CN106344933A - Establishment method and application of anxiety disorder or related disease animal model of non-human mammals - Google Patents
Establishment method and application of anxiety disorder or related disease animal model of non-human mammals Download PDFInfo
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- CN106344933A CN106344933A CN201610711516.6A CN201610711516A CN106344933A CN 106344933 A CN106344933 A CN 106344933A CN 201610711516 A CN201610711516 A CN 201610711516A CN 106344933 A CN106344933 A CN 106344933A
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Abstract
The invention provides an establishment method and application of an anxiety disorder or related disease animal model of non-human mammals and in particular provides a preparation method of the anxiety disorder or related disease animal model of the non-human mammals. The preparation method comprises the following steps: (a) providing cells of the non-human mammals and inactivating Glce genes in the cells to obtain non-human mammal cells with inactivated Glce genes; (b) preparing the anxiety disorder or related disease animal model with the inactivated Glce genes through utilizing the cells with the inactivated Glce genes, which are prepared in the step (a). The animal model provided by the invention is an effective anxiety disorder or related disease animal model and can be widely used for researching diseases including generalized anxiety, acute anxiety attack, phobic disorder, post-traumatic stress disorder, acute stress disorder, obsessive-compulsive disorder and the like, and can be used for screening and testing specific drugs.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to a kind of non-human mammal anxiety neurosis or its relevant disease
Method for building up of animal model and application thereof.
Background technology
Anxiety neurosis, are also called anxiety neurosises, are modal one kind in this big class disease of neurosiss, with anxiety feelings
Thread is principal character, and its core symptom is worry.Should after main inclusion generalized anxiety disorder, acute anxiety attack, phobia, wound
Sharp obstacle, acute stress disorder, obsessive-compulsive disorder.It is mainly shown as the nervous worry of no clear and definite objective objects, fidgety, plant
Nervous symptoms (cardiopalmus, hand shaking, perspiration, frequent micturition etc.).
World Health Organization (WHO) (who) statistics shows, from nineteen ninety to 2003, suffers from the painful people of depressive and anxiety neurosis
Number increases to 6.15 hundred million from 4.16 hundred million, has nearly reached the 10% of population in the world.And, mental disorder has accounted for non-lethal disease
The 30% of disease.It is estimated that in the personnel that emergency occurs, just having 1 to suffer from depressive and anxiety neurosis in every 5.The whole world is every
Burden about 1 trillion dollars are needed to treat depression and anxiety neurosis in year.
Because the pathogenesis of anxiety neurosis are extremely complex, the pathogenesis of anxiety neurosis not yet study clear, shortage peace so far
Effective treatment meanss entirely, therefore its pathogeny of further investigated are the important foundations for the treatment of anxiety neurosis.Mouse disease model exists
Very important effect is served in the pathogenesis of research human diseasess and drug screening.Probe into the mankind cognitive function,
The aspects such as neurodegenerative diseases, neuropsychiatric disease, mouse model has big advantage.
Gene knockout is the complicated Protocols in Molecular Biology growing up the eighties in last century, and it is based on Development of Mouse Embryos
Tire stem cell dna homologous recombination principle, also referred to as " gene targeting " technology.Thereafter this technique construction several thousands genes are utilized
Mutant mice, these genetic engineering mice not only bring breakthrough, also in new drug development for the research of bioscience and medical science
Play vital effect.
The method for building up of existing mouse anxiety disease model mainly includes two classes at present: a class is the row based on unconditional
For model, exploratory behavior model and social behavior's model can be divided into according to its feature again.However, this class model is only limitted to body substantially
Body stress, lacks effective etiology monitoring meanss.Another kind of Anxiety Model is the model based on trained reflex, main bag
Include drinking-water conflict model, conditionality electric shock model etc..However, this class model includes the composition of this kind of mechanical stress that shocks by electricity, make
The ratio increase obtaining Somatic Stress in psychological stress is its major defect.
Therefore, this area in the urgent need to exploitation one kind can preferable simulation clinically idiopathic anxiety neurosis, individual diversity
Different less, its genetic background is highly consistent, can be used as the research pathogeny of anxiety neurosis and the powerful of new medicament screen
Animal model, and the animal model of structure is used for studying the pathogeny of anxiety neurosis and screening or identification treatment anxiety neurosis
Material.
Content of the invention
It is an object of the invention to provide one kind can preferable simulation clinically idiopathic anxiety neurosis, individual variation is relatively
Little, its genetic background is highly consistent, can be dynamic as the powerful of the pathogeny of research anxiety neurosis and new medicament screen
Thing model, and the animal model of structure is used for the thing studying the pathogeny of anxiety neurosis and screening or identification treatment anxiety neurosis
Matter.
A first aspect of the present invention provides a kind of anxiety neurosis of non-human mammal or its relevant disease animal model
Preparation method, comprises the following steps:
A () provides the cell of non-human mammal, the glce gene inactivation in described cell obtains glce gene inactivation
Nonhuman mammalian cells；
B () utilizes the cell of the glce gene inactivation obtaining in step (a), prepare the anxiety neurosis of glce gene inactivation
Or the animal model of its relevant disease.
In another preference, in step (a), also comprise the steps:
(a1) utilize dna homologous recombination technique, by one of exon 3 in described glce gene to exon 5 or
Multiple exons are rejected or are interrupted, and are replaced with selection markers, obtain the nonhuman mammalian cells of glce gene inactivation.
In another preference, in step (b), also comprise the steps:
(b1) nonhuman mammalian cells of the glce gene inactivation obtaining in step (a) are utilized to prepare chimeric inhuman
Mammal；
(b2) will be numerous to the chimeric non-human mammal obtaining and normal wild type mating non-human mammals in step (b1)
Educate, screening in offspring obtains the heterozygote non-human mammal of glce gene inactivation；
(b3) pass through for the mutual copulation of heterozygote non-human mammal obtaining in step (b2) to obtain glce gene inactivation
Homozygote non-human mammal, thus obtaining the animal model of the non-human mammal of glce gene inactivation.
In another preference, in step (b3), also include step (b4): will be inhuman for the homozygote of glce gene inactivation
Mammal is hybridized with the neuronal specificity knockout instrument non-human mammal of same species, thus it is special to obtain neuron
The animal model of the non-human mammal of glce gene inactivation of the opposite sex.
In another preference, described by glce gene inactivation include gene knockout, gene disruption or gene insertion.
In another preference, described gene inactivation includes glce gene and does not express, or expression does not have activated glce egg
In vain.
In another preference, described glce gene inactivation is to be inactivated by lacking or knocking out the exon 3 of glce.
In another preference, described glce gene inactivation is the glce gene inactivation of neuronal specificity.
In another preference, described non-human mammal be rodent or primate, be preferably comprised mice,
Rat, rabbit and/or monkey.
In another preference, described non-human mammal is mice, and glce loxp/ in step (b4)
Loxp mice is copulationed with instrument Mus nse (neuron-specific enolase)-cre, that is, obtain in neuronal cell specificity
Glce knock out mice abbreviation cko mice (i.e. neuronal specificity glce inactivated mice).
In another preference, described selection markers are selected from the group: resistant gene, fluorescence protein gene or a combination thereof.
In another preference, described selection markers include neo gene.
In another preference, the animal mould of the non-human mammal of glce gene inactivation obtaining in described step (b)
In type, compared with wild-type control animals, there are the one or more features being selected from the group:
(t1) behavior of anxiety sample increases；
(t2) anxiety degree increases；
(t3) spacious field level of activation reduces；
(t4) desire exploring strange environment reduces；
(t5) behavior depression increases；
(t6) Degree of Depression increases；
(t7) frightened sample behavior increases；
(t8) frightened degree increases；
(t9) cognitive disorder increases；
(t10) incidence rate of apoplexy increases；
(t11) obese degree of form increases；
(t12) incidence rate of obesity symptom increases；
(t13) fatty tissue weight in wet base increases.
In another preference, described anxiety-like behavior is selected from the group: explores the time of middle section in spacious field experiment
Reduce, show the desire reduction exploring strange environment, the time minimizing exploring open arms in elevated plus-maze test, stop
The time staying closure arm increases, and shows anxiety-like behavior or a combination thereof.
In another preference, described spacious field level of activation is selected from the group the distance of spacious field activity, activity time, activity speed
Degree or a combination thereof.
In another preference, described behavior depression is selected from the group: explores the time of middle section in spacious field experiment
Reduce, show desire reduction, the dead time increase in forced swim test exploring strange environment, show behavior exhausted
Hope or a combination thereof.
In another preference, described fatty tissue weight in wet base is selected from the group: visceral adipose tissue weight in wet base, subcutaneus adipose tissue
Weight in wet base or a combination thereof.
In another preference, described visceral adipose tissue weight in wet base is selected from the group: bilateral gonad peripheral adipose tissue weight in wet base,
Bilateral perirenal adipose tissue weight in wet base or a combination thereof.
In another preference, described subcutaneus adipose tissue weight in wet base includes bilateral inguinal fatty tissue weight in wet base.
Second aspect present invention provides a kind of non-human mammal model of first aspect present invention methods described preparation
Purposes, by this model be used as research anxiety neurosis or its relevant disease animal model.
In another preference, described anxiety neurosis or its relevant disease are selected from the group: generalized anxiety disorder, acute anxiety are sent out
Work, phobia, posttraumatic stress disorder, acute stress disorder, obsessive-compulsive disorder or a combination thereof.
In another preference, described chronic anxiety includes generalized anxiety disorder.
In another preference, described acute anxiety includes panic disorder.
Third aspect present invention provides a kind of non-human mammal model of first aspect present invention methods described preparation
Purposes, can mitigate or treat the material (therapeutic agent) of anxiety neurosis or its relevant disease for screening or identifying.
In another preference, described anxiety neurosis or its relevant disease are selected from the group: generalized anxiety disorder, acute anxiety are sent out
Work, phobia, posttraumatic stress disorder, acute stress disorder, obsessive-compulsive disorder or a combination thereof.
In another preference, described chronic anxiety includes generalized anxiety disorder.
In another preference, described acute anxiety includes panic disorder.
Fourth aspect present invention provides a kind of screening or identification treatment or alleviates the potential of anxiety neurosis or its relevant disease
The method of therapeutic agent, comprises the following steps:
A (), in test group, in the presence of test compound, test compound is applied to first aspect present invention institute
State the non-human mammal model of method preparation, the behavior to described animal model carries out Behavioral assessment；And do not applying
In described test compound and other conditions identical matched group, the behavior to described animal model carries out Behavioral assessment；
B () is compared to the behavior of test group and control animals model, wherein, compared with matched group, if applied
Characterize anxiety behavior in the animal model of test compound to be improved, then show that this test compound can be used as anxiety neurosis
Potential therapeutic agent.
In another preference, described behavior analysis include: autonomic activitieses, spacious field experiment and/or elevated plus fan
Palace is tested.
In another preference, described method is nondiagnostic and non-therapeutic.
In another preference, described improvement is the improvement statistically with significant.
In another preference, methods described includes step (c), and the potential therapeutic agent of step (b) screening or identification is applied
For the non-human mammal model of first aspect present invention methods described preparation, thus measuring it to described animal model anxiety
The impact of the order of severity of disease or its relevant disease.
Fifth aspect present invention provides a kind of non-human mammal model, with first aspect present invention methods described system
Standby.
In another preference, for glce gene inactivation, described non-human mammal model be heterozygosis or
Homozygosis.
In another preference, described glce gene inactivation is the glce gene inactivation of neuronal specificity.
Sixth aspect present invention provides a kind of purposes of cell, the glucuronic acid c5 isomerase in described cell
(glucuronyl c5-epimerase, glce) gene inactivation or downward, build the anxiety neurosis of non-human mammal for preparation
Or the biological preparation of its relevant disease animal model.
In another preference, described biological preparation is liquid formulation.
Seventh aspect present invention provides a kind of purposes of the deactivator of glce gene or its albumen, builds non-for preparation
The anxiety neurosis of people mammal or the preparation of its relevant disease animal model.
In another preference, described deactivator includes inhibitor.
In another preference, described deactivator is selected from the group: antibody, micromolecular compound, nucleic acid or a combination thereof.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, thus constituting new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description
Fig. 1 shows glce gene mutation sequential design strategy.
Fig. 2 shows glce gene knockout targeting vector plasmid map.
Fig. 3 shows the enzyme action qualification result of glce gene knockout targeting vector plasmid dna.
Fig. 4 shows the electrophoresis result of 5 ' the end pcr products of es colone genome dna.
Fig. 5 shows the electrophoresis result of 3 ' the end pcr products of es colone genome dna.
Fig. 6 shows glce transgenic mice (loxp/+) series jump site pcr result.
Fig. 7 shows glce no mutant homozygote (loxp/loxp cre), heterozygous mutation (loxp/+cre) and wild
The series jump site pcr result of type (+/+cre).
Fig. 8 shows spontaneous activity distance, time and the speed of glce no mutant homozygote mice.
Fig. 9 shows glce no mutant homozygote mice in the activity distance of spacious field middle section, time and speed.
Figure 10 shows that glce no mutant homozygote mice probes into open arms, central area in elevated plus-maze test respectively
Domain and the time of closure arm.
Figure 11 shows spontaneous activity distance, time and the speed of glce no mutant homozygote mice.
Figure 12 shows activity total distance in spacious field experiment for the glce no mutant homozygote mice, time and speed.
Figure 13 shows glce no mutant homozygote mice in the activity distance of spacious field middle section, time and speed.
Figure 14 shows the activity distance in spacious field neighboring area for the glce no mutant homozygote mice, time and speed.
Figure 15 shows the dead time in forced swim test for the glce no mutant homozygote mice.
Figure 16 shows the form contrast of glce no mutant homozygote mice and c57 mice.Wherein, left figure: above for c57 female
Mice, lower is glce no mutant homozygote female mice；Right figure: above for c57 male mice, lower is that glce no mutant homozygote male is little
Mus.
Figure 17 shows that glce no mutant homozygote mice and the body weight of c57 mice contrast situation.
Figure 18 shows that glce no mutant homozygote mice and the body fat weight in wet base of c57 mice contrast situation.
Figure 19 shows that glce no mutant homozygote mice and the bilateral gonad peripheral adipose tissue weight in wet base of c57 mice contrast feelings
Condition.
Figure 20 shows that glce no mutant homozygote mice and the bilateral perirenal adipose tissue weight in wet base of c57 mice contrast situation.
Figure 21 shows that glce no mutant homozygote mice and the visceral adipose tissue weight in wet base of c57 mice contrast situation.
Figure 22 shows that glce no mutant homozygote mice and the bilateral inguinal fatty tissue weight in wet base of c57 mice contrast feelings
Condition.
Figure 23 shows that apoplexy Behavioral assessment is tested.
Figure 24 shows ttc dyeing detection apoplexy Mice brain tissues infraction situation.
Specific embodiment
The present inventor through extensively in-depth study, establish the stable anxiety neurosis of a kind of inheritance stability, phenotype or its
Relevant disease model, it be glce gene disallowable or inactivation mice or other non-human mammals.The animal mould of the present invention
Type is a kind of effective anxiety neurosis or its relevant disease animal model, can be used for studying anxiety neurosis or its relevant disease (as extensively
Property anxiety) it is possible to for the screening of certain drug and testing experiment.Complete the present invention on this basis.
Additionally, the present invention has been unexpectedly discovered that, the animal model rejected or inactivate obtained by glce gene can also be simultaneously
For studying the diseases such as apoplexy, obesity, depression.Complete the present invention on this basis.
Glce gene
Heparan sulfate is a kind of polysaccharide being widely present in cell surface and cytoplasmic matrix, negative as a band
The linear macromolecule of electricity, many cytokines, somatomedin, chemotactic factor and interleukin can specifically combine therewith, and then
The physiological process such as fetal development, cell growth, inflammatory reaction, blood coagulation, neoplasm metastasis and virus infection play a role1.
Glucuronic acid c5 isomerase (glce) is that one of heparan sulfate proteoglycan sugar chain building-up process is closed
Key enzyme, the d- glucuronic acid in sugar chain can be tautomerized to l- iduronic acid by it2, substantially increase answering of Heparan sulfate
Miscellaneous degree, and provide more flexibilities for sugar chain, l- iduronic acid is that Heparan sulfate identifies numerous protein moleculars
Individual indispensable site3.
Find in the research to 21 normal galactophore tissues and 74 breast tumor tissues, the human milk adenoncus of 82%-84%
In tumor tissue, glce significantly lowers or completely loses in the expression of mrna level and protein level4.Additionally, in breast carcinoma
The propagation of small cell lung cancer and breast cancer cell can be suppressed with overexpression glce in lung cancer cell line, this points out our glce possible
It is a potential antioncogene5,6.
Glce gene is located on No. 9 chromosomes of mouse genome, total length 618 (ensemblgene id:
Ensmusg00000032252, genebank accession number: 93683).Glce genome sequence include 4 introns and 5 show outward
Son, glce gene expression albumen has 3 transcripts, and transcript 1 has 3 exons, 2 introns, and transcript 2 has 5
Exon, 4 introns, transcript 3 has 2 exons, 1 intron.These sequence informations can be found in document or
The public databases such as ensemblgene, genebank.
The glce gene of other species such as the mankind also can be found in the common datas such as document or ensemblgene, genebank
Storehouse.
It should be understood that term " glce " also includes the variant form of various naturally occurring glce genes.Representational example
Including: the nucleotide sequence with wild type identical glce albumen, encoding wild type glce is encoded because of the degeneracy of codon
The nucleotide sequence of conservative variation's polypeptide of albumen.Additionally, during for other mammals outside mice, this term refers to
Homologue in this mammal for the glce gene.For example for people, this term refers to glce (the known mice glce base of people
Because the cdna homology degree with mankind's glce gene is 91.4%, the homology degree of aminoacid sequence is 97.4%).
Glce gene or the deactivator of its albumen
In the present invention, the deactivator of described glce includes all inactivating or part inactivates.
The deactivator of the glce albumen of the present invention includes (a) inhibitor, and the example of described inhibitor includes (but not limiting
In): micromolecular compound, antibody, antisensenucleic acidses, mirna, sirna or a combination thereof；The knockout agent of (b) glce gene.
Anxiety neurosis or its relevant disease
Anxiety neurosis, are also called anxiety neurosises, are modal one kind in this big class disease of neurosiss, with anxiety feelings
Thread is principal character, and its core symptom is worry.Stress hinder including after generalized anxiety disorder, acute anxiety attack, phobia, wound
Hinder, acute stress disorder, obsessive-compulsive disorder etc..It is mainly shown as the nervous worry of no clear and definite objective objects, fidgety, plant god
Through symptom (cardiopalmus, hand shaking, perspiration, frequent micturition etc.).
The feature of generalized anxiety disorder mainly has: (1) emotional symptoms.It is mainly shown as in the case of not having obvious inducement,
Often the excessive worry not being inconsistent, anxiety with real situation and fears, patient feels oneself to be constantly in a kind of anxiety not in patient
Peace, on tenterhooks, frightened, fear, in worried inner experience.(2) vegetative nerve symptom.It is mainly shown as dizzy, the uncomfortable in chest, heart
Unbearably, rapid breathing, xerostomia, frequent micturition, urgent micturition, the symptom of the body aspect such as perspire, tremble.(3) mobility is uneasy.It is mainly shown as
It is on tenterhooks, feel restless, irritated, it is difficult to get down.
The feature of panic disorder mainly has: (1) dying sense or sense out of control.In normal daily life, patient is almost with just
Ordinary person is the same, and when showing effect (have has certain trigger situation), extremely frightened psychology suddenly in patient, experiences and is on the point of
Dead sense or sense out of control.
(2) autonomic nervous system symptom.Occur as uncomfortable in chest, nervous, dyspnea simultaneously, perspire, shake all over.
(3) a few minutes are typically continued to a few hours.Outbreak starts suddenly, Clear consciousness during outbreak.
(4) easily mistaken diagnosis.During outbreak, patient often dials " 120 " emergency call, goes to see the emergency treatment of Cardiological.Although patient
Look that symptom is very heavy, but coherence check result is mostly normal, therefore often acatalepsia is true.
The feature of phobia (including social phobia, topophobia, specifically terror) mainly has: the core manifestation of phobia
The same with acute anxiety attack is all panic attack.Difference be the anxiety attack of phobia be by some specific places or
Person's situation causes, and patient is not at when these particular places or situation causing anxiety.
In the present invention, anxiety neurosis or its relevant disease include but is not limited to: generalized anxiety disorder, acute anxiety attack,
Phobia, posttraumatic stress disorder, acute stress disorder and/or obsessive-compulsive disorder.
Gene inactivation
Research for Unknown Function gene can be adopted in many ways, for example, make the gene inactivation requiring study, and analyzes institute
The character mutation of the genetic modification obtaining, and then obtain the function information of this gene.Another advantage of this research method is permissible
Gene function and disease are associated, thus also can obtain while obtaining gene function this gene as potential drug or
Disease information and disease animal model that person's drug target can be treated.The method of gene inactivation can pass through gene knockout, gene
Interrupt or gene insertion mode completing.Wherein, gene knochout technique is the non-of research function in entirety for the human gene
Often strong means.
Animal model
In the present invention, there is provided the non-human mammal model of a kind of very effective anxiety neurosis or its relevant disease.
In the present invention, the example of non-human mammal includes (but being not limited to): mice, rat, rabbit, monkey etc., more preferably
Ground is rat and mice.
As used herein, term " glce gene inactivation " includes the situation that one or two glce gene is deactivated, that is, wrap
With including glce genetic heterozygosis and homozygosis inactivate.For example, the mice of glce gene inactivation can be the mice of heterozygosis or homozygosis.
In the present invention, can gene knockout or proceed to exogenous gene (or fragment) and make the methods such as glce gene inactivation prepare
The non-human mammal (as mice) of glce gene inactivation.In the art, by gene knockout or proceed to exogenous gene and make
The technology of target gene inactivation is known, and these routine techniquess can be used in the present invention.
In another preference of the present invention, the inactivation of glce gene is realized by gene knockout.
In another preference of the present invention, the inactivation of glce gene be by insertion exogenous gene in glce gene (or
Fragment) and realize.
In an instantiation of the present invention, a construction containing external source Insert Fragment can be built, this construction contains
With the homology arm of the flanking sequence homology of the both sides of the insertion point of target gene (glce), such that it is able to by homologous recombination high frequency
External source Insert Fragment (or gene) is inserted into glce genome sequence (especially exon region) by ground, causes mice glce base
The frameshit of cause, in advance termination or knockout, thus leading to glce disappearance or inactivating.
The homozygosis being obtained with the inventive method or the mice of heterozygosis can educate, and develop normal.The glce gene of inactivation can be with Meng
Dare law heredity is to progeny mice.
In a preference, the invention provides a kind of Mice homozygous animal pattern of disappearance glce gene.
Drug candidate or therapeutic agent
In the present invention, a kind of animal model of the utilization present invention, screening treatment anxiety neurosis or its related disease are additionally provided
The drug candidate of disease or the method for therapeutic agent.
In the present invention, drug candidate or therapeutic agent refer to known to have certain pharmacological activity or be detected can
Can have the material of certain pharmacological activity, including but not limited to nucleic acid, albumen, saccharide, the small molecule of chemosynthesis or big point
Sub- compound, cell etc..The administering mode of drug candidate or therapeutic agent can be administered orally, intravenous injection, lumbar injection, subcutaneous note
Penetrate, canalis spinalis is administered or direct intracerebral injection.
Main advantages of the present invention include:
(1) present invention can preferable simulation clinically idiopathic anxiety neurosis；
(2) individual variation is less, and its genetic background is highly consistent；
(3) can be used as the research pathogeny of anxiety neurosis and the powerful of new medicament screen.
(5) the anxiety neurosis model inheritance stability of the present invention, phenotype are stable.
(6) homozygosis being obtained with the inventive method or the animal model of heterozygosis can be educated.Transgenic chimeric mice has reproduction
Ability, the glce gene of inactivation can be with Mendel's law heredity to progeny mice.
(7) anxiety neurosis of the present invention or its relevant disease animal model show anxiety symptom, therefore can be widely applied to
The drug screening of anxiety neurosis relevant disease and test.
(8) the invention firstly discloses reject or inactivation glce gene obtained by animal model can also be simultaneously used for grinding
Study carefully the diseases such as apoplexy, obesity, depression.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as sambrook et al., molecular cloning: laboratory manual (new york:cold spring harbor
Laboratory press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, no
Then percentage ratio and number are percentage by weight and parts by weight.
If no special instructions, the material used by embodiment is commercially available prod.
Embodiment 1 sets up the mouse model that glce gene knocks out in neuron-specific condition.
Build glce mutant gene sequence (Fig. 1) first.As shown in figure 1, design glce gene knockout targeting vector sequence,
Loxp/loxp allele is inserted into glce gene 3 exon both sides, in 3 ' end insertion neo genes, 5 ' end arms
3125bp, 3 ' end arms 3718bp.
Fig. 2 is glce gene knockout targeting vector plasmid map.1. obtain the homologous fragment of genes of interest (glce), by this
Dna fragment is cloned in plasmid vector；2. excise most of homology dna sequence of genes of interest from recombiant plasmid, only stay portion
The two ends of the online plasmid vector of sub-sequence；3. by neo gene cloning in the linear plasmid with genes of interest DNA sequence homologous,
It is allowed to the centre positioned at residual genes of interest DNA sequence homologous；4. the outside linearisation recombiant plasmid in genes of interest DNA sequence homologous carries
Body, by hsv-tk gene cloning in this linear carrier.Gene locis are as shown in table 1.
Table 1
Note: gene location reference numerals are according to " 10kb up and down of glce gene ".
Fig. 3 is the enzyme action identification of glce gene knockout targeting vector plasmid dna, using 1kb dna ladder.To target practice
Carrier carries out linearisation: 100 μ g glce-cko plasmid dna noti (enzyme dosage: 150u) linearisations, and enzyme action system is 150 μ
L, overnight, after equal-volume phenol chloroform, chloroform are processed, dehydrated alcohol precipitates, and the aseptic pbs of 100 μ l is resuspended standby for 37 DEG C of digestion.Es is thin
Born of the same parents practice shooting: es cell scr012 derives from 129sv/evThe embryo of strain male mice (purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center)
Tire stem cell, linearisation dna measures: 35 μ g, electroporation model: bio-rad gene pulser (cat.no.165-2105), electricity
Perforation condition: voltage 240v, electric capacity 500 μ f, actual conduction time 10.5ms, virtual voltage 256v, colony screening condition: 300 μ
G/ml g418 and 2 μm of ganc screens 8 days.Picking resistance clone and totally 96 parts of dna sample of offer.
Positive es cellular genome authentication method:
1.5 ' arm pcr identifications
P1 primer is located at outside 5 ' arm, and p2 primer is located at neo recombination region, apart from arm outer primer 8.2kb.
P1 and p2 primer sequence:
p1:ggcatttgcactcacatacacaaccca(gene site:15824-15850)(seq id no.:1)
P2:gtgccactcccactgtcctttcc (seq id no.:2) pcr reaction system (ul):
Pcr reaction condition:
Pcr instrument: eppendorf ag 22331hamburg
Reagent: takara la taq treasured biological engineering (Dalian) company limited (cat:drr002b)
Molecular weight marker:mbi generuler 1kb dna ladder (brilliant U.S. biological, cat:sm0311)
2.3 ' arm pcr identifications
P4 primer is located at outside 3 ' arm, and p3 primer is located at neo recombination region, apart from arm outer primer 4.7kb.
P3 and p3 primer sequence:
p4:gagaggcttggaggcggtgctgatctt(gene site:29603-29629)(seq id no.:3)
P3:gatatactatgccgatgattaattgtc (seq id no.:4) pcr reaction system (ul):
Pcr reaction condition:
Pcr instrument: eppendorf ag 22331hamburg
Reagent: takara la taq treasured biological engineering (Dalian) company limited (cat:drr002b)
Molecular weight marker:mbi generuler 1kb dna ladder (brilliant U.S. biological, cat:sm0311)
Es cell clone qualification result pcr identifies drug resistance es cell clone 96, and wherein 19 es clone's generations are double
Arm homologous recombination.Pcr product is further characterized by through dna sequencing.Fig. 4 shows that 5 ' the end pcr of es colone genome dna produce
The electrophoresis result of thing.Fig. 5 shows the electrophoresis result of 3 ' the end pcr products of es colone genome dna.
Positive es cloned blastocysts injection:
Microinjection blastaea is originated: the super several rows of c57bl/6j mice (purchased from Shanghai Slac Experimental Animal Co., Ltd.)
Ovum, is developed to blastocyst stage in natural conception body.60 pieces of embryos of injection, are implanted into 3 pseudopregnant mouse receptor uterus, are subject to after injection
Body is the first-filial generation of c57bl/6j (♂) and cba (♀) (purchased from Shanghai Slac Experimental Animal Co., Ltd.).Little from birth
Select the Mouse feeder more than 50% for the chimeric rate in Mus to growing up, copulationed with c57bl/6j female mice, offspring's Lycoperdon polymorphum Vitt is little
Mus extracted coda gene group dna carries out pcr identification (identification strategy is ibid), and result is as shown in fig. 6, obtain both arms positive f1 for little
Mus (loxp/+).
By f1 for Mouse feeder to growing up, with nse-cre instrument Mus (purchased from Shanghai Slac Experimental Animal Co., Ltd.)
Hybridization, obtains f2 for loxp/+cre mice.By f2 for Mouse feeder to grow up, f2 for the mutual copulation of interior male and female, according to Mendel
Law heredity, obtains f3 for no mutant homozygote (loxp/loxp cre): heterozygous mutation (loxp/+cre): wild type (+/
+ cre) ratio be about 1:2:1.Extracted coda gene group dna carries out pcr identification (identification strategy is ibid), result such as Fig. 7 institute
Show.No mutant homozygote mice (loxp/loxp cre) is used for follow-up Animal Behavior Science experiment.
The behavior analysis of embodiment 2glce no mutant homozygote mice
The glce gene mutation homozygote mice (hereinafter referred to as glce no mutant homozygote mice) carrying cre recombinase is put
Raise in cleaning feeding environment.At mice adolescence (> 3 monthly age) by a series of Animal Behavior Science experiment include autonomic activitieses,
The anxiety neurosis sample symptom of the experimental evaluation mouse models such as spacious field experiment, Elevated plus-maze.
2.1 autonomic activitieses
Mice is positioned in dark experimental box (110mm x 110mm x 330mm), the spontaneous activity of test mice
5min.Shoot the active situation of mice by infrared photography.Video tracking using Jiliang Software Sci-Tech Co., Ltd., Shanghai is soft
Part and analysis software are analyzed to mice event trace and activity time.
Research find, compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice spontaneous
There were significant differences (Fig. 8) for the total distance of activity, activity time and moving speed.
Result shows, the born habit of adolescence glce no mutant homozygote mice compared with c57bl/6 normal mouse simultaneously
It is not significantly different from.
2.2 spacious field experiments
Mice is positioned in bright spaciousness experimental box (500mm x 500mm x 590mm), the exploration of test mice is lived
Dynamic 5min.Shoot the active situation of mice by infrared photography.Video tracking using Jiliang Software Sci-Tech Co., Ltd., Shanghai
Software and analysis software are analyzed to mice event trace and activity time.
Research finds, compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice is in spacious field
The distance of middle section activity, activity time and moving speed are all substantially reduced (Fig. 9).
Result shows, adolescence glce no mutant homozygote mice explores strange environment compared with c57bl/6 normal mouse
Activity substantially reduce, and produce stronger anxiety.
2.3 elevated plus-maze test
Mice is positioned over the long 30cm of open arms single armed, wide 6cm, the long 30cm of closure arm single armed, wide 6cm, high 14.5cm
In Elevated plus-maze, height about 50cm from the ground, by the activity in Elevated plus-maze in photography mice 5min
Situation.Activity to mice is analyzed finding, compared with adolescence c57bl/6 normal mouse, adolescence glce mutation is pure
The time that zygote mice probes into open arms in elevated plus-maze test substantially reduces, and the time resting on closure arm significantly increases
Plus, it is not significantly different from (Figure 10) in middle section residence time.
Result shows, compared with c57bl/6 normal mouse, anxiety degree is obvious for adolescence glce no mutant homozygote mice
Increase.Male adolescence glce no mutant homozygote mice anxiety compared with female adolescence glce no mutant homozygote mice
Become apparent from.In addition, the adolescence glce no mutant homozygote mice producing to a certain degree anxiety also produces to a certain degree simultaneously
Depression.
The Depressive behavior credit analysis of embodiment 3glce no mutant homozygote mice
3.1 autonomic activitieses
The glce gene mutation homozygote mice carrying cre recombinase is positioned over (110mm x in dark experimental box
110mm x 330mm), the spontaneous activity 5min of test mice.Shoot the active situation of mice by infrared photography.Using Shanghai
The video tracking software of Ji Liang software Science and Technology Ltd. and analysis software are carried out to mice event trace and activity time point
Analysis.
Research display, compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice spontaneous
There were significant differences (Figure 11) for the total distance of activity, activity time and moving speed.
Result shows, the born habit of adolescence glce no mutant homozygote mice compared with c57bl/6 normal mouse simultaneously
It is not significantly different from.
3.2 spacious field experiments
Mice is positioned in bright spaciousness experimental box (500mm x 500mm x 590mm), the exploration of test mice is lived
Dynamic 5min.Shoot the active situation of mice by infrared photography.Video tracking using Jiliang Software Sci-Tech Co., Ltd., Shanghai
Software and analysis software are analyzed to mice event trace and activity time.
Result shows, compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice is in spacious field
In experiment, the total distance of activity, activity time and moving speed are all substantially reduced (Figure 12).Result shows, adolescence glce dashes forward
Become that homozygote mice is more movable compared with c57bl/6 normal mouse substantially reduces.
Compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice is in spacious field middle section
The distance of activity, activity time and moving speed are all substantially reduced (Figure 13), and result shows, adolescence glce no mutant homozygote
The activity that mice explores strange environment compared with c57bl/6 normal mouse substantially reduces.
Compared with adolescence c57bl/6 normal mouse, adolescence glce no mutant homozygote mice is in spacious field neighboring area
The distance of activity, activity time and moving speed are all substantially reduced (Figure 14), and result shows adolescence glce no mutant homozygote
Mice is more movable compared with c57bl/6 normal mouse to be substantially reduced.
Generally speaking, compared with adolescence c57bl/6 normal mouse, the activity of adolescence glce no mutant homozygote mice
Substantially reduce, and explore the activity of strange environment and substantially reduce, show that adolescence glce no mutant homozygote mice has certain journey
The depression of degree.
3.3 forced swimming
Mice is positioned over diameter 12cm, in the water vat of height 25cm (water temperature 21-22 DEG C), mice is forced in water temperature relatively
Low water went swimming, the movable 6min of test mice, record the dead time in rear 4min for the mice.By photography mice
Active situation.The mice activity time is analyzed.Research finds, compared with adolescence c57bl/6 normal mouse, young
Dead time in forced swim test for the phase glce no mutant homozygote mice dramatically increases (Figure 15).
Result shows, adolescence glce no mutant homozygote mice has a certain degree of compared with c57bl/6 normal mouse
Depressed.
The morphological analysis of embodiment 4glce no mutant homozygote mice
The birth ratio of glce no mutant homozygote mice meets Mendel's law, and by detecting the body weight of mice, result is such as
Shown in Figure 16.Result shows, compared with adult c57bl/6 normal mouse, the shape of adult glce neuron-specific knock-out mice
State is fatter.
The body weight analysis of embodiment 5glce no mutant homozygote mice
Glce no mutant homozygote mice is placed on electronic balance and weighs, research is grown up glce no mutant homozygote mice and become
The weight differences of year c57bl/6 normal mouse.
Research finds (Figure 17), and compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice is fat
The incidence rate of symptom is higher, and body weight has statistically-significant difference compared with adult c57bl/6 normal mouse.
Result shows, is considered fertile with the standard exceeding more than the 20% of adult c57bl/6 normal mouse Weight averages
Fat, the weight average of female adult c57bl/6 normal mouse is 28.7g, the body weight of female adult glce no mutant homozygote mice
Average out to 34.6g, the incidence rate of female adult glce no mutant homozygote mice obesity symptom is about 50%, with female adult
C57bl/6 normal mouse compares with statistically-significant difference (p < 0.01)；The body weight of male adult c57bl/6 normal mouse
Average out to 32.2g, the weight average of male adult glce no mutant homozygote mice is 39.5g, male adult glce mutant homozygous
The incidence rate of sub- mice obesity symptom is about 66%, has statistically significant compared with male adult c57bl/6 normal mouse
Difference (p < 0.01)；The weight average of adult c57bl/6 normal mouse (comprising male and female) is 30.7g, adult glce mutant homozygous
The weight average of sub- mice (comprising male and female) is 37.8g, and the incidence rate of adult glce no mutant homozygote mice obesity symptom amounts to
It is about 60%, there is compared with adult c57bl/6 normal mouse statistically-significant difference (p < 0.01).
The above results fully show, compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice is fertile
The incidence rate of fat symptom is higher.
The body fat analysis of embodiment 6glce no mutant homozygote mice
Fatty tissue is taken to include bilateral gonad peripheral adipose tissue, Bilateral Renal week after glce no mutant homozygote mice is dissected
Fatty tissue, bilateral inguinal fatty tissue are placed in and weigh on electronic balance, and research is grown up glce no mutant homozygote mice and become
The fatty tissue weight in wet base difference of year c57bl/6 normal mouse.
Research finds (Figure 18), compared with adult c57bl/6 normal mouse, the body of adult glce no mutant homozygote mice
Fat weight in wet base increases, and has statistically-significant difference.
Result shows, the body fat weight in wet base average out to 0.94g of female adult c57bl/6 normal mouse, female adult glce dash forward
Become the body fat weight in wet base average out to 8.50g of homozygote mice, there are compared with female adult c57bl/6 normal mouse statistics and show
Write difference (p < 0.01)；The body fat weight in wet base average out to 2.98g of male adult c57bl/6 normal mouse, male adult glce mutation
The body fat weight in wet base average out to 7.52g of homozygote mice, has statistically significant compared with male adult c57bl/6 normal mouse
Difference (p < 0.01)；The body fat weight in wet base average out to 2.21g of adult c57bl/6 normal mouse (comprising male and female), adult glce mutation
The body fat weight in wet base average out to 8.01g of homozygote mice (comprising male and female), has statistics compared with adult c57bl/6 normal mouse
Learn significant difference (p < 0.01).
The above results fully show, adult c57bl/6 normal mouse compares, the body of adult glce no mutant homozygote mice
Fat weight in wet base increases.
The fatty tissue analysis of embodiment 7glce no mutant homozygote mice
The bilateral gonad peripheral adipose tissue analysis of 7.1glce no mutant homozygote mice
Take bilateral gonad peripheral adipose tissue to be placed on electronic balance after glce no mutant homozygote mice is dissected to weigh, grind
Study carefully the bilateral gonad peripheral adipose tissue weight in wet base difference of adult glce no mutant homozygote mice and adult c57bl/6 normal mouse.
Compared with adult c57bl/6 normal mouse, the bilateral gonad peripheral adipose of adult glce no mutant homozygote mice
Tissue wet dramatically increases (Figure 19).
Result shows, the bilateral gonad peripheral adipose tissue weight in wet base average out to of female adult c57bl/6 normal mouse
0.54g, the bilateral gonad peripheral adipose tissue weight in wet base average out to 3.10g of female adult glce no mutant homozygote mice, with female
Adult c57bl/6 normal mouse compares with statistically-significant difference (p < 0.01)；Male adult c57bl/6 normal mouse
Bilateral gonad peripheral adipose tissue weight in wet base average out to 1.66g, around the bilateral gonad of male adult glce no mutant homozygote mice
Fatty tissue weight in wet base average out to 2.85g, have compared with male adult c57bl/6 normal mouse statistically-significant difference (p <
0.01)；The bilateral gonad peripheral adipose tissue weight in wet base average out to 1.24g of adult c57bl/6 normal mouse (comprising male and female), grows up
The bilateral gonad peripheral adipose tissue weight in wet base average out to 2.98g of glce no mutant homozygote mice (comprising male and female), with grow up
C57bl/6 normal mouse compares with statistically-significant difference (p < 0.01).
The above results fully show, compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice
Bilateral gonad peripheral adipose tissue weight in wet base dramatically increases.
The bilateral perirenal adipose tissue analysis of 7.2glce no mutant homozygote mice
Take bilateral perirenal adipose tissue to be placed on electronic balance after glce no mutant homozygote mice is dissected to weigh, study into
The bilateral perirenal adipose tissue weight in wet base difference of year glce no mutant homozygote mice and adult c57bl/6 normal mouse.
Compared with adult c57bl/6 normal mouse, the bilateral perirenal adipose tissue of adult glce no mutant homozygote mice
Weight in wet base dramatically increases (Figure 20).
Result shows, the bilateral perirenal adipose tissue weight in wet base average out to 0.36g of female adult c57bl/6 normal mouse, female
Property grow up glce no mutant homozygote mice bilateral perirenal adipose tissue weight in wet base average out to 2.06g, with female adult c57bl/6 just
Often mice compares with statistically-significant difference (p < 0.01)；The bilateral perirenal fat of male adult c57bl/6 normal mouse
Tissue wet average out to 1.18g, the bilateral perirenal adipose tissue weight in wet base average out to of male adult glce no mutant homozygote mice
1.61g, there is compared with male adult c57bl/6 normal mouse statistically-significant difference (p < 0.01)；Adult c57bl/6 is just
The often bilateral perirenal adipose tissue weight in wet base average out to 0.87g of mice (comprising male and female), adult glce no mutant homozygote mice (comprises
Male and female) bilateral perirenal adipose tissue weight in wet base average out to 1.83g, compared with adult c57bl/6 normal mouse, there are statistics
Significant difference (p < 0.01).
The above results fully show, compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice
Bilateral perirenal adipose tissue weight in wet base dramatically increases.
The visceral adipose tissue analysis of 7.3glce no mutant homozygote mice
Bilateral gonad peripheral adipose tissue and bilateral perirenal adipose tissue is taken to put after glce no mutant homozygote mice is dissected
Weigh on electronic balance, visceral adipose tissue weight in wet base gross weight is bilateral gonad peripheral adipose tissue and bilateral perirenal adipose tissue
Weight in wet base sum, the adult glce no mutant homozygote mice of research is poor with the visceral adipose tissue weight in wet base of adult c57bl/6 normal mouse
Different.
Compared with adult c57bl/6 normal mouse, the visceral adipose tissue weight in wet base of adult glce no mutant homozygote mice
Dramatically increase (Figure 21).
Result shows, the visceral adipose tissue weight in wet base average out to 0.90g of female adult c57bl/6 normal mouse, and female becomes
The visceral adipose tissue weight in wet base average out to 5.16g of year glce no mutant homozygote mice, with female adult c57bl/6 normal mouse phase
Relatively there is statistically-significant difference (p < 0.01)；The visceral adipose tissue weight in wet base of male adult c57bl/6 normal mouse is average
For 2.84g, the visceral adipose tissue weight in wet base average out to 4.45g of male adult glce no mutant homozygote mice, and male adult
C57bl/6 normal mouse compares with statistically-significant difference (p < 0.01)；Adult c57bl/6 normal mouse (comprising male and female)
Visceral adipose tissue weight in wet base average out to 2.11g, the visceral adipose tissue of adult glce no mutant homozygote mice (comprising male and female)
Weight in wet base average out to 4.81g, has statistically-significant difference (p < 0.01) compared with adult c57bl/6 normal mouse.
The above results fully show, compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice
Visceral adipose tissue weight in wet base dramatically increases.
Subcutaneus adipose tissue (as the bilateral inguinal fatty tissue) analysis of 7.4glce no mutant homozygote mice
Take bilateral inguinal fatty tissue to be placed on electronic balance after glce no mutant homozygote mice is dissected to weigh, subcutaneous
Fatty tissue weight in wet base is bilateral inguinal fatty tissue weight in wet base sum, and research is grown up glce no mutant homozygote mice and grown up
The subcutaneus adipose tissue weight in wet base difference of c57bl/6 normal mouse.
Compared with adult c57bl/6 normal mouse, the bilateral inguinal fat group of adult glce no mutant homozygote mice
Knit (representing subcutaneus adipose tissue) weight in wet base and dramatically increase (Figure 22).
Result shows, the bilateral inguinal fatty tissue weight in wet base average out to 0.04g of female adult c57bl/6 normal mouse,
The bilateral inguinal fatty tissue weight in wet base average out to 3.34g of female adult glce no mutant homozygote mice, with female adult
C57bl/6 normal mouse compares with statistically-significant difference (p < 0.01)；The bilateral of male adult c57bl/6 normal mouse
Groin fatty tissue weight in wet base average out to 0.13g, the bilateral inguinal fatty tissue of male adult glce no mutant homozygote mice
Weight in wet base average out to 3.07g, has statistically-significant difference (p < 0.01) compared with male adult c57bl/6 normal mouse；Become
The bilateral inguinal fatty tissue weight in wet base average out to 0.10g in year c57bl/6 normal mouse (comprising male and female), adult glce mutation is pure
The bilateral inguinal fatty tissue weight in wet base average out to 3.20g of zygote mice (comprising male and female), with adult c57bl/6 normal mouse phase
Relatively there is statistically-significant difference (p < 0.01).
The above results fully show, compared with adult c57bl/6 normal mouse, adult glce no mutant homozygote mice
Bilateral inguinal fatty tissue (representing subcutaneus adipose tissue) weight in wet base dramatically increases.
The apoplexy behavior analysis of embodiment 8glce no mutant homozygote mice
Glce gene mutation homozygote mice with cre recombinase is placed in cleaning feeding environment raise.This mouse species
Model is entering (about 1.5 years) geratic period generation apoplexy sample symptom.Evaluate mouse species by a series of apoplexy Behaviors survey
The apoplexy sample symptom of model and the order of severity, experimental technique is published in the evaluation rat stroke symptom on stroke with reference to calendar year 2001
A series of Behaviors survey (chen j, et al.stroke.2001) of systems of the order of severity, including exercise test, sensation
Test, balanced capacity test, the reflection measurement of body, the test of abnormal mobility etc., specially keep flat test, propose tail survey
Examination, visual tactile test, proprioceptive sensation test, balance beam test, auricle reflex, eyelid closure reflex, startle reflection, tic, spasm, flesh
The performance testings such as dystonia, the behavior to mice is scored.
There is apoplexy in glce gene mutation homozygote mice group 40 only 10, apoplexy incidence rate is in one's old age
25%；Matched group 40 mices of c57bl/6 mice group have 2 apoplexy occurs in one's old age, and apoplexy incidence rate is 5%, result
Show, glce gene mutation homozygote mice is more easy to apoplexy compared with c57bl/6 mice.
8.1 exercise test.
(1) keep flat test: mice is positioned on level land, observes whether mice can normally walk, normal walking scores 0
Point；Whether can keep straight on it is impossible to 1 point of score of keeping straight on, otherwise 0 point of score；Whether turn-take around damage side, turn-take around damaging side
1 point of score, otherwise 0 point of score；Whether fall to damage inclination, roll 1 point of score, otherwise 0 point of score to damaging.
(2) propose tail test: lift the tail of mice, observe the situation of mouse limb activity, if forelimb curves inwardly, pawl
Son is firmly grasped, if mice forelimb curves inwardly, claw firmly grasps 1 point of score, otherwise 0 point of score；Whether hind leg curves inwardly, claw is grabbed
Tightly, if mouse hind leg curves inwardly, claw firmly grasps 1 point of score, otherwise 0 point of score；In 30 seconds, head lifts and vertical axises
Angle is more than 10 degree, if head lifts and vertical axises angle is more than 1 point of 10 degree of scores, otherwise 0 point of score in mice 30 seconds.
8.2 sensory test.
(1) visual tactile test: grasp mouse body, make mice forelimb freely movable, make mice towards estrade
Edge or cage edge, whether by mice quickly near table edge or cage edge, observing mice forelimb can be quick
Accurately catch table edge or cage edge to protracting and opening claw in time.If mice forelimb can not quickly to extension,
And open claw in time and accurately catch table edge or 1 point of cage marginal score, otherwise 0 point of score.
(2) proprioceptive sensation test: grasp mouse body, make mouse hind leg freely movable, mice forelimb is positioned over
Whether table edge or cage edge, hind leg are hanging, press from both sides mouse hind leg leg muscle using tweezers, observe mouse hind leg and can
Snapback.If mouse hind leg is unable to 1 point of snapback score, otherwise 0 point of score.
8.3 balanced capacity tests.
Mice is positioned over balance beam one end, observes the situation of mice freely activity on balance beam, main inclusion following 7
The situation of kind: whether (1) can keep one's balance, freely walk on balance beam, if mice can keep one's balance, flat
0 point of the upper free walking scores of weighing apparatus wood.
(2) whether catch balance beam edge, if mice catches 1 point of balance beam marginal score.
(3) embrace balance beam, but have a hind leg to drop, if mice embraces balance beam, but have a hind leg to drop score 2
Point.
(4) embrace balance beam, but have two hind legs to drop, or on balance beam rotate, and on balance beam when
Between be more than 60 seconds, if mice embraces balance beam, but have two hind legs to drop, or rotate on balance beam, and in balance beam
On time be more than 3 points of 60 seconds scores.
(5) attempt balance is kept on balance beam but finally drop, the time on balance beam is more than 40 seconds, if mice
Attempt balance is kept on balance beam but finally drops, the time on balance beam is more than 4 points of 40 seconds scores.
(6) attempt balance is kept on balance beam but finally drop, the time on balance beam is more than 20 seconds, if mice
Attempt balance is kept on balance beam but finally drops, the time on balance beam is more than 5 points of 20 seconds scores.
(7) it is not attempt to the desire keeping balance on balance beam or holding balance beam tightly, the time on balance beam is little
Dropped in 20 seconds, if mice is not attempt to the desire keeping balance on balance beam or holding balance beam tightly, on balance beam
Time dropped less than 20 seconds 6 points of score.
The mobility test of the reflection measurement of 8.4 bodies and exception.
(1) auricle reflex: stimulate mice auditory meatus using cotton swab, observe whether mice gets rid of head reaction, if being described mice
There is auricle reflex, if no explanation mice does not have 1 point of auricle reflex, score.
(2) eyelid closure reflex: stimulating mice iris using cotton swab, observing whether mice has the reaction closing eyelid, if having
Bright mice has eyelid closure reflex, if no explanation mice does not have 1 point of eyelid closure reflex, score.
(3) startle reflection: manufactures a big noise, such as water bottle drops, observes whether mice has frightened jumping up
Reaction, if being described mice there is the reflection that startles, if no explanation mice does not startle 1 point of reflection, score.
(4) observe mice whether have a convulsion, the phenomenon such as spasm, myodystonia.If mice is had a convulsion, spasm, flesh
The phenomenons such as dystonia, 1 point of score.
Calculate the PTS of each addition, PTS 1-6 is divided into minor injury, and PTS 7-12 is divided into moderate lesion,
PTS 13-18 is divided into severe injury.
For example, the mice in Figure 23 can not keep straight on 1 point of score in keeping flat test, turn-takes 1 point of score around damaging side, to
Damage and roll 1 point of score；In proposing tail test, forelimb curves inwardly, claw firmly grasps 1 point of score, and hind leg curves inwardly, claw
Promptly 1 point of score, in 30 seconds, head lifts and vertical axises angle is more than 1 point of 10 degree of scores；Forelimb energy in visual tactile test
Quickly accurately catch table edge or 0 point of cage marginal score to protracting and opening claw in time；In proprioceptive sensation test
0 point of hind leg energy snapback score；In balance beam test, but mice is attempted keeping balance on balance beam finally drops,
Time on balance beam is more than 4 points of 40 seconds scores；Mice has 0 point of auricle reflex score；There is 0 point of eyelid closure reflex score；Tool
Startle 0 point of score of reflection；Mice is had a convulsion 1 point of score.Must being divided into of this mice: 1+1+1+1+1+1+0+0+4+0+0+
0+1=11 divides, and is evaluated as moderate apoplexy.
The cerebral tissue infraction analysis of embodiment 9glce no mutant homozygote mice
Detect the infraction situation of apoplexy Mice brain tissues by ttc (2,3,5 triphenyltetrazolium chloride) colouring method.
Operating procedure is: directly takes brain after anesthesia, about quick-freezing 5-10 minute in -20 degree refrigerators, is easy to cut into slices.Section: cut every 1mm
A piece of.Section is placed in ttc, normal concentration is 2%.After being covered with masking foil, put into 37 degree of incubator 15-30min, frequently turn over
Beat one's brains piece, makes brain piece uniform contact to dyeing liquor.Then fix 30min using 4% paraformaldehyde.Take pictures.
Ttc is fat-soluble photaesthesia complex, can be used to dye the ischemia infraction of detection mammalian tissues.It is breathing
Dehydrogenase reaction in the proton acceptor of pyridine-nucleotide structure enzyme system, with normal structure in chain and take on a red color, and ischemic tissue
Interior dehydrogenase activity declines it is impossible to react, therefore will not produce change in pale.
As shown in figure 24, result shows result, and after ttc dyes, the normal cerebral tissue of apoplexy mice takes on a red color, and
The cerebral tissue of infraction is in pale.Observe olfactory bulb, prefrontal lobe, corpus callosum, hippocampal tissue, striatum, corpus amygdaloideum, hypothalamuses, temporo
Leaf, cerebellum, pons, oblongata etc. all have infraction phenomenon.
The embodiment 10 drug verification medicine sorting platform for the treatment of anxiety neurosis (as generalized anxiety disorder)
In the present embodiment, the medicine of anxiety neurosis in the animal pattern injected in mice Present clinical treatment building to embodiment 1
Thing paroxetine or lycium barbarum polysaccharide, are estimated to the anxiety order of severity of animal pattern mice immediately.
Result shows, medicine paroxetine or lycium barbarum polysaccharide can significantly reduce the serious journey of the anxiety neurosis of animal pattern mice
Degree, specifically, can significantly reduce the incidence rate of the anxiety neurosis of animal pattern mice.
Embodiment 11 is waited using the medicine sorting platform screening for the treatment of anxiety neurosis or its relevant disease (as generalized anxiety disorder)
Select medicine
In the present embodiment, the animal pattern injected in mice anxiety neurosis by building or its related disease are planned to embodiment 1
The medicine of disease, is estimated to the order of severity of the anxiety symptom of animal pattern mice immediately.
By comparing the difference of the order of severity of anxiety symptom with the animal pattern mice to placebo, can significantly reduce
The drug candidate of the incidence rate of anxiety neurosis, is the potential medicine of this anxiety neurosis or its relevant disease.
Other anxiety neurosis or its relevant disease can also be tight to the anxiety symptom of animal pattern mice with reference to said method
Weight degree is estimated, thus screening drug candidate.
The all documents referring in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
List of references:
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sulfate and proteins:the concept of specificity.the journal of cell
biology2006；174:323-7.
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zhang x,lindahl u.targeted disruption of a murine glucuronyl c5-epimerase
gene results in heparan sulfate lacking l-iduronic acid and in neonatal
lethality.the journal of biological chemistry 2003；278:28363-6.
3.jia j,maccarana m,zhang x,bespalov m,lindahl u,li jp.lack of l-
iduronic acid in heparan sulfate affects interaction with growth factors and
cell signaling.the journal of biological chemistry 2009；284:15942-50.
4.grigorieva e,eshchenko t,rykova vi,chernakov a,zabarovsky e,sidorov
sv.decreased expression of human d-glucuronyl c5-epimerase in breast
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5.prudnikova ty,mostovich la,domanitskaya nv,pavlova tv,kashuba vi,
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6.grigorieva ev,prudnikova ty,domanitskaya nv,mostovich la,pavlova
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Claims (10)
1. the preparation method of a kind of anxiety neurosis of non-human mammal or its relevant disease animal model is it is characterised in that include
Following steps:
A () provides the cell of non-human mammal, the glce gene inactivation in described cell obtains the non-of glce gene inactivation
People's mammalian cell；
B () utilizes the cell of glce gene inactivation obtaining in step (a), prepare the anxiety neurosis of glce gene inactivation or its
The animal model of relevant disease.
2. the method for claim 1 is it is characterised in that in step (a), also comprise the steps:
(a1) utilize dna homologous recombination technique, by one or more of exon 3 in described glce gene to exon 5
Exon is rejected or is interrupted, and is replaced with selection markers, obtains the nonhuman mammalian cells of glce gene inactivation.
3. the method for claim 1 is it is characterised in that in step (b), also comprise the steps:
(b1) nonhuman mammalian cells of the glce gene inactivation obtaining in step (a) are utilized to prepare chimeric inhuman suckling
Animal；
(b2) the chimeric non-human mammal obtaining and normal wild type mating non-human mammals in step (b1) are bred,
In offspring, screening obtains the heterozygote non-human mammal of glce gene inactivation；
(b3) pass through for the mutual copulation of heterozygote non-human mammal obtaining in step (b2) to obtain the pure of glce gene inactivation
Zygote non-human mammal, thus obtain the animal model of the non-human mammal of glce gene inactivation.
4. method as claimed in claim 3 is it is characterised in that in step (b3), also include step (b4): by glce gene
The homozygote non-human mammal of inactivation and the neuronal specificity of same species knock out instrument non-human mammal and are hybridized,
Thus obtaining the animal model of the non-human mammal of glce gene inactivation of neuronal specificity.
5. the method for claim 1 it is characterised in that the glce gene inactivation that obtains in described step (b) inhuman
In the animal model of mammal, compared with wild-type control animals, there are the one or more features being selected from the group:
(t1) behavior of anxiety sample increases；
(t2) anxiety degree increases；
(t3) spacious field level of activation reduces；
(t4) desire exploring strange environment reduces；
(t5) behavior depression increases；
(t6) Degree of Depression increases；
(t7) frightened sample behavior increases；
(t8) frightened degree increases；
(t9) cognitive disorder increases；
(t10) incidence rate of apoplexy increases；
(t11) obese degree of form increases；
(t12) incidence rate of obesity symptom increases；
(t13) fatty tissue weight in wet base increases.
6. a kind of purposes of the non-human mammal model of claim 1 methods described preparation is it is characterised in that use this model
Make the animal model of research anxiety neurosis or its relevant disease.
7. a kind of non-human mammal model of claim 1 methods described preparation purposes it is characterised in that for screening or
Identification can mitigate or treat the material (therapeutic agent) of anxiety neurosis or its relevant disease.
8. the method for the potential therapeutic agent of a kind of screening or identification treatment or alleviation anxiety neurosis or its relevant disease, its feature exists
In comprising the following steps:
A (), in test group, in the presence of test compound, test compound is applied to claim 1 methods described system
Standby non-human mammal model, the behavior to described animal model carries out Behavioral assessment；And do not applying described test
In compound and other conditions identical matched group, the behavior to described animal model carries out Behavioral assessment；
B () is compared to the behavior of test group and control animals model, wherein, compared with matched group, if application of survey
Characterize anxiety behavior in the animal model of examination compound to be improved, then show that this test compound can be potential as anxiety neurosis
Therapeutic agent.
9. a kind of purposes of cell is it is characterised in that glucuronic acid c5 isomerase (glucuronyl c5- in described cell
Epimerase, glce) gene inactivation or downward, build the anxiety neurosis of non-human mammal for preparation or its relevant disease moves
The biological preparation of thing model.
10. a kind of purposes of the deactivator of glce gene or its albumen is it is characterised in that build non-human mammal for preparation
Anxiety neurosis or its relevant disease animal model preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036490A1 (en) * | 2016-08-23 | 2018-03-01 | 中国科学院上海药物研究所 | Method and application for building animal model of non-human mammal neuropsychiatric disorder |
| CN109258573A (en) * | 2018-11-21 | 2019-01-25 | 梧州学院 | Natural enemy mewing audio induces construction method and the application of Animal Models of Anxiety |
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|---|---|---|---|---|
| CN104450602A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院遗传与发育生物学研究所 | Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104450602A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院遗传与发育生物学研究所 | Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof |
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| Title |
|---|
| JIE LI ET AL.: "GLCE regulates PC12 cell neuritogenesis induced by nerve growth factor through activating SMAD/ID3 signalling", 《BIOCHEM. J.》 * |
| 涂海华等: "远志抗抑郁有效部位中寡糖酯单体的分离及活性研究", 《中国中药杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036490A1 (en) * | 2016-08-23 | 2018-03-01 | 中国科学院上海药物研究所 | Method and application for building animal model of non-human mammal neuropsychiatric disorder |
| CN109258573A (en) * | 2018-11-21 | 2019-01-25 | 梧州学院 | Natural enemy mewing audio induces construction method and the application of Animal Models of Anxiety |
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