CN114778860A - Method for measuring regulation and control effect of miR181a on insulin resistance - Google Patents
Method for measuring regulation and control effect of miR181a on insulin resistance Download PDFInfo
- Publication number
- CN114778860A CN114778860A CN202210452745.6A CN202210452745A CN114778860A CN 114778860 A CN114778860 A CN 114778860A CN 202210452745 A CN202210452745 A CN 202210452745A CN 114778860 A CN114778860 A CN 114778860A
- Authority
- CN
- China
- Prior art keywords
- insulin
- mir181a
- regulation
- effect
- insulin resistance
- 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.)
- Pending
Links
- 230000000694 effects Effects 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 48
- 108091031103 miR-181a stem-loop Proteins 0.000 title claims abstract description 45
- 108091046591 miR-181a-4 stem-loop Proteins 0.000 title claims abstract description 45
- 108091049627 miR-181a-5 stem-loop Proteins 0.000 title claims abstract description 45
- 206010022489 Insulin Resistance Diseases 0.000 title claims abstract description 43
- 208000001072 type 2 diabetes mellitus Diseases 0.000 title claims abstract description 41
- 230000033228 biological regulation Effects 0.000 title claims abstract description 32
- 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 abstract description 230
- 102000004877 Insulin Human genes 0.000 claims abstract description 127
- 108090001061 Insulin Proteins 0.000 claims abstract description 127
- 229940125396 insulin Drugs 0.000 claims abstract description 115
- 239000000243 solution Substances 0.000 claims abstract description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 16
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 239000012488 sample solution Substances 0.000 claims description 39
- OMLWNBVRVJYMBQ-YUMQZZPRSA-N Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OMLWNBVRVJYMBQ-YUMQZZPRSA-N 0.000 claims description 16
- 108010068380 arginylarginine Proteins 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- 239000002243 precursor Substances 0.000 claims description 16
- 239000012086 standard solution Substances 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 13
- 102000007079 Peptide Fragments Human genes 0.000 claims description 12
- 108010033276 Peptide Fragments Proteins 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 12
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 12
- 239000005089 Luciferase Substances 0.000 claims description 10
- 108700008625 Reporter Genes Proteins 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 230000004044 response Effects 0.000 claims description 9
- 230000000638 stimulation Effects 0.000 claims description 9
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000001262 western blot Methods 0.000 claims description 6
- 150000001413 amino acids Chemical group 0.000 claims description 5
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 4
- AYKYXWQEBUNJCN-UHFFFAOYSA-N 3-methylfuran-2,5-dione Chemical compound CC1=CC(=O)OC1=O AYKYXWQEBUNJCN-UHFFFAOYSA-N 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 4
- 230000000903 blocking effect Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 4
- 229960002442 glucosamine Drugs 0.000 claims description 4
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 238000002203 pretreatment Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 108060001084 Luciferase Proteins 0.000 claims description 3
- 108091080995 Mir-9/mir-79 microRNA precursor family Proteins 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000001502 gel electrophoresis Methods 0.000 claims description 3
- 238000003384 imaging method Methods 0.000 claims description 3
- 108091047084 miR-9 stem-loop Proteins 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 15
- 238000004458 analytical method Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 230000004907 flux Effects 0.000 abstract description 2
- 210000003494 hepatocyte Anatomy 0.000 abstract description 2
- 238000004750 isotope dilution mass spectroscopy Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229920002527 Glycogen Polymers 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 229940096919 glycogen Drugs 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 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 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000004136 fatty acid synthesis Effects 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
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/62—Insulins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of medicines, and discloses a method for measuring the regulation and control effect of miR181a on insulin resistance, wherein miR-181a can be successfully induced and adsorbed by a miR-181a adsorption sponge carrier strategy, the constructed pEGFP-C2-181a-SP carrier gene remarkably increases the insulin sensitivity of HepG2 cells, a potential solution is provided for improving hepatocyte insulin resistance caused by miR-181a, and the method is likely to become a new strategy for treating type 2 diabetes; meanwhile, the insulin content is determined by adopting an isotope dilution mass spectrometry reference technology to directly quantify the insulin in the sample, and the quantitative result is accurate and reliable; in the determination process, the insulin does not need to be subjected to enzyme digestion, so that the influence of enzyme digestion efficiency is avoided; the sample is directly measured without separation, and the analysis speed and the analysis flux are obviously superior to those of the common method.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for measuring the regulation and control effect of miR181a on insulin resistance.
Background
Insulin is a protein hormone secreted by the beta cells of the islets of langerhans in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body which reduces blood sugar and promotes the synthesis of glycogen, fat and protein. Exogenous insulin is mainly used for treating diabetes. The main physiological role of insulin is to regulate metabolic processes. For sugar metabolism: promoting glucose uptake and utilization of tissue cells, promoting glycogen synthesis, inhibiting gluconeogenesis, and lowering blood sugar; for fat metabolism: promote fatty acid synthesis and fat storage, reduce lipolysis; for proteins: promote amino acid to enter cell and promote protein synthesis. The overall effect is to promote anabolism. Insulin is the only hormone in the body for reducing blood sugar and the only hormone for promoting the synthesis of glycogen, fat and protein. The mechanism of action belongs to the receptor tyrosine kinase mechanism. However, the existing method for measuring the regulation and control effect of miR181a on insulin resistance cannot effectively improve insulin sensitivity; at the same time, the insulin content cannot be determined accurately.
In summary, the problems of the prior art are: the existing method for measuring the regulation and control effect of miR181a on insulin resistance cannot effectively improve insulin sensitivity; at the same time, the insulin content cannot be determined accurately.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for measuring the regulation and control effect of miR181a on insulin resistance.
The invention is realized in such a way that a method for measuring the regulation and control effect of miR181a on insulin resistance comprises the following steps:
designing an antisense sequence mutated at 9-12 positions for a sequence of mature human miR-181a, repeating 7 copies, connecting in series into a pEGFP-C2 eukaryotic expression vector and a pGL3-Promoter vector, and verifying the effect of the adsorption sponge through a dual-luciferase reporter gene system;
transfecting miR-181a adsorption sponge carrier in a glucosamine-induced HepG2 cell insulin resistance model, and detecting the response effect of cells to insulin stimulation;
and step three, determining the content of insulin.
Further, the specific method for constructing the adsorption sponge comprises the steps of carrying out double enzyme digestion on pUC57-18la-SP and pEGFP-C2 empty vectors by EcoRi and BamHi, carrying out DNA gel electrophoresis, recovering a 215bp small fragment and a molecular fragment obtained by carrying out double enzyme digestion on pEGFP-C3, and connecting the two fragments at 4 ℃ overnight by using T4 ligase.
Further, the effect of the adsorption sponge is verified by a dual-luciferase reporter gene system, and the constructed pEGFP-C2-181aSP luciferase reporter gene vector is co-transfected into HEK293T cells.
Further, the effect of the response of the cells to the insulin stimulation was detected by Western blot, and the change in the bands was compared with the control group to draw a conclusion.
Further, the Western blot detection is carried out, and the gray value of each protein band is analyzed by ImageJ software after imaging.
Further, the method for measuring the insulin content comprises the following steps:
(1) reversibly blocking a lysine residue in an Arg-Arg precursor insulin of the contained amino acid sequence, comprising contacting the Arg-Arg precursor insulin with a solution at pH6-10 comprising a buffer and citraconic anhydride; partially cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin with trypsin, thereby obtaining insulin; synthesizing an isotope-labeled insulin B chain peptide fragment and preparing an isotope-labeled insulin B chain peptide fragment solution as an internal standard;
(2) an insulin standard solution and an insulin sample solution are prepared and subjected to mass spectrometry.
(3) And calculating the content of the insulin in the insulin sample solution.
Further, pretreating the insulin standard solution and the insulin sample solution;
further, the pretreatment method comprises the following steps:
1) weighing 1mL of the insulin sample solution, adding 1mL of the isotope-labeled insulin B chain peptide fragment solution, weighing, uniformly mixing to obtain a labeling solution, reducing to obtain a reduced insulin sample solution, and pretreating according to the mass fraction of insulin in the insulin sample solution;
2) carrying out solid phase extraction on the reduced insulin sample solution, uniformly mixing the solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally drying the mixture;
3) when the mass fraction of the insulin in the insulin sample solution is more than or equal to 10-7g/g, uniformly mixing the reduced insulin sample solution with a MALDI-TOF matrix, spotting on a target plate, and naturally drying; or performing solid phase extraction, mixing with MALDI-TOF matrix, spotting on target plate, and naturally air drying; 1mL of the insulin standard solution was pretreated in the same manner as described above.
Furthermore, the miR-9 possibly exerts the same mechanism, and the obtained result is also applicable.
The invention has the advantages and positive effects that: the miR-181a can be successfully induced and adsorbed by a miR-181a adsorption sponge carrier strategy, the constructed pEGFP-C2-181a-SP carrier gene remarkably increases the insulin sensitivity of HepG2 cells, a potential solution is provided for improving hepatocyte insulin resistance caused by miR-181a, and the method is likely to become a new strategy for treating type 2 diabetes; meanwhile, the insulin content is measured by adopting an isotope dilution mass spectrometry reference technology to directly quantify the insulin in the sample, and the quantitative result is accurate and reliable; in the determination process, the insulin does not need to be subjected to enzyme digestion, so that the influence of enzyme digestion efficiency is avoided; the sample is directly measured without separation, and the analysis speed and the analysis flux are obviously superior to those of the common method.
Drawings
FIG. 1 is a flow chart of a determination method for regulating the effect of miR181a on insulin resistance, which is provided by the implementation of the invention.
FIG. 2 is a flow chart of a method for determining insulin content according to the present invention.
FIG. 3 is a flow chart of a pre-processing method provided in the practice of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
As shown in fig. 1, the method for determining the regulation effect of miR181a on insulin resistance provided by the embodiments of the present invention includes the following steps:
s101, designing an antisense sequence mutated at positions 9-12 for a sequence of mature human miR-181a, repeating 7 copies, connecting the antisense sequence in series into a pEGFP-C2 eukaryotic expression vector and a pGL3-Promoter vector, and verifying the effect of the adsorption sponge through a dual-luciferase reporter gene system;
s102, transfecting a miR-181a adsorption sponge carrier in a glucosamine-induced HepG2 cell insulin resistance model, and detecting the response effect of cells on insulin stimulation;
and S103, measuring the content of the insulin (by adopting the prior art).
As shown in fig. 2, the method for measuring insulin content provided by the present invention comprises the following steps:
s201, reversibly blocking a lysine residue in an Arg-Arg precursor insulin of an amino acid sequence contained therein, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-10; partially cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin with trypsin, thereby obtaining insulin; synthesizing an isotope-labeled insulin B chain peptide fragment and preparing an isotope-labeled insulin B chain peptide fragment solution as an internal standard; preparing an insulin standard solution and an insulin sample solution;
s202, pretreating the insulin standard solution and the insulin sample solution (the prior art can be adopted);
s203, performing mass spectrometry on the pretreated insulin standard solution and the insulin sample solution;
and S204, calculating the content of the insulin in the insulin sample solution (the prior art can be adopted).
As shown in fig. 3, the pretreatment method provided by the present invention is as follows:
s301, taking 1mL of the insulin sample solution and weighing, adding 1mL of the isotope-labeled insulin B chain peptide fragment solution and weighing, uniformly mixing to obtain a labeling solution, then reducing to obtain a reduced insulin sample solution, and pretreating according to the mass fraction of insulin in the insulin sample solution;
s302, performing solid phase extraction on the reduced insulin sample solution, uniformly mixing the reduced insulin sample solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally drying the mixture;
s303, when the mass fraction of the insulin in the insulin sample solution is more than or equal to 10-7g/g, uniformly mixing the reduced insulin sample solution with a MALDI-TOF matrix, spotting on a target plate, and naturally drying; or performing solid phase extraction, mixing with MALDI-TOF matrix, spotting on target plate, and naturally air drying; 1mL of the insulin standard solution was subjected to pretreatment in the same manner as described above.
Example 2
The embodiment of the invention provides a method for measuring the regulation and control effect of miR181a on insulin resistance, which comprises the following steps:
designing an antisense sequence mutated at positions 9-12 for a sequence of mature human miR-181a, repeating 7 copies, connecting the antisense sequence in series into a pEGFP-C2 eukaryotic expression vector and a pGL3-Promoter vector, and verifying the effect of the adsorption sponge through a dual-luciferase reporter gene system;
transfecting miR-181a adsorption sponge carrier in a glucosamine-induced HepG2 cell insulin resistance model, and detecting the response effect of cells to insulin stimulation;
and step three, determining the content of insulin.
Example 3
Based on the method for measuring the regulation and control effect of miR181a on insulin resistance provided in embodiment 2, as a preferred embodiment, in the first step, the specific method for constructing the adsorption sponge comprises the steps of carrying out double enzyme digestion on pUC57-18la-SP and pEGFP-C2 empty vectors by EcoRi and BamHI, carrying out DNA gel electrophoresis, and recovering a 215bp small fragment and a pEGFP-C3 double enzyme digestion vector.
Example 4
Based on the determination method of the regulation effect of miR181a on insulin resistance provided in example 2, as a preferred example, the obtained molecular fragments were ligated with T4 ligase at 4 ℃ overnight.
Example 4
Based on the determination method of the regulation and control effect of miR181a on insulin resistance provided in example 2, as a preferred example, in step one, the effect of the adsorption sponge is verified by a dual-luciferase reporter gene system, and the constructed pEGFP-C2-181aSP luciferase reporter gene vector is co-transfected into HEK293T cells.
Example 5
Based on the method for measuring the regulation and control effect of miR181a on insulin resistance provided in example 2, as a preferred embodiment, in step two, the response effect of cells to insulin stimulation is detected, and Western blot detection is used to draw a conclusion by comparing the change of the bands with the control group.
Example 6
Based on the method for measuring the regulation and control effect of miR181a on insulin resistance provided in example 2, as a preferred embodiment, in step two, the response effect of cells to insulin stimulation is detected, and the gray value of each protein band is analyzed by ImageJ software after imaging for Western blot detection.
Example 7
Based on the determination method of the regulation and control effect of the miR181a on insulin resistance provided in example 2, as a preferred embodiment, in the third step, the determination method of the insulin content is as follows:
(1) reversibly blocking a lysine residue in an Arg-Arg precursor insulin of the amino acid sequence comprised, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-10; partially cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin with trypsin, thereby obtaining insulin; synthesizing isotope-labeled insulin B chain peptide fragment and preparing isotope-labeled insulin B chain peptide fragment solution as an internal standard;
(2) insulin standard solutions and insulin sample solutions were prepared and subjected to mass spectrometry (available in the art).
(3) The insulin content of the insulin sample solution is calculated (using known techniques).
Example 8
Based on the determination method of the regulation effect of miR181a on insulin resistance provided in example 2, as a preferred embodiment, in the third step, the insulin standard solution and the insulin sample solution are pretreated.
Example 9
Based on the determination method of the regulation and control effect of miR181a on insulin resistance provided in example 2, as a preferred embodiment, the pretreatment method is as follows:
1) weighing 1mL of the insulin sample solution, adding 1mL of the isotope-labeled insulin B chain peptide fragment solution, weighing, uniformly mixing to obtain a labeling solution, reducing to obtain a reduced insulin sample solution, and pretreating according to the mass fraction of insulin in the insulin sample solution;
2) performing solid-phase extraction on the reduced insulin sample solution, uniformly mixing the insulin sample solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally drying the mixture;
3) when the mass fraction of the insulin in the insulin sample solution is more than or equal to 10-7When the concentration is g/g, uniformly mixing the reduced insulin sample solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally airing; or performing solid phase extraction, mixing with MALDI-TOF matrix, spotting on target plate, and naturally air drying; 1mL of the insulin standard solution was subjected to pretreatment in the same manner as described above.
The sequence or vector-based method related to the embodiment of the invention can be obtained through websites and published documents, and the innovation point of the invention is how to utilize a new determination program to determine the related biological information by means of a storage medium with an operation function.
Application example
The method for measuring the regulation and control effect of miR181a on insulin resistance provided by the above embodiment of the invention can be applied to the measurement of the regulation and control effect of miR-9 on insulin resistance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The method for measuring the regulation and control effect of the miR181a on the insulin resistance is characterized in that the method for measuring the regulation and control effect of the miR181a on the insulin resistance comprises the following steps:
designing an antisense sequence mutated at 9-12 positions for a sequence of mature human miR-181a, repeating 7 copies, connecting in series into a pEGFP-C2 eukaryotic expression vector and a pGL3-Promoter vector, and verifying the effect of the adsorption sponge through a dual-luciferase reporter gene system;
transfecting a miR-181a adsorption sponge carrier in a glucosamine-induced HepG2 cell insulin resistance model, and detecting the response effect of cells on insulin stimulation;
and step three, determining the content of the insulin.
2. The method for determining the regulation and control effect of miR181a on insulin resistance, according to claim 1, wherein in step one, the specific method for constructing the adsorption sponge comprises the steps of carrying out double enzyme digestion on pUC57-18la-SP and pEGFP-C2 empty vectors by EcoRI and BamHI, carrying out DNA gel electrophoresis, and recovering a 215bp small fragment and the pEGFP-C3 double enzyme digestion vector.
3. The method for determining the regulation effect of miR181a on insulin resistance according to claim 2, wherein the obtained molecular fragments are ligated with T4 ligase at 4 ℃ overnight.
4. The method for determining the regulation and control effect of miR181a on insulin resistance, which is characterized in that in the first step, the effect of the adsorption sponge is verified through a dual-luciferase reporter gene system, and the constructed pEGFP-C2-181aSP luciferase reporter gene vector is co-transfected into HEK293T cells.
5. The method for determining the regulation effect of miR181a on insulin resistance according to claim 1, wherein in the second step, the effect of the cells on the response to insulin stimulation is detected by Western blot, and the change of the bands is compared with that of the control group to conclude.
6. The method for determining the regulation and control effect of the miR181a on insulin resistance according to claim 5, wherein in the second step, the response effect of the cells to insulin stimulation is detected, and the Western blot detection is performed, and then ImageJ software is used for analyzing the gray value of each protein band after imaging.
7. The method for measuring the regulation and control effect of the miR181a on insulin resistance according to claim 1, wherein in the third step, the method for measuring the insulin content comprises the following steps:
(1) reversibly blocking a lysine residue in an Arg-Arg precursor insulin of the amino acid sequence comprised, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-10; partially cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin with trypsin, thereby obtaining insulin; synthesizing an isotope-labeled insulin B chain peptide fragment and preparing an isotope-labeled insulin B chain peptide fragment solution as an internal standard;
(2) preparing an insulin standard solution and an insulin sample solution, and performing mass spectrometry;
(3) and calculating the content of the insulin in the insulin sample solution.
8. The method for determining the regulation effect of the miR181a on insulin resistance according to claim 1, wherein in the third step, the insulin standard solution and the insulin sample solution are subjected to pretreatment.
9. The method for determining the regulation effect of miR181a on insulin resistance according to claim 8, wherein the pretreatment method comprises the following steps:
1) weighing 1mL of the insulin sample solution, adding 1mL of the isotope-labeled insulin B chain peptide fragment solution, weighing, uniformly mixing to obtain a labeled solution, reducing to obtain a reduced insulin sample solution, and pretreating according to the mass fraction of insulin in the insulin sample solution;
2) carrying out solid phase extraction on the reduced insulin sample solution, uniformly mixing the solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally drying the mixture;
3) when the mass fraction of the insulin in the insulin sample solution is more than or equal to 10-7When the concentration is g/g, uniformly mixing the reduced insulin sample solution with a MALDI-TOF matrix, spotting the mixture on a target plate, and naturally airing; or performing solid phase extraction, mixing with MALDI-TOF matrix, spotting on target plate, and naturally air drying; 1mL of the insulin standard solution was pretreated in the same manner as described above.
10. The method for measuring the regulation and control effect of miR181a on insulin resistance according to claim 1, which is used for measuring the regulation and control effect of miR-9 on insulin resistance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210452745.6A CN114778860A (en) | 2022-04-27 | 2022-04-27 | Method for measuring regulation and control effect of miR181a on insulin resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210452745.6A CN114778860A (en) | 2022-04-27 | 2022-04-27 | Method for measuring regulation and control effect of miR181a on insulin resistance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114778860A true CN114778860A (en) | 2022-07-22 |
Family
ID=82433995
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210452745.6A Pending CN114778860A (en) | 2022-04-27 | 2022-04-27 | Method for measuring regulation and control effect of miR181a on insulin resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114778860A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981242A (en) * | 2013-02-07 | 2014-08-13 | 华凌科技有限公司 | Preparation method of insulin |
| CN104458890A (en) * | 2014-12-17 | 2015-03-25 | 中国计量科学研究院 | Method for determination of insulin content by virtue of isotopic dilution mass spectrometry |
-
2022
- 2022-04-27 CN CN202210452745.6A patent/CN114778860A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981242A (en) * | 2013-02-07 | 2014-08-13 | 华凌科技有限公司 | Preparation method of insulin |
| CN104458890A (en) * | 2014-12-17 | 2015-03-25 | 中国计量科学研究院 | Method for determination of insulin content by virtue of isotopic dilution mass spectrometry |
Non-Patent Citations (3)
| Title |
|---|
| B. ZHOU 等: "Downregulation of miR-181a upregulates sirtuin-1 (SIRT1) and improves hepatic insulin sensitivity", 《DIABETOLOGIA》, vol. 55, 4 April 2012 (2012-04-04), pages 2032 - 2043 * |
| 李伟 等: "2型糖尿病患者胰岛素抵抗与外周血单个核细胞miR-181a、miR-9的关系", 《疑难病杂志》, vol. 19, no. 8, 31 August 2020 (2020-08-31), pages 763 * |
| 牛瑜 等: "miR-181a吸附海绵载体改善肝细胞胰岛素抵抗作用研究", 《陕西医学杂志》, vol. 45, no. 7, 31 July 2016 (2016-07-31), pages 781 - 781 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bruderer et al. | Analysis of 1508 Plasma Samples by Capillary-Flow Data-Independent Acquisition Profiles Proteomics of Weight Loss and Maintenance'[S] | |
| Gobom et al. | Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry | |
| Sjöstedt et al. | A specific antibody to detect transcription factor T-Pit: a reliable marker of corticotroph cell differentiation and a tool to improve the classification of pituitary neuroendocrine tumours | |
| Okita et al. | Modified Western blotting for insulin and other diabetes-associated peptide hormones | |
| Citartan et al. | Monitoring recombinant human erythropoietin abuse among athletes | |
| Reverter-Branchat et al. | Determination of recent growth hormone abuse using a single dried blood spot | |
| Kritmetapak et al. | Chemical characterization and quantification of circulating intact PTH and PTH fragments by high-resolution mass spectrometry in chronic renal failure | |
| Hu et al. | Corin is downregulated in renal ischemia/reperfusion injury and is associated with delayed graft function after kidney transplantation | |
| Bielohuby et al. | Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits | |
| Cox et al. | Sensitive quantification of IGF-1 and its synthetic analogs in dried blood spots | |
| Kam et al. | Serum insulin-like growth factor I quantitation by mass spectrometry: insights for protein quantitation with this technology | |
| Jasuja et al. | Accurate measurement and harmonized reference ranges for total and free testosterone levels | |
| Wang et al. | Differentiation and quantification of endogenous and recombinant-methionyl human leptin in clinical plasma samples by immunocapture/mass spectrometry | |
| US20160018420A1 (en) | Methods and systems for the diagnosis and treatment of androgen disorders | |
| Pinyot et al. | Growth hormone secretagogues: out of competition | |
| Ding et al. | Perspective: proteomic approach to detect biomarkers of human growth hormone | |
| CN114778860A (en) | Method for measuring regulation and control effect of miR181a on insulin resistance | |
| Mizuguchi et al. | Prediction of response to medical therapy by serum soluble (pro) renin receptor levels in Graves’ disease | |
| Cawley et al. | Biomarker ratios | |
| Lo et al. | Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method | |
| Bouwman et al. | Novel markers for tying-up in horses by proteomics analysis of equine muscle biopsies | |
| Abdulwahab et al. | Association of TATA box-binding protein-associated factor RNA polymerase I subunit C (TAF1C) with T2DM | |
| Cho et al. | Regional differences of proteins expressing in adipose depots isolated from cows, steers and bulls as identified by a proteomic approach | |
| Hamza | Biological assay of insulin: an old problem rediscovered | |
| Chilelli et al. | In vitro chronic glycation induces AGEs accumulation reducing insulin-stimulated glucose uptake and increasing GLP1R in adipocytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |