CN101130769B - Nucleotide molecule SR4B and application of the same in producing antidiabetic medicament - Google Patents
Nucleotide molecule SR4B and application of the same in producing antidiabetic medicament Download PDFInfo
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- CN101130769B CN101130769B CN2006100302539A CN200610030253A CN101130769B CN 101130769 B CN101130769 B CN 101130769B CN 2006100302539 A CN2006100302539 A CN 2006100302539A CN 200610030253 A CN200610030253 A CN 200610030253A CN 101130769 B CN101130769 B CN 101130769B
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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 SR4B 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 item key 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, I type) can occur in any age, but pilosity is born in blue or green childhood.Clinical characters is that onset is anxious, and many foods, diuresis, symptom such as drink more, 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 pancreas islet release test is lower than normally, and glucose stimulates back insulin secretion curve low flat, shows insulin deficit.
Non insulin dependent diabetes (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 symptom relatively light or lack as.No ketoacidosis tendency, but under certain inducement effect, also ketoacidosis or hyperosmolar coma can take place.Rely on Regular Insulin, but when diet and oral antidiabetic drug result of treatment are not good enough, or, also need sometimes to control hyperglycemia with Regular Insulin because of the existence of complication and concomitant disease.
Yet up to the present, the someone reports and is used for antidiabetic 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: CAUCCGCAUCGCAGACUUU.Be called SR4B.
In the present invention, term " nucleic acid molecule SR4B " refers to have and suppresses the BRSK2 protein-active or increase amount of insulin secretion, and contains and CAUCCGCAUCGCAGACUUU sequence height homologous nucleotide sequence.This term also comprises the homology at least 70% with CAUCCGCAUCGCAGACUUU, preferably at least 80%, and at least 90% nucleotide sequence more preferably.
This term also comprises the nucleotide sequence variation form of CAUCCGCAUCGCAGACUUU.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 (deoxythymidine) back forms behind CAUCCGCAUCGCAGACUUU.
In the present invention, can be behind SR4B (3 ' end) connect dTdT, thus form CAUCCGCAUCGCAGACUUUdTdT.
Also provide nucleic acid molecule in the present invention, its sequence comprises CAUCCGCAUCGCAGACUUU and AAAGCUGUUCUUGAUGGAG.
Also provide nucleic acid molecule in the present invention, its sequence comprises CAUCCGCAUCGCAGACUUUdTdT and dTdT AAAGUCUGCGAUGCGGAUG.
The present invention also provides a kind of carrier, and it contains above-mentioned nucleic acid molecule SR4B.
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 CAUCCGCAUCGCAGACUUUdTdT that will contain SR4B nucleotide sequence of the present invention then operationally is connected in expression regulation sequence, can form recombinant vectors.
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 leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means adjacent and do not influence function for nucleic acid molecule 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 prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, 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 nucleic acid molecule SR4B, and the sequence of promptly pressing CAUCCGCAUCGCAGACUUU is with each ribonucleic acid molecule dehydrating condensation successively.
Nucleic acid molecule SR4B of the present invention can adopt preparation method's preparation of various routines.Nucleic acid molecule SR4B sequence of the present invention can obtain with the method for enzymolysis process or synthetic usually.
The present invention also provides the above-mentioned application of nucleic acid molecule SR4B in the preparation antidiabetic medicine.
To contain in the CAUCCGCAUCGCAGACUUUdTdT of SR4B nucleotide sequence of the present invention and the beta Cell of islet (NIT) that dTd TAAAGUCUGCGAUGCGGAUG is transfected into mouse with the method for siRNA, radioimmunoassay method detected 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 mouse, 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 amount 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 mouse.From the result of immunohistochemical methods as seen, BRSK2 mainly expresses in the pancreas islet of pancreatic tissue.
Neurone differentiation associated gene BRSK2 (sequence number of Nucleotide is AF533880 among the NCBI, and the sequence number of Nucleotide is AAP97727) has very high expression amount in pancreas, even surpasses the expression amount in brain.Further Showed by immune group result, BRSK2 is high expression level in the pancreas islet of pancreas mainly.With BRSK2 overexpression in the beta Cell of islet (NIT) of mouse, 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 mouse on the other hand with the method for siRNA, same radioimmunoassay method detected result shows, content of insulin increase in the cells and supernatant.
Nucleic acid molecule SR4B of the present invention and analogue thereof when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, intracutaneous or topical.
With nucleic acid molecule SR4B of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains compound and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Nucleic acid molecule SR4B of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, 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 nucleic acid molecule SR4B of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, 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 nucleic acid molecule SR4B that contains effective therapeutic dose and carrier pharmaceutically or vehicle.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 item key 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.
Nucleic acid molecule SR4B of the present invention adds in the beta Cell of islet (NIT) of mouse, and content of insulin increases in the cells and supernatant.Therefore think that nucleic acid molecule SR4B of the present invention has the function of insulin secretion in the beta Cell of islet (NIT) that promotes mouse, can be used as new antidiabetic medicine or develop, for alleviating and the treatment diabetes provide a kind of new approach and means.
Embodiment
Embodiment 1 siRNA disturbs endogenous BRSK2 to express later influence to insulin secretion
(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 (siRNA interferential negative control), siRNA (dTdTAAAGUCUGCGAUGCGGAUG and CAUCCGCAUCGCAGACUUUdTdT)-5 1 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 perfect medium (DMEM high glucose medium+15% foetal calf serum) into and continue to cultivate 24 hours.
(4) change perfect 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 mouse of usefulness siRNA, and same radioimmunoassay method detected result shows that content of insulin increases in the cells and supernatant.
Embodiment 2 BRSK2 overexpressions are to the influence of insulin secretion
(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.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 perfect medium (DMEM high glucose medium+15% foetal calf serum) into and continue to cultivate 24 hours.
(4) change perfect 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 Regular Insulin reduces among the mouse islets Beta cell NIT.
Claims (8)
1. an isolated nucleic acid molecule is characterized in that, its sequence is CAUCCGCAUCGCAGACUUU.
2. a nucleic acid molecule is characterized in that, its sequence is CAUCCGCAUCGCAGACUUUdTdT.
3. a carrier is characterized in that, it contains claim 1 or 2 described nucleic acid molecule.
4. host cell that contains the described carrier of claim 3.
5. the preparation method as claim 1 or 2 described nucleic acid molecule is characterized in that, by the sequence of claim 1 or 2 described nucleic acid molecule, with each Nucleotide group dehydrating condensation successively.
6. the application in the preparation antidiabetic medicine as claim 1 or 2 described nucleic acid molecule.
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 molecule and carrier pharmaceutically or vehicle.
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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| CN2006100302539A CN101130769B (en) | 2006-08-22 | 2006-08-22 | Nucleotide molecule SR4B and application of the same in producing antidiabetic medicament |
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| CN2006100302539A CN101130769B (en) | 2006-08-22 | 2006-08-22 | Nucleotide molecule SR4B and application of the same in producing antidiabetic medicament |
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| CN101130769A CN101130769A (en) | 2008-02-27 |
| CN101130769B true CN101130769B (en) | 2011-06-15 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738648A (en) * | 2003-01-17 | 2006-02-22 | 佛罗里达大学研究基金会有限公司 | Small interference RNA gene therapy |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738648A (en) * | 2003-01-17 | 2006-02-22 | 佛罗里达大学研究基金会有限公司 | Small interference RNA gene therapy |
Non-Patent Citations (1)
| Title |
|---|
| 无.基因"沉默"疗法:治疗糖尿病和癌症的希望.《健康大视野》.2005,(第3期),26－27. * |
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Granted publication date: 20110615 Termination date: 20160822 |