CN101683354B - Liver damage relevant drug target and application thereof - Google Patents
Liver damage relevant drug target and application thereof Download PDFInfo
- Publication number
- CN101683354B CN101683354B CN2008102004800A CN200810200480A CN101683354B CN 101683354 B CN101683354 B CN 101683354B CN 2008102004800 A CN2008102004800 A CN 2008102004800A CN 200810200480 A CN200810200480 A CN 200810200480A CN 101683354 B CN101683354 B CN 101683354B
- Authority
- CN
- China
- Prior art keywords
- scd1
- hepatic injury
- sirna molecule
- sirna
- antagonist
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a liver damage relevant drug target and application thereof. The drug target is stearoyl coenzyme A desaturase (Scd1), and liver damage of mammals can be obviously relieved by descending the expression of Scd1. The invention also discloses application of antagonist of Scd1 in preparing a composite for inhibiting liver damage of mammals. The invention further discloses a method for screening a potential material for inhibiting liver damage of mammals.
Description
Technical field
The invention belongs to the gene technology field, more specifically, the present invention relates to a kind of hepatic injury relevant drug target and application thereof.
Background technology
Hepatitis is the major disease of serious harm human health.Hepatitis particularly viral hepatitis continues in higher level at the sickness rate of China; With regard to hepatitis B, the whole world 2,000,000,000 people infect hepatitis B virus, and wherein 6.9 hundred million in China; There are HBsAg (HBsAg) carrier 3.5 hundred million people in the whole world, and wherein 1/3rd in China; The whole world has 750,000 people to die from hepatitis B virus to dye the disease that causes every year, and wherein 280,000 people are from China.
Hepatitis is seen from the cause of disease and is mainly contained viral hepatitis, alcoholic hepatitis, autoimmune hepatitis etc.But discover, no matter its initial cause of disease how, common in the pathological process that inflammation and immune-mediated hepatocyte and liver tissue injury are these diseases must be through process and the main cause that influences disease severity and prognosis.Yet this area is deep not enough for which factor or molecule and inflammation actually and immune-mediated hepatocyte and the closely-related research of liver tissue injury.
Therefore, this area is necessary to find the relevant new drug target of hepatic injury very much, for the treatment of hepatic injury or relevant disease provides new approach.
Summary of the invention
The object of the present invention is to provide the purposes of the antagonist of a kind of stearyl-coenzyme A desaturase 1 (Scd1), be used to prepare the compositions that suppresses the mammal hepatic injury.
Another object of the present invention is to provide a kind of method of screening the potential material that suppresses the mammal hepatic injury.
In first aspect of the present invention, the purposes of the antagonist of a kind of stearyl-coenzyme A desaturase 1 (Scd1) is provided, be used to prepare the compositions that suppresses the mammal hepatic injury.
In another preference, described hepatic injury is inflammation and immune-mediated hepatocyte and liver tissue injury.
In another preference, described hepatic injury comprises: hepatitis, liver organization is downright bad.
In another preference, described compositions also is used for
Reduce the mammiferous gpt activity of hepatic injury; Or
Reduce the concentration of inflammatory cytokine in the hepatic injury mammal.
In another preference, described inflammatory cytokine is selected from: TNF-α, or IFN-γ.
In another preference, the antagonist of described stearyl-coenzyme A desaturase 1 is selected from:
Specificity disturbs the siRNA molecule or the GEM 132 of stearyl-coenzyme A desaturase 1 gene expression; Or
Specificity and stearyl-coenzyme A desaturase 1 bonded antibody or part.
In another preference, the antagonist of described stearyl-coenzyme A desaturase 1 is:
(a) siRNA molecule of nucleotide sequence shown in the SEQ ID NO:1; And/or
(b) nucleotide sequence and the complementary siRNA molecule of SEQ ID NO:1.
In another preference, described siRNA molecule is a duplex molecule, and its forward chain has nucleotide sequence shown in the SEQID NO:1; Reverse strand has nucleotide sequence shown in the SEQ ID NO:2.
In second aspect of the present invention, a kind of siRNA molecule that is used to suppress the mammal hepatic injury is provided, described siRNA molecule is:
(a) siRNA molecule of nucleotide sequence shown in the SEQ ID NO:1; And/or
(b) nucleotide sequence and the complementary siRNA molecule of SEQ ID NO:1.
In the third aspect of the invention, a kind of compositions that is used to suppress the mammal hepatic injury is provided, described compositions contains:
(1) the described siRNA molecule of effective dose; With
(2) pharmaceutically acceptable carrier.
In fourth aspect of the present invention, a kind of method of screening the potential material that suppresses the mammal hepatic injury is provided, said method comprises:
(1) handles the system of expressing stearyl-coenzyme A desaturase 1 with candidate substances; With
(2) expression or the activity of stearyl-coenzyme A desaturase 1 in the said system of detection;
Wherein, if said candidate substances can reduce the expression or the activity of stearyl-coenzyme A desaturase 1, show that then this candidate substances is to suppress the potential material of mammal hepatic injury.
In another preference, step (1) comprising: in testing group, candidate substances is joined in the system of expressing stearyl-coenzyme A desaturase 1; And/or
Step (2) comprising: detect the expression or the activity of stearyl-coenzyme A desaturase 1 in the system of testing group, and with matched group relatively, wherein said matched group is the system of not adding the expression stearyl-coenzyme A desaturase 1 of said candidate substances;
(preferably significantly be lower than, more than 20%, preferable is low more than 50% as low if the expression of stearyl-coenzyme A desaturase 1 or activity are lower than on statistics in the testing group; Better is low more than 80%) matched group, just show that this material standed for is the potential material that suppresses the mammal hepatic injury.
In another preference, described system is selected from: cell system (as expressing the liver cell of Scd1); (or cell culture objects system), subcellular fraction system, solution system, organizational framework, organ systems or animal system.
In another preference, described method also comprises: the potential material to obtaining carries out further cell experiment and/or animal experiment, from candidate substances, further to select and to confirm for suppressing the useful material of mammal hepatic injury.
On the other hand, the present invention also provides a kind of method that suppresses the mammal hepatic injury, and said method comprises: expression or the activity of reducing stearyl-coenzyme A desaturase 1 in the said mammalian body.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1. normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage.Get serum after 17 hours, the concentration of tumor necrosis factor-alpha and gamma interferon in the detection serum.
The A.ELISA method detects the concentration of gamma interferon in the serum;
The B.ELISA method detects the concentration of tumor necrosis factor-alpha in the serum.
Fig. 2. normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage, get the determination of serum glutamate pyruvate transaminase after 17 hours and live.Data are represented with the form of average ± standard deviation.
Fig. 3. the H&E dyeing of liver and TUNEL dyeing.Normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage.After 17 hours, get liver tissue slices H&E dyeing or TUNEL dyeing.
A. normal mouse is induced H&E coloration result behind the CIH;
The B.Scd1 deficient mice is induced H&E coloration result behind the CIH;
C. normal mouse is induced TUNEL coloration result behind the CIH;
D.Scd1 deficient mice TUNEL coloration result.
Fig. 4. tail vein injection dosage is the mice of the ConA of 15mg/kg behind the injecting normal saline, and tail vein injection dosage is that the mice (siRNA) of the ConA of 15mg/kg is got the determination of serum glutamate pyruvate transaminase in ConA injection respectively and lives after 17 hours behind the injection siRNA.
expression is not injected the pyruvic transaminase of the normal mouse of ConA and is lived.Data are represented with the form of " average ± standard deviation ".
The specific embodiment
The inventor discloses the generation of stearyl-coenzyme A desaturase 1 (Scd1) and hepatic injury first or develops closely related through extensive and deep research.After in advance in the chromosome of liver damage animal model, knocking out the Scd1 gene; The symptom of Scd1 deficient animals generation hepatic injury significantly alleviates; Therefore can know that Scd1 albumen is a kind of factor that in the hepatic injury pathogenesis, plays a significant role, thereby can be used as the medicine of drug target exploitation prevention or treatment hepatic injury.
Scd1 and uses thereof
The Scd gene code be positioned the Stearoyl Coenzyme A dehydrogenase on the endocytoplasmic reticulum; It can catalysis source food or the dehydrogenation of de novo synthesis satisfied fatty acid generate monounsaturated fatty acid; Being lipometabolic key enzyme, is the important regulatory factor in the energy metabolism therefore.In mice; Scd1 is the Scd gene that in the lipid metabolism of adult mice, plays main effect; Two Scd genes all have the homology more than 85% with the Scd1 gene of mice in the human genome, and research in the past shows that Scd1 is having important effect aspect body weight and the insulin sensitivity regulating.
Any suitable Scd1 albumen includes in the present invention.Described Scd1 albumen comprises Scd1 albumen or its bioactive fragment of total length.Preferably, the proteic aminoacid sequence of described Scd1 can with the GenBank accession number: the sequence shown in the NP_033153.2 is substantially the same.Encode the proteic nucleotide sequence of described Scd1 can with the GenBank accession number: the sequence of the degeneracy of the sequence shown in the NM_009127.3 or this sequence is substantially the same.
The proteic aminoacid sequence of Scd1 that passes through replacement, disappearance or the interpolation of one or more amino acid residues and form is also included among the present invention.Scd1 albumen or its bioactive fragment comprise the alternative sequence of a part of conserved amino acid, and said sequence through the aminoacid replacement does not influence its activity or kept the activity of its part.For example, described Scd1 albumen can come from Mus (like mice), also can be come from the people or other is mammiferous, aminoacid sequence near or essentially identical (greater than 70%, preferable homogeny is greater than 80% like homogeny; Better homogeny is greater than 85%; Best homogeny is greater than 90%) and the albumen of biological activity identical with the proteic biological activity of mice Scd1 (generation or development as for hepatic injury have important function).
The inventor utilizes a kind of mice of Scd1 gene generation deletion mutation; Utilize internationally recognized liver damage animal model; Research has confirmed that the downward modulation (or disappearance) of Scd1 gene has significant protective effect to immune-mediated hepatic injury; The activity of serum glutamic pyruvic transminase (ALT) is that (normal mouse ALT value is about 50U/L below the 500U/L behind the mice induction of immunity liver damage of Scd1 gene function downward modulation (or disappearance); The ALT meansigma methods is more than 5000U/L during normal mouse induction of immunity liver damage); The pathological analysis of liver also shows simultaneously, and the mice of Scd1 gene function disappearance almost can't see the destruction and the male apoptotic cell of TUNEL of tangible liver normal configuration.
The deep work of the inventor shows that also the disappearance of the function of Scd1 makes the multiple inflammatory cytokine of mediation hepatic injury, comprises the remarkable decline that IFN-and tumor necrosis factor are expressed, thereby the liver protecting is avoided damage.
Can know that through above-mentioned research work Scd1 is new, as liver inflammation and relevant disease to be had a protective effect drug target.Can be used as the novelty and effective means of treatment liver inflammation and relevant disease to the various treatment meanss of Scd1.
Antagonist of Scd1 and uses thereof
Based on the inventor's above-mentioned new discovery, the invention provides the purposes of the antagonist of a kind of Scd1, be used to prepare the compositions that suppresses the mammal hepatic injury.Described hepatic injury is inflammation and immune-mediated hepatocyte and liver tissue injury preferably.Described hepatic injury includes but not limited to: hepatitis, liver organization is downright bad.The antagonist of described Scd1 also is used to reduce the mammiferous gpt activity of hepatic injury.The antagonist of described Scd1 also is used for reducing the concentration of hepatic injury mammal inflammatory cytokine.Described inflammatory cytokine comprises: TNF-α or IFN-γ.
As used herein, described Scd1 gene or proteic antagonist have comprised inhibitor, adjustment down, blocker, blocker etc.
Described Scd1 gene or proteic antagonist are meant the proteic activity of any Scd1 of reduction, reduce the material of Scd1 gene or proteic stability, the proteic expression of downward modulation Scd1, minimizing Scd1 albumen effective acting time or inhibition Scd1 gene transcription and translation; These materials all can be used for the present invention; As for downward modulation Scd1 useful material, thereby can be used for prevention or treatment hepatic injury.For example, described antagonist is: specificity disturbs the siRNA molecule or the GEM 132 of Scd1 gene expression; Or specificity and bonded antibody of Scd1 or part.
As a kind of optimal way of the present invention, the antagonist of described Scd1 is a species specificity and the bonded antibody of Scd1.Described antibody can be monoclonal antibody or polyclonal antibody.Available Scd1 protein immune animal, like rabbit, mice, rat are waited and produce polyclonal antibody; Multiple adjuvant can be used for the enhance immunity reaction, includes but not limited to Freund adjuvant etc.Similarly, expressing Scd1 or its has antigenic segmental cell and can be used to immune animal and produce antibody.Described antibody also can be monoclonal antibody, and this type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
As a kind of optimal way of the present invention, the antagonist of described Scd1 is the specific siRNA molecule of a kind of Scd1 (siRNA).As used herein; Described " siRNA (small interferingRNA; siRNA) " be meant a kind of short segments double stranded rna molecule, can be the specific mRNA of target degraded with the mRNA of homology complementary series, this process is exactly that RNA disturbs (RNA interference) process.
SiRNA can be prepared into the form of double-strandednucleic acid, and it contains a positive-sense strand and an antisense strand, and these two chains only form double-stranded under the condition of hybridization.A double-stranded RNA complex can be prepared by positive-sense strand that is separated from each other and antisense strand.Therefore, by way of example, complementary positive-sense strand and antisense strand are chemosynthesis, can produce synthetic double-stranded RNA complex through annealing hybridization thereafter.
As preferred especially mode of the present invention, a kind of respond well siRNA molecule is provided, described siRNA molecule can specific interference Scd1 expression of gene, does not have significant homology with other human nucleic acid sequence; And empirical tests, it has the effect that good interference Scd1 expresses.Described siRNA molecule is the siRNA molecule with the nucleotide sequence shown in the SEQ ID NO:1.
The present invention has no particular limits the method for preparing of siRNA, includes but not limited to: chemical synthesis, in vitro transcription method etc.Should be understood that those skilled in the art after the dependency that gets cicada Scd1 and hepatic injury, can prepare described siRNA easily with various approach, thereby be used to suppress hepatic injury.Described siRNA can be transported to (like the liver position) in the body through adopting suitable transfection reagent, or also can adopt multiple technologies known in the art to be transported in the body.
In addition, the positive-sense strand and the antisense strand that comprise of siRNA can prepare through the expression cassette of one or more coding positive-sense strands and antisense strand.When positive-sense strand and antisense strand during by an independent expression cassette coding, they can form isolating positive-sense strand and antisense strand from the transcript cleaved that generates, and hybridization thereafter generates double-chain small disturbance RNA.
Described siRNA can be transported in the cell through adopting suitable transfection reagent, or also can adopt multiple technologies known in the art to be transported in the cell.
The present invention also provides a kind of compositions, and it contains effective dose (like 0.000001-50wt%; Preferable 0.00001-20wt%; Better, the antagonist of described Scd1 0.0001-10wt%), and pharmaceutically acceptable carrier.Described compositions can be used for suppressing hepatic injury.The antagonist of any aforesaid Scd1 all can be used for preparation of compositions.
As used herein, said " effective dose " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.Said " pharmaceutically acceptable carrier " refers to be used for the carrier of therapeutic agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.Acceptable carrier can contain liquid on combination of Chinese medicine is learned, like water, saline, buffer.In addition, also possibly there is complementary material in these carriers, like filler, lubricant, fluidizer, wetting agent or emulsifying agent, pH buffer substance etc.Can also contain cell transfecting reagent in the described carrier.
After the purposes of the said Scd1 gene of cicada or proteic antagonist, can adopt several different methods well known in the art that described antagonist or its encoding gene or its pharmaceutical composition are delivered medicine to mammal.Include but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, slow release give etc.; Preferably, said administering mode is that non-intestinal gives.
Preferably, can adopt the means of gene therapy to carry out.Such as, can be directly with the antagonist of Scd1 through delivering medicine to the experimenter such as methods such as injections; Perhaps; The ceneme that can will carry the antagonist of Scd1 through certain approach is (such as expression vector or virus etc.; Or siRNA) is delivered on the target spot; And make it the Scd1 antagonist of expression activity, and concrete condition need be looked the type of described antagonist and decided, and these all are well-known to those skilled in the art.
The effective dose of the antagonist of Scd1 of the present invention can change with the order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be confirmed (for example through clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization rate of described Scd1 gene or proteic antagonist, metabolism, half-life etc.; The patient the order of severity, patient's body weight, patient's immune state, the approach of administration etc. of the disease that will treat.Usually, the dosage when antagonist every day of Scd1 of the present invention with about 0.00001mg-50mg/kg the weight of animals (preferable 0.0001mg-10mg/kg the weight of animals) gives, and can obtain gratifying effect.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
Drug screening
After the closely related property that gets cicada Scd1 and hepatic injury, can screen expression or the active material that suppresses Scd1 based on this characteristic.Can from described material, find for prevention or the real useful medicine of treatment hepatic injury.
Therefore, the present invention provides a kind of method of screening the potential material that suppresses hepatic injury, and described method comprises: handle the system of expressing Scd1 with candidate substances; With the expression or the activity that detect Scd1 in the said system; If said candidate substances can suppress expression or the activity of Scd1, show that then this candidate substances is to suppress the potential material of hepatic injury.The system of described expression Scd1 for example can be cell (or cell culture) system, and described cell can be the cell of endogenous expression Scd1; It maybe can be the cell of recombinant expressed Scd1.The system of described expression Scd1 can also be subcellular fraction system, solution system, organizational framework, organ systems or animal system (like animal model, the animal model of preferred non-human mammal is like Mus, rabbit, sheep, monkey etc.) etc.
In optimal way of the present invention, when screening,, also matched group can be set in order to be easier to observe expression or the active change of Scd1, described matched group can be the system of not adding the expression Scd1 of said candidate substances.
As optimal way of the present invention, described method also comprises: the potential material to obtaining carries out further cell experiment and/or animal experiment, with further selection and definite for suppressing the real useful material of hepatic injury.
The present invention has no particular limits for the detection method of the proteic expression of Scd1, activity, amount or secretion situation.Can adopt conventional protein quantification or half-quantitative detection technology, for example (but being not limited to): SDS-PAGE method, Western-Blot method etc.
On the other hand, the present invention also provides the potential material of the inhibition hepatic injury of adopting said screening technique acquisition.The material that these Preliminary screening go out can constitute a screening storehouse so that people finally can therefrom filter out can be for the expression and the activity that suppress Scd1, and then suppress the useful material of hepatic injury.
Major advantage of the present invention is:
(1) disclose first the generation of Scd1 and hepatic injury or develop closely related, thereby can be used as the medicine of drug target exploitation prevention or treatment hepatic injury.
(2) found based on the inventor's new discovery that can to suppress Scd1 be antagonist, it can suppress the expression of Scd1 very effectively, thereby suppresses the generation of hepatic injury.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: the described condition of lab guide (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Experimental group: (1) matched group (wild type) mice;
(2) SCD1 deficient mice.
The BIAO and BEN that is used to estimate its therapeutic effect comprises serum, liver tissue slices, and liver organization RNA etc.
Embodiment 1.Scd1 defective is to the influence of hepatic injury mice serum cytokine
The report of Lu in 2004 etc. a kind of mice ab of gene mutation
Xyk(referring to Mol Gen Genomics (2004) 272:129-137).This mice and Balb/c mice were backcrossed after 2 generations,, be used for research of the present invention as the Scd1 deficient mice.
Normal (wild type) mice: the Balb/c mice, available from Shanghai Slac Experimental Animal Co., Ltd..
In order to study the relevant disease of liver, there is nearly decades the relevant animal model of multiple liver to be established.The animal model of setting up the immunity hepatopathy mainly is to stimulate activating immune cell through multiple means, makes multiple inflammatory cytokine up-regulated, thereby causes the hepatic parenchymal cells apoptosis dead.In these means of stimulating activity immunocyte, tail vein injection ConA is a kind of effective means of generally acknowledging, can simulate the pathogenic process of the multiple mankind's hepatic disease.Induced the back 8 to 24 hours at liver injury model, the serum glutamic pyruvic transminase of animal (ALT) activity sharply increases, and the visible liver normal configuration of HE dyeing is destroyed, and a large amount of hepatic parenchymal cells apoptosis is dead.In this model, cytokine comprises TNF α, IFN γ, and il-1 2, interleukin-4 has played pivotal role to the damage of liver.
Normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage.Get serum after 2 hours and 8 hours, adopt the ELISA method to detect the concentration of tumor necrosis factor-alpha and gamma interferon in the serum respectively.
ELISA detects use Duoset ELISA kit, and (R&D, MN USA), operate according to the description that the manufacturer provides.Concise and to the point step is:
100 μ L one are anti-, and (USA) wrapper sheet spends the night for R&D, MN, washes behind the plate sealing of 300 μ L sealing buffer 2 hours; Wash and add 100 microlitre sample and standard substance behind the plate, hatched 2 hours, add two anti-(R&D after washing plate; MN USA) is hatched 2 hours, washes to add enzyme mark streptavidin behind the plate; Add the substrate colour developing, sulphuric acid is ended, ELIASA 450nm reading (reference wavelength 540nm).Concentration according to each cytokine in the standard curve calculation sample.
The result sees Fig. 1.A is that ELISA detects tumor necrosis factor-alpha (TNF-α) concentration result in the serum; B is that ELISA detects swollen gamma interferon (IFN-γ) concentration result in the serum.
Visible by The above results, the content of multiple inflammatory cytokine significantly is lower than matched group in the Scd1 deficient mice serum.
Embodiment 2.Scd1 defective reduces hepatic injury mice gpt activity
Normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage, induce hepatic injury, get serum after 17 hours, and conventional method is measured gpt activity in the serum.
The result sees Fig. 2, and visible Scd1 deficient mice serum glutamic pyruvic transminase activity significantly is lower than the normal mouse control mice.
Therefore, downward modulation Scd1 can significantly reduce the gpt activity of hepatic injury mice in mice.
Embodiment 3.Scd1 deficient mice liver tissue slices is observed
Normal mouse and Scd1 deficient mice are the ConA of 15mg/kg by tail vein injection dosage.After 17 hours, get liver tissue slices and carry out H&E dyeing, perhaps carry out TUNEL dyeing.The result sees Fig. 3.
Fig. 3 A is that normal mouse is induced H&E coloration result behind the CIH, wherein can be observed liver organization and the downright bad phenomenon of large tracts of land occurs, and normal configuration is by heavy damage.
Fig. 3 B is that the Scd1 deficient mice is induced H&E coloration result behind the CIH, does not see inflammatory lesions and necrotic zone in the liver organization.
Fig. 3 C is that normal mouse is induced TUNEL coloration result behind the CIH, wherein can be observed a large amount of male apoptotic cells of TUNEL to occur; Corresponding with the result of Fig. 3 A.
Fig. 3 D is a Scd1 deficient mice TUNEL coloration result, does not wherein also find the TUNEL positive cells; Corresponding with the result of Fig. 3 B.
The preparation of the proteic antibody of embodiment 4. specific anti Scd1
Immune animal is NZw, and is male, and body weight is 2-2.5kg.The immunity flow process: (its aminoacid sequence is referring to the Genbank accession number: NP_033153.2) mixes with Freund ' s adjuvant 1:1, after the emulsifying, subcutaneous and Intradermal multi-point injection carries out immunity with the Scd1 albumen that adopts conventional recombinant expression method to express to obtain.The proteic dosage of Scd1 is 0.9mg/ time/rabbit.First immunisation is used complete Freund ' s adjuvant, once more immunity full Freund ' the s adjuvant that toos many or too much for use.Be 3 weeks the blanking time of immunity.In 1 week of back of immunity for the third time, antiserum is collected in the rabbit blood-letting, obtains anti-Scd1 protein immunization multi-resistance.Utilize Scd1 albumen as antigen, carry out antigen antibody reaction with the anti-Scd1 protein immunization multi-resistance of aforementioned acquisition, the result finds that said anti-Scd1 protein immunization multi-resistance can combine Scd1 albumen by specificity.
Normal mouse is divided into two groups, and one group is matched group (handling with normal saline), and one group is the antibody treatment group.Before inducing hepatic injury with ConA, matched group gives normal saline, and the antibody treatment group gives the proteic antibody of specific anti Scd1 of above-mentioned preparation.In 1 week of successive administration, after every day 1 time, two groups of mices are the ConA of 15mg/kg by tail vein injection dosage.After 17 hours, get liver tissue slices and carry out H&E dyeing, perhaps carry out TUNEL dyeing.
The result finds that compare with matched group, the hepatic injury symptom of antibody treatment group mice is significantly slighter.
Embodiment 5. utilizes siRNA to suppress the compliance test result of Scd1
Sequential design according to the Scd1 gene has synthesized the siRNA of specificity to Scd1, and the double-stranded siRNA sequence of design is:
Group 1 (siRNA1):
Forward chain: 5 ' GCGCAUCUCUAUGGAUAUC3 ' (SEQ ID NO:3);
Reverse strand: 5 ' GAUAUCCAUAGAGAUGCGC3 ' (SEQ ID NO:4).
Group 2 (siRNA2):
Forward chain: 5 ' GAGAUCUCCAGUUCUUACA3 ' (SEQ ID NO:1);
Reverse strand: 5 ' UGUAAGAACUGGAGAUCUC3 ' (SEQ ID NO:2).
When synthetic above-mentioned each bar chain, add respectively that at 3 ' end of forward chain and reverse strand two bases " TT " are as protecting base.
Normal mouse is divided into two groups, and one group is matched group (handling with normal saline), and one group is siRNA processed group 1 (siRNA1); Another group is siRNA processed group 2 (siRNA2).The siRNA processed group gives the synthetic specificity 0.75nmole/g of tail vein injection body weight Scd1siRNA.Scd1siRNA is dissolved in the 2ml normal saline, accomplishes injection in 4 seconds.Matched group gives normal saline with the same manner.
Two groups of mices are the ConA of 15mg/kg by tail vein injection dosage after 48 hours.Measure the serum alt level after 17 hours.
The result is as shown in Figure 4, and the siRNA of visible siRNA processed group 2 has suppressed the expression of Scd1 effectively, thereby has reduced the serum alt level effectively, so this kind siRNA has the inhibitory action of highly significant for hepatic injury.
Get the liver tissue slices of each treated animal and carry out H&E dyeing and also discovery of TUNEL dyeing, the hepar damnification level of the animal of the siRNA of use siRNA processed group 2 is starkly lower than the siRNA that uses siRNA processed group 1, more is lower than matched group.
Embodiment 6 drug screenings
1. cellular level screening
Get the liver cell of normal mouse, but this cell endogenous expression Scd1 albumen.With said cell culture in the CM culture medium.With this kind cell as the cell model that is used to screen the medicine that suppresses the mammal hepatic injury.
Testing group: the culture of the above-mentioned cell of handling with candidate substances;
Matched group: the culture of the above-mentioned cell of handling without candidate substances.
Appropriate time after processing adopts conventional method to measure the proteic expression of Scd1, activity, amount or the secretion situation of said cell.If compare with matched group, the proteic expression of the Scd1 in the testing group, activity, amount or secretion significantly descend more than 30%, explain that then this candidate substances is the material of potential inhibition mammal hepatic injury.
Adopt the proteic antibody of specific anti Scd1 of embodiment 4 preparations to test as candidate substances, the result shows the proteic activity inhibited of Scd1, thereby this antibody is the material of potential inhibition mammal hepatic injury.
2. animal level screening
With normal mouse as animal model.
Testing group: give (also can give) mice through filling stomach, intravenous injection, intramuscular injection, hypodermic mode with the candidate substances lumbar injection;
Matched group: give mice without candidate substances.
Appropriate time after processing adopts conventional method to measure the proteic expression of Scd1 in the mouse liver, activity, amount or secretion situation.If compare with matched group, the proteic expression of the Scd1 in the testing group, activity, amount or secretion significantly descend more than 30%, explain that then this candidate substances is the material of potential inhibition mammal hepatic injury.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Shanghai Inst. of Life Science, CAS
< 120>relevant drug target and the application thereof of a kind of hepatic injury
<130>085292
<160>4
<170>PatentIn?version?3.3
<210>1
<211>19
<212>RNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>siRNA
<400>1
<210>2
<211>19
<212>RNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>siRNA
<400>2
<210>3
<211>19
<212>RNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>siRNA
<400>3
<210>4
<211>19
<212>RNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>siRNA
<400>4
Claims (6)
1. the purposes of the antagonist of a stearyl-coenzyme A desaturase 1 is used to prepare the compositions that suppresses the mammal hepatic injury; Described antagonist is the siRNA molecule that specificity disturbs stearyl-coenzyme A desaturase 1 gene expression; Be selected from:
(a) siRNA molecule of nucleotide sequence shown in the SEQ ID NO:1; And/or
(b) nucleotide sequence and the complementary siRNA molecule of SEQ ID NO:1.
2. purposes as claimed in claim 1 is characterized in that, described hepatic injury comprises: hepatitis or liver organization are downright bad.
3. purposes as claimed in claim 1 is characterized in that, described compositions also is used for:
Reduce the mammiferous gpt activity of hepatic injury; Or
Reduce the concentration of inflammatory cytokine in the hepatic injury mammal.
4. purposes as claimed in claim 3 is characterized in that, described inflammatory cytokine is selected from: TNF-α, or IFN-γ.
5. a siRNA molecule that is used to suppress the mammal hepatic injury is characterized in that, described siRNA molecule is:
(a) siRNA molecule of nucleotide sequence shown in the SEQ ID NO:1; And/or
(b) nucleotide sequence and the complementary siRNA molecule of SEQ ID NO:1.
6. a compositions that is used to suppress the mammal hepatic injury is characterized in that, described compositions contains:
(1) the described siRNA molecule of the claim 5 of effective dose; With
(2) pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102004800A CN101683354B (en) | 2008-09-25 | 2008-09-25 | Liver damage relevant drug target and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102004800A CN101683354B (en) | 2008-09-25 | 2008-09-25 | Liver damage relevant drug target and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101683354A CN101683354A (en) | 2010-03-31 |
| CN101683354B true CN101683354B (en) | 2012-02-22 |
Family
ID=42046885
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102004800A Expired - Fee Related CN101683354B (en) | 2008-09-25 | 2008-09-25 | Liver damage relevant drug target and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101683354B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361352A (en) * | 2013-07-31 | 2013-10-23 | 扬州大学 | Goose primary hepatocyte SCD1 gene silencing kit and applications thereof |
| WO2017150595A1 (en) * | 2016-03-02 | 2017-09-08 | 東レ株式会社 | Immunity inducer |
| CN116144751A (en) * | 2022-11-18 | 2023-05-23 | 中国人民解放军军事科学院军事医学研究院 | Early hepatotoxicity prediction biomarker for traditional Chinese medicine, use method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829690A (en) * | 2003-07-29 | 2006-09-06 | 泽农医药公司 | Pyridyl derivatives and their use as therapeutic agents |
-
2008
- 2008-09-25 CN CN2008102004800A patent/CN101683354B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829690A (en) * | 2003-07-29 | 2006-09-06 | 泽农医药公司 | Pyridyl derivatives and their use as therapeutic agents |
Non-Patent Citations (2)
| Title |
|---|
| 田枫等.固醇辅酶A去饱和酶1 ( scd1)基因突变对老年小鼠血脂、血糖的影响.《中国老年学杂志》.2007,第27卷(第2期),149－150页. * |
| 霍立.硬脂酰辅酶A 去饱和酶-1 在肝脏脂质代谢中的作用及其调节.《 国外医学·消化系疾病分册》.2005,第25卷(第1期),20-22. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101683354A (en) | 2010-03-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bao et al. | A long‐acting FGF21 alleviates hepatic steatosis and inflammation in a mouse model of non‐alcoholic steatohepatitis partly through an FGF21‐adiponectin‐IL17A pathway | |
| She et al. | PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target | |
| JP6728352B2 (en) | How to treat disorders related to bile acids | |
| Lentscher et al. | Chikungunya virus replication in skeletal muscle cells is required for disease development | |
| Pena-Leon et al. | Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21 | |
| Bernotiene et al. | Delayed resolution of acute inflammation during zymosan-induced arthritis in leptin-deficient mice | |
| Lacraz et al. | Deficiency of interleukin-15 confers resistance to obesity by diminishing inflammation and enhancing the thermogenic function of adipose tissues | |
| Zychowska et al. | Spinal CCL1/CCR8 signaling interplay as a potential therapeutic target–evidence from a mouse diabetic neuropathy model | |
| Wu et al. | Mesenteric adipose tissue contributes to intestinal barrier integrity and protects against nonalcoholic fatty liver disease in mice | |
| Nechemia-Arbely et al. | Early hepatocyte DNA synthetic response posthepatectomy is modulated by IL-6 trans-signaling and PI3K/AKT activation | |
| Jones et al. | A selective inhibitor of human C-reactive protein translation is efficacious in vitro and in C-reactive protein transgenic mice and humans | |
| Han et al. | Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression | |
| Kim et al. | Protection against collagen-induced arthritis by intramuscular gene therapy with an expression plasmid for the interleukin-1 receptor antagonist | |
| Zhang et al. | IGFBPrP1 induces liver fibrosis by inducing hepatic stellate cell activation and hepatocyte apoptosis via Smad2/3 signaling | |
| CN102137682B (en) | Anti-hepatitis c virus composition | |
| CN101683354B (en) | Liver damage relevant drug target and application thereof | |
| Chen et al. | Overexpression of hepcidin alleviates steatohepatitis and fibrosis in a diet-induced nonalcoholic steatohepatitis | |
| WO2019126524A1 (en) | Therapeutic targets for nash-induced hepatocellular carcinoma | |
| Huang et al. | Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice | |
| CN103690928B (en) | Novel cancer antigen eEF2 | |
| US10640768B2 (en) | Method of treating pain with an antibody against netrin-4, UNC5B or neogenin | |
| US20210238295A1 (en) | Compositions and methods for treating abdominal aortic aneurysm | |
| US20220259276A1 (en) | Vgll4 with ucp-1 cis-regulatory element and method of use thereof | |
| CN101935687B (en) | Application of IL-12p40 siRNA to promoting liver regeneration | |
| EP2931293B1 (en) | Compositions and methods for regulating erythropoiesis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120222 Termination date: 20160925 |