CN101586103B - RNA Interference Sequence of Glucagon Receptor Gene - Google Patents

Abstract

The invention relates to an RNA interference sequence of glucagon receptor gene in the technical field of gene, wherein the first RNA interference sequence is a double-stranded RNA molecule, and the sense strand of the RNA molecule has the sequence shown in SEQ ID NO: 1, and the antisense strand has the sequence shown in SEQ ID NO: 2; the second RNA interference sequence is a double-stranded RNA molecule, the sense strand of which has the sequence of SEQ ID NO: 3, and the antisense strand has a sequence shown in SEQ ID NO: 4; the third RNA interference sequence is a double-stranded RNA molecule, the sense strand of which has the sequence of SEQ ID NO: 5, and the antisense strand has the sequence shown in SEQ ID NO: 6. The interfering RNA sequence of the invention can effectively reduce the blood sugar concentration of a diabetes model mouse and improve the glucose utilization capability of the mouse.

Description

The RNA interference sequence of glucagon receptor gene

Technical field

What the present invention relates to is the RNA interference sequence in a kind of gene engineering field, is specifically related to the RNA interference sequence of a kind of glucagon receptor (GCGR) gene.

Background technology

RNA disturbs (RNA interference, RNAi) be to suppress a kind of phenomenon that specific gene is expressed in the organism, it is meant when existing with endogenous mRNA coding region homologous double-stranded RNA in the cell, degraded takes place and causes the phenomenon of genetic expression silence in this mRNA, this phenomenon occurs in post-transcriptional level, is called PTGS again.The RNA interference effect is to realize by the more stable intermediate medium of a class.Study on plants is proved the double-stranded RNA complex body is degraded into the small RNA molecular about 35nt earlier, they combine with mRNA by sequence is complementary then, thereby cause the mRNA degraded.To studies have shown that of fruit bat, length is that the small RNA molecular of 21～23nt is the immediate cause that causes the RNA interference phenomenon, this small RNA molecular be referred to as siRNA (smallinterfering RNA, siRNA).SiRNA is considered to a kind of method of regulating and control gene expression in vivo fast, efficiently, thereby has great application prospect aspect gene therapy.

Clinical study is thought, is main for insulin resistant, or hypoinsulinism is the type ii diabetes illness due to the main companion, can be used for reaching the purpose of stablizing glucose level in patient's body by what regulate hyperglycemic-glycogenolytic factor.On the one hand, hyperglycemic-glycogenolytic factor has intensive and promotes glyconeogenesis and glycogenolytic effect, therefore can be used for relief of symptoms by the rising blood sugar of glucagon suppression; On the other hand, to the adjusting of hyperglycemic-glycogenolytic factor hormone will be indirect the insulin secretion situation that has influence on, reach the purpose of relief of symptoms.Glucagon receptor molecule GCGR has the g protein coupled receptor family of wearing membrane structure for seven times, mainly is distributed in organs such as liver, kidney, pancreas.Wherein, the glucagon receptor of liver organ distribution and the interaction between the hyperglycemic-glycogenolytic factor have great importance for blood glucose regulation.Blood sugar concentration obviously reduces in the gene knock-out mice model of discovery GCGR such as R.W.Gelling, and the sugar tolerance of mouse has obtained good raising.Small-molecule drug glucagon receptor (GCGR) antagonist is reported in that the diabetic mice model is medium also a good hypoglycemic stabilization in recent years.And the Antisense Suppression Nucleotide of GCGR also is proved the hyperglycemia that can effectively improve in the diabetic mice model.It is a main Remedies for diabetes target spot that these researchs have disclosed liver GCGR gene, and the effect that suppresses liver GCGR acceptor can be alleviated the symptom of blood sugar increasing, the treatment diabetes.

Find still do not have the relevant report of the RNA interference sequence of hyperglycemic-glycogenolytic factor acceptor gene through literature search to prior art.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of RNA interference sequence of glucagon receptor gene is provided.RNA interference sequence of the present invention can effectively reduce the blood sugar concentration of diabetic mice.

The present invention realizes by following technical scheme:

The RNA interference sequence of the first kind of glucagon receptor gene that the present invention relates to, this sequence are double stranded rna molecule, and the positive-sense strand of this RNA molecule has the sequence shown in the SEQ ID NO:1, and antisense strand has the sequence shown in the SEQID NO:2.

The RNA interference sequence of the second kind of glucagon receptor gene that the present invention relates to, this sequence are double stranded rna molecule, and the positive-sense strand of this RNA molecule has the sequence shown in the SEQ ID NO:3, and antisense strand has the sequence shown in the SEQID NO:4.

The RNA interference sequence of the third glucagon receptor gene that the present invention relates to, this sequence are double stranded rna molecule, and the positive-sense strand of this RNA molecule has the sequence shown in the SEQ ID NO:5, and antisense strand has the sequence shown in the SEQID NO:6.

The present invention has following beneficial effect: interference RNA sequence of the present invention comparatively effectively reduces the blood sugar concentration of diabetic mice, by the analysis to area AUC under blood sugar-concentration curve, the model mice after the administration is being greatly improved aspect the glucose utilization ability.

Description of drawings

Fig. 1 is diabetic mice model sugar tolerance test blood sugar concentration-time curve synoptic diagram after the administration;

Fig. 2 is that diabetic mice model blood-sugar content changes synoptic diagram after the administration of different siRNA group.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

Reagent: tetraoxypyrimidine Alloxan monohydrate (the HPLC pure reagent is available from U.S. sigma company, production code member A7413);

Glucose assays test kit---glucose oxidase-peroxidase method (available from Shanghai Rongsheng Bioisystech Co., Ltd, production code member 361510, lot number 20030101);

Laboratory animal: healthy male ♂ Kunming mouse in six ages in week is provided by Shanghai Si Laike experimental animal responsibility company limited that (credit number SCXK (Shanghai)-2007-0005) experimentizes after cultivating a week by adaptability in 20 ℃ of light and shade alternate environments is pre-.

Experimentation comprises the steps:

Step 1, the siRNA design is synthetic

The siRNA of design mouse glucagon receptor (GCGR), refseq sequence NM_008101 according to GCGR, use the siRNA design software of masschusetts, u.s.a Whitehead biomedical research institute, format is the siRNA of AA19NTT, and it is as follows to select three results:

GCGR

SiRNA_1 target sequence AAAGCTCTTCAGGAGGAAAGGTT (1451-1473),

Positive-sense strand 5 '-AGCUCUUCAGGAGGAAAGGUU

Antisense strand 3 '-UUUCGAGAAGUCCUCCUUUCC;

SiRNA_2 target sequence AAAGTGCAGCACCGCCTAGTGTT (455-477),

Positive-sense strand 5 '-AGUGCAGCACCGCCUAGUGUU,

Antisense strand 3 '-UUUCACGUCGUGGCGGAUCAC;

SiRNA_3 target sequence AACTACATCCATGGGAACCTGTT (707-729),

Positive-sense strand 5 '-CUACAUCCAUGGGAACCUGUU

Antisense strand 3 '-UUGAUGUAGGUACCCUUGGAC.

The non-target siRNA of control group

Positive-sense strand 5 '-UUCUCCGAACGUGUCACGUTT,

Antisense strand 3 '-TTAAGAGGCUUGCACAGUGCA.

The synthetic of siRNA finished by Shanghai JiMa pharmacy Technology Co., Ltd.

Step 2, the foundation of diabetic mice model and model stability property testing

20 of Kunming mouses, body weight and blood glucose value after adapting to one week of feeding environment and surveying normal mouse empty stomach 4h; After water 12h is can't help in the mouse fasting, prepare fresh tetraoxypyrimidine inductor solution, administering mode is disposable mouse peritoneal injection (ip), and dosage is 200mgKg^-1The mouse body weight.Be that mouse is replenished drinking-water and food after injection finishes, and change bedding and padding.Behind the injection inductor 72h, measure later mouse body weight and the blood sugar of 4h on an empty stomach, detection is modeled as power and divides into groups.In 16 days, detect mouse body weight situation after the modeling to understand the model state of health.

Step 3, the blood sugar test standard method is set up

The mouse fasting be can't help water on an empty stomach after the 4h, keeps mouse mood calmness as far as possible, gets about blood 100ul (3 bleed to 4) in the mouse eyeground with glass capillary rapidly, places the 0.5ml centrifuge tube.Blood sample in 4 ℃ of refrigerator and cooled hide be placed to the centrifuge tube bottom and coagulation of blood occurs after, centrifugal (3000rpm 5min) gets upper serum and makes glucose content and detect in 4 ℃ immediately.

R1 in the glucose detection test kit (Shanghai Rongsheng Bioisystech Co., Ltd, article No. 361510) is mixed as the glucose test fluid with the R2 equal proportion.Get test sample book 4ul and add in the test fluid of 1ml, fully mixing places 37 ℃ of water-bath 15min, makes it colour developing.At ultraviolet wavelength 505nm place,, read the absorbance A of sample tube with the zeroing of skip test liquid adding distil water.Simultaneously,preparation 5～30mmolL^-1The glucose reference liquid of gradient concentration, thus the production standard curve reads the glucose concn of sample, typical curve R²＞0.99.

Step 4 is administered systemically in the mouse model siRNA body

With becoming the mouse of mould to be divided into 4 groups according to the blood sugar test situation, be respectively siRNA_1, siRNA_2, siRNA_3 group and control group, administering mode adopts the quick push injection of tail vein high pressure.Synthetic siRNA is dissolved among the PBS that DEPC handled, carries out administration according to 0.1nmol/g mouse body weight: the liquor capacity that every mouse model is injected fast is 8% of a mouse body weight, is about the PBS-siRNA solution about 3ml.Close observation mouse after administration finishes is in time replenished drinking-water and feed.

Step 5, carbohydrate tolerance test

Carbohydrate tolerance test: 2d after administration, fasting 4h get blood (when being zero), and abdominal injection glucose (2g/kg) is measured and given 0.5,1,1.5,2 hours the blood glucose value in sugar back and calculate area under the blood glucose curve then.

Step 6, mouse model change of blood sugar curve

Respectively at 1d after the administration, 2d, 4d, 8d measures each mouse blood sugar value in 4 groups according to the blood sugar test standard method in the step 3.Obtain the mouse model blood sugar-time curve in 8 days after the administration.

Test-results and analysis

(1) foundation of diabetic mice model

MBG 7.1 ± 3.0mmolL before 20 mouse are induced^-1, induce dead two of back, blood sugar is greater than 15mmolL^-1Mouse is 12 (MBG 34.6 ± 3.9mmolL^-1); Inducing and being modeled as power is 60%.

(2) sugar tolerance test after the siRNA administration

Calculate four groups (1) siRNA-1 as shown in Figure 1, (2) siRNA-2, (3) siRNA-3, area (AUC) is respectively under (4) non-target contrast siRNA blood sugar-time curve: 964.5; 3216.1; 2606.9 and 4173.9minmmolL^-1AUC has reflected the utilize degree of animal to glucose, and the area of AUC is more little, proves that it can effectively utilize glucose more, wherein (1), (2), the AUC area of (3) group is respectively 23.11%, 77.1% and 62.5% of contrast (4) group.

(3) blood sugar-time changing curve after the siRNA administration

8 days model mice blood sugar-time curves after the administration as shown in Figure 2: induce successfully the diabetic mice model to be divided into 4 groups for 12:

1. blood sugar 38.4 ± 3.3mmolL before the siRNA-1 slice groups, administration^-1, first day blood sugar 5.4 ± 1.5mmolL after the administration^-1, before administration, reduced by 86.0%; After this blood-sugar content gos up to some extent in several days, but the 8th day blood-sugar content still is lower than the preceding blood sugar of administration, is 11.7 ± 3.5mmolL^-1, before administration, reduced by 69.4%;

2. blood sugar 29.2 ± 4.6mmolL before the siRNA-2 slice groups, administration^-1, first day blood sugar 17.1 ± 1.4mmolL after the administration^-1, blood sugar has reduced by 41.4% before administration, and several days blood sugar after this go up;

3. blood sugar 34.8 ± 3.8mmolL before the siRNA-3 slice groups, administration^-1, first day blood sugar 27.1 ± 7.8mmolL after the administration^-1, blood sugar has reduced by 22.2% before administration; After this several days blood sugar go up;

4. blood sugar 36.1 ± 2.1mmolL before the non-target siRNA sequence control group, administration^-1, first day blood sugar 37.8 ± 4.2mmolL after the administration^-1, blood sugar has raise 4.7% before administration; After this several days blood sugar raise gradually, and model blood sugar has raise 32% in the time of the 8th day before administration.

Sequence table

＜110〉Shanghai Communications University

＜120〉the RNA interference sequence of glucagon receptor gene

<160>6

<170>PatentIn?version?3.3

<210>1

<211>21

<212>RNA

＜213〉artificial sequence

<400>1

agcucuucag?gaggaaaggu?u 21

<210>2

<211>21

<212>RNA

＜213〉artificial sequence

<400>2

ccuuuccucc?ugaagagcuu?u 21

<210>3

<211>21

<212>RNA

＜213〉artificial sequence

<400>3

agugcagcac?cgccuagugu?u 21

<210>4

<211>21

<212>RNA

＜213〉artificial sequence

<400>4

cacuaggcgg?ugcugcacuu?u 21

<210>5

<211>21

<212>RNA

＜213〉artificial sequence

<400>5

cuacauccau?gggaaccugu?u 21

<210>6

<211>22

<212>RNA

＜213〉artificial sequence

<400>6

cagguuccca?uggauguag?uu 21

Claims (1)

1. the RNA interference sequence of a glucagon receptor gene is characterized in that, this sequence is a double stranded rna molecule, and the positive-sense strand of this RNA molecule is shown in SEQ ID NO:1, and antisense strand is shown in SEQ ID NO:2.

Images