100A16, realize the insulin resistant therapeutical effect, therefore we can be according to the S100A16 gene order, the medicine that corresponding exploitation and S100A16 act on mutually, or, have extremely important application value and commercial promise as the target spot of screening treatment insulin resistant medicine.
This result of study has disclosed S100A16 first and insulin resistant has direct correlation; as new drug target; the S100A16 gene comprises by S100A16 gene upstream and downstream target spot preparing patent protection in the insulin resistant related drugs in the application of preparation treatment insulin resistant related drugs.
Description of drawings
Figure 1A extracts albumen after showing 3T3-L1 cell transfecting S100A16 high-expression plasmid, adopts Western blot analysis verification S100A16 high-expression plasmid transfection effect..Lane 1: normal 3T3-L1 cell; Lane 2: empty carrier pcDNA3.1 plasmid; Lane 3:pcDNA3.1-S100A16-3T3-L1; Figure A protein expression is carried out quantitative analysis obtain the gray analysis figure B (data from three groups of independent trialss) that S100A16 expresses.The result represents with mean+standard deviation.Compare with contrast 3T3-L1 * P＜0.01 or * P＜0.05;
Fig. 2 shows that S100A16 is to the glucose uptake of 3T3-L1 cell differentiation later stage insulin stimulating and to phosphorylation in the 3T3-L1 cell differentiation procedure;
After wherein Fig. 2 A shows that 3T3-L1, pcDNA3.1 empty carrier and pcDNA3.1-S100A16-3T3-L1 cell (S100A16 overexpressing cell) are induced differentiation, detect the glucose uptake situation of differentiation different number of days insulin stimulating, with the detected value of normal 3T3-L1 cell as radix, other detected values are relatively represented with relative ratio with it, results suggest S100A16 crosses the picked-up that expression can obviously suppress 3T3-L1 cell differentiation later stage glucose, the result represents with mean ± standard deviation, n=3.*P＜0.05, compare with normal 3T3-L1 * * P＜0.01;
Fig. 2 B shows that S100A16 crosses expression plasmid transfection 3T3-L1 cell, induce differentiation to adopt insulin of different concentration (0,5,10,20,50nM) to stimulate after ten minutes and collect albumen in back 10 days, detect p-Akt with Western blot method and express, results suggest S100A16 crosses the phosphorylation that expression can suppress 3T3-L1 cell differentiation later stage Akt;
Fig. 3 shows Ca in the 3T3-L1 cell
2+The nuclear output and the consideration convey of-dependence are gone into, and the 3T3-L1 cell adopts 1.5mM Ca respectively
2+And 1 μ M ionomycin stimulates 0,5,10,15 minutes, perhaps using calcium couplant 3mM EGTA stimulated 0,10,20,30 minutes, detect S100A16 expression in total protein of cell and the nucleoprotein, the results suggest high calcium can promote S100A16 albumen to cytoplasmic output, and low calcium can promote S100A16 albumen to change over to nuclear.
Fig. 4 shows that S100A16 is in the obesity of normal and diet induced and the expression in the ob/ob Adips Mus fat tissue, from the wild Mus of C57BL/6J of 16 week sizes and 12 week size the high fat diets fat Mus and the ob/ob Adips Mus fat tissue extraction albumen of feeding, adopt the expression of Western blot methods analyst S100A16 at above-mentioned three kinds of tissues, results suggest S100A16 crosses expression in fat Mus that high fat diet is fed and ob/ob Adips Mus fat tissue.α-tubulin in contrast.
The specific embodiment
(the 15th chapter is at the expression in escherichia coli cloned gene for the 3rd of reference experiment guide in the present embodiment; Import cloned gene in the 16th chapter mammal cultured cell; The buffer that uses in appendix 1 molecular cloning and the preparation of reagent; Common technology in appendix 8 molecular clonings), modern practical cell and molecular biology experiment technology (chapter 1 cell culture processes; Chapter 8, cellular morphology and cytochemistry quantitative analysis tech).
Before adipose cell 3T3-L1 available from Shanghai cell biological institute, 14C labelled glucose [3H] 2-deoxy-D-glucose is available from Kemei Dongya Biological Technology Co., Ltd., Beijing,
One, adipose cell 3T3-L1 confirms the effect of S100A16 in fat insulin resistant before cellular level adopts mice.
1. make up S100A16 and cross expression plasmid transfection 3T3-L1 cell, carry out three stimulations and induce differentiation, glucose uptake test and phosphorylation by insulin stimulating detect the phosphorylation of observation S100A16 to Akt in the 3T3-L1 cell differentiation procedure, disclose the effect of S100A16 in insulin resistant.
2. the 3T3-L1 cell model that adopts high calcium and low calcium to stimulate is inquired into the calcium concentration variation consideration convey of S100A16 gene is gone into influence.
Concrete grammar such as follows
1, the foundation of Jing Dian 3T3-L1 cell induction differentiation model:
The 3T3-L1 cell culture is (10% hyclone) in the DMEM culture fluid, 5%CO
2, 37 ℃ of incubators.When cell grow to fully merge back 48h after, adding 0.5mM3-isobutyl-1-methyxan-thine (MIX), (Sigma USA) induces differentiation for 1ug/ml insulin and 1uM dexamethasone.Use the complete medium that only contains the 1ug/ml insulin behind the 48h instead, changed liquid up to the 8th day complete differentiation and maturation of cell in later per two days.
2, S100A16 crosses the structure and the cell transfecting of expression plasmid
According to " molecular biosciences cloning experimentation guide ", import cloned gene scheme 1 fat in the 16th chapter mammal cultured cell and dye the DNA transfection method of mediation,
With mouse liver cDNA is masterplate; With primer S100a16-F, its sequence is Seq NO.2; S100A16-R, its sequence is Seq NO.3; Amplification S100A16 open reading frame; The PCR condition: the 100ul system, 56 ° of annealing, 72 ° are extended 30s, and the purpose fragment is obtained in 33 circulations; Adopt PCR purification kit (TakaRa DNA Fragment Purification Kit) purification purpose fragment; Adopt NheI and EcoRI to carry out enzyme action, the enzyme action afterproduct is cut glue purification; Purified product is connected on the carrier pcDNA3.1; Get 5UL and connect product transformed competence colibacillus cell, after the conversion bacterium liquid is applied to the Amp plate, put 37 degree incubators and cultivated 16 hours, in super-clean bench, choose monoclonal and add 4ml LB+40ul Amp and put shaking table 16 hours, get 1ul bacterium liquid and make PCR when masterplate; The bacterium liquid 3ml of positive colony takes out plasmid for a short time and send order-checking.After order-checking comparison is entirely true, in take out plasmid transfection 3T3-L1 cell selection clonal cell line and cultivate stand-by.By western blot method validation transfection success (accompanying drawing 1).The 3T3-L1 cell (pcDNA3.1-S100A16-3T3-L1) of transfection S100A16 is cultivated according to above-mentioned 1 method and is induced differentiation.
3, adopt the influence of the glucose absorption laboratory observation S100A16 pair cell glucose uptake of insulin stimulating:
Promptly use the method for [3H] 2-deoxy-D-glucose to detect the ability that difference differential period adipose cell is handled glucoses.
3T3-L1 cell (normally reach S100A16 and cross 3T3-L1 cell after the expression plasmid transfection) and pcDNA3.1 empty carrier (positive control), method according to step 1 is induced differentiation, inducing differentiation back different number of days (0,2,4,6,8,10days), with KRP liquid (NaCl 128mM, KCl 4.7mM, CaCl2 1.65mM, MgSO42.5mM, Na2HPO4 5mM, pH 7.4) washed twice, incubated altogether 10 minutes with 100nM procine insulin insulin, adding then and contain 0.2%BSA, 1 μ Ci/ml[3H] KRP of 2-deoxy-D-glucose and 5mM glucose incubated 37 ℃ again 10 minutes; Sample with containing 0.5M NaOH and 0.1%SDS dissolving, is determined the radioactivity absorbing state of cell with cold KRP liquid washing three times with scintillation counter then.
Results suggest S100A16 crosses the picked-up that expression can obviously suppress 3T3-L1 cell differentiation later stage glucose; After (2A) 3T3-L1, pcDNA3.1 empty carrier and the differentiation of pcDNA3.1-S100A16-3T3-L1 cell induction in the accompanying drawing, detect the glucose uptake situation of differentiation different number of days insulin stimulating, as radix, other detected values are relatively represented with relative ratio with it with the detected value of normal 3T3-L1 cell.The result represents with mean ± standard deviation.N=3.*P＜0.05, compare with normal 3T3-L1 * * P＜0.01.
4, adopt Western blot method to detect the influence of Akt phosphorylation in the S100A16 pair cell:
3T3-L1, pcDNA3.1 empty carrier and pcDNA3.1-S100A16 overexpressing cell are planted 6 orifice plates respectively, three stimulate and to induce differentiation, and the common culture fluid (containing 0.1% BSA) that is changed to serum-free and does not have an insulin to the tenth day culture fluid is cultivated after 2 hours with insulin of different concentration (0,5,10,20,50nM) stimulated ten minutes, collect albumen, detect p-Akt with Western blot method, total-Akt in contrast.The IP3K/Akt signal path is the main signal path of insulin action, and Akt (serine/threonine kinase) accepts IP3K (phosphinositides 3 kinases) and regulates, after IP3K activates Akt, and can be by following three kinds of approach performance biological action.(1) the dystopy effect of glucose transporter Glut4 in the pair cell makes the effect of its performance transhipment glucose; (2) promote glycogen synthetic by glycogen synthase kinase 3 (GSK 3); (3) at adipose cell, insulin suppresses steatolysis by the signal pathway that IP3K and Akt rely on.And the Akt phosphorylation is the key step that starts above-mentioned signal path, and the Akt phosphorylation activity descends and then suppresses the IP3K/Akt signal path, thus the absorption of affecting glucose.Studies show that, at the insulin resistant state, the effect of the IP3K/Akt signal transduction path that insulin stimulating causes descends, and we cross expression to activating the influence of IP3K/Akt signal path by detecting S100A16, inquire into the effect of S100A16 in insulin resistant.
Results suggest S100A16 crosses the phosphorylation that expression can suppress 3T3-L1 cell differentiation later stage Akt.S100A16 crosses expression plasmid transfection 3T3-L1 cell shown in accompanying drawing 2B, and (0,5,10,20,50nM) stimulation was collected albumen after ten minutes, detected p-Akt with Western blot method and expressed to induce differentiation to adopt insulin of different concentration in back 10 days.
5, adopt Western blot method to detect the influence of intracellular Ca2+ level variation to S100A16
The 3T3-L1 cell adopts 1.5mM Ca respectively
2+And Calcium ionophore 1 μ M ionomycin stimulates the rising that promoted the concentration of cellular calcium in 0,5,10,15 minutes; Perhaps use calcium couplant 3mM EGTA and stimulate 3T3- L1 cell 0,10,20,30 minutes, detect S100A16 expression in total protein of cell and the nucleoprotein, as shown in Figure 3, the results suggest high calcium can promote S100A16 albumen to cytoplasmic output, and low calcium can promote S100A16 albumen to change over to nuclear.
Two, adopt the fat Mus of diet induced and transgenic to confirm the effect of S100A16 in fat insulin resistant in the animal level.
The foundation of the fat mouse model of diet induced: 30 of male SD rats (available from Jiangsu Province's Experimental Animal Center), sub-cage rearing, 1/cage, body weight 90 120 grams.Raise with normal feedstuff, freely take the photograph water feed thing, 21 ℃-23 ℃ of ambient temperatures, rat conformed after a week, weighed in, and was divided into two groups, and one group is continued to feed normal feedstuff, other one group of (diet-induced obesity; DIO) change hello high lipid food (the laboratory animal feedstuff is purchased the collaborative animal feed company in Nanjing).Feed formula: normal feedstuff nutritional labeling proportioning, carbohydrate 65%, fat 20%, protein 15%; The high lipid food nutrition-allocated proportion, carbohydrate 20%, fat 60%, protein 20%), it is long to write down body weight, body length and tail every day, and the body weight of the high fat group in two week backs obviously increases, and continues to feed for six weeks.Fasting is 12 hours before putting to death, and the anesthesia of lumbar injection 1% pentobarbital sodium is weighed, and opens heart extracting blood 5-10 milliliter, separation of serum, and-70 ℃ are frozen standby, claim interior fat weight simultaneously, and it is frozen standby to get-70 ℃ in tissues such as lactone, sebum, skeletal muscle, brain.The ob/ob mouse model: available from Nanjing University model animal center, the conventional raising got fatty tissue and extracted Protein Detection S100A16 expression of gene after the execution.
Separate the mice of high lipid food nursing and the fatty tissue of normal control Mus, detect the expression of S100A16 in above-mentioned tissue, and the spontaneous fat model mouse ob/ob of purchase leptin receptor defects, detect the expression of S100A16 gene in ob/ob Adips Mus fat tissue.
Results suggest S100A16 crosses in fat Mus that high fat diet is fed and ob/ob Adips Mus fat tissue and expresses, and proves that S100A16 is relevant with insulin resistant with obesity.As fat Mus and the ob/ob Adips Mus fat tissue extraction albumen that accompanying drawing 4 is fed from the wild Mus of C57BL/6J and the big or small high fat diets of 12 weeks of 16 all sizes, employing Western blot methods analyst S100A16 is in the expression of above-mentioned three kinds of tissues.α-tubulin in contrast.The above-mentioned specific embodiment does not limit technical scheme of the present invention in any form, and the technical scheme that mode obtained that every employing is equal to replacement or equivalent transformation all drops on protection scope of the present invention.
Claims (1)
1.S100A16 the application of gene in preparation treatment insulin resistant medicine.
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| WO2006042005A2 (en) * | 2004-10-07 | 2006-04-20 | Immunivest Corporation | Global gene expression profiling of circulating tumor cells |
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Non-Patent Citations (3)
| Title |
|---|
| Marenholz等.S100A16 a ubiquitously expressed EF-hand protein which is up-regulated in tumors.《Biochemical and Biophysical Research Communications》.2004 |
| Marenholz等.S100A16, a ubiquitously expressed EF-hand protein which is up-regulated in tumors.《Biochemical and Biophysical Research Communications》.2004,第313卷237-244. * |
| 陈建魁等.神经生化标志物S100蛋白的分子生物学基础及临床应用.《军事医学科学院院刊》.2003,第27卷(第01期),70-72. * |
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