CN101130774B - Nucleotide molecule SR5B and its application in preparation of antidiabetic medicine - Google Patents
Nucleotide molecule SR5B and its application in preparation of antidiabetic medicine Download PDFInfo
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- CN101130774B CN101130774B CN2006100303029A CN200610030302A CN101130774B CN 101130774 B CN101130774 B CN 101130774B CN 2006100303029 A CN2006100303029 A CN 2006100303029A CN 200610030302 A CN200610030302 A CN 200610030302A CN 101130774 B CN101130774 B CN 101130774B
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Abstract
The invention discloses a nucleic acid molecule and the application to prepare antidiabetic in the biological medical domain, wherein the diabetes mellitus is a group of clinical syndrome interacted by genetic and environmental factor, which bases on hyperglycemia as main mark to lead the harm of multiple systems due to lingering illness; the nucleic acid molecule SR5B is added into Langerhans' islet beta-cell to increase the content of insulin in the cell culture supernatant. The invention can accelerate the secretory function of insulin as new antidiabetic, which provides a new path and method to release and treat diabetes.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of nucleic acid molecule and the application in the preparation antidiabetic medicine thereof.
Background technology
Diabetes (diabetes mellitus) are one group of clinical syndromes that is caused by the h and E factor interaction.Absolute or relative deficiency and target tissue cell reduce insulin sensitivity because of insulin secretion, cause a series of metabolism disorders such as sugar, albumen, fat, power and water Xie Zhi.Clinical is outstanding feature with the hyperglycemia, and prolonged illness can cause a plurality of system damages.The state of an illness punish severely or stress the time acute metabolism disorder such as ketoacidosis etc. can take place.
Diabetes major clinical types insulin dependent diabetes mellitus (IDDM) (IDDM, I type) can occur in any age, but pilosity is born in blue or green childhood.Clinical characters is that onset is anxious, and polyphagia, polyuria, polydipsia, symptom such as lose weight are more obvious, and the tendency that ketoacidosis takes place is arranged, and must rely on insulinize and earn a bare living.Islet cells autoantibody positive rate height in the onset initial stage blood.The visible basal insulin level of oral glucose islets of langerhans release test is lower than normally, and glucose stimulates back insulin secretion curve low flat, shows insulin deficit.
Non-insulin-dependent diabetes mellitus (NIDDM, II type) also can occur in any age, but in being more common in after 40 years old, old age.The most patient onset is slow, clinical symptoms relatively light or lack as.No ketoacidosis tendency, but under certain inducement effect, also ketoacidosis or hyperosmolar coma can take place.Rely on insulin, but when diet and oral antidiabetic drug therapeutic effect are not good enough, or, also need sometimes to control hyperglycemia with insulin because of the existence of complication and concomitant disease.
Yet up to the present, the someone reports and is used for the diabetes genomic medicine as yet.
Summary of the invention
The purpose of this invention is to provide a kind of nucleic acid molecule that is used to prepare antidiabetic medicine.
Another object of the present invention provides the application of above-mentioned nucleic acid molecule.
The invention provides a kind of isolated nucleic acid molecule, its sequence comprises: GCUAGAGCACAUUCAGAAA.Be called SR5B.
In the present invention, term " nucleotide molecule SR 5 B " refers to have and suppresses the BRSK2 protein active or increase amount of insulin secretion, and contains and the homologous nucleotide sequence of GCUAGAGCACAUUCAGAAA sequence height.This term also comprises the homology at least 70% with GCUAGAGCACAUUCAGAAA, preferably at least 80%, and at least 90% nucleotide sequence more preferably.
This term also comprises the nucleotide sequence variation form of GCUAGAGCACAUUCAGAAA.These variant forms comprise (but being not limited to): several (are generally 1-15, preferably 1-10,1-5 more preferably) disappearance, insertion and/or the replacement of nucleotide, and add several (being generally in 10, preferably is in 5) nucleotide at 5 ' and/or 3 ' end.For example, (3 ' end) add the sequence that some dT (deoxyribosylthymine) back forms behind GCUAGAGCACAUUCAGAAA.
In the present invention, can be behind SR5B (3 ' end) connect dTdT, thus form GCUAGAGCACAUUCAGAAAdTdT.
Also provide nucleic acid molecule in the present invention, its sequence comprises GCUAGAGCACAUUCAGAAA and UUUCUGAAUGUGCUCUAGC.
Also provide nucleic acid molecule in the present invention, its sequence comprises GCUAGAGCACAUUCAGAAAdTdT and dTdT UUUCUGAAUGUGCUCUAGC.
The present invention also provides a kind of carrier, and it contains above-mentioned nucleic acid molecule, i.e. SR5B.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, selecting commercially available carrier for use, the GCUAGAGCACAUUCAGAAAdTdT that will contain SR5B nucleotide sequence of the present invention then operationally is connected in expression regulation sequence, can form recombinant vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion targeting sequencing) DNA operationally is connected in polypeptid DNA so; If transcribing of promoter control sequence, it is operationally to be connected in coded sequence so; When if ribosome binding site is placed in the position that can make its translation, it is operationally to be connected in coded sequence so.Generally, " operationally being connected in " means adjoining, then means adjacent and do not influence function for nucleic acid molecules of the present invention.
The present invention also provides above-mentioned carrier transformed host cells.
In the present invention, term " host cell " comprises prokaryotic cell and eukaryotic cell.The example of prokaryotic host cell commonly used comprises escherichia coli, bacillus subtilis etc.Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS, NIT cell etc.
On the other hand, the present invention also provides the preparation method of above-mentioned nucleotide molecule SR 5 B, and the sequence of promptly pressing GCUAGAGCACAUUCAGAAA is with each ribonucleic acid molecule dehydrating condensation successively.
Nucleotide molecule SR 5 B of the present invention can adopt the preparation method preparation of various routines.Nucleotide molecule SR 5 B sequence of the present invention can obtain with the method for enzymatic isolation method or synthetic usually.
The present invention also provides the above-mentioned application of nucleotide molecule SR 5 B in the preparation antidiabetic medicine.
To contain in the GCUAGAGCACAUUCAGAAAdTdT of SR5B nucleotide sequence of the present invention and the beta Cell of islet (NIT) that dTdUUUCUGAAUGUGCUCUAGC is transfected into mice with the method for siRNA, radioimmunoassay method testing result shows that content of insulin increases in the cells and supernatant.
Studies show that further in the above-mentioned siRNA interference experiment, principle is the expression of endogenous BRSK2 in the beta Cell of islet (NIT) that has disturbed mice, just cause content of insulin increase in the cells and supernatant.
From the result of Northern as seen, BRSK2 mainly expresses in brain and pancreas, especially with the expression in the pancreas for the highest.The result of Western is consistent with the result of front Northern, and BRSK2 also mainly expresses at brain and pancreas in mice.From the result of SABC as seen, BRSK2 mainly expresses in the islets of langerhans of pancreatic tissue.
Neuron differentiation associated gene BRSK2 (serial number of nucleotide is AF533880 among the NCBI, and the serial number of nucleotide is AAP97727) has very high expression in pancreas, even surpasses the expression in brain.Further Showed by immune group result, BRSK2 is high expressed in the islets of langerhans of pancreas mainly.With BRSK2 overexpression in the beta Cell of islet (NIT) of mice, radioimmunoassay method detects content of insulin in the cells and supernatant, and the result shows that insulin secretion reduces; Disturb the expression activity of endogenous BRSK2 in the beta Cell of islet (NIT) of mice on the other hand with the method for siRNA, same radioimmunoassay method testing result shows, content of insulin increase in the cells and supernatant.
Nucleotide molecule SR 5 B of the present invention and analog thereof when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.
With nucleotide molecule SR 5 B of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains chemical compound and the pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Nucleotide molecule SR 5 B of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, BRSK2 of the present invention also can use with the other treatment agent.
When nucleotide molecule SR 5 B of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to mammal, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Among the present invention, the pharmaceutical composition that described antidiabetic medicine is made up of the nucleotide molecule SR 5 B that contains effective therapeutic dose and carrier pharmaceutically or excipient.Described pharmaceutical composition can be injection or tablet.Its effective therapeutic dose can for every day 1 microgram/kilogram to 5 micrograms/kg body weight.
Among the present invention, described medicine is injection or tablet.
Diabetes (diabetes mellitus) are one group of clinical syndromes that is caused by the h and E factor interaction.Absolute or relative deficiency and target tissue cell reduce insulin sensitivity because of insulin secretion, cause a series of metabolism disorders such as sugar, albumen, fat, power and water Xie Zhi.Clinical is outstanding feature with the hyperglycemia, and prolonged illness can cause a plurality of system damages.The state of an illness punish severely or stress the time acute metabolism disorder such as ketoacidosis etc. can take place.
Nucleotide molecule SR 5 B of the present invention adds in the beta Cell of islet (NIT) of mice, and content of insulin increases in the cells and supernatant.Therefore think that nucleotide molecule SR 5 B of the present invention has the function of insulin secretion in the beta Cell of islet (NIT) that promotes mice, can be used as new antidiabetic medicine or develop, for alleviating and the treatment diabetes provide a kind of new approach and means.
Description of drawings
Fig. 1 is the influence figure of BRSK2 overexpression to insulin secretion.Among this figure, block diagram from left to right is followed successively by insulin assay concentration, cell number and relative insulin secretion value to blank and the influence that adds pair cell behind the BRSK2.Scheme as seen from the western of the relative insulin secretion value figure and the rightmost side, the BRSK2 overexpression will cause insulin secretion to reduce.
Fig. 2 is that BRSK2 is by the endogenous figure that influences that disturbs the back to insulin secretion.Among this figure, block diagram from left to right is followed successively by insulin assay concentration, cell number and relative insulin secretion value to blank and the influence that adds siRNA (1 μ l, 3 μ l, 5 μ l) back pair cell.From the western of the relative insulin secretion value figure and rightmost side figure as seen, BRSK2 will cause the insulin secretion rising after by endogenous interference.
The specific embodiment
(1) with the NIT cell with 1 * 10
5The density in individual/hole is seeded on 24 orifice plates, and cell is put into CO
2Cultivated 18-24 hour for 37 ℃ in the incubator, make the cell abundance reach 70-80%.
(2) carry out transfection with Invitrogen lipofectamine 2000 eucaryon transfection reagent boxes.Difference transfection Nonsilence 5 μ l (the interferential negative control of siRNA), siRNA (dTdUUUCUGAAUGUGCUCUAGC and GCUAGAGCACAUUCAGAAAdTdT)-51 μ l, 3 μ l and 5 μ l (concentration of every μ l is 20 μ M).Every kind of transfection repeats 4 holes.
(3) after the transfection 16 hours, change complete medium (DMEM high glucose medium+15% hyclone) into and continue to cultivate 24 hours.
(4) change complete medium (sugared concentration is 25mM) again, cultivate and collect culture supernatant after 20 minutes.
(5) adopt radio immunoassay to measure concentration of insulin in the culture supernatant, detection kit is provided by the institute of biological products, Shanghai City, and measured value is criticized interior CV＜4.2%, CV＜7.6% between batch.
(6) count with cell counting count board after the cell tryptase enzymic digestion on the culture plate.
(7) cell of collecting after counting is done the expression that Western blot detects endogenous BRSK2.
(8) Treatment Analysis data.
The result shows, endogenous BRSK2 in the beta Cell of islet (NIT) of the method interference mice of usefulness siRNA, and same radioimmunoassay method testing result shows that content of insulin increases in the cells and supernatant.
(1) with the NIT cell with 1 * 10
5The density in individual/hole is seeded on 24 orifice plates, and cell is put into CO
2Cultivated 18-24 hour for 37 ℃ in the incubator, make the cell abundance reach 7080%.
(2) carry out transfection with Invitrogen lipofectamine 2000 eucaryon transfection reagent boxes.Respectively transfection Pcmv-Myc empty carrier and two kinds of plasmids of Pcmv-Myc-BRSK2, every kind of plasmid is according to 4 holes of each transfection of concentration in 200ng/ hole.
(3) after the transfection 16 hours, change complete medium (DMEM high glucose medium+15% hyclone) into and continue to cultivate 24 hours.
(4) change complete medium (sugared concentration is 25mM) again, cultivate and collect culture supernatant after 20 minutes.
(5) adopt radio immunoassay to measure concentration of insulin in the culture supernatant, detection kit is provided by the institute of biological products, Shanghai City, and measured value is criticized interior CV＜4.2%, CV＜7.6% between batch.
(6) count with cell counting count board after the cell tryptase enzymic digestion on the culture plate.
(7) cell of collecting after counting is done the expression that Western blot detects BRSK2-Myc.
(8) Treatment Analysis data.
The result of overexpression shows that after the overexpression BRSK2, the relative secretory volume of insulin reduces among the mouse islets Beta cell NIT.
Claims (8)
1. an isolated nucleic acid molecules is characterized in that, its sequence is GCUAGAGCACAUUCAGAAA.
2. a nucleic acid molecules is characterized in that, its sequence is GCUAGAGCACAUUCAGAAAdTdT.
3. a carrier is characterized in that, it contains claim 1 or 2 described nucleic acid molecules.
4. host cell that contains the described carrier of claim 3.
5. the preparation method as claim 1 or 2 described nucleic acid molecules is characterized in that, by claim 1 or 2 described sequence of nucleic acid molecules, with each nucleotide group dehydrating condensation successively.
6. the application in the preparation antidiabetic medicine as claim 1 or 2 described nucleic acid molecules.
7. application as claimed in claim 6 is characterized in that the pharmaceutical composition that described antidiabetic medicine is made up of the claim 1 that contains effective therapeutic dose or 2 described nucleic acid molecules and carrier pharmaceutically or excipient.
8. application as claimed in claim 6 is characterized in that, described medicine is injection or tablet.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006100303029A CN101130774B (en) | 2006-08-22 | 2006-08-22 | Nucleotide molecule SR5B and its application in preparation of antidiabetic medicine |
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| CN2006100303029A CN101130774B (en) | 2006-08-22 | 2006-08-22 | Nucleotide molecule SR5B and its application in preparation of antidiabetic medicine |
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| CN101130774A CN101130774A (en) | 2008-02-27 |
| CN101130774B true CN101130774B (en) | 2010-10-06 |
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| CN2006100303029A Expired - Fee Related CN101130774B (en) | 2006-08-22 | 2006-08-22 | Nucleotide molecule SR5B and its application in preparation of antidiabetic medicine |
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Non-Patent Citations (4)
| Title |
|---|
| J. M. Lizcano et al..LKB1 is a master kinase that activates 13 kinases oftheAMPK subfamily, including MARK/PAR-1.EMBO Journal23 4.2004,23(4),833-843. * |
| J.M.Lizcanoetal..LKB1isamasterkinasethatactivates13kinasesoftheAMPKsubfamily including MARK/PAR-1.EMBO Journal23 4.2004 |
| L. Sabater et al..BR serine/threonine kinase 2: A new autoantigeninparaneoplastic limbic encephalitis.Journal of Neuroimmunology170 1－2.171 ,170(1－2),186-190. |
| L. Sabater et al..BR serine/threonine kinase 2: A new autoantigeninparaneoplastic limbic encephalitis.Journal of Neuroimmunology170 1-2.171 ,170(1-2),186-190. * |
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