CN105092860A - Method for detecting correct folding rate of proinsulin in pancreatic islet beta cells - Google Patents
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Abstract
The invention discloses a method for detecting correct folding rate of proinsulin in pancreatic islet beta cells by utilizing a molecular biology method. The method includes the steps of culture and treatment of the pancreatic islet beta cells, extraction of intracellular protein, reducing western blotting experimenting of plasmosin, non-reducing western blotting experimenting of plasmosin of the pancreatic islet beta cells and detection of the correct folding rate of proinsulin in the pancreatic islet beta cells. Autoradiography immunoblotting technique is utilized in current foreign study of folding rate of proinsulin in the pancreatic islet beta cells but has the problems of radioactive pollution and expensive instrument equipment, so that a new idea is provided for detecting state of proinsulin in the pancreatic islet beta cells by utilizing molecular biology technology to detect the correct folding rate of proinsulin.
Description
Technical field
The invention belongs to the technical field of diabetes study, especially about the detection method of the correct folding ratio of proinsulin in beta Cell of islet.
Background technology
Diabetes take hyperglycaemia as the metabolic disease of feature, its incidence of disease rises year by year, the serious threat health of the mankind.Hyperglycaemia is that islet beta cell function is diabetes study focus caused by islet beta cell function reduction or insulin resistance, also may be the Effective target site for the treatment of.
Islet beta cell function reduction shows the synthesis of beta Cell of islet insulin and the minimizing of secretory volume, and whether insulin synthesis and secretion reduce and insulin precurosor---whether proinsulin is correctly folding closely related.Insulin is correctly folded by proinsulin and shears generation, and proinsulin correctly folds with the synthesis of insulin and secretes closely related.If proinsulin cannot correctly fold, the proinsulin of false folding can form multimeric forms, cannot be converted into the insulin of function, only has when proinsulin is correctly folded the insulin monomer that just can be cut into and have biological function.
In research at home and abroad, scholar is had to utilize the folding ratio of radioautograph immunoblotting analysis technical research proinsulin in beta Cell of islet, but there is the problem such as radioactive contamination, instrument and equipment costliness in radioautograph immunoblotting analysis technology, therefore cannot popularize.
Summary of the invention
The technical matters solved
Research for the correct folding ratio of proinsulin is domestic is still blank now, The present invention gives the method for the scientific and reasonable correct folding ratio of detection proinsulin.
The technical scheme adopted
In beta Cell of islet, the detection method of the correct folding ratio of proinsulin comprises the steps:
The cultivation (for murine insulinoma cell MIN6, purchased from American ATCC) of a, beta Cell of islet:
Recovery freeze-stored cell, with the DMEM high glucose medium containing 10% (v/v) hyclone, 100U/ml penicillin and 100U/ml streptomysin, at 37 DEG C, 5% (v/v) CO
2constant incubator in cultivate, within every 1 ~ 3 day, change liquid once, routine passage;
The process of b, beta Cell of islet:
After step a, MIN6 is divided into 4 groups to continue to cultivate, after grouping during good, the medium density of cell attachment, change the DMEM high glucose medium containing ciclosporin A (ciclosporin A can former correct folding of interfere with insulin) respectively into, drug concentration is respectively 0 μm of ol/L, 0.1 μm of ol/L, 1 μm of ol/L and 10 μm ol/L, intervenes MIN6 cell 48 hours;
The extraction of albumen in c, beta Cell of islet:
By the cell pyrolysis liquid cracking of the cell after step b process, supernatant is extracted after the centrifugal 30min of 12000rpm, obtain beta Cell of islet plasmosin, the albumen of extraction is carried out protein quantification (BCA albuminimetry), different histone solution is adjusted to same concentration;
The reductibility westernblotting experiment of d, beta Cell of islet plasmosin:
Get the protein solution 16ul adjusting concentration, add 4ul5 × reductibility protein electrophorese damping fluid (containing mercaptoethanol), 5min is boiled in boiling water, carry out SDS-PAGE protein electrophorese (12% separation gel, 5% concentrated glue), by the pvdf membrane of protein delivery in glue to 5 × 8cm size after electrophoresis, after transferring film completes, use closes 2h containing the PBST room temperature of 5% skimmed milk power, 4 DEG C are spent the night and incubate insulin primary antibodie (CST#8138), incubate primary antibodie PBST and wash film 3 times, each 10min, room temperature 2h incubates mouse-anti two anti-(CST#7076), incubate two anti-PBST and wash film 3 times, each 10min, film is washed once again with PBS, wash post-exposure development, obtain insulin westernblotting result,
The irreducibility westernblotting experiment of e, beta Cell of islet plasmosin:
Get the protein solution 16ul adjusting concentration, add 4ul5 × non-reducing protein electrophoretic buffer (not containing mercaptoethanol), do not boil and directly carry out SDS-PAGE protein electrophorese (12% separation gel, 5% concentrated glue), by the pvdf membrane of protein delivery in glue to 5 × 8cm size after electrophoresis, after transferring film completes, use closes 2h containing the PBST room temperature of 5% skimmed milk power, 4 DEG C are spent the night and incubate C peptide primary antibodie (abcam14181), incubate primary antibodie PBST and wash film 3 times, each 10min, room temperature 2h incubates rabbit anti-two anti-(CST#7074), incubate two anti-PBST and wash film 3 times, each 10min, film is washed once again with PBS, wash post-exposure development, obtain proinsulin westernblotting result,
The detection of the correct folding ratio of proinsulin in f, beta Cell of islet:
Utilize QuantityOne software to carry out gray analysis to westernblotting result, calculate the correct folding ratio of proinsulin.
Beneficial effect
The present invention utilizes Horseradish Peroxidase Conjugates to replace radioautograph antibody, the correct proinsulin with false folding that folds can be detected equally, avoid radioactive contamination simultaneously, reduce price needed for equipment, add the popularization of laboratory study, road has been paved in the research carrying out diabetes for scholar.
Accompanying drawing explanation
Fig. 1 is insulin reductibility westernblotting result.
Fig. 2 is proinsulin irreducibility westernblotting result.
Fig. 3 is the correct folding ratio result of proinsulin.
Specific embodiment
It is below complete embodiment
The first step: the cultivation of MIN6 cell
MIN6 cell, purchased from American ATCC.Recovery freeze-stored cell, with the DMEM high glucose medium containing 10% (v/v) hyclone, 100U/ml penicillin and 100U/ml streptomysin, at 37 DEG C, 5% (v/v) CO
2constant incubator in cultivate, within every 1 ~ 3 day, change liquid once, routine passage.
Second step: the process of MIN6 cell
In 6 orifice plates during good, the medium density of cell attachment, change the DMEM high glucose medium containing ciclosporin A (ciclosporin A can former correct folding of interfere with insulin) respectively into, drug concentration is respectively 0 μm of ol/L, 0.1 μm of ol/L, 1 μm of ol/L and 10 μm ol/L, intervenes MIN6 cell 48 hours.
3rd step: the extraction of albumen in beta Cell of islet
After drug treating cell 48h, extract plasmosin.Concrete steps are as follows:
1) remove cell culture medium, clean cell surface 2 times with the PBS of precooling, clean residual liquid, add 100ul cell pyrolysis liquid, place half an hour on ice;
2) the cell cell scraper after step 1) being placed scrapes gently, is collected in 1.5ml centrifuge tube, high speed centrifugation 12000rpm, 4 DEG C of centrifugal 30min;
3) by step 2) centrifugal after supernatant be drawn in a new 1.5ml centrifuge tube, in centrifuge tube, supernatant is MIN6 plasmosin.
The mensuration (BCA method) of MIN6 plasmosin concentration
1) complete soluble protein standard items (0.5 μ g/ μ l), and press AB liquor ratio 50:1 preparation BCA working fluid;
2) by standard items by 0,1,2,4,8,12,16,20ml is added in 96 orifice plates respectively, add aqua sterilisa and supply 20ml;
3) be added in 96 orifice plates by appropriate volume sample, often group arranges 2 multiple holes, adds aqua sterilisa and supplies 20ml;
4) each hole adds 200mlBCA working fluid, puts into 37 DEG C of shaking table 100rpm and mixes 30min;
5) each hole 572nm absorbance is measured by microplate reader;
6) drawing standard curve, calculates the protein concentration in sample according to typical curve, each extraction protein concentration is about 3mg/ml.All groupings are adjusted to same concentration protein solution, and add reductibility albumen sample-loading buffer (carrying out reductibility electrophoresis) or irreducibility albumen sample-loading buffer (carrying out irreducibility electrophoresis) prepares electrophoresis.
4th step: the reductibility westernblotting experiment of beta Cell of islet plasmosin
Westernblotting experiment detects reductibility insulin.
1) preparative separation glue and concentrated glue:
Prepared by A separation gel (12%):
Distilled water 1.0ml
30%Arc-Bis（29：1）2.0ml
1MTris，pH8.81.9ml
10%SDS50μl
10% ammonium persulfate 50 μ l
TEMED0.2μl
Cumulative volume: 5ml
B concentrates the preparation of glue (5%):
Distilled water 1.4ml
30%Arc-Bis（290.33ml
1MTris，pH6.80.25ml
10%SDS20μl
10% ammonium persulfate 20 μ l
TEMED2μl
Cumulative volume: 2ml
2) SDS-PAGE electrophoresis:
After gelling to be concentrated is solid, ready sample (reductibility electrophoresis sample) and Protein Marker is added loading hole, in electrophoresis tank, adds Tris-glycine running buffer start electrophoresis.Start electrophoresis apparatus constant voltage 50V, enter after separation gel until sample, high voltage, to 100V, stops electrophoresis when the bromophenol blue indicator in albumen sample-loading buffer moves to bottom offset plate.
3) transferring film:
After electrophoresis terminates, take off offset plate, shear the pvdf membrane of 5 × 8cm, immersion methanol solution activates, after film soaks into, transferring film folder, filter paper, glue and pvdf membrane are prepared into " sandwich " structure, glue at positive pole, puts into transferring film groove constant current 100mA transferring film 2h in negative electrode film.
4) close:
After transferring film completes, visible Protein Marker on pvdf membrane, in glue, Protein Marker disappears, and prove transferring film success, the PBST room temperature be placed in by pvdf membrane containing 5% skimmed milk power shakes closed 2 hours slowly.
5) primary antibodie is hatched:
C peptide, insulin antibody or β-actin antibody incubation pvdf membrane that rear primary antibodie diluted to be closed is anti-, put 4 ° of C and spend the night.
6) hatch two to resist:
Film is put into PBST after hatching primary antibodie and wash 3 times, each 10min.Wash sheep anti mouse or goat anti-rabbit igg antibody that to be put into by film after film completes and mark with the horseradish peroxidase (HorseradishPeroxidase, HRP) diluted containing 3% skimmed milk power PBS, incubated at room 2h.
7) exposure imaging:
After hatching two and be anti-, film is put into PBST and wash 3 times, wash 10min at every turn, wash 1 10min with PBS afterwards, then with the super quick luminous substrate exposure imaging of ECL.
As shown in Figure 1: Fig. 1 is insulin reductibility westernblotting result, in figure, NC is Normal group, 0.1,1,10 be respectively 0.1 μm of ol/L, 1 μm of ol/L and 10 μm of ol/L ciclosporin A processed group, under ciclosporin A effect, insulin synthesis level obviously declines.
5th step: the irreducibility westernblotting experiment of beta Cell of islet plasmosin
Westernblotting experiment detects irreducibility proinsulin.Preparative separation glue and concentrated glue, SDS-PAGE electrophoresis, transferring film, close, hatch primary antibodie, two anti-and exposure imaging processes of hatching with reductibility westernblotting experiment, electrophoresis sample is irreducibility electrophoresis sample.
As shown in Figure 2: Fig. 2 is proinsulin irreducibility westernblotting result, in figure, NC is Normal group, 0.1,1,10 0.1 μm of ol/L, 1 μm of ol/L and 10 μm of ol/L ciclosporin A processed group is respectively, under ciclosporin A effect, incorrect the folding of proinsulin is increased, and polymer band increases.
6th step: the detection of the correct folding ratio of proinsulin in beta Cell of islet
1) westernblotting is tested acquired results scanner scanning and obtain Tiff format image files;
2) picture QuantityOne software is carried out gray-scale value analysis, obtain the gray-scale value of proinsulin albumen under the gray-scale value of insulin protein under reductibility electrophoresis and irreducibility electrophoresis;
3) the correct folding ratio of insulin protein is calculated:
In normal islets β cell, the correct folding ratio of proinsulin is 70%, standard value is calculated with normal group, standard value=70% ÷ (normal group insulin protein gray-scale value/normal group proinsulin albumen gray-scale value), other group proinsulin correct folding ratio=(insulin protein gray-scale value/proinsulin albumen gray-scale value) × standard values;
As shown in Figure 3, Fig. 3 is the correct folding ratio result of calculation of proinsulin, in figure, NC is Normal group, 0.1,1,10 0.1 μm of ol/L, 1 μm of ol/L and 10 μm of ol/L ciclosporin A processed group is respectively, Normal group mark turns to 70% folding ratio, and the correct folding ratio of ciclosporin A processed group proinsulin is respectively 19%, 6% and 1%.
Sum up
The correct folding ratio of Late Cambrian proinsulin of the present invention can detect with Protocols in Molecular Biology, for state-detection insulinogenic in beta Cell of islet provides new approaches.
Claims (1)
1. the detection method of the correct folding ratio of proinsulin in beta Cell of islet, specifically comprises the following steps:
The cultivation of a, beta Cell of islet:
Get recovery freeze-stored cell, with the DMEM high glucose medium containing 10% (v/v) hyclone, 100U/ml penicillin and 100U/ml streptomysin, at 37 DEG C, 5% (v/v) CO
2constant incubator in cultivate, within every 1 ~ 3 day, change liquid once, routine passage;
The process of b, beta Cell of islet:
After step a, MIN6 is divided into 4 groups to continue to cultivate, after grouping during good, the medium density of cell attachment, change the DMEM high glucose medium containing ciclosporin A respectively into, drug concentration is respectively 0 μm of ol/L, 0.1 μm of ol/L, 1 μm of ol/L and 10 μm ol/L, intervenes MIN6 cell 48 hours;
The extraction of albumen in c, beta Cell of islet:
By the cell pyrolysis liquid cracking of the cell after step b process, extract supernatant after the centrifugal 30min of 12000rpm, obtain beta Cell of islet plasmosin, the albumen of extraction is carried out protein quantification, different histone solution is adjusted to same concentration;
The reductibility westernblotting experiment of d, beta Cell of islet plasmosin:
Get the protein solution 16ul adjusting concentration, add 4ul5 × reductibility protein electrophorese damping fluid, 5min is boiled in boiling water, carry out SDS-PAGE protein electrophorese, wherein 12% separation gel, 5% concentrated glue, by the pvdf membrane of protein delivery in glue to 5 × 8cm size after electrophoresis, after transferring film completes, use closes 2h containing the PBST room temperature of 5% skimmed milk power, 4 DEG C are spent the night and incubate insulin primary antibodie, incubate primary antibodie PBST and wash film 3 times, each 10min, room temperature 2h is incubated mouse-anti two and is resisted, incubate two anti-PBST and wash film 3 times, each 10min, film is washed once again with PBS, wash post-exposure development, obtain insulin westernblotting result,
The irreducibility westernblotting experiment of e, beta Cell of islet plasmosin:
Get the protein solution 16ul adjusting concentration, add 4ul5 × non-reducing protein electrophoretic buffer, do not boil and directly carry out SDS-PAGE protein electrophorese, by the pvdf membrane of protein delivery in glue to 5 × 8cm size after electrophoresis, after transferring film completes, use closes 2h containing the PBST room temperature of 5% skimmed milk power, 4 DEG C are spent the night and incubate C peptide primary antibodie, incubate primary antibodie PBST and wash film 3 times, each 10min, room temperature 2h is incubated rabbit anti-two and is resisted, incubate two anti-PBST and wash film 3 times, each 10min, film is washed once again with PBS, wash post-exposure development, obtain proinsulin westernblotting result,
The detection of the correct folding ratio of proinsulin in f, beta Cell of islet:
Utilize QuantityOne software to carry out gray analysis to westernblotting result, calculate the correct folding ratio of proinsulin.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1290299A (en) * | 1997-12-29 | 2001-04-04 | 株式会社钟根堂 | Process for preparing human proinsulin |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1290299A (en) * | 1997-12-29 | 2001-04-04 | 株式会社钟根堂 | Process for preparing human proinsulin |
Non-Patent Citations (3)
| Title |
|---|
| JIE WANG 等: "A mutation in the insulin 2 gene induces diabetes with severe pancreatic β-cell dysfunction in the Mody mouse", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| JIE WANG 等: "Control of Precursor Maturation and Disposal Is an Early Regulative Mechanism in the Normal Insulin Production of Pancreatic β-Cells", 《PLOS ONE》 * |
| 赵峰 等: "重组人白细胞介素-12(CHO细胞)的纯化及其活性", 《中国生物制品学杂志》 * |
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Application publication date: 20151125 |