CN1363838A - Process for testing bioactivity of insulinotropic hormone secretion peptide - Google Patents
Process for testing bioactivity of insulinotropic hormone secretion peptide Download PDFInfo
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- CN1363838A CN1363838A CN 01126695 CN01126695A CN1363838A CN 1363838 A CN1363838 A CN 1363838A CN 01126695 CN01126695 CN 01126695 CN 01126695 A CN01126695 A CN 01126695A CN 1363838 A CN1363838 A CN 1363838A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000028327 secretion Effects 0.000 title claims abstract description 16
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- 239000005556 hormone Substances 0.000 title claims description 9
- 230000002473 insulinotropic effect Effects 0.000 title claims description 9
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- 238000012360 testing method Methods 0.000 title claims description 8
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- 108010011459 Exenatide Proteins 0.000 claims description 21
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Abstract
The invention relates to a method for measuring biological activity of peptide for secernent of insulin. The method of injecting peptide for secernent of insulin into C57/BL/6J chmice is adopted. The effect for promoting secretion of insulin caused by the peptide for secernent of insulin is observed through measuring density of insulin and C peptide in blood at different times. The effect for reducing blood sugar caused by the peptide for secernent of insulin is observed by injection glucose and peptide for secernent of insulin into C57/BL/6J chmice, and through measuring density of blood sugar at different times. With being obtained and bred easily, C57/BL/6J chmice possess features of longer time for tolerating glucose and more obvious effect for promoting secretion of insulin nad reducing blood sugar with said medicine injected than other model animal. Comapring with methods that uses NOD chmice, db/db or ob/ob chmice, diabetes and fatness Zucker rat or diabetes, rhesus, the invented method provides merits of lower cost, easy to implement and more obvious effect.
Description
Technical field
The present invention relates to biological activity determination method, more specifically to the biological activity determination method of insulin secretion accelerating peptide.
Background technology
Animal pattern is very important in the diabetes medicament research process.For type i diabetes medicine such as insulin, its biological activity determination can use the mouse blood sugar descent method (" Chinese pharmacopoeia, version was two ones in 1995,99 pages of appendix), or with operation the rat pancreas is partly excised (The Journal ofBiological Chemistry, 1999, Vol.274, No.20 p14122-14121), also can destroy pancreatic (Diabetes with medicine, 1990, Vol.39, p226-233), insulin injection is observed its hypoglycemic activity then.Type ii diabetes patient's pancreas also has the ability of excreting insulin, but the insulin secretion quantity not sufficient is secreted time lag.Therefore, for insulin secretion accelerating peptide, above-mentioned animal pattern is also inapplicable, because the mechanism of action of this class polypeptide is the synthetic mRNA of pancreatic that promotes the type ii diabetes patient, thereby promote the synthetic and secretion of insulin, this just needs the pancreas of animal pattern also to remain with the function of excreting insulin.Usually use non-obese diabetes (NOD) mouse (Diabetes at present, 2000, Vol.49, p1992-1997), the fat db/db of diabetes, the ob/ob mouse, the fat Zucker rat of diabetes and diabetes rhesus monkey (Diabetes, 1999, Vol.48, p1026-1034) be used as the animal model of insulin secretion accelerating peptide Exendin4 and GLP-1 biological activity determination, but exist be difficult for obtaining, shortcoming such as cost height.Discovering in recent years, a series of active peptides have the function that promotes the pancreas excreting insulin, are referred to as insulin secretion accelerating peptide.Insulin secretion accelerating peptide might become the medicine of treatment type ii diabetes.
Exendin4 and GLP-1 are the typical insulin secretion accelerating peptides that has been found that.GLP-1 (Glucagon Like Peptide-1, glucagon-like peptide) is the polypeptide of human body self secretion.Exendin4 is the polypeptide that obtains from Heloderma suspectum Yi salivary secretion thing.Discover, to both molecular structures modify or minor alteration (such as both derivants, perhaps a few amino acid in both amino acid sequences is changed, perhaps increases and decreases a few amino acid), the polypeptide that obtains may still maintain the ability that promotes insulin secretion.Therefore, insulin secretion accelerating peptide involved in the present invention means the active peptides that all can promote insulin secretion.
Summary of the invention
The assay method that the purpose of this invention is to provide a kind of bioactivity of insulinotropic hormone secretion peptide convenient and practical, with low cost.
The present invention adopts healthy C
57/ BL/6J mouse is observed the insulin secretion accelerating and the hypoglycemic activity of insulin secretion accelerating peptide as animal pattern.
C
57The background of/BL/6J mouse
C
57/BL/6J
Another name: B6; BLACK 6; C
57BLACK
Outward appearance: black
Genotype: α/α Ahl/Ahl
H2 haplotype (monotype): h
Use:
1. cardiovascular research: food causes arteriosclerosis (disease occurred frequently)
2. Neurobiology research: vestibular and sense of hearing mistake are scarce, and (the senile sense of hearing is lost and is lacked; The 12-18 month)
3. sensory nerve research: the vestibular and the sense of hearing are lost and are lacked
The present invention is achieved through the following technical solutions.
Process for testing bioactivity of insulinotropic hormone secretion peptide provided by the invention is used C
57/ BL/6J mouse feeds insulin secretion accelerating peptide in the mouse body as animal pattern, to observe the insulin secretion accelerating and the hypoglycemic activity of insulin secretion accelerating peptide.
Process for testing bioactivity of insulinotropic hormone secretion peptide provided by the invention preferably feeds C with insulin secretion accelerating peptide
57Approach in the/BL/6J mouse body is a lumbar injection.
Process for testing bioactivity of insulinotropic hormone secretion peptide provided by the invention, preferred insulin secretion accelerating peptide is GLP-1 or Exendin4.
The promoting insulin secretion assay method of insulin secretion accelerating peptide provided by the invention is: get healthy C
57/ BL/6J mouse, the injection insulin secretion accelerating peptide is got hematometry insulin and C peptide concentration constantly in difference.The control group injecting normal saline.Contrast and experiment is to observe the promoting insulin secretion of insulin secretion accelerating peptide.
The hypoglycemic activity assay method of insulin secretion accelerating peptide provided by the invention is: get healthy C
57/ BL/6J mouse, injectable dextrose monohydrate and insulin secretion accelerating peptide are in difference moment measuring blood sugar of blood extracting concentration.A control group injectable dextrose monohydrate is not given insulin secretion accelerating peptide.Contrast and experiment is to observe the hypoglycemic activity of insulin secretion accelerating peptide.
C
57/ BL/6J mouse obtains easily, raises easily, and long to the glucose tolerance time, insulin secretion accelerating and hypoglycemic activity are more more obvious than other several animal patterns after the administration.Therefore, insulin secretion accelerating peptide activity determination method provided by the invention is cheaper than present widely used method cost, enforcement is more convenient, effect is more obvious.
Description of drawings
Fig. 1 is the promoting insulin secretion experimental result of GLP-1.
Fig. 2 is the short C peptide secretion experimental result of GLP-1.
Fig. 3 is the promoting insulin secretion experimental result of Exendin4.
Fig. 4 is the short C peptide secretion experimental result of Exendin4.
Fig. 5 is the hypoglycemic activity experimental result of GLP-1.
Fig. 6 is the hypoglycemic activity experimental result of Exendin4.
The present invention is further elaborated below in conjunction with specific embodiment, but the present invention is not imposed any restrictions.
Embodiment 1
With C
57/ BL/6J mouse is the promoting insulin secretion that animal pattern is measured GLP-1
Experiment material and method:
● healthy C
57/ BL/6J mouse fasting standby after 2 hours (Chinese Academy of Sciences's Shanghai animal center provides)
● 40% glucose solution
● 0.9%NaCl solution
● radio-immunity insulin assay kit (health ministry Shanghai institute of Biological Products product)
● radio-immunity C-peptide is measured kit (health ministry Shanghai institute of Biological Products product)
●GLP-1：Peninsula?Laboratories，code?7168，(1996/1997?Catalog)
Healthy C
57The fasting of/BL/6J mouse is after 2 hours, lumbar injection 10 microgram GLP-1, use scale kapillary (earlier with 1mg/mL heparin rinse capillary tube inner wall) to get blood 30 microlitres at once from the eye hole, insert mixing in the 300 microlitre physiological saline, 3000 rev/mins of centrifugal red blood cells of removing, serum is used to measure insulin and C-peptide concentration.Respectively at 5,15, got blood, separation of serum by same operation in 30,60 minutes.The control group injecting normal saline is got blood by the identical time interval.Two groups of blood samples are all used the radio-immunity kit method and are measured insulin and C-peptide concentration, with the promoting insulin secretion of check GLP-1.
Experimental result as depicted in figs. 1 and 2.
Insulin and C peptide concentration remain unchanged in the control group blood; And insulin and all obviously increases of C peptide concentration in the administration group blood.The promoting insulin secretion of provable GLP-1 thus.
Embodiment 2
With C
57/ BL/6J mouse is the promoting insulin secretion that animal pattern is measured Exendin4
Experiment material, method are identical with example 1 operation, inject 1 microgram Exendin4 and replace GLP-1, measure promoting insulin secretion.
The result as shown in Figure 3 and Figure 4.
Insulin and C peptide concentration remain unchanged in the control group blood; And insulin and all obviously increases of C peptide concentration in the administration group blood.The promoting insulin secretion of provable Exendin4 thus.
Embodiment 3
With C
57/ BL/6J mouse is the hypoglycemic activity that animal pattern is measured GLP-1
Experiment material and method:
● healthy C
57/ BL/6J mouse fasting standby after 2 hours (Chinese Academy of Sciences's Shanghai animal center provides)
● 40% glucose solution
● 0.9%NaCl solution
● blood sugar detection kit (health ministry Shanghai institute of Biological Products product)
Healthy C
57The fasting of/BL/6J mouse is after 2 hours, lumbar injection 200 microlitres, 40% glucose and 10 microgram GLP-1, use scale kapillary (handling with heparin before the injection) to get blood 30 microlitres at once from the eye hole, insert mixing in the 300 microlitre physiological saline, 3000 rev/mins of centrifugal red blood cells of removing, serum is used for glucose concentration determination.Respectively at 30,60, got blood, separation of serum by same operation in 120 minutes.A control group injectable dextrose monohydrate is not given GLP-1, gets blood by the identical time interval.Two groups of blood samples are measured blood sugar concentration according to the described method of kit, with the hypoglycemic activity of check GLP-1.
The result as shown in Figure 5.
After the control group blood sugar concentration raises significantly, fall back to normal level gradually; And remarkable rising does not appear in administration group blood sugar concentration, remains near the normal level always.This is to have promoted insulin secretion owing to after the administration, thereby has avoided the fluctuation of blood sugar concentration.The hypoglycemic activity of provable short GLP-1 thus.
With C
57/ BL/6J mouse is the hypoglycemic activity that animal pattern is measured Exendin4
Experiment material and method:
● healthy C
57/ BL/6J mouse fasting standby after 2 hours (Chinese Academy of Sciences's Shanghai animal center provides)
● 40% glucose solution
● 0.9%NaCl solution
● blood sugar detection kit (health ministry Shanghai institute of Biological Products product)
●Exendin-4：Peninsula?Laboratories，code?7089，(1996/1997?Catalog)
Get healthy C
57The fasting of/BL/6J mouse is after 2 hours, press described five kinds of mode lumbar injection glucose of table 1 and Exendin4, use scale kapillary (handling with heparin before the injection) to get blood 30 microlitres at once from the eye hole, insert mixing in the 300 microlitre physiological saline, 3000 rev/mins of centrifugal red blood cells of removing, serum is used to measure blood sugar concentration.Respectively at 30,60, got blood, separation of serum by same operation in 120 minutes.Five groups of blood samples are all measured blood sugar concentration according to kit method, with the hypoglycemic activity of check Exendin4.
Table 1
| Group number | Administration |
| ????1 | 20 microlitres, 40% glucose |
| ????2 | 20 microlitres, 40% glucose+0.5 microgram Exendin4 |
| ????3 | 20 microlitres, 40% glucose+1 microgram Exendin4 |
| ????4 | 20 microlitres, 40% glucose+2 microgram Exendin4 |
| ????5 | 20 microlitres, 40% glucose+5 microgram Exendin4 |
The result as shown in Figure 6.
After first group of mouse blood sugar concentration raises significantly, fall back to normal level gradually; Reduction has in various degree all appearred in 2-5 group blood sugar concentration, but blood sugar concentration reduction amplitude is little.This is because insulin secretion accelerating peptide only promotes insulin secretion when blood sugar concentration is high, blood sugar lowering, and blood sugar concentration just fails when returning to normal concentration.The experiment structure proves that not only Exendin4 has hypoglycemic activity, proves that also Exendin4 can not cause hypoglycemia.
Claims (3)
1. a process for testing bioactivity of insulinotropic hormone secretion peptide is characterized in that: use C
57/ BL/6J mouse feeds insulin secretion accelerating peptide in the mouse body as animal pattern, measures the insulin secretion accelerating and the hypoglycemic activity of insulin secretion accelerating peptide.
2. process for testing bioactivity of insulinotropic hormone secretion peptide according to claim 1 is characterized in that: insulin secretion accelerating peptide is fed C
57Approach in the/BL/6J mouse body is a lumbar injection.
3. process for testing bioactivity of insulinotropic hormone secretion peptide according to claim 1 is characterized in that: described insulin secretion accelerating peptide is GLP-1 or Exendin4.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01126695 CN1131433C (en) | 2001-09-07 | 2001-09-07 | Process for testing bioactivity of insulinotropic hormone secretion peptide |
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|---|---|---|---|
| CN 01126695 CN1131433C (en) | 2001-09-07 | 2001-09-07 | Process for testing bioactivity of insulinotropic hormone secretion peptide |
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| Publication Number | Publication Date |
|---|---|
| CN1363838A true CN1363838A (en) | 2002-08-14 |
| CN1131433C CN1131433C (en) | 2003-12-17 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293315A (en) * | 2012-06-14 | 2013-09-11 | 中国食品药品检定研究院 | RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog |
| CN101548189B (en) * | 2006-12-06 | 2017-03-08 | 花王株式会社 | The evaluation methodology of obesity controller |
-
2001
- 2001-09-07 CN CN 01126695 patent/CN1131433C/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101548189B (en) * | 2006-12-06 | 2017-03-08 | 花王株式会社 | The evaluation methodology of obesity controller |
| CN103293315A (en) * | 2012-06-14 | 2013-09-11 | 中国食品药品检定研究院 | RGA (Report Gene Assay) method for detecting biological activity of exendin-4-HAS Byetalog |
| CN103293315B (en) * | 2012-06-14 | 2016-05-25 | 中国食品药品检定研究院 | RGA method detects insulin secretion accelerating peptide fusion BA |
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