CN101884794B - S100a16基因在制备治疗胰岛素抵抗药物中的应用 - Google Patents
S100a16基因在制备治疗胰岛素抵抗药物中的应用 Download PDFInfo
- Publication number
- CN101884794B CN101884794B CN2010102187476A CN201010218747A CN101884794B CN 101884794 B CN101884794 B CN 101884794B CN 2010102187476 A CN2010102187476 A CN 2010102187476A CN 201010218747 A CN201010218747 A CN 201010218747A CN 101884794 B CN101884794 B CN 101884794B
- Authority
- CN
- China
- Prior art keywords
- cell
- gene
- insulin
- expression
- calcium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000003814 drug Substances 0.000 title claims abstract description 14
- 101150094306 S100A16 gene Proteins 0.000 title abstract description 21
- 208000001072 type 2 diabetes mellitus Diseases 0.000 title abstract description 13
- 206010022489 Insulin Resistance Diseases 0.000 title abstract description 11
- 101000693050 Homo sapiens Protein S100-A16 Proteins 0.000 claims abstract description 68
- 102100026296 Protein S100-A16 Human genes 0.000 claims abstract description 68
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 140
- 102000004877 Insulin Human genes 0.000 claims description 71
- 108090001061 Insulin Proteins 0.000 claims description 71
- 229940125396 insulin Drugs 0.000 claims description 70
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000011160 research Methods 0.000 abstract description 10
- 208000030159 metabolic disease Diseases 0.000 abstract description 5
- 238000010171 animal model Methods 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract 2
- 230000009456 molecular mechanism Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 53
- 239000011575 calcium Substances 0.000 description 38
- 230000014509 gene expression Effects 0.000 description 33
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 30
- 229910052791 calcium Inorganic materials 0.000 description 30
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 230000004069 differentiation Effects 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 230000026731 phosphorylation Effects 0.000 description 15
- 238000006366 phosphorylation reaction Methods 0.000 description 15
- 235000001727 glucose Nutrition 0.000 description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 13
- 239000008103 glucose Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 210000001789 adipocyte Anatomy 0.000 description 11
- 230000024245 cell differentiation Effects 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 230000009471 action Effects 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 230000004936 stimulating effect Effects 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 208000008589 Obesity Diseases 0.000 description 7
- 235000005911 diet Nutrition 0.000 description 7
- 239000013613 expression plasmid Substances 0.000 description 7
- 230000004190 glucose uptake Effects 0.000 description 7
- 235000020824 obesity Nutrition 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 230000037213 diet Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 229910001424 calcium ion Inorganic materials 0.000 description 5
- 235000009200 high fat diet Nutrition 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000004132 lipogenesis Effects 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- VRYALKFFQXWPIH-PBXRRBTRSA-N (3r,4s,5r)-3,4,5,6-tetrahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)CC=O VRYALKFFQXWPIH-PBXRRBTRSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108090000312 Calcium Channels Proteins 0.000 description 3
- 102000003922 Calcium Channels Human genes 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 101000685712 Homo sapiens Protein S100-A1 Proteins 0.000 description 3
- 102100025087 Insulin receptor substrate 1 Human genes 0.000 description 3
- 101710201824 Insulin receptor substrate 1 Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 102100023097 Protein S100-A1 Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- PMMURAAUARKVCB-UHFFFAOYSA-N alpha-D-ara-dHexp Natural products OCC1OC(O)CC(O)C1O PMMURAAUARKVCB-UHFFFAOYSA-N 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108091006300 SLC2A4 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 210000003050 axon Anatomy 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 208000016097 disease of metabolism Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000037149 energy metabolism Effects 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 201000008980 hyperinsulinism Diseases 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 2
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 210000003360 nephrocyte Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108091052347 Glucose transporter family Proteins 0.000 description 1
- 102000042092 Glucose transporter family Human genes 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 102000002254 Glycogen Synthase Kinase 3 Human genes 0.000 description 1
- 108010014905 Glycogen Synthase Kinase 3 Proteins 0.000 description 1
- 101000852815 Homo sapiens Insulin receptor Proteins 0.000 description 1
- 101000653788 Homo sapiens Protein S100-A11 Proteins 0.000 description 1
- 101000693054 Homo sapiens Protein S100-A13 Proteins 0.000 description 1
- 101000685719 Homo sapiens Protein S100-A5 Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 102100036721 Insulin receptor Human genes 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 102100031775 Leptin receptor Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101100181592 Mus musculus Lep gene Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 102100029811 Protein S100-A11 Human genes 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 102100025670 Protein S100-A13 Human genes 0.000 description 1
- 102100023089 Protein S100-A2 Human genes 0.000 description 1
- 102100023090 Protein S100-A3 Human genes 0.000 description 1
- 102100023087 Protein S100-A4 Human genes 0.000 description 1
- 102100023088 Protein S100-A5 Human genes 0.000 description 1
- 102100032446 Protein S100-A7 Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 102000013674 S-100 Human genes 0.000 description 1
- 108700021018 S100 Proteins 0.000 description 1
- 108010005256 S100 Calcium Binding Protein A7 Proteins 0.000 description 1
- 108010085149 S100 Calcium-Binding Protein A4 Proteins 0.000 description 1
- 108700016890 S100A12 Proteins 0.000 description 1
- 101150110386 SLC2A4 gene Proteins 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 241000786103 Steatomys pratensis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 229960005084 calcitriol Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- HAUBPZADNMBYMB-UHFFFAOYSA-N calcium copper Chemical compound [Ca].[Cu] HAUBPZADNMBYMB-UHFFFAOYSA-N 0.000 description 1
- 239000003710 calcium ionophore Substances 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008727 cellular glucose uptake Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- CJAONIOAQZUHPN-KKLWWLSJSA-N ethyl 12-[[2-[(2r,3r)-3-[2-[(12-ethoxy-12-oxododecyl)-methylamino]-2-oxoethoxy]butan-2-yl]oxyacetyl]-methylamino]dodecanoate Chemical compound CCOC(=O)CCCCCCCCCCCN(C)C(=O)CO[C@H](C)[C@@H](C)OCC(=O)N(C)CCCCCCCCCCCC(=O)OCC CJAONIOAQZUHPN-KKLWWLSJSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 150000002304 glucoses Chemical class 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 101150090422 gsk-3 gene Proteins 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 108010019813 leptin receptors Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000001818 nuclear effect Effects 0.000 description 1
- 230000005937 nuclear translocation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002374 sebum Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明涉及S100A16基因及其相关通路在代谢性疾病药物研发中的应用，S100A16基因为与胰岛素抵抗相关的一个新基因，为胰岛素抵抗的分子机制研究提供基础，通过细胞及动物模型的构建首次揭示S100A16在胰岛素抵抗中的重要作用，为胰岛素抵抗新药的研发提供理论基础及可行性依据可用于作为新的药物靶点。
Description
技术领域
本发明涉及S100A16基因在制备治疗胰岛素抵抗药物中的应用。
背景技术
神经组织蛋白S100家族是一类广泛分布于不同组织的EF-hand型酸性可溶性钙结合蛋白，其中S即soluble，100表示该蛋白在饱和硫酸氨中的溶解度为100％，S100蛋白含2个与钙亲和力不同的EF螺旋结构，C-末端EF螺旋结构由12个氨基酸组成典型的Ca2+结合环，N-末端EF螺旋结构包含14个具有S100蛋白特异性的氨基酸。
人S100蛋白的基因在染色体1q21区，肿瘤在此区的基因常频繁重组，易引起S100基因表达失控，因此，肿瘤晚期和发生肿瘤转移时有S100异常表达，特别是神经外胚层起源的肿瘤组织中有异常表达，且与肿瘤分期和预后有相关性。S100蛋白在哺乳动物分布有特异性：S100A1主要在神经细胞，骨骼肌细胞，心肌细胞和肾细胞的胞浆；S100A2在肺和肾细胞的胞浆和核中；S100A3和S100A5的分布尚不明确；S100A4在成纤维细胞，肌上皮细胞和肿瘤细胞胞浆和胞外液中；S100A12分布与海马细胞；S100A13在甲状腺细胞最丰富，特别是核周区；S100A16分布广泛，见于多种肿瘤组织。
研究显示S100蛋白可通过调节钙离子及糖基化受体RAGE参与细胞的增生、分化、迁移凋亡及认知[1]。如整个S100家族对神经轴突的生长起调节作用[2]，而S100A7，S100A 13，S100A 14等在肿瘤中差异表达，已被用做肿瘤诊断与预后的标志基因[3，4]。
S100A16基因是我们应用全基因组微阵列芯片技术，筛查代谢性疾病相关基因的研究中获得的差异表达新基因，是钙调蛋白S100家族的新成员，除了具有S100家族的基本结构特征，如：是一类较小的Ca2+、Zn2+结合蛋白，含2个与钙亲和力不同的EF螺旋结构，C-末端EF螺旋结构由12个氨基酸组成典型的Ca2+结合环，尚具有以下特异性：
1、不同于传统的S100蛋白的EF端由14个氨基酸组成，S100A16蛋白的EF端由15个氨基酸组成loop环；
2、在EF端最后位置缺少谷氨酸盐残基，这对于协调钙的位点是非常重要的；所以提示S100A16基因存在更复杂的调节机制。
S100家族在细胞的增生、分化、迁移与凋亡，神经轴突的生长与认知形成及肿瘤发生发展中均发挥着重要角色。关于其新成员S100A16基因与疾病发生发展的研究，由于研究尚浅，目前尚无明确定论。我们研究小组于2005年起即以其为靶标开展系列研究，初步阐明了其在代谢性疾病的发生发展中发挥的作用。
申请人早先的研究表明S100A16 mRNA在正常食道、脂肪及结肠组织中过表达，有研究表明S100A16也过表达在膀胱、肺、甲状腺、胰腺及卵巢肿瘤中[5]，提示其生物学功能的多样性。最新研究表明[1]，细胞内Ca2+浓度可有效调节S100A16蛋白的入核出核信号，细胞内Ca2+浓度增高，S100A16蛋白主要分布在胞浆，而随细胞内Ca2+浓度减低，S100A16蛋白的的核转运增强并在核中大量积累，并与核仁紧密相关，提示S100A16蛋白在核糖核蛋白复合体多相过程、基因沉默或细胞分裂中可能起作用。S100A16钙依赖的异位可能受最近描述[6，7]的核浆网状调节，在核附近终止，负责游离钙释放到局部固定的亚核位置。与S100家族其他成员一样，S100A16缺乏明显的入核信号肽，其入核可能与S100A11类似，需要与转运蛋白反应[6]或者像钙铜调节那样可能通过选择性的促进传播路径发生[7]。这些结果提示S100A16基因在特定的一些组织中，在钙信号通路调控或其他尚未鉴定的通路调控下，发挥着重要的生物学功能。但是S100A16在核周的作用及S100A16-Ca2+依赖的原因及功能意义，是否与脂肪细胞分化及胰岛素分泌有关需进一步阐明，目前国内外未见报道。
胰岛素抵抗(Insulin Resistance，IR)是指胰岛素作用的靶器官对胰岛素作用的敏感性下降，即正常剂量的胰岛素产生低于正常生物学效应的一种状态。在脂肪细胞内，胰岛素抗性导致储存的甘油三酸酯的水解，进而提高血浆内自由脂肪酸的含量。在肌肉细胞内，胰岛素抗性降低葡萄糖的吸收；而在肝细胞内，降低葡萄糖的储备，两者共同导致血糖含量的提高。胰岛素抗性引起的血浆中高胰岛素和高糖含量经常导致代谢综合征和2型糖尿病。胰岛素抵抗是基因与外环境共同作用的结果，细胞内钙离子在代谢紊乱相关的胰岛素抵抗中起重要作用[8]。近来，钙与胰岛素抵抗的关系逐渐成为一个研究热点，为寻找控制胰岛素抵抗及其相关疾病的治疗方法开拓了一片崭新的视野。研究表明，细胞内钙离子浓度在与胰岛素抵抗相关的能量代谢失衡中起着重要作用[9]。细胞内钙离子浓度的增加导致脂肪合成基因的表达和脂肪合成的增加、抑制脂肪分解，最终增加脂肪积聚而导致胰岛素抵抗。低钙饮食可导致1，25-二羟维生素D3的产生增加，刺激钙离子向细胞内流动从而促进胰岛素抵抗的发生[10，11]。在这个过程中，钙离子通道介导的钙离子细胞内流是一个重要环节。因此围绕着钙离子通道及其调控因素开展进一步的研究将具有重要的意义。
本专利申请首次证明S100A16对胰岛素抵抗具有治疗作用。
参考文献
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.
发明内容
技术目的
本发明的一个目的是提供S100A16基因制备治疗胰岛素抵抗药物中的应用。
技术方案
S100A16基因在制备治疗胰岛素抵抗药物中的应用，通过如下技术方案得到证实：
一、采用小鼠前脂肪细胞3T3-L1细胞模型，应用同位素标志的葡萄糖C14通过检测胰岛素刺激的葡萄糖摄取试验证实S100A16过表达对葡萄糖摄取的抑制作用。
二、采用小鼠前脂肪细胞3T3-L1细胞模型，应用Western blot方法检测S100A16对细胞内Akt磷酸化的影响，揭示S100A16影响胰岛素作用的机制。
三、在动物水平采用饮食诱导的肥胖鼠及转基因证实S100A16在肥胖胰岛素抵抗中的作用。
总而言之S100A16基因表达与胰岛素抵抗有直接关联，即S100A16过表达加剧胰岛素抵抗，S100A16低表达减弱胰岛素抵抗，从而为开发抗胰岛素抵抗药物提供一种选择的靶点。
其中S100A16基因的核苷酸序列为Seq NO.1
前脂肪细胞3T3-L1细胞模型为经典的检测胰岛素抵抗的模型。
有益效果
1、大量的研究表明，肥胖尤其是内脏型肥胖是产生胰岛素抵抗(Insulin Resistance，IR)的重要因素。胰岛素抵抗(Insulin Resistance，IR)是指胰岛素作用的靶器官对胰岛素作用的敏感性下降，即正常剂量的胰岛素产生低于正常生物学效应的一种状态。在脂肪细胞内，胰岛素抵抗导致储存的甘油三酸酯的水解，进而提高血浆内自由脂肪酸的含量。在肌肉细胞内，胰岛素抵抗降低葡萄糖的吸收；而在肝细胞内，降低葡萄糖的储备，两者共同导致血糖含量的提高。胰岛素抵抗引起的血浆中高胰岛素和高糖含量经常导致代谢综合征和2型糖尿病。我们的实验结果提示S100A16在高脂饮食喂养的肥胖鼠及ob/ob鼠脂肪组织中过表达，提示与肥胖及胰岛素抵抗有关。
2、胰岛素抵抗是基因与外环境共同作用的结果，细胞内钙离子在代谢紊乱相关的胰岛素抵抗中起重要作用。近来，钙与胰岛素抵抗的关系逐渐成为一个研究热点，为寻找控制胰岛素抵抗及其相关疾病的治疗方法开拓了一片崭新的视野。研究表明，细胞内钙离子浓度在与胰岛素抵抗相关的能量代谢失衡中起着重要作用。细胞内钙离子浓度的增加导致脂肪合成基因的表达和脂肪合成的增加、抑制脂肪分解，最终增加脂肪积聚而导致胰岛素抵抗。我们的研究结果发现高钙可促进S100A16基因表达的蛋白向细胞浆的输出，低钙可促进S100A16基因表达的蛋白向细胞核的转入。提示S100A16可能通过钙离子通道介导的钙离子细胞内流与胰岛素抵抗有关。
3、磷酸化途径在胰岛素抵抗中起重要的作用，胰岛素受体具有内在的酪氨酸蛋白激酶活性，通过使其自身及胰岛素受体底物-1(IRS-1)磷酸化而介导细胞对胰岛素的反应，磷酸化了的IRS-1相当于第二信使，它与富含SH2的蛋白质磷脂酰肌醇-3激酶(PI-3k)结合，使之激活，促进葡萄糖转运体-4(GLUT-4)的合成及由细胞内池迁移至细胞膜，启动细胞对葡萄糖的摄取。我们的研究结果发现在脂肪细胞分化过程中S100A16过表达对于磷酸化途径，可抑制3T3-L1细胞分化后期Akt的磷酸化作用，从而抑制3T3-L1细胞分化后期胰岛素刺激的葡萄糖摄取。因此，S100A16通过抑制磷酸化作用在胰岛素抵抗中发挥生物学功能，即通过抑制S100A16过表达或S100A16表达下调，来实现胰岛素抵抗治疗作用，因此我们可以根据S100A16基因序列，相应开发与S100A16相作用的药物，或是作为筛选治疗胰岛素抵抗药物的靶点，具有非常重要应用意义和商业前景。
本研究结果首次揭示了S100A16与胰岛素抵抗有直接关联，作为新的药物靶点，S100A16基因在制备治疗胰岛素抵抗相关药物的应用中，包括通过S100A16基因上下游靶点制备胰岛素抵抗相关药物中申请专利保护。
附图说明
图1A显示3T3-L1细胞转染S100A16高表达质粒后提取蛋白，采用Western blot 分析验证S100A16高表达质粒转染效果。.Lane 1：正常3T3-L1细胞；lane 2：空载体pcDNA3.1质粒；lane 3：pcDNA3.1-S100A16-3T3-L1；对图A蛋白表达进行定量分析获得S100A16表达的灰度分析图B(数据来源于三组独立试验)。结果以均数+标准差表示。**P＜0.01或*P＜0.05与对照3T3-L1比较；
图2显示S100A16对3T3-L1细胞分化后期胰岛素刺激的葡萄糖摄取及对3T3-L1细胞分化过程中磷酸化作用；
其中图2A显示3T3-L1、pcDNA3.1空载体及pcDNA3.1-S100A16-3T3-L1细胞(S100A16过表达细胞)诱导分化后，检测分化不同天数胰岛素刺激的葡萄糖摄取情况，以正常3T3-L1细胞的检测值作为基数，其他检测值与之比较以相对比值表示，结果提示S100A16过表达可明显抑制3T3-L1细胞分化后期葡萄糖的摄取，结果以均数±标准差表示，n＝3.*P＜0.05，**P＜0.01与正常3T3-L1比较；
图2B显示S100A16过表达质粒转染3T3-L1细胞，诱导分化后10天采用不同浓度胰岛素(0、5、10、20、50nM)刺激十分钟后收集蛋白，用Western blot方法检测p-Akt表达，结果提示S100A16过表达可抑制3T3-L1细胞分化后期Akt的磷酸化作用；
图3显示3T3-L1细胞中Ca2+-依赖的核输出及核转入，3T3-L1细胞分别采用1.5mM Ca2+及1μM ionomycin刺激0，5，10，15分钟，或者应用钙耦合剂3mM EGTA刺激0，10，20，30分钟，检测细胞总蛋白及核蛋白中S100A16表达，结果提示高钙可促进S100A16蛋白向细胞浆的输出，低钙可促进S100A16蛋白向细胞核的转入。
图4显示S100A16在正常及饮食诱导的肥胖及ob/ob鼠脂肪组织中的表达，从16周大小的C57BL/6J野生鼠及12周大小的高脂饮食喂养的肥胖鼠及ob/ob鼠脂肪组织提取蛋白，采用Western blot方法分析S100A16在上述三种组织的表达，结果提示S100A16在高脂饮食喂养的肥胖鼠及ob/ob鼠脂肪组织中过表达。α-tubulin作为对照。
具体实施方式
实施例1
本实施例中参考实验指南第三册(第15章在大肠杆菌中表达克隆化基因；第16章哺乳动物培养细胞中导入克隆化基因；附录1分子克隆中使用的缓冲液和试剂的配制；附录8分子克隆中的常用技术)，现代实用细胞与分子生物学实验技术(第一章细胞培养方法；第八章细胞形态与细胞化学定量分析技术)。
前脂肪细胞3T3-L1购自上海细胞生物研究所，14C标记葡萄糖[3H]2-deoxy-D-glucose购自北京科美东雅生物技术有限公司，
一、在细胞水平采用小鼠前脂肪细胞3T3-L1证实S100A16在肥胖胰岛素抵抗中的作用。
①构建S100A16过表达质粒转染3T3-L1细胞，进行三联刺激诱导分化，通过胰岛素刺激的葡萄糖摄取试验及磷酸化检测观察S100A16对3T3-L1细胞分化过程中Akt的磷酸化作用，揭示S100A16在胰岛素抵抗中的作用。
②采用高钙及低钙刺激的3T3-L1细胞模型，探讨钙浓度变化对S100A16基因的核转入影响。
具体方法如如下
1、经典的3T3-L1细胞诱导分化模型的建立：
3T3-L1细胞培养在DMEM培养液中(10％胎牛血清)，5％CO2、37℃培养箱。当细胞生长至完全融合后48h后，加入0.5mM3-isobutyl-1-methyxan-thine(MIX)，1ug/ml胰岛素及1uM地塞米松(Sigma，USA)诱导分化。48h后换用仅含1ug/ml胰岛素的完全培养基，以后每两天换液直到第八天细胞完全分化成熟。
2、S100A16过表达质粒的构建及细胞转染
根据《分子生物克隆实验指南》，第16章哺乳动物培养细胞中导入克隆化基因方案1脂染介导的DNA转染方法，
以小鼠肝脏cDNA为模版；以引物S100a16-F，其序列为Seq NO.2；S100A16-R，其序列为Seq NO.3；扩增S100A16开放阅读框；PCR条件：100ul体系，56°退火，72°延伸30s，33个循环，获取目的片段；采用PCR纯化试剂盒(TakaRa DNA Fragment Purification Kit)纯化目的片段；采用NheI和EcoRI进行酶切，对酶切后产物进行切胶纯化；将纯化产物连接至载体pcDNA3.1上；取5UL连接产物转化感受态细胞，转化后将菌液涂至Amp板，放37度孵箱培养16小时，在超净台内挑单克隆加4ml LB+40ul Amp放摇床16小时，取1ul菌液当模版作PCR；阳性克隆的菌液3ml小抽质粒并送测序。测序比对完全正确后，中抽质粒转染3T3-L1细胞挑选克隆细胞株培养待用。通过western blot方法验 证 转 染 成 功 ( 附 图 1 ) 。转 染 S100A16 的 3T3-L1 细 胞(pcDNA3.1-S100A16-3T3-L1)按照上述1的方法培养诱导分化。
3、采用胰岛素刺激的葡萄糖吸收实验观察S100A16对细胞葡萄糖摄取的影响：
即用[3H]2-deoxy-D-glucose的方法检测不同分化阶段脂肪细胞处理葡萄糖的能力。
3T3-L1细胞(正常及S100A16过表达质粒转染后3T3-L1细胞)和pcDNA3.1空载体(阳性对照)，按照步骤1的方法诱导分化，在诱导分化后不同天数(0，2，4，6，8，10days)，用KRP液(NaCl 128mM，KCl 4.7mM，CaCl2 1.65mM，MgSO42.5mM，Na2HPO4 5mM，pH 7.4)洗涤两次，与100nM procine insulin胰岛素共孵10分钟，然后加入含有0.2％BSA，1μCi/ml[3H]2-deoxy-D-glucose及5mM glucose的KRP再孵10分钟，37℃；然后样本用冷的KRP液洗涤三次，用含有0.5M NaOH及0.1％SDS溶解，用闪烁计数仪确定细胞的放射性吸收情况。
结果提示S100A16过表达可明显抑制3T3-L1细胞分化后期葡萄糖的摄取；如附图中(2A)3T3-L1、pcDNA3.1空载体及pcDNA3.1-S100A16-3T3-L1细胞诱导分化后，检测分化不同天数胰岛素刺激的葡萄糖摄取情况，以正常3T3-L1细胞的检测值作为基数，其他检测值与之比较以相对比值表示。结果以均数±标准差表示。n＝3.*P＜0.05，**P＜0.01与正常3T3-L1比较。
4、采用Western blot方法检测S100A16对细胞内Akt磷酸化的影响：
3T3-L1，pcDNA3.1空载体及pcDNA3.1-S100A16过表达细胞分别种6孔板，三联刺激诱导分化，到第十天培养液换为无血清无胰岛素的普通培养液(含0.1％的BSA)培养2小时后用不同浓度胰岛素(0，5，10，20，50nM)刺激十分钟，收集蛋白，用Western blot方法检测p-Akt，total-Akt作为对照。IP3K/Akt信号通路是胰岛素作用的主要信号通路，Akt(丝氨酸/苏氨酸激酶)接受IP3K(磷酯酰肌醇3激酶)调节，IP3K激活Akt后，可通过以下三种途径发挥生物学作用。(1)对细胞内葡萄糖运载体Glut4的异位作用，使其发挥转运葡萄糖的作用；(2)通过糖原合成酶激酶3(GSK 3)促进糖原合成；(3)在脂肪细胞，胰岛素通过IP3K和Akt依赖的信号途径抑制脂肪分解。而Akt磷酸化是启动上述信号通路的主要步骤，Akt磷酸化活性下降则抑制IP3K/Akt信号通路，从而影响葡萄糖的吸收。研究表明，在胰岛素抵抗状态，胰岛素刺激引起的IP3K/Akt信号传导途径的作用下降，我们通过检测S100A16过表达对激活IP3K/Akt信号通路的影响，探讨S100A16在胰岛素抵抗中的作用。
结果提示S100A16过表达可抑制3T3-L1细胞分化后期Akt的磷酸化作用。如附图2B所示S100A16过表达质粒转染3T3-L1细胞，诱导分化后10天采用不同浓度胰岛素(0，5，10，20，50nM)刺激十分钟后收集蛋白，用Western blot 方法检测p-Akt表达。
5、采用Western blot方法检测细胞内钙水平变化对S100A16的影响
3T3-L1细胞分别采用1.5mM Ca2+及钙离子载体1μM ionomycin刺激0，5，10，15分钟促进胞内钙的浓度的升高；或者应用钙耦合剂3mM EGTA刺激3T3-L1细胞0，10，20，30分钟，检测细胞总蛋白及核蛋白中S100A16表达，如附图3所示，结果提示高钙可促进S100A16蛋白向细胞浆的输出，低钙可促进S100A16蛋白向细胞核的转入。
二、在动物水平采用饮食诱导的肥胖鼠及转基因证实S100A16在肥胖胰岛素抵抗中的作用。
饮食诱导肥胖鼠模型的建立：雄性SD大鼠30只(购自江苏省实验动物中心)，分笼饲养，1只/笼，体重90 120克。饲以基础饲料、自由摄水进食物，环境温度21℃-23℃，大鼠适应环境一周后，称量体重，分为两组，一组继续喂养基础饲料，另外一组(diet-induced obesity；DIO)改喂高脂饲料(实验动物饲料购置南京协同动物饲料公司)。饲料配方：基础饲料营养成分配比，碳水化合物65％，脂肪20％，蛋白质15％；高脂饲料营养配比，碳水化合物20％，脂肪60％，蛋白质20％)，每天记录体重、体长及尾长，两周后高脂组的体重明显增加，继续喂养六周。处死前禁食12小时，腹腔注射1％戊巴比妥钠麻醉，称体重，打开心脏取血5-10毫升，分离血清，-70℃冻存备用，同时称内脏脂肪重量，取内脂、皮脂、骨骼肌、脑等组织-70℃冻存备用。ob/ob鼠模型：购自南京大学模式动物中心，常规饲养，处死后取脂肪组织提取蛋白检测S100A16基因的表达。
分离高脂饲料喂养的小鼠及正常对照鼠的脂肪组织，检测S100A16在上述组织中的表达，并购买leptin受体缺陷的自发性肥胖模型鼠ob/ob，检测S100A16基因在ob/ob鼠脂肪组织中的表达。
结果提示S100A16在高脂饮食喂养的肥胖鼠及ob/ob鼠脂肪组织中过表达，证明S100A16与肥胖和胰岛素抵抗有关。如附图4从16周大小的C57BL/6J野生鼠及12周大小的高脂饮食喂养的肥胖鼠及ob/ob鼠脂肪组织提取蛋白，采用Western blot方法分析S100A16在上述三种组织的表达。α-tubulin作为对照。上述具体实施方式不以任何形式限制本发明的技术方案，凡是采用等同替换或等效变换的方式所获得的技术方案均落在本发明的保护范围。
Claims (1)
1.S100A16基因在制备治疗胰岛素抵抗药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102187476A CN101884794B (zh) | 2010-07-02 | 2010-07-02 | S100a16基因在制备治疗胰岛素抵抗药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102187476A CN101884794B (zh) | 2010-07-02 | 2010-07-02 | S100a16基因在制备治疗胰岛素抵抗药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101884794A CN101884794A (zh) | 2010-11-17 |
| CN101884794B true CN101884794B (zh) | 2011-08-31 |
Family
ID=43070990
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102187476A Expired - Fee Related CN101884794B (zh) | 2010-07-02 | 2010-07-02 | S100a16基因在制备治疗胰岛素抵抗药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101884794B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006042005A2 (en) * | 2004-10-07 | 2006-04-20 | Immunivest Corporation | Global gene expression profiling of circulating tumor cells |
-
2010
- 2010-07-02 CN CN2010102187476A patent/CN101884794B/zh not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006042005A2 (en) * | 2004-10-07 | 2006-04-20 | Immunivest Corporation | Global gene expression profiling of circulating tumor cells |
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. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101884794A (zh) | 2010-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Myokine IL-15 regulates the crosstalk of co-cultured porcine skeletal muscle satellite cells and preadipocytes | |
| Douard et al. | Regulation of the fructose transporter GLUT5 in health and disease | |
| Tang et al. | Genes involved in fatty acid metabolism: molecular characterization and hypothalamic mRNA response to energy status and neuropeptide Y treatment in the orange-spotted grouper Epinephelus coioides | |
| CN103638531A (zh) | Lgr4基因用于筛选促进白色脂肪棕色化的药物 | |
| Li et al. | ACE2 suppresses the inflammatory response in LPS-induced porcine intestinal epithelial cells via regulating the NF-κB and MAPK pathways | |
| CN109310740A (zh) | 使用slit－robo系统的预防或治疗肌肉减少症的组合物 | |
| Li et al. | Age‐related intestinal monosaccharides transporters expression and villus surface area increase in broiler and layer chickens | |
| Oh et al. | Culturing characteristics of Hanwoo myosatellite cells and C2C12 cells incubated at 37 C and 39 C for cultured meat | |
| Takahashi et al. | The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn | |
| Song et al. | Traditional Chinese medicine decoction enhances growth performance and intestinal glucose absorption in heat stressed pigs by up-regulating the expressions of SGLT1 and GLUT2 mRNA | |
| Chen et al. | AQP7 mediates post-menopausal lipogenesis in adipocytes through FSH-induced transcriptional crosstalk with AP-1 sites | |
| CN114340677B (zh) | Jwa基因以及相关化合物的抗衰老用途 | |
| Zhang et al. | Tumor‐derived semaphorin 3D promoting cancer cachexia via regulating hypothalamic pro‐opiomelanocortin neurons | |
| Jung et al. | Nanomolar melatonin influences insulin synthesis and secretion in rat insulinoma INS-1E cells | |
| CN111793686A (zh) | luminal型和HER2型乳腺癌诊断及预后的标志物、治疗用PPARγ抑制剂 | |
| KR101303462B1 (ko) | Ｄｒｇ2 유전자의 신규한 용도 | |
| Tsagkaraki et al. | Crosstalk between corepressor NRIP1 and cAMP signaling on adipocyte thermogenic programming | |
| CN101884794B (zh) | S100a16基因在制备治疗胰岛素抵抗药物中的应用 | |
| Reindl et al. | Differential ligand binding and agonist-induced regulation characteristics of the two rainbow trout GH receptors, Ghr1 and Ghr2, in transfected cells | |
| CN101890172B (zh) | S100a16基因在制备治疗肥胖症药物中的应用 | |
| KR20200112743A (ko) | Sgk3 유전자를 이용한 뇌신경계 질환의 진단 및 치료 | |
| CN101912617B (zh) | Mg53基因在治疗胰岛素抵抗和ⅱ型糖尿病及其相关病症中的应用 | |
| CN117100772A (zh) | 植物乳杆菌shy21-2在增加耐力缓解疲劳中的应用 | |
| Holik et al. | Impact of free N ε-carboxymethyllysine, its precursor glyoxal and AGE-modified BSA on serotonin release from human parietal cells in culture | |
| Zhao et al. | Effects of L-arginine supplementation on glucose and nitric oxide (NO) levels and activity of NO synthase in corticosterone-challenged broiler chickens (Gallus gallus) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110831 Termination date: 20210702 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |