CN109030804A - The insulin secretion rating model of glucose stimulation - Google Patents
The insulin secretion rating model of glucose stimulation Download PDFInfo
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- CN109030804A CN109030804A CN201810933693.8A CN201810933693A CN109030804A CN 109030804 A CN109030804 A CN 109030804A CN 201810933693 A CN201810933693 A CN 201810933693A CN 109030804 A CN109030804 A CN 109030804A
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Abstract
The invention discloses the insulin secretion rating models of glucose stimulation.Determine the culture administration time for 24 hours for beta Cell of islet；Insulin concentration is measured using ELISA method；OD value is measured by CCK8 method to reflect cell quantity indirectly；Select HBSS solution as later extracellular buffer carry out using；Insulin secretion accelerating positive drug glibenclamide is selected to promote the secretion of beta Cell of islet insulin.The model that the present invention establishes is able to reflect the difference of the sugared different stimulated of high sugar and basis, is able to reflect the effect that stimulates insulin secretion of positive drug glibenclamide under the conditions of high pass and basis sugar.
Description
Technical field
The invention belongs to medicine technology fields, are related to the insulin secretion rating model of glucose stimulation.
Background technique
Diabetes B is the main Types of diabetes, accounts for 90%~95%, be a kind of insulin resistance with insulin not
Foot, defect of insulin secretion are with or without chronic hyperglycemia disease caused by insulin resistance.The generation of diabetes B
Insulin is generated with beta Cell of islet, blood circulation system transports insulin and target cell receives insulin and plays physiological action
Etc. closely related, these three processes are to research and develop the foundation of diabetes B, and wherein insulin secretion accelerating class drug is anti-2 type glycosurias
The important kind of medicine research and development.Insulinotropic hormone excretion is mainly using sulfonylureas receptor as the sulfonylurea of target spot and Ge Lie
How class drug, but there are offer limited effectiveness, be also easy to produce the problems such as toxic side effect.Therefore, small novel of good effect, toxic side effect is researched and developed
Insulin secretion accelerating class antidiabetic medicine is one of the hot spot of field of medicaments research.Promote the main function of insulin secretion medicine
Mechanism is to promote beta Cell of islet release insulin, and increase the sensibility that beta Cell of islet stimulates blood glucose.Clinic has been answered
Drugs Promoting Insulin Secretion is broadly divided into sulfonylurea drug and non-sulfonylurea drug, and there are larger for these two types of pharmaceutical chemistry structures
Difference, but there is similar promoting insulin secretion mechanism.
Drug screening is that the steps necessary that medicament research and development starts by the detection to biological effector molecule is found to have life
The active natural products of object and artificial-synthetic compound.With scientific and technical continuous development, drug screening technology also generates huge
Big progress, gradually from animal model development to cell, molecular level, good insulin secretion model is for improving anti-sugar
Urine medicine research and development speed guarantees that drug screening quality, reduction medicament research and development cost have important practical significance.
Summary of the invention
The purpose of the present invention is to provide the insulin secretion rating models of glucose stimulation.
The technical scheme adopted by the invention is that being carried out according to the following conditions:
Condition 1. determines the culture administration time for beta Cell of islet for 24 hours；
Condition 2. measures insulin concentration using ELISA method；
Condition 3. measures OD value by CCK8 method to reflect cell quantity indirectly；
Condition 4. select HBSS solution as later extracellular buffer carry out using；
Condition 5. selects insulin secretion accelerating positive drug glibenclamide to promote the secretion of beta Cell of islet insulin.
Further, in step 2, the rat Langerhans islet of anti-diabetic related reagent specialized company Sweden Mercodia production is selected
Plain assay kit carries out insulin assay.
Further, in step 3, the time that 0.5h is incubated for as cell count cell and CCK8 is selected.
Research and utilization porous plate technology and ELISA sandwich method of the present invention combine, thin by exploring optimization porous plate pancreas islet β
Born of the same parents be inoculated with quantity, cell culture condition, method for cell count, outer buffer formulation and ELISA reagent type, determination condition,
Extension rate etc. finally establishes beta Cell of islet insulin secretion (GSIS) model of glucose stimulation.The result shows that foundation
GSIS model is able to reflect the difference of the sugared different stimulated of high sugar and basis, is able to reflect positive drug glibenclamide in high pass and basis
The effect to stimulate insulin secretion under the conditions of sugar
Detailed description of the invention
Fig. 1 be in the cell inoculation to 96 orifice plates of different densities for 24 hours after the microphoto that shoots under 10 times of object lens；
Fig. 2 is the cell growth curve of different densities measured in 54h every 8h；
Fig. 3 is insulin assay standard curve；
Fig. 4 is the optimization of sample extension rate；
Fig. 5 is the optimization of incubation time；
Fig. 6 is the stimulation of beta Cell of islet glucose or HACAT cell, TM cell culture fluid insulin content；
Fig. 7 is the secretion of beta Cell of islet insulin under different extracellular buffer conditions；
Fig. 8 is the investigation of insulin secretion accelerating positive drug.
Specific embodiment
The present invention is described in detail With reference to embodiment.
The optimization of 1 cell density and the determination of administration time
Since cell density has large effect to cell growth state and growth tendency, to being inoculated in 96 orifice plates
Beta Cell of islet density (according to preliminary result, is provided with 2000/hole, 4000/hole, 6000/hole, 8000/hole etc. 4
A Graded Density, n=3) it is optimized, beta Cell of islet is examined in terms of cell growth state and growth tendency two
It examines.
As a result as depicted in figs. 1 and 2, in 96 orifice plates, cell density is from 2000/hole, 4000/hole, 6000/hole
To 8000/hole, after cultivating for 24 hours, with the increase of cell density, the growth conditions of cell are better, and 8000/hole is
Occur a degree of to converge (Fig. 1)；From the point of view of cell growth curve, density is that the vitro growth rates in 8000/hole are very fast,
When incubation time from 16h to for 24 hours when, the speed of growth is most fast, compared with the index of coincidence phase growth trend.Accordingly, it is determined that being for 24 hours pancreas islet
Culture administration time of the β cell in 96 orifice plates.
The optimization of 2 insulin assay conditions
We have primarily looked at the measuring method of insulin, and final choice measures insulin concentration using ELISA method.Through
The comparison of preliminary experiment is crossed, the rat insulin for selecting anti-diabetic related reagent specialized company Sweden Mercodia production measures examination
Agent box carries out insulin assay.The standard curve of kit measurement as shown in figure 3, carried out data fitting using polynomial equation,
3 equation of n th order n have been obtained, have carried out the calculating of insulin concentration by the OD value of measurement with this equation.
Since the calculating that ultraviolet-visible spectrum measurement OD value carries out material concentration has certain range of linearity, in order to measure
As a result accuracy, we measure insulin concentration to obtained sample segment solution preliminary experiment first, further according to tentatively obtaining
Result determine suitable extension rate, then (Fig. 4) is measured to whole samples.
The optimization of 3 cell count conditions
Before cell inoculation to 96 orifice plates, cell count is carried out by haemocytometer, then presses cell in 96 orifice plates
Same cell density bed board, after culture, there is difference in cell quantity, in order to correct this species diversity, terminates in drug treatment
Afterwards, OD value can be measured by CCK8 method to reflect cell quantity indirectly.Firstly, cell and CCK8 are incubated for time (0.5h,
1h, 2h, 4h) it is optimized, as shown in figure 5, the results show that 0.5h to 4h is incubated with the extension of time, OD value constantly increases
It educates the time, OD value about increases to 0.40 or so from 0.25, comprehensively considers time and response measurement data, and finally selected 0.5h makees
The time being incubated for for cell count cell and CCK8.
The insulin secretion of 4 glucose stimulation and the investigation of measuring method specificity
We by beta Cell of islet itself secretion insulin, by the secretomotor insulin of glucose and other two kinds
The culture solution of cell compares, as shown in fig. 6, beta Cell of islet itself can generate certain insulin, stimulates item in glucose
Under part, the insulin of higher concentration can be generated, and HACAT cell, culture solution of the TM cell in culture bottle essentially free of
Insulin, the specificity that can illustrate that measuring method itself detects insulin are stronger.
The investigation of 5 heterogeneity extracellular fluids
In the secretomotor insulin assay of beta Cell of islet glucose, the extracellular buffer comprising glucose is to reality
The critical function tested, therefore two kinds of buffers of HBSS, KRB have been investigated, their ingredient is as shown in table 1.
The extracellular buffer components table of table 1
Using two different buffers, the insulin secretion experiment of beta Cell of islet glucose stimulation, such as Fig. 7 have been carried out
It is shown, the experimental results showed that, the amount for the insulin that two kinds of buffers are secreted after stimulating beta Cell of islet glucose, no matter in nothing
Under the conditions of sugar or under the conditions of high sugar, significant difference is all not present, illustrates that both buffers stimulate beta Cell of islet glucose
Insulin secretion influence less, can use, select HBSS solution as later extracellular buffer carry out using.
The investigation of 6 insulin secretion accelerating positive drugs
Insulin secretion accelerating positive drug glibenclamide is investigated to the facilitation of the secretion of beta Cell of islet insulin, with inspection
The validity of insulin secretion accelerating model is tested, is prepared for screening insulin secretion accelerating positive drug.As a result as shown in figure 8, low
A certain amount of insulin of islet β cell under the conditions of sugar and high sugar, after 10uM glibenclamide is added, no matter in low sugar or height
Under the conditions of sugar, the secretion of beta Cell of islet insulin can be remarkably promoted.The result shows that the beta Cell of islet glucose of foundation stimulates
Insulin secretion model can cash out the insulin secretion accelerating effect of insulin secretion accelerating positive drug glibenclamide, for based on
The screening of the lead compound of the anti-diabetes B insulinotropic hormone excretion of this pharmacological mechanism lays the foundation.
The above is only not to make limit in any form to the present invention to better embodiment of the invention
System, any simple modification that embodiment of above is made according to the technical essence of the invention, equivalent variations and modification,
Belong in the range of technical solution of the present invention.
Claims (3)
1. the insulin secretion rating model of glucose stimulation, it is characterised in that carried out according to the following conditions:
Condition 1. determines the culture administration time for beta Cell of islet for 24 hours；
Condition 2. measures insulin concentration using ELISA method；
Condition 3. measures OD value by CCK8 method to reflect cell quantity indirectly；
Condition 4. select HBSS solution as later extracellular buffer carry out using；
Condition 5. selects insulin secretion accelerating positive drug glibenclamide to promote the secretion of beta Cell of islet insulin.
2. the insulin secretion rating model stimulated according to glucose described in claim 1, it is characterised in that: in the step 2,
The rat insulin assay kit of selected anti-diabetic related reagent specialized company Sweden Mercodia production carries out insulin
Measurement.
3. the insulin secretion rating model stimulated according to glucose described in claim 1, it is characterised in that: in the step 3,
The time that selected 0.5h is incubated for as cell count cell and CCK8.
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| CN201810933693.8A CN109030804A (en) | 2018-08-16 | 2018-08-16 | The insulin secretion rating model of glucose stimulation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048540A (en) * | 2020-09-14 | 2020-12-08 | 北京大学 | Method for detecting insulin secretion in pancreatic islet and application thereof |
| CN114736845A (en) * | 2022-03-23 | 2022-07-12 | 浙江树人学院 | Kit for preparing high-sugar low-sugar solution for insulin secretion test stimulated by insulin from islet cell mass glucose |
| CN114868736A (en) * | 2022-03-08 | 2022-08-09 | 四川中科奥格生物科技有限公司 | Islet cell stabilizing solution and preparation method and application thereof |
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| CN103599340A (en) * | 2013-09-10 | 2014-02-26 | 杨中林 | Pharmaceutical composition used for treating and preventing diabetes and eye complications thereof, and applications of the pharmaceutical composition |
| CN104399059A (en) * | 2014-10-31 | 2015-03-11 | 辽宁大学 | Use of antimicrobial peptide AWRK6 in preparation of drug for treating type 2 diebetes |
| CN105092490A (en) * | 2014-05-04 | 2015-11-25 | 重庆派金生物科技有限公司 | External biological activity determination method for human insulin and analog or conjugate |
| WO2017078439A1 (en) * | 2015-11-03 | 2017-05-11 | 재단법인대구경북과학기술원 | Pharmaceutical composition for treating diabetes, comprising pancreatic islet cells and elastin-like artificial extracellular matrix |
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2018
- 2018-08-16 CN CN201810933693.8A patent/CN109030804A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599340A (en) * | 2013-09-10 | 2014-02-26 | 杨中林 | Pharmaceutical composition used for treating and preventing diabetes and eye complications thereof, and applications of the pharmaceutical composition |
| CN105092490A (en) * | 2014-05-04 | 2015-11-25 | 重庆派金生物科技有限公司 | External biological activity determination method for human insulin and analog or conjugate |
| CN104399059A (en) * | 2014-10-31 | 2015-03-11 | 辽宁大学 | Use of antimicrobial peptide AWRK6 in preparation of drug for treating type 2 diebetes |
| WO2017078439A1 (en) * | 2015-11-03 | 2017-05-11 | 재단법인대구경북과학기술원 | Pharmaceutical composition for treating diabetes, comprising pancreatic islet cells and elastin-like artificial extracellular matrix |
Non-Patent Citations (1)
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| MARCO TOZZI 等: ""The P2X7 receptor and pannexin-1 are involved in glucose-induced autocrine regulation in β-cells"", 《SCIENTIFIC REPORTS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112048540A (en) * | 2020-09-14 | 2020-12-08 | 北京大学 | Method for detecting insulin secretion in pancreatic islet and application thereof |
| CN114868736A (en) * | 2022-03-08 | 2022-08-09 | 四川中科奥格生物科技有限公司 | Islet cell stabilizing solution and preparation method and application thereof |
| CN114868736B (en) * | 2022-03-08 | 2022-12-09 | 四川中科奥格生物科技有限公司 | Islet cell stabilizing solution and preparation method and application thereof |
| CN114736845A (en) * | 2022-03-23 | 2022-07-12 | 浙江树人学院 | Kit for preparing high-sugar low-sugar solution for insulin secretion test stimulated by insulin from islet cell mass glucose |
| CN114736845B (en) * | 2022-03-23 | 2024-03-05 | 浙江树人学院 | High-sugar low-sugar solution preparation kit for insulin secretion test stimulated by islet cell mass glucose |
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Application publication date: 20181218 |