CN101890172B - Application of S100A16 gene in preparing drug for treating obesity - Google Patents
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Abstract
The invention relates to an S100A16 gene and an application of relevant access in developing a drug for treating metabolic disease. The invention provides a new gene relevant to obesity, provides a basis for research of molecular mechanism of obesity, reveals the important function of the S100A16 in obesity for the first time through construction of cell and animal model, provides a theoretical basis and feasibility in developing new drugs for treating obesity through research of S100A16 interacted protein and upstream-downstream gene on a signal access, and can be used as a new drug target.
Description
Technical field
The present invention relates to the application of S100A16 gene in preparation treatment of obesity medicine.
Background technology
Nervous tissue's Protein S 100 families are that one type of acid solubility calcium of EF-hand type that is distributed widely in different tissues is conjugated protein; 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; Be prone to cause that S100 gene expression is out of control, therefore, the S100 unconventionality expression 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 distributes extensively, is shown in the kinds of tumors tissue.
Research shows that S100 albumen can be through regulating calcium ion and glycosylation receptor RAGE participation proliferation of cells, differentiation, migration apoptosis and cognition
[1]Like 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 one type of less Ca
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 a2+ coupling collar by 12 aminoacid, 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, had tentatively illustrated its effect of in the incidence and development of metabolic disease, bringing into play.
The previous research of applicant shows S100A16 mRNA high expressed in normal esophagus, fat and colon, there are some researches show S100A16 also high expressed in bladder, lung, thyroid, pancreas and ovarian tumor
[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 possibly work in ribonucleoprotein complex complex process, gene silencing or cell division.The calcium dependent dystopy of S100A16 possibly receive 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 lack of significant is gone into the nuclear signal peptide, and it goes into to endorse can be similar with S100A11, need react with transport protein
[6]Perhaps, calcium copper possibly take place as regulating through 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.
Correlational study has shown that the lipogenesis process receives the transcription factor of a series of complicacies to regulate the adjusting like C/EBP, PPAR and ADD/SREBP-1c etc.
[8,9]The adipose cell process of differentiation receives the influence of signal conduction in a series of extraneous factors and the cell
[10]Present part Study shows intracellular calcium concentration, and differentiation plays an important role for adipose cell; This process more complicated; The rising of intracellular calcium concentration; Can suppress the synthetic of adipose cell in early days at the 3T3-L1 cell differentiation, but but break up the expression that the later stage increases the marker gene of adipose cell differentiation and maturation
[11]Whether S100A16 can pass through Ca
2+The differentiation of adjusting, PPAR γ and C/EBP alpha signal path affecting lipocyte, S100A16 is at the nuclear effect and the S100A16-Ca in week
2+Whether the reason and the functional meaning that rely on break up and then relevant need further the illustrating of causeing fat with adipose cell, do not appear in the newspapers both at home and abroad at present.
Present patent application proves the facilitation of S100A16 to lipogenesis first, and then promotes the synthetic of fat, thereby proof S100A16 treatment of obesity has using value.
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?S100?proteins?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?ofCirculating?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?ofCa2+-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.Fajas，L.，Fruchart，J.C.，and?Auwerx，J.Transcriptional?control?of?adipogenesis.Current?opinion?in?cell?biology.1998；10(2)：165-173
9.Rosen，E.D.，and?Spiegelman，B.M.Molecular?regulation?of?adipogenesis.Annual?review?of?cell?and?developmental?biology.2000；16：145-171.
10.MacDougald?OA，Mandrup?S.Adipogenesis：forces?that?tip?the?scales.Trends?Endocrinol?Metab.2002，13(1)：5-11.
11.Shi?H，Halvorsen?YD，Ellis?PN，Wilkison?WO，Zemel?MB.Role?of?intracellular?calcium?in?human?adipocyte?differentiation.Physiol?Genomics.2000，3(2)：75-82.
Summary of the invention
Technical purpose
An object of the present invention is to provide the S100A16 gene and prepare the application in the treatment of obesity medicine.
Technical scheme
The application of S100A16 gene in preparation treatment of obesity medicine is confirmed through following technical scheme:
One, adipose cell 3T3-L1 confirms the effect of S100A16 in obesity before cellular level adopts mice.
Two, adopt the fat Mus of diet induced and transgenic to confirm the effect of S100A16 in obesity in the animal level.
Wherein the nucleotides sequence of S100A16 gene is classified Seq NO.1 as
Beneficial effect
1, correlational study has shown that the lipogenesis process is regulated by the transcription factor of a series of complicacies, like the adjusting of C/EBP, PPAR-gama and ADD/SREBP-1c etc.Our result of study prompting S100A16 possibly be that the lipogenesis of a novelty promotes the factor, regulates the effect that performance promotes lipogenesis through above-mentioned transcription factor.Our nearest extensive yeast two-hybrid result shows that it can interact with S100A14 and heat shock protein.Known S100B, S100A2 and S100A4 can with a series of intracellular protein effect, especially P53, a tumor suppressor protein equally also is a lipogenesis negativity regulatory factor.S100A4, S100A2 block the generation of PPAR-gama body of gland and then the generation of blocking-up fat through P53 blocking-up cell cycle in the adipose cell atomization.And the S100B effect is just in time opposite, strengthens the effect of P53.S100A16 is the same with S100A4 to form the common transcriptional units of regulating by 3 exons, and S100A16 is potential tissue-specificly to be expressed in different transcriptions and to be similar to S100A4.
Therefore, thereby we have confirmed that through the co-immunoprecipitation method S100A16 and S100A4 have identical effect and suppress the inhibitory action of P53 minimizing P53 to lipogenesis, and then promote the synthetic of fat, thereby proof S100A16 treatment of obesity has using value.
This result of study has disclosed the effect of S100A16 in obesity reaches first; As new drug target; The S100A16 gene comprises through S100A16 gene upstream and downstream target spot preparing patent protection in the fat related drugs in the application of the fat insulin resistant related drugs of preparation treatment.
Description of drawings
Fig. 1 shows expression and the outgrowth influence of pair cell thereof in the S100A16 albumen pro-adipose cell atomization;
Figure 1A shows 3T3-L1 cell induction differentiation back 0,2,4; Collected albumen in 6,8,10 days; Adopt Western blot method to measure the expression of S100A16 albumen in cell differentiation procedure, the result finds S100A16 along with atomization is expressed rising, and α-tubulin is as contrast;
Figure 1B shows the effect of Western blot analysis verification S100A16 high expressed and interference plasmid transfection; Lane 1: normal 3T3-L1 cell; Lane 2: empty carrier pcDNA3.1 plasmid; Lane 3:pcDNA3.1-S100A16-3T3-L1; Lane 4:shRNA negative control; Lane 5:3T3-L1-S100A16-shRNA-1; Lane 6:3T3-L1-S100A16-shRNA1-2;
Figure 1A and Figure 1B are data from three groups of independent trialss;
Fig. 1 C carries out quantitative analysis to Figure 1A protein expression to obtain the gray analysis figure that S100A16 expresses;
Fig. 1 D carries out quantitative analysis to Figure 1B protein expression to obtain the gray analysis figure that S100A16 expresses;
Fig. 1 E showed the outgrowth influence of expression S100A16 pair cell;
Fig. 1 F shows the outgrowth influence of interference S100A16 pair cell;
The result of Fig. 1 E and Fig. 1 F representes with the form of mean ± standard deviation; Compare with contrast 3T3-L1 * P＜0.01 or * P＜0.05;
Fig. 2 shows that S100A16 expresses the influence to adipose cell differentiation function before the 3T3-L1;
Wherein Fig. 2 A induces differentiation after showing 3T3-L1 cell transfecting S100A16 high expressed and interference plasmid; Adopt oil red dyeing to observe the formation situation that fat drips; The result finds that the S100A16 high expressed can promote the formation that fat drips in the 3T3-L1 cell differentiation procedure, and S100A16 disturbs can suppress the formation that fat drips; (amplification coefficient 200 * (LycraUSA));
Fig. 2 B shows the influence that S100A16 forms triglyceride (TG), and the result shows that the S100A16 high expressed can obviously increase the influence that triglyceride (TG) is formed; Adopt spectrophotometer to measure absorption value at wavelength 510nm place and calculate TG content, the result representes (n=3) with the form of mean ± standard deviation, and * * P＜0.01 and corresponding matched group are relatively;
Fig. 2 C shows the influence of S100A16 to correlating markings gene in the 3T3-L1 cell differentiation procedure; Extract 3T3-L1 cell differentiation different number of days albumen and adopt western blot method to detect the marker gene expression, results suggest S100A16 high expressed can raise the expression of leptin, adiponectin, aP2; The expression that S100A16 can reduce said gene after disturbing;
Fig. 2 D shows the influence of S100A16 to correlating markings gene in the 3T3-L1 cell differentiation procedure; The albumen that extracts 3T3-L1 cell differentiation different number of days adopts western blot method to detect the marker gene expression; Results suggest S100A16 disturbs can raise ppar-gama, the expression of C/EBP-a; The expression that S100A16 can reduce said gene after disturbing;
Fig. 3 shows that S100A16 is to the proteic inhibitory action of P53;
Collecting cell extracted albumen after Fig. 3 A demonstration 3T3-L1, S100A16 crossed expression and S100A16 interference cell bed board 48h, adopted the co-immunoprecipitation method one anti-P53 antibody of using, and whether detection S100A16 is through combining to bring into play its biological function with P53; The result shows that S100A16 crosses the proteic effect of P53 that obviously suppresses of expressing, and the S100A16 interference can promote the effect of P53 albumen;
Fig. 3 B shows 3T3-L1; Collecting cell extracted albumen after S100A16 crossed expression and S100A16 interference cell bed board 48h; Western blot method detects the S100A16 expression, and S100A16 overexpressing cell strain S100A16 albumen high expressed is compared in result's demonstration with parental cell 3T3-L1; S100A16 down-regulated expression in the strain of S100A16 interference cell has successfully made up S100A16 and has crossed expression and the strain of S100A16 interference cell in the illustrative experiment;
Fig. 3 C shows that 3T3-L1, S100A16 cross expression and the S100A16 interference cell is spread 12 orifice plates, transfection p53-luciferase plasmid behind the 18h, fluorescence intensity behind the 72h; Result's demonstration is compared with parental cell 3T3-L1, and fluorescence volume reduces in the strain of S100A16 overexpressing cell, and fluorescent value raises in the strain of S100A16 interference cell; Explanation is in the strain of S100A16 overexpressing cell, and S100A16 combines with p53 to reduce its activity, and in the strain of S100A16 interference cell; Discharge p53, cause its active rise;
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 is high expressed in fat Mus that high fat diet is fed and ob/ob Adips Mus fat tissue.α-tubulin is as 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).
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 and RNA interference slow virus plasmid transfection 3T3-L1 cell, carry out three stimulations and induce differentiation, measure observation S100A16 drips formation to fat in the 3T3-L1 cell differentiation procedure influence through oil red dyeing and TG.
2. adopt Real time PCR and Western blot to detect Leptin in the above-mentioned cell differentiation procedure, Adiponectin, LPL, the influence of S100A16 to marker gene in the 3T3-L1 cell differentiation procedure inquired in the variation of marker gene such as PPAR-gamma.
Concrete grammar is following
1, adopt the influence of classical 3T3-L1 cell induction differentiation model research S100A16 to 3T3-L1 cell differentiation function:
The 3T3-L1 cell (normally reach S100A16 cross express and after RNA disturbs transfection) cultivate in the DMEM culture fluid (10% hyclone) 5%CO
2, 37 ℃ of incubators.When cell grow to merge fully after two days (day0) add 0.5mM3-isobutyl-1-methyxan-thine (MIX), (Sigma USA) induces differentiation for 1ug/ml insulin and 1uM dexamethasone.(day2) uses the complete medium that only contains the 1ug/ml insulin instead behind the 48h, changes liquid up to the 8th day complete differentiation and maturation of cell in later per two days.The cell RNA and the albumen that extract the differentiation different number of days are used for the index of correlation detection.
The result finds that S100A16 express to raise along with atomization, be undifferentiated 2 times in the expression of four days S100A16 of cell differentiation to the, and having arrived the 10th day differentiation later stage is 5 times, explains that S100A16 obviously promotes the differentiation of the preceding adipose cell of 3T3-L1.Wherein (1A) shown in Fig. 1 3T3-L1 cell induction breaks up back 0,2,4; 6; Collect albumen in 8,10 days, adopt Western blot method to measure the expression of S100A16 albumen in cell differentiation procedure; The result finds S100A16 along with atomization is expressed rising, and prompting S100A16 possibly be fat relevant new marker gene.α-tubulin is as contrast.(1B) Western blot analysis verification S100A16 high expressed and interference plasmid transfection effect..Lane 1: normal 3T3-L1 cell; Lane 2: empty carrier pcDNA3.1 plasmid; Lane 3:pcDNA3.1-S100A16-3T3-L1; Lane 4:shRNA negative control; Lane5 and lane 6,3T3-L1-S100A16-shRNA-1 and 3T3-L1-S100A16-shRNA1-2; (1C & 1D) carries out quantitative analysis to (A & B) protein expression and obtains the gray analysis figure (data from three groups of independent trialss) that S100A16 expresses.
2, adopt oil red dyeing and TG Determination on content to observe the influence of S100A16 to 3T3-L1 cell differentiation function:
Cell between different differentiation phases is carried out oil red dyeing; Be cell through 10% buffered formalin (pH 7.4) fixedly behind the 30min; With DPBS washing three times; Add 60% isopropyl alcohol and oil red O dyestuff application liquid is hatched 5min, inhale and abandon liquid and rinse well with tap water, inverted microscope is observed fat down and is dripped the formation situation; Adopt TG to measure the content of test kit through spectrophotometric determination OD value (510nM) quantitative analysis TG.
The result shows that the S100A16 high expressed can promote the formation that fat drips in the 3T3-L1 cell differentiation procedure; The S100A16 high expressed can obviously increase the influence that triglyceride (TG) forms; Lipogenesis receives the influence of a series of transcription regulaton factors, comprises C/EBP α, C/EBP β; C/EBP δ; PPAR γ etc., S100A16 cross the obvious formation prompting S100A16 that promotes fat to drip of expression can promote lipogenesis, but how to promote the needs of lipogenesis to do further research.Induce differentiation behind (2A) 3T3-L1 cell transfecting S100A16 high expressed and the interference plasmid in the accompanying drawing 2, adopt oil red dyeing to observe fat and drip the formation situation.The result finds that the S100A16 high expressed can promote the formation that fat drips in the 3T3-L1 cell differentiation procedure, and S100A16 disturbs can suppress the formation that fat drips.(amplification coefficient 200 * (Lycra USA)).(2B) S100A16 is to the influence of triglyceride (TG) formation, and the result finds that the S100A16 high expressed can obviously increase the influence that triglyceride (TG) forms.Adopt spectrophotometer to measure absorption value at wavelength 510nm place and calculate TG content.The result representes (n=3) with mean ± standard deviation, and compare with corresponding matched group * * P＜0.01.
3, adopt cell proliferation experiment to observe the influence of S100A16 to the 3T3-L1 cell proliferation:
Adopt WST-1 assay kit (Roche) to carry out cell proliferation experiment.Cell is layered on (cell density is 1 * 10 in 96 orifice plates
3/ hole) at 5%CO
237 ℃ of incubators hatch 12,24,36,48 respectively, after 60,72 hours, add 10 μ, 1 WST-1 mixture and mix gently, place incubator again and hatch 4 hours, measure absorption value in the 450nm wavelength.
The S100A16 back that express to raise obviously promotes the 3T3-L1 proliferation of cells, cultivates that the cell enlargement number is 1.4 times of beginning after 24 hours, cultivates that the cell enlargement number has been nearly 1.7 times that begin after 72 hours.On the contrary, S100A16 express to disturb the back obviously to suppress the 3T3-L1 proliferation of cells, cultivates that cell enlargement has begun to be suppressed after 24 hours, cultivates that the cell enlargement number has suppressed nearly 80% after 72 hours.Like the outgrowth influence of (1E &1F) S100A16 pair cell in the accompanying drawing 1.The result representes with mean ± standard deviation.Compare with contrast 3T3-L1 * P＜0.01 or * P＜0.05.
4, adopt Real-time PCR and Western Blot to detect index of correlation to estimate the influence of S100A16 to adipose cell differentiation, insulin resistant function.：
Extract the RNA of cell and tissue according to description, ultraviolet spectrophotometer carries out after RNA quantitatively reaches purity analysis, and reverse transcription cDNA is a template; With relevant obesity and insulin resistant marker gene is primer; Amplification PCR product detects PPAR γ and C/EBP α, Adiponectin, LPL, FAS; AP2, the variation of glycolipid metabolism genes such as Pref-1, Resistin, Leptin.Extract the albumen of cell and tissue equally according to description, measure concentration, with the proteic expression of associated antibodies testing goal.
(2C & 2D) S100A16 is to the influence of correlating markings gene in the 3T3-L1 cell differentiation procedure in result such as the accompanying drawing 2.Extract 3T3-L1 cell differentiation different number of days albumen and adopt western blot method to detect the marker gene expression, results suggest S100A16 high expressed can raise adiponectin, leptin, aP2, and ppar-gama, the expression of C/EBP-a.
5, adopt the co-immunoprecipitation method to observe S100A16 albumen and P53 protein-interacting:
3T3-L1, zero load and S100A16 overexpressing cell are planted the 10cm plate respectively, collecting cell behind the 48h; Add 200ul cell lysis buffer solution (containing protease inhibitor), cracking 30min on ice, 12000rpm; 4 ℃ centrifugal; Get supernatant, add S100A16 antibody (1: 500), 4 ℃ are slowly rocked incubated overnight.Get 10 μ l protein A sepharose 4B pretreatment, pretreated protein A sepharose 4B is joined in the cell pyrolysis liquid that above-mentioned and antibody incubation spend the night, 4 ℃ are slowly rocked and hatch 2-4h, and antibody and protein A sepharose 4B are connected by chance.After the immunoprecipitation, 4 ℃ with 3, the centrifugal 3min of 000rpm speed, sepharose 4B is centrifugal to managing the end; The careful suction of supernatant gone, and sepharose 4B is washed 3-4 time with the 1ml lysis buffer; 2 * SDS the sample-loading buffer that adds 15 μ l at last, boiling water boiled 5 minutes, detected with western blot, and one is anti-with P53 antibody (1: 500), whether detected S100A16 through combine its biological function of performance with P53.Available P53 antibody carries out co-immunoprecipitation simultaneously, carries out Western blot with S100A16 antibody and detects, and further proves both interactions conversely.
The result shows
As in the accompanying drawing 3 A.3T3-L1, S100A16 cross express and S100A16 interference cell bed board 48h after collecting cell extract albumen, adopt the co-immunoprecipitation method one anti-P53 antibody of use, whether detect S100A16 through combine to bring into play its biological function with P53.The result shows that S100A16 crosses the proteic effect of P53 that obviously suppresses of expressing, and the S100A16 interference can promote the effect of P53 albumen.
As in the accompanying drawing 3 B.3T3-L1, S100A16 cross express and S100A16 interference cell bed board 48h after collecting cell extract albumen, western blot method detects the S100A16 expression.Result's demonstration is compared with parental cell 3T3-L1, S100A16 overexpressing cell strain S100A16 albumen high expressed, and S100A16 down-regulated expression in the strain of S100A16 interference cell has successfully made up S100A16 and has crossed expression and the strain of S100A16 interference cell in the illustrative experiment.
As in the accompanying drawing 3 C.3T3-L1, S100A16 crosses and expresses and the S100A16 interference cell is spread 12 orifice plates, transfection p53-luciferase plasmid behind the 18h, fluorescence intensity behind the 72h.Result's demonstration is compared with parental cell 3T3-L1; Fluorescence volume reduces in the strain of S100A16 overexpressing cell, and fluorescent value raises in the strain of S100A16 interference cell, explains in the strain of S100A16 overexpressing cell; S100A16 combines to reduce its activity with p53; And in the strain of S100A16 interference cell, discharge p53, cause its active rise.
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) (the laboratory animal feedstuff is purchased the collaborative animal feed company in Nanjing to change hello high lipid food.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 subsequent use, claim interior fat weight simultaneously, and it is frozen subsequent use 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 buy the expression of spontaneous fat model mouse ob/ob detection S100A16 gene in ob/ob Adips Mus fat tissue of leptin receptor defects.
The fat Mus and the ob/ob Adips Mus fat tissue extraction albumen of feeding from the wild Mus of C57BL/6J and the big or small high fat diets of 12 weeks of 16 all sizes in result such as the accompanying drawing 4; Adopt the expression of Western blot methods analyst S100A16 at above-mentioned three kinds of tissues, results suggest S100A16 is high expressed in fat Mus that high fat diet is fed and ob/ob Adips Mus fat tissue.α-tubulin is as 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.
SEQUENCE?LISTING
< 110>cloud, Liu
< 120>the S100A16 gene is in the application of preparation treatment of obesity medicine
<130>
<160>1
<170>PatentIn?version?3.3
<210>1
<211>375?<212>DNA
< 213>artificial sequence
<400>1
atggctgact?gctatacaga?gctggagaag?gcagttgttg?tcctggtgga?aaacttttat 60
aaatatgtat?ccaagcacag?cctggtcaaa?aacaagatca?gcaagtccag?cttccgaaag 120
atgctccaga?gagagttgaa?ccacatgctg?acggacacag?ggaaccgaaa?ggcagctgac 180
aagctcatcc?agaacctgga?tgccaaccac?gacgggcgca?tctgctttga?cgaatactgg 240
accatgattg?gtggaatcac?cagccccatg?gccaacctca?tccgccaaca?ggagtgccag 300
caggagagcc?agcaggagtg?ccagcaggag?agccagcagg?agagccagca?ggagagccag 360
cagggcagca?gctag 375
Claims (1)
1.S100A16 gene promotes the application in the lipogenesis medicine in preparation, the nucleotides sequence of described S100A16 gene is classified Seq NO.1 as.
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| CN2010102187599A CN101890172B (en) | 2010-07-02 | 2010-07-02 | Application of S100A16 gene in preparing drug for treating obesity |
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