CN109406769A - The application of AKR insulin secretion Mechanism Study in MIN6 cell - Google Patents

The application of AKR insulin secretion Mechanism Study in MIN6 cell Download PDF

Info

Publication number
CN109406769A
CN109406769A CN201811142521.5A CN201811142521A CN109406769A CN 109406769 A CN109406769 A CN 109406769A CN 201811142521 A CN201811142521 A CN 201811142521A CN 109406769 A CN109406769 A CN 109406769A
Authority
CN
China
Prior art keywords
added
cell
min6
insulin
insulin secretion
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
Application number
CN201811142521.5A
Other languages
Chinese (zh)
Inventor
李军
李思源
张君
李含晖
尹亮
火睿
李佳佳
胡颖
王双
石艳秋
胡倩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CN201811142521.5A priority Critical patent/CN109406769A/en
Publication of CN109406769A publication Critical patent/CN109406769A/en
Pending legal-status Critical Current

Links

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
    • G01N33/53—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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses a kind of applications of AKR insulin secretion Mechanism Study in MIN6 cell, under different glucose environment, the GC that various concentration level is arranged intervenes, by stimulating PKA signal path, and before and after adding stimulant ISO, utilize the content of FRET technology real-time quantitative measurement PKA, how discussion GC passes through second messenger PKA signal path plays a role in beta Cell of islet insulin secretion, to further supplement our about the shortcoming in the study of incident mechanism of diabetes B early periods, and the foundation in terms of for the research and development of new diabetes target therapeutic agent providing laboratory.

Description

The application of AKR insulin secretion Mechanism Study in MIN6 cell
Technical field
The invention belongs to medicine technology fields, specifically, being related to AKR insulin secretion Mechanism Study in MIN6 cell Application.
Background technique
The correlative study of early period shows in the case where different concentration of glucose, the GC of various concentration by cAMP, DAG, The signal paths approach such as PKC, raises its content in the cell, is known as facilitation to islet β cell pancreas islet, still Whether the downstream signaling molecule PKA of cAMP signal path influences effect of the GC in terms of the secretion of beta Cell of islet insulin, if Influence to be again how to influence the problem of GC plays a role to need to be further clarified.
Summary of the invention
The purpose of the present invention is to provide a kind of applications of AKR insulin secretion Mechanism Study in MIN6 cell, not With under concentration of glucose environment, the GC of setting various concentration level intervenes, and pierces by stimulating PKA signal path, and in addition Before and after swashing agent ISO, using the content of FRET technology real-time quantitative measurement PKA, it is logical how discussion GC passes through second messenger's PKA signal Road plays a role in beta Cell of islet insulin secretion, to further supplement in the study of incident mechanism about diabetes B Shortcoming, and the foundation in terms of providing laboratory for the research and development of new diabetes target therapeutic agent.
Itself the specific technical proposal is:
A kind of method of AKR insulin secretion Mechanism Study in MIN6 cell, comprising the following steps:
Step 1, cell culture
With containing 15% fetal calf serum (FBS), 4.5g/L glucose, 2% mycillin, 1%L- glutamine and 1% β-mercapto The DMEM culture medium of base ethyl alcohol is cultivated in 37 DEG C of incubators containing 5%CO2, it is alternative in experiment to be passaged to 4~15.
Step 2, grouping
By MIN6 cell with 4 × 105/ hole is cultivated in 6 well culture plates, in logarithmic growth phase for testing.One group is used for ELISA detection MIN6 is intracellular to be not added with ISO insulin release, and one group is detected MIN6 addition ISO insulin intracellular for ELISA Burst size, cell is divided into behind 3 parts to be cultivated under sugar-free, low sugar, high saccharide ring border respectively, give later various concentration Glg (0, 500,1000ng/L) intervention processing；One group horizontal for FRET technology detection PKA.
Step 3, method
3.1 are based on FRET technology Real_time quantitative detection PKA in the case where different glucose and GC are intervened；
The burst size of 3.2ELISA method detection each group MIN6 cell insulin；
3.2.1 MIN6 cell insulin sample is acquired；
(1) passage of MIN6 cell is in good condition to its adherent stabilization in 56 well culture plates；
(2) culture medium is carefully sucked out, change contains the culture medium of 2%FBS (other ingredients and ratio are identical), continues at 37 DEG C It is cultivated under environment；
(3) original culture medium is sucked out after cultivating about 12h, is gently rinsed 1 time with Krebs liquid, 1ml is added in every hole culture dish Krebs liquid is incubated for 2h under 37 DEG C of environment；
(4) original Krebs liquid is sucked out, is separately added into treatment fluid 1ml in Xiang Gekong culture dish, blank control group is directly added into 1ml
Kerbs, 37 DEG C of incubators are incubated for 1h；
(5) cell supernatant in every hole is drawn respectively, moves into 1.5ml centrifuge tube, marks rear clip sealed membrane sealing, It is to be measured to be stored in -20 DEG C of refrigerators；
3.2.2 insulin in MIN6 cell conditioned medium is measured
(1) prepare kit:
(2) program is detected:
(3) result calculates and judges:
1. with 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ ml、
The standard items of 0pg/ml isoconcentration draw canonical plotting using corresponding OD value as ordinate for abscissa；
2. calculating corresponding insulin content on standard curve according to the cell supernatant sample OD value measured, being multiplied by Insulin content in sample is obtained after extension rate.
Step 4, statistical method
Data processing is carried out using SPSS17.0 statistical software, measurement data is with mean ± standard deviationIt indicates, it is more The comparison of mean uses one-way analysis of variance between group, and using two-sided test, P < 0.05 thinks that difference has statistical significance.
Preferably, step 3.1 specifically:
(1) the MIN6 cell after fluorescence microscopy under the microscope transfection 48h, fluorescent protein expression is good, transfection efficiency About 80%-90%；
(2) original culture medium is carefully sucked out, Krebs liquid rinses 2 times；
(3) 0.5ml Krebs liquid is added in each culture dish, puts back to incubator；
(4) appoint and take a culture dish, be placed under laser confocal microscope, use oil mirror observation instead after positioning individual cells；
(5) excitation wavelength and launch wavelength range are set on laser co-focusing computer software, start to acquire image；
(6) each culture dish sequentially adds drug according to experimental group；
It (7) is that ordinate is drawn using the time as abscissa, CFP/YFP ratio.
Further, the preparation kit specifically:
1. opening 10 × sample diluent mother liquor packaging, according to 10 × sample diluent: distilled water=1:10 ratio adds Enter distilled water dilution sample diluent；
2. the preparation of standard items liquid: standard items liquid packaging is opened, 500 μ l distilled waters are added with micropipette rifle and mix, it will Its solution for being configured to 40ng/ml.Prepare 8 clean centrifuge tubes, marks 1-8 respectively, it is dilute that 900 μ l samples are added in Xiang Guan 1 Liquid is released, 500 μ l sample diluents are respectively added into pipe 8 for pipe 2.The standard solution that 100 μ l 40ng/ml are added into pipe 1 is mixed It is even, move to pipe 2 from 500 μ l are wherein sucked out with micropipette rifle, operate repeatedly, each pipe solution is diluted, finally from 500 μ l are sucked out in pipe 7 to discard, pipe 8 is used as blank control；
3. according to cleaning solution mother liquor liquid: distilled water=1:10 ratio dilutes cleaning solution；
4. according to sample: distilled water=1:20 ratio dilutes the cell supernatant sample being collected into.
Further, the detection program specifically:
1. drawing the standard items of 100 μ l respectively, it is added in each reacting hole, mixes well, is stood under 37 DEG C of environment 40min；
2. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted about 5 times；
3. respectively add 50 μ l distilled waters and first antibody working solution in addition to blank group to each reacting hole, mix well, 37℃
20min is stood under environment；
4. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted about 5 times；
5. drawing the enzyme labelled antibody working solution of 100 μ l respectively, it is added in each reacting hole, mixes well, in 37 DEG C of ring 10min is stood under border；
6. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted about 5 times；
7. drawing the substrate working solution of 100 μ l respectively, it is added in each reacting hole, mixes well, is placed on 37 DEG C of environment Under be protected from light 15min；
8. drawing the terminate liquid of 100 μ l respectively, it is added in each reacting hole, mixes well；
9. surveying OD value at 450nm with Bio-RAD microplate reader, this operation must be completed in 30min.
10. same method detects sample to be tested.
The method of AKR of the present invention insulin secretion Mechanism Study in MIN6 cell is in diabetes target therapeutic agent Application in preparation process.
Compared with prior art, beneficial effects of the present invention:
The present invention is under different glucose environment, and the GC of setting various concentration level intervenes, by stimulating PKA signal Access, and before and after adding stimulant ISO, using the content of FRET technology real-time quantitative measurement PKA, inquire into how GC passes through Second messenger PKA signal path plays a role in beta Cell of islet insulin secretion, to further supplement about 2 type glycosurias Shortcoming in the study of incident mechanism of disease, and for the research and development of new diabetes target therapeutic agent provide in terms of laboratory according to According to.
Detailed description of the invention
MIN6 cell after transfection AKR 48h is changed liquid, added by detection of the Fig. 1 without the low intracellular PKA of GC group MIN6 of glucose Enter 0.5ml Krebs liquid, be placed under laser confocal microscope, uses oil mirror observation instead after positioning the individual cells visual field, setting swashs Hair wavelength and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scan to 75s, this is space management Stage adds 0.5ml krebs liquid in culture dish, and holding concentration of glucose is 0mmol/L, scans to 385s, this is 0ng/L The GC stage is equal to the space management stage, and the GC liquid of the final concentration of 500ng/L of 0.5ml is added, and scans to 745s, this is Stimulant ISO is added in the 500ng/L GC stage, about stops scanning to 1010s, wherein A, B, C are respectively CFP, YFP, FRET existing The fluorescent image of elephant, the overlapping image of D CFP, YFP, FRET, E are the scanning screenshot of dynamic FRET phenomenon；
Situation of change of the Fig. 2 without the low GC group MIN6 cell CFP/YFP of glucose, compared with the space management stage, #P ﹤ 0.05；Compared with the 500ng/L GC stage,*P ﹤ 0.05.
MIN6 cell after transfection AKR 48h is changed liquid, added by detection of the Fig. 3 without the high intracellular PKA of GC group MIN6 of glucose Enter 0.5ml Krebs liquid, be placed under laser confocal microscope, uses oil mirror observation instead after positioning the individual cells visual field, setting swashs Hair wavelength and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scan to 75s, this is space management Stage adds 0.5ml krebs liquid in culture dish, and holding concentration of glucose is 0mmol/L, scans to 385s, this is 0ng/L The GC stage is equal to the space management stage, and the GC liquid of the final concentration of 1000ng/L of 0.5ml is added, and scans to 745s, this is Stimulant ISO is added in the 1000ng/L GC stage, about stops scanning to 1010s, wherein A, B, C are respectively CFP, YFP, FRET The fluorescent image of phenomenon, the overlapping image of D CFP, YFP, FRET, E are the scanning screenshot of dynamic FRET phenomenon；
Fig. 4 is the situation of change of the high GC group MIN6 cell CFP/YFP of no glucose, compared with the space management stage, #P ﹤ 0.05；Compared with the 1000ng/L GC stage,*P ﹤ 0.05.
MIN6 cell after transfection AKR 48h is changed liquid, added by the detection of the low intracellular PKA of GC group MIN6 of Fig. 5 low glucose Enter 0.5ml Krebs liquid, be placed under laser confocal microscope, it is determined as uses oil mirror observation after individual cells instead, excitation wave is set Long and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scanning to 75s, this is space management rank The glucose of the final concentration of 2.8mmol/L of 0.5ml is added in culture dish in section, and holding concentration of glucose is 2.8mmol/L, is swept It retouches to 385s, this is the 0ng/L GC stage, and the GC liquid of the final concentration of 500ng/L of 0.5ml is added, and is scanned to 745s, this is Stimulant ISO is added in the 500ng/L GC stage, about stops scanning to 1010s, wherein A, B, C are respectively CFP, YFP, FRET existing The fluorescent image of elephant, the overlapping image of D CFP, YFP, FRET, E are the scanning screenshot of dynamic FRET phenomenon；
The situation of change of the low GC group MIN6 cell CFP/YFP of Fig. 6 low glucose, compared with the space management stage, #P ﹤ 0.05；Compared with the 0ng/L GC stage,*P ﹤ 0.05；Compared with the 500ng/L GC stage, △ P ﹤ 0.05.
MIN6 cell after transfection AKR 48h is changed liquid, added by the detection of the high intracellular PKA of GC group MIN6 of Fig. 7 low glucose Enter 0.5ml Krebs liquid, be placed under laser confocal microscope, uses oil mirror observation instead after positioning the individual cells visual field, setting swashs Hair wavelength and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scan to 75s, this is space management The glucose of the final concentration of 2.8mmol/L of 0.5ml is added in culture dish in stage, and holding concentration of glucose is 2.8mmol/L, To 385s, this is the 0ng/L GC stage, and the GC liquid of the final concentration of 1000ng/L of 0.5ml is added, scans to 745s, this is for scanning Stimulant ISO is added in the 1000ng/L GC stage, about stops scanning to 1010s, about stops scanning to 1010s, wherein A, B, C The respectively fluorescent image of CFP, YFP, FRET phenomenon, the overlapping image of D CFP, YFP, FRET, E are dynamic FRET phenomenon Scan screenshot；
The situation of change of the high GC group MIN6 cell CFP/YFP of Fig. 8 low glucose, compared with the space management stage, #P ﹤ 0.05；Compared with the 0ng/L GC stage,*P ﹤ 0.05；Compared with the 1000ng/L GC stage, △ P ﹤ 0.05.
MIN6 cell after transfection AKR 48h is changed liquid, added by the detection of the low intracellular PKA of GC group MIN6 of Fig. 9 high glucose Enter 0.5ml Krebs liquid, be placed under laser confocal microscope, uses oil mirror observation instead after positioning the individual cells visual field, setting swashs Hair wavelength and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scan to 75s, this is space management The glucose of the final concentration of 16.7mmol/L of 0.5ml is added in culture dish in stage, and holding concentration of glucose is 16.7mmol/ L, scanning to 385s, this is the 0ng/L GC stage, and the GC liquid of the final concentration of 500ng/L of 0.5ml is added, scans to 745s, this is Stimulant ISO is added in the 500ng/L GC stage, about stops scanning to 1010s, about stops scanning to 1010s, wherein A, B, C points Not Wei CFP, YFP, FRET phenomenon fluorescent image, the overlapping image of D CFP, YFP, FRET, E is that dynamic FRET phenomenon is swept Retouch screenshot；
The situation of change of the low GC group MIN6 cell CFP/YFP of Figure 10 high glucose, note: compared with the space management stage, #P ﹤ 0.05；Compared with the 0ng/L GC stage,*P ﹤ 0.05；Compared with the 500ng/L GC stage, △ P ﹤ 0.05.
MIN6 cell after transfection AKR 48h is changed liquid by the detection of the high intracellular PKA of GC group MIN6 of Figure 11 high glucose, 0.5ml Krebs liquid is added, is placed under laser confocal microscope, uses oil mirror observation, setting instead after positioning the individual cells visual field Excitation wavelength and launch wavelength range, sweep time are set as 25s/ times, start Image Acquisition, scan to 75s, this is blank space The glucose of the final concentration of 16.7mmol/L of 0.5ml is added in culture dish in the reason stage, and holding concentration of glucose is 16.7mmol/L, scanning to 385s, this is the 0ng/L GC stage, the GC liquid of the final concentration of 1000ng/L of 0.5ml is added, scanning is extremely 745s, this is the 1000ng/L GC stage, and stimulant ISO is added, about stops scanning to 1010s, about stops scanning to 1010s, In, A, B, C are respectively the fluorescent image of CFP, YFP, FRET phenomenon, and the overlapping image of D CFP, YFP, FRET, E is dynamic The scanning screenshot of FRET phenomenon；
The situation of change of the high GC group MIN6 cell CFP/YFP of Figure 12 high glucose, note: compared with the space management stage, #P ﹤ 0.05；Compared with the 0ng/L GC stage,*P ﹤ 0.05；Compared with the 1000ng/L GC stage, △ P ﹤ 0.05.
Figure 13 various concentration GC generates the comparison influenced to PKA, and "-" indicates that, without addition ISO, "+" indicates addition ISO； Compared with 500ng/L GC, #P ﹤ 0.05；Compared with 500ng/LGC ISO+,*P ﹤ 0.05.
The comparison of different disposal group amount of insulin secretion when Figure 14 is without ISO, with compared with group 0ng/L GC level, #P ﹤ 0.05；With compared with group 500ng/L GC level,*P ﹤ 0.05.
The comparison of different disposal group amount of insulin secretion when Figure 15 adds ISO, with compared with group 0ng/L GC level, #P ﹤ 0.05；With compared with group 500ng/L GC level,*P ﹤ 0.05.
Figure 16 adds the comparison of different disposal group Δ amount of insulin secretion before and after ISO, and Δ amount of insulin secretion is addition thorn Swash the incrementss of the front and back agent ISO amount of insulin secretion；Compared with 0ng/L GC level, #P ﹤ 0.05；With the horizontal phase of 500ng/L GC Than,*P ﹤ 0.05.
Specific embodiment
Technical solution of the present invention is described in more detail with specific embodiment with reference to the accompanying drawing.
1. experimental material
Beta Cell of islet system MIN6 cell, PCDNA3.1 and AKR plasmid.Main agents: isoprenaline hydrochloride (ISO), 0.25% trypsin solution containing EDTA-, DMEM culture medium, mouse INS-ELISA kit, cAMP-ELISA kit, plasmid Big extraction reagent kit etc..
2, cell culture
With containing 15% fetal calf serum (FBS), 4.5g/L glucose, 2% mycillin, 1%L- glutamine and 1% β-mercapto The DMEM culture medium of base ethyl alcohol is cultivated in 37 DEG C of incubators containing 5%CO2, it is alternative in experiment to be passaged to 4~15.
3, it is grouped
By MIN6 cell with 4 × 105/ hole is cultivated in 6 well culture plates, in logarithmic growth phase for testing.One group is used for ELISA detection MIN6 is intracellular to be not added with ISO insulin release, and one group is detected MIN6 addition ISO insulin intracellular for ELISA Burst size, cell is divided into behind 3 parts to be cultivated under sugar-free, low sugar, high saccharide ring border respectively, give later various concentration Glg (0, 500,1000ng/L) intervention processing；One group horizontal for FRET technology detection PKA, and following (table 1,2,3) is arranged in grouping.
Grouping situation of the table 1 based on FRET technology the real time measure PKA level
Note: "-" indicates that, without addition ISO, "+" indicates addition ISO
Detect the grouping of insulin release when table 2 does not add ISO into the cell in MIN6
The grouping of insulin release is detected into the cell in MIN6 after the addition of table 3 ISO
4, statistical method
Data processing is carried out using SPSS17.0 statistical software, measurement data is with mean ± standard deviationIt indicates, it is more The comparison of mean uses one-way analysis of variance between group, and using two-sided test, P < 0.05 thinks that difference has statistical significance.
It is different under Real_time quantitative detection different glucose environment in MIN6 cell that embodiment 1. is based on FRET technology Influence of the concentration GC level to PKA content
Each group A, B, C are respectively the fluorescent image of CFP, YFP, FRET phenomenon, the overlapping image of D CFP, YFP, FRET, E For the scanning screenshot of dynamic FRET phenomenon.
1.1 detections without the low intracellular PKA of GC group MIN6 of glucose
As the result is shown: under sugar-free environment, being added after 500ng/L GC compared with the space management stage, on CFP/YFP ratio Rise (P < 0.05), after ISO stimulation is added, CFP/YFP ratio rise compared with space management stage and 500ng/L GC stage (P < 0.05) (Fig. 2).
1.2 detections without the high intracellular PKA of GC group MIN6 of glucose
As the result is shown: under sugar-free environment, being added after 1000ng/L GC stimulation compared with the space management stage, CFP/YFP ratio Value rises (P < 0.05), and after ISO stimulation is added, CFP/YFP ratio rises compared with space management stage and 1000ng/L GC stage (P < 0.05) (Fig. 4).
The detection of the low intracellular PKA of GC group MIN6 of 1.3 low glucoses
As the result is shown: under low sugar environment, being added after low sugar stimulation compared with the space management stage, CFP/YFP ratio rises (P < 0.05) is added after 500ng/L GC stimulation compared with space management stage and 0ng/L GC stage, and CFP/YFP ratio rises (P < 0.05), after ISO stimulation is added, CFP/YFP ratio is compared with space management stage, 0ng/L GC stage and 500ng/L GC stage Obviously rise (P < 0.05) (Fig. 6).
The detection of the high intracellular PKA of GC group MIN6 of 1.4 low glucoses
As the result is shown: under low sugar environment, being added after low sugar stimulation compared with the space management stage, CFP/YFP ratio rises (P < 0.05) is added after 1000ng/L GC stimulation compared with space management stage and 0ng/L GC stage, on CFP/YFP ratio It rises (P < 0.05)；After ISO stimulation is added, CFP/YFP ratio is compared with space management stage, 0ng/L GC stage and 1000ng/L GC Stage obviously rises (P < 0.05) (Fig. 8).
The detection of the low intracellular PKA of GC group MIN6 of 1.5 high glucoses
As the result is shown: under high saccharide ring border, being added after high sugar stimulation compared with the space management stage, CFP/YFP ratio rises (P < 0.05) is added after 500ng/L GC stimulation compared with space management stage and 0ng/L GC stage, and CFP/YFP ratio rises (P<0.05)；After ISO stimulation is added, CFP/YFP ratio is compared with space management stage, 0ng/L GC stage and 500ng/L GC stage Obviously rise (P < 0.05) (Figure 10).
The intracellular PKA content detection of the high GC group MIN6 of 1.6 high glucoses
As the result is shown: under high saccharide ring border, being added after high sugar stimulation compared with the space management stage, CFP/YFP ratio rises (P < 0.05) is added after 1000ng/L GC stimulation compared with space management stage and 0ng/L GC stage, on CFP/YFP ratio It rises (P < 0.05)；After ISO stimulation is added, CFP/YFP ratio is compared with space management stage, 0ng/L GC stage and 1000ng/L GC Stage obviously rises (P < 0.05) (Figure 12).
Different level GC, which intervenes, under 1.7 different concentration of glucose generates the comparison influenced to PKA
Under 0mmol/L concentration environment, 500ng/L GC stage CFP/YFP ratio rises 9.73%, 1000ng/L GC rank Duan Shangsheng 10.26%, indifference between two intervention stages (P equal > 0.05), the 500ng/L GC rank after adding stimulant ISO Section CFP/YFP ratio rises 34.36%, the 1000ng/L GC stage and rises 34.28%, between two stages still indifference (P is equal > 0.05)；Under 2.8mmol/L concentration environment, 500ng/L GC stage CFP/YFP ratio rises 17.04%, 1000ng/L GC Stage rises 26.12%, 1000ng/L GC stage climbing and is higher than 500ng/L GC stage (P < 0.05), after stimulant is added 500ng/L GC stage CFP/YFP ratio rising 52.59%, the 1000ng/L GC stage rises 69.31%, 1000ng/L GC rank Section climbing is higher than 500ng/L GC stage (P < 0.05)；Under 16.7mmol/L concentration environment, 500ng/L GC stage CFP/ YFP ratio rising 42.53%, the 1000ng/L GC stage rises 23%, 1000ng/L GC stage climbing higher than 500ng/L In the GC stage (P < 0.05), 500ng/L GC stage CFP/YFP ratio rises 75.37%, 1000ng/L GC rank after stimulant is added It is higher than 500ng/L GC stage (P < 0.05) (Figure 13) on Duan Shangsheng 104.59%, 1000ng/L the GC stage.
2.ELISA method detects influence of the various concentration GC to each group MIN6 cell insulin secretion amount
2.1 detect amount of insulin secretion in the case where no addition stimulant ISO
In 1 group of sugar-free, when amount of insulin secretion is higher than 0ng/L GC after 500ng/L GC intervenes (P < 0.05), 1000ng/L When amount of insulin secretion is higher than 500ng/L GC and 0ng/L GC after GC intervenes (P < 0.05)；1 group of low sugar, 500ng/L GC intervenes When amount of insulin secretion is higher than 0ng/L GC afterwards (P < 0.05), amount of insulin secretion is higher than 500ng/L after 1000ng/L GC intervenes When GC and 0ng/L GC (P < 0.05)；High 1 group of sugar, when amount of insulin secretion is higher than 0ng/L GC after 500ng/L GC intervenes (P < 0.05) when amount of insulin secretion is higher than 500ng/L GC and 0ng/L GC after, 1000ng/L GC intervenes (P < 0.05) (Figure 14).
2.2 detect amount of insulin secretion in the case where adding stimulant ISO
As the result is shown: 2 groups of sugar-free, when amount of insulin secretion is higher than 0ng/L GC after 500ng/L GC intervenes (P < 0.05), When amount of insulin secretion is higher than 500ng/L GC and 0ng/L GC after 1000ng/L GC intervenes (P < 0.05)；2 groups of low sugar, When amount of insulin secretion is higher than 0ng/L GC after 500ng/L GC intervenes (P < 0.05), insulin point after 1000ng/L GC intervenes When the amount of secreting is higher than 500ng/L GC and 0ng/L GC (P < 0.05)；High 2 groups of sugar, amount of insulin secretion is high after 500ng/L GC intervenes When 0ng/L GC (P < 0.05), when amount of insulin secretion is higher than 500ng/L GC and 0ng/L GC after 1000ng/L GC intervenes (P < 0.05) (Figure 15).
The comparison of amount of insulin secretion before and after 2.3 addition stimulant ISO
Under sugar-free environment, indifference (P > 0.05) after 500ng/L GC intervenes after intervening with 0ng/L GC, 1000ng/L GC Indifference (P > 0.05) after intervening after intervention with 500ng/L GC, 1000ng/L GC have difference after intervening after intervening with 0ng/L GC It is different, and 1000ng/L GC intervene after amount of insulin secretion incrementss be higher than 0ng/L GC when (P < 0.05)；Under low sugar environment, 500ng/L GC intervene after with 0ng/L GC intervene after it is variant, and 500ng/L GC intervene after amount of insulin secretion incrementss When higher than 0ng/L GC (P<0.05), indifference (P>0.05) after 1000ng/L GC intervenes after intervening with 500ng/L GC, 1000ng/L GC intervene after with 0ng/L GC intervene after it is variant, and 1000ng/L GC intervene after amount of insulin secretion increase When amount is higher than 0ng/L GC (P < 0.05)；Under high saccharide ring border, 500ng/L GC is variant after intervening after intervening with 0ng/L GC, and When the incrementss of amount of insulin secretion are higher than 0ng/L GC after 500ng/L GC intervenes (P < 0.05), after 1000ng/L GC intervenes After intervening with 500ng/L GC and 0ng/L GC intervene after variant, and amount of insulin secretion after 1000ng/L GC intervention When incrementss are higher than 500ng/L GC and 0ng/L GC (P < 0.05) (Figure 16).
Conclusion
1.GC promotes the secretion of insulin by increasing the concentration of the intracellular PKA of MIN6 in the form of concentration gradient；
2. adding, GC after stimulant ISO increases PKA and the effect of insulin secretion is more obvious, illustrates that GC promotes insulin Secretion acts through the realization of second messenger's signal system PKA access approach；
3.GC promotes the effect of insulin secretion to have certain glucose dependency by increasing the concentration of PKA.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to Altered or equivalence replacement are fallen within the protection scope of the present invention.

Claims (5)

1. a kind of application of AKR insulin secretion Mechanism Study in MIN6 cell, which comprises the following steps:
Step 1, cell culture
With containing 15% fetal calf serum, 4.5g/L glucose, 2% mycillin, 1%L- glutamine and 1% beta -mercaptoethanol DMEM culture medium contains 5%CO at 37 DEG C2Incubator in cultivate, it is alternative in experiment to be passaged to 4~15；
Step 2, grouping
By MIN6 cell with 4 × 105/ hole is cultivated in 6 well culture plates, in logarithmic growth phase for testing；One group is used for ELISA Detection MIN6 is intracellular to be not added with ISO insulin release, and one group is detected MIN6 addition ISO insulin releasing intracellular for ELISA Amount, cell is divided into behind 3 parts to be cultivated under sugar-free, low sugar, high saccharide ring border respectively, give later various concentration Glg:0,500, 1000ng/L, intervention processing；One group horizontal for FRET technology detection PKA；
Step 3, method
3.1 are based on FRET technology Real_time quantitative detection PKA in the case where different glucose and GC are intervened；
The burst size of 3.2ELISA method detection each group MIN6 cell insulin；
3.2.1 MIN6 cell insulin sample is acquired；
(1) passage of MIN6 cell is in good condition to its adherent stabilization in 56 well culture plates；
(2) culture medium is carefully sucked out, changes to the culture medium containing 2%FBS, continuation is cultivated under 37 DEG C of environment；
(3) original culture medium is sucked out after cultivating 12h, is gently rinsed 1 time with Krebs liquid, 1ml Krebs is added in every hole culture dish Liquid is incubated for 2h under 37 DEG C of environment；
(4) original Krebs liquid is sucked out, is separately added into treatment fluid 1ml in Xiang Gekong culture dish, blank control group is directly added into 1ml Kerbs, 37 DEG C of incubators are incubated for 1h；
(5) cell supernatant in every hole is drawn respectively, moves into 1.5ml centrifuge tube, is marked rear clip sealed membrane sealing, is saved It is to be measured in -20 DEG C of refrigerators；
3.2.2 insulin in MIN6 cell conditioned medium is measured
(1) prepare kit:
(2) program is detected:
(3) result calculates and judges:
1. with 4000pg/ml, 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, The standard items of 0pg/ml concentration draw canonical plotting using corresponding OD value as ordinate for abscissa；
2. calculating corresponding insulin content on standard curve according to the cell supernatant sample OD value measured, being multiplied by dilution Insulin content in sample is obtained after multiple；
Step 4, statistical method
Data processing is carried out using SPSS17.0 statistical software, measurement data is indicated with mean ± standard deviation (x ± s), between multiple groups The comparison of mean uses one-way analysis of variance, and using two-sided test, P < 0.05 thinks that difference has statistical significance.
2. the application of AKR according to claim 1 insulin secretion Mechanism Study in MIN6 cell, which is characterized in that Step 3.1 specifically:
(1) the MIN6 cell after fluorescence microscopy under the microscope transfection 48h, fluorescent protein expression is good, transfection efficiency 80%- 90%；
(2) original culture medium is carefully sucked out, Krebs liquid rinses 2 times；
(3) 0.5ml Krebs liquid is added in each culture dish, puts back to incubator；
(4) appoint and take a culture dish, be placed under laser confocal microscope, use oil mirror observation instead after positioning individual cells；
(5) excitation wavelength and launch wavelength range are set on laser co-focusing computer software, start to acquire image；
(6) each culture dish sequentially adds drug according to experimental group；
It (7) is that ordinate is drawn using the time as abscissa, CFP/YFP ratio.
3. the application of AKR according to claim 1 insulin secretion Mechanism Study in MIN6 cell, which is characterized in that The preparation kit specifically:
1. opening 10 × sample diluent mother liquor packaging, according to 10 × sample diluent: distilled water=1:10 ratio is added double It steams water and dilutes sample diluent；
2. the preparation of standard items liquid: opening standard items liquid packaging, 500 μ l distilled waters are added with micropipette rifle and mix, are matched The solution of 40ng/ml is made；Prepare 8 clean centrifuge tubes, mark 1-8 respectively, 900 μ l sample diluents are added in Xiang Guan 1, 500 μ l sample diluents are respectively added into pipe 8 for pipe 2；The standard solution that 100 μ l 40ng/ml are added into pipe 1 mixes, and use is micro- It measures liquid-transfering gun and moves to pipe 2 from 500 μ l are wherein sucked out, operate repeatedly, each pipe solution is diluted, is finally inhaled from pipe 7 500 μ l are discarded out, and pipe 8 is used as blank control；
3. according to cleaning solution mother liquor liquid: distilled water=1:10 ratio dilutes cleaning solution；
4. according to sample: distilled water=1:20 ratio dilutes the cell supernatant sample being collected into.
4. the application of AKR according to claim 1 insulin secretion Mechanism Study in MIN6 cell, which is characterized in that The detection program specifically:
1. drawing the standard items of 100 μ l respectively, it is added in each reacting hole, mixes well, stands 40min under 37 DEG C of environment；
2. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted, 5 times；
3. respectively adding 50 μ l distilled waters and first antibody working solution in addition to blank group to each reacting hole, mixing well, at 37 DEG C 20min is stood under environment；
4. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted 5 times；
5. drawing the enzyme labelled antibody working solution of 100 μ l respectively, it is added in each reacting hole, mixes well, under 37 DEG C of environment Stand 10min；
6. the moisture on reaction plate is blotted with filter paper with the abundant washing reaction plate of the cleaning solution diluted 5 times；
7. drawing the substrate working solution of 100 μ l respectively, it is added in each reacting hole, mixes well, is placed under 37 DEG C of environment and keeps away Light reaction 15min；
8. drawing the terminate liquid of 100 μ l respectively, it is added in each reacting hole, mixes well；
9. surveying OD value at 450nm with Bio-RAD microplate reader, this operation must be completed in 30min；
10. same method detects sample to be tested.
5. the method for the insulin secretion Mechanism Study in MIN6 cell of AKR described in claim 1 is in diabetes targeted therapy medicine Application in object preparation process.
CN201811142521.5A 2018-09-28 2018-09-28 The application of AKR insulin secretion Mechanism Study in MIN6 cell Pending CN109406769A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811142521.5A CN109406769A (en) 2018-09-28 2018-09-28 The application of AKR insulin secretion Mechanism Study in MIN6 cell

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811142521.5A CN109406769A (en) 2018-09-28 2018-09-28 The application of AKR insulin secretion Mechanism Study in MIN6 cell

Publications (1)

Publication Number Publication Date
CN109406769A true CN109406769A (en) 2019-03-01

Family

ID=65466433

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811142521.5A Pending CN109406769A (en) 2018-09-28 2018-09-28 The application of AKR insulin secretion Mechanism Study in MIN6 cell

Country Status (1)

Country Link
CN (1) CN109406769A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050872A2 (en) * 1999-02-26 2000-08-31 Cellomics, Inc. A system for cell-based screening
DE10306085A1 (en) * 2002-09-06 2004-03-25 Forschungsverbund Berlin E.V. New nucleic acid encoding splice variant of protein kinase A anchor protein, useful e.g. for identifying agents for treating diabetes or hypertension, also new polypeptide
CN1643134A (en) * 2002-03-22 2005-07-20 拜尔药品公司 Method for producing pseudo islets
US20060154330A1 (en) * 2002-09-06 2006-07-13 Enno Klussmann Akap18 delta, a novel splicing variant of a protein kinase a anchor protein and the use of the same
US20070003532A1 (en) * 2001-03-30 2007-01-04 Biostratum Ab Novel mechanism for identifying drugs for the treatment of type II diabetes
US20140357556A1 (en) * 2012-03-01 2014-12-04 The Regents Of The University Of California Novel Target for Diagnosis and Treatment of Diabetes and Cardiovascular Diseases

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050872A2 (en) * 1999-02-26 2000-08-31 Cellomics, Inc. A system for cell-based screening
US20070003532A1 (en) * 2001-03-30 2007-01-04 Biostratum Ab Novel mechanism for identifying drugs for the treatment of type II diabetes
CN1643134A (en) * 2002-03-22 2005-07-20 拜尔药品公司 Method for producing pseudo islets
DE10306085A1 (en) * 2002-09-06 2004-03-25 Forschungsverbund Berlin E.V. New nucleic acid encoding splice variant of protein kinase A anchor protein, useful e.g. for identifying agents for treating diabetes or hypertension, also new polypeptide
US20060154330A1 (en) * 2002-09-06 2006-07-13 Enno Klussmann Akap18 delta, a novel splicing variant of a protein kinase a anchor protein and the use of the same
US20140357556A1 (en) * 2012-03-01 2014-12-04 The Regents Of The University Of California Novel Target for Diagnosis and Treatment of Diabetes and Cardiovascular Diseases

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KATSUYA DEZAKI等: "Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet beta-Cells" *
丁艳洁等: "胰高血糖素对不同葡萄糖培养条件下胰岛β细胞分泌胰岛素的影响及机制" *
常子涛: "胰高血糖素通过cAMP信号通路对胰岛β细胞胰岛素分泌的调节作用及机制的研究" *
李江妹等: "胰高血糖素通过环磷酸腺苷信号通路调节胰岛素分泌机制的研究" *
石艳秋等: "荧光共振能量转移技术在检测环磷酸腺苷信号通路对于MIN6细胞胰岛素分泌机制中的作用" *

Similar Documents

Publication Publication Date Title
CN101788480B (en) Hepatoxic substance sieving and evaluating method based on fluorescence labeling
CN109917134A (en) A kind of calibration object stabilizer, the detection kit and detection method for measuring C peptide
CN111537706B (en) Immune lotion for electrochemical luminescence immunoassay instrument
Suhling et al. Fluorescence lifetime imaging of molecular rotors in living cells
Cichoń et al. Contact dermatitis to diabetes medical devices
Huang et al. High content drug screening of primary cardiomyocytes based on microfluidics and real-time ultra-large-scale high-resolution imaging
Colton et al. Characterization of islet preparations
Vhora et al. Recent advances in in-vitro assays for type 2 diabetes mellitus: an overview
Amatore et al. Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells
CN109406769A (en) The application of AKR insulin secretion Mechanism Study in MIN6 cell
Mozneb et al. Non-invasive plasmonic-based real-time characterization of cardiac drugs on cardiomyocytes functional behavior
Jin et al. Time-and gender-dependent alterations in mice during the aging process
CN102943066B (en) Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody
JP2010540963A (en) Single cell label-free assay
CN104694624A (en) Screening method of anticancer medicine
CN106546577B (en) The bioluminescent assay kit and its application method of human carboxylatase 1 and application
CN109142298B (en) Quantitative determination method for sweetness at cell level
CN103018447A (en) Enzyme-linked immunoassay kit for salbutamol detection, and applications thereof
CN1963505A (en) Chemiluminescent examining method for insulin content and C-peptide content in human blood serum
CN106198959A (en) Serum hyaluronic acid chemiluminescence immune detection reagent kit and preparation method thereof
Salih Ex-vivo model systems of cancer-bone cell interactions
CN110187106A (en) A kind of Multilayer film dry plate and its measuring method quantitative determining alpha-amylase activity
CN106226511B (en) A kind of recombinant human horny cell growth factor-2 biological activity detection method
Landmann et al. A semiquantitative scoring system for histopathological and immunohistochemical assessment of lesions and tissue tropism in avian influenza. Viruses. 2021
CN103645320A (en) Furaltadone metabolite chemiluminescent enzyme linked immune sorbent rapid analysis kit

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
RJ01 Rejection of invention patent application after publication

Application publication date: 20190301

RJ01 Rejection of invention patent application after publication