WO2018036490A1 - 一种非人哺乳动物神经精神疾病动物模型的建立方法及其用途 - Google Patents

一种非人哺乳动物神经精神疾病动物模型的建立方法及其用途 Download PDF

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WO2018036490A1
WO2018036490A1 PCT/CN2017/098535 CN2017098535W WO2018036490A1 WO 2018036490 A1 WO2018036490 A1 WO 2018036490A1 CN 2017098535 W CN2017098535 W CN 2017098535W WO 2018036490 A1 WO2018036490 A1 WO 2018036490A1
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glce
mice
gene
animal model
human mammal
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丁侃
李艳玲
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中国科学院上海药物研究所
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for establishing an animal model of neuropsychiatric diseases in non-human mammals and uses thereof.
  • Depression also known as depressive disorder, is a typical neuropsychiatric disease.
  • the clinical manifestations are mainly core symptoms such as significant and persistent low mood, lack of power, and behavioral despair.
  • Depression has become one of the most common and most disabling neuropsychiatric diseases because of its high morbidity, high recurrence rate and high disability rate.
  • the mouse disease model plays a very important role in studying the pathogenesis and drug screening of human diseases.
  • the mouse model has great advantages in exploring human cognitive functions, neurodegenerative diseases, and neuropsychiatric diseases.
  • Gene knockout is a complex molecular biology technique developed in the 1980s based on the principle of DNA homologous recombination of mouse embryonic stem cells, also known as "gene targeting" technology. Since then, thousands of genetically modified mice have been constructed using this technology. These genetically engineered mice have not only brought breakthroughs in biological science and medical research, but also played a vital role in the development of new drugs.
  • the object of the present invention is to provide a kind of depression which can better simulate clinical primary depression, and there is no difference in surgical operation or drug dosage between individuals, and the genetic background is highly consistent and can be made.
  • An animal model of neuropsychiatric disorders in non-human mammals screened for the pathogenesis of depression and new drugs.
  • a first aspect of the invention provides a method of preparing an animal model of a neuropsychiatric disease in a non-human mammal, comprising the steps of:
  • the method further includes the following steps:
  • step (b) the method further includes the following steps:
  • step (b2) mating the chimeric non-human mammal obtained in the step (b1) with a normal wild-type non-human mammal, and screening the progeny to obtain a heterozygous non-human mammal in which the Glce gene is inactivated;
  • step (b3) further comprising the step (b4): homozygous non-human mammal inactivating the Glce gene and hybridizing with a neuron-specific knockout tool of the same species as a non-human mammal
  • step (b4) homozygous non-human mammal inactivating the Glce gene and hybridizing with a neuron-specific knockout tool of the same species as a non-human mammal
  • the inactivating the Glce gene includes gene knockout, gene disruption or gene insertion.
  • the gene inactivation comprises the Glce gene not expressing, or expressing the inactive Glce protein.
  • the inactivation of the Glce gene is inactivated by deletion or knockout of exon 3 of Glce.
  • the Glce gene inactivation is a neuron-specific Glce gene inactivation.
  • the non-human mammal is a rodent or primate, preferably a mouse, a rat, a rabbit, and/or a monkey.
  • the non-human mammal is a mouse
  • the GlceLoxp/Loxp mouse is mated with the neuron-specific enolase-Cre in step (b4) to obtain the neuronal cell.
  • Specific Glce knockout mice are referred to as cKO mice (ie, neuron-specific Glce inactivated mice).
  • the selection marker is selected from the group consisting of a resistance gene, a fluorescent protein gene, or a combination thereof.
  • the selection marker comprises a neo gene.
  • the animal model of the non-human mammal in which the Glce gene obtained in the step (b) is inactivated has one or more of the following characteristics compared to the wild type control animal:
  • the open field activity level is selected from the group consisting of: a distance of the open field activity, an activity time, an activity speed, or a combination thereof.
  • the depression-like behavior is selected from the group consisting of a decrease in the time to explore the central region in the open field experiment, a decrease in the desire to explore a new environment, and an increase in the immobility time in the forced swimming experiment. Demonstrate behavioral despair, or a combination thereof.
  • the wet weight of the adipose tissue is selected from the group consisting of wet weight of visceral adipose tissue, wet weight of subcutaneous fat tissue, or a combination thereof.
  • the wet weight of the visceral adipose tissue is selected from the group consisting of wet weight of bilateral adipose tissue around the gonads, wet weight of bilateral perirenal adipose tissue, or a combination thereof.
  • the subcutaneous adipose tissue wet weight comprises bilateral wet weight of the inguinal adipose tissue.
  • a second aspect of the invention provides the use of a non-human mammalian model prepared by the method of the first aspect of the invention, the model being used as an animal model for studying neuropsychiatric disorders.
  • the neuropsychiatric disorder comprises: schizophrenia, bipolar disorder, depression, anxiety, phobia, autism spectrum disorder, stroke, and/or Alzheimer's disease.
  • a third aspect of the invention provides the use of a non-human mammalian model prepared by the method of the first aspect of the invention, wherein the model is used to screen or identify a substance (therapeutic agent) that can alleviate or treat neuropsychiatric disorders .
  • the neuropsychiatric disease is a disease associated with a decrease in neurite outgrowth of a newborn neuron.
  • the neonatal neuron's neurite outbreak related diseases include schizophrenia, bipolar disorder, depression, anxiety, phobia, autism spectrum disorder, stroke, and/or senile dementia disease.
  • a fourth aspect of the invention provides a method of screening or identifying a potential therapeutic agent for treating or ameliorating a neuropsychiatric disorder, comprising the steps of:
  • test compound In a test group, a test compound was applied to a non-human mammal model prepared by the method of the first aspect of the invention in the presence of a test compound, and behavioral analysis of the behavior of the animal model of the test group was performed. And behavioral analysis of the behavior of the animal model of the control group in a control group in which the test compound was not administered and other conditions were the same;
  • the behavioral analysis includes: autonomous activity, open field experiment, forced swimming test, elevated plus maze test, conditional fear test, Y maze test, new object recognition experiment, water maze test, or combination.
  • the method is non-diagnostic and non-therapeutic.
  • the method comprises the step (c) of applying a potential therapeutic agent screened or identified in step (b) to a non-human mammalian model prepared by the method of the first aspect of the invention, thereby determining its The effects of the behavior of the animal model.
  • the improvement is a statistically significant improvement.
  • a fifth aspect of the invention provides a non-human mammalian model prepared by the method of the first aspect of the invention.
  • the non-human mammalian model is heterozygous or homozygous for the Glce gene inactivation.
  • the Glce gene inactivation is a neuron-specific Glce gene inactivation.
  • a sixth aspect of the invention provides the use of a cell in which a Glucuronyl C5-epimerase (Glce) gene is inactivated or down-regulated for the preparation of a neuropsychiatric construct of a non-human mammal Biological preparation for disease animal models.
  • Glce Glucuronyl C5-epimerase
  • the biological preparation is a liquid preparation.
  • a seventh aspect of the invention provides the use of an inactivating agent of the Glce gene or a protein thereof for the preparation of a preparation for constructing an animal model of neuropsychiatric disease in a non-human mammal.
  • the inactivating agent comprises an inhibitor.
  • the inactivating agent is selected from the group consisting of an antibody, a small molecule compound, a nucleic acid, or a combination thereof.
  • Figure 1 shows the Glce gene mutation sequence design strategy.
  • Figure 2 shows the plasmid map of the Glce knockout targeting vector.
  • Figure 3 shows the results of restriction enzyme digestion of the Glce gene knockout targeting vector plasmid DNA.
  • Figure 4 shows the results of electrophoresis of the 5'-end PCR product of ES cloned genomic DNA.
  • Figure 5 shows the results of electrophoresis of the 3'-end PCR product of ES cloned genomic DNA.
  • Figure 6 shows the PCR results of the Glce transgenic mouse (Loxp/+) sequence mutation site.
  • Figure 7 shows the PCR results of sequence mutation sites of Glce mutant homozygotes (Loxp/Loxp Cre), mutant heterozygotes (Loxp/+Cre) and Wild type (+/+Cre).
  • Figure 8 shows the spontaneous activity, time and speed of Glce mutant homozygous mice.
  • Figure 9 shows the total distance, time and velocity of Glce mutant homozygous mice in open field experiments.
  • Figure 10 shows the travel distance, time and velocity of Glce mutant homozygous mice in the central region of the open field.
  • Figure 11 shows the travel distance, time and velocity of Glce mutant homozygous mice in the surrounding area of the open field.
  • Figure 12 shows the immobility time of Glce mutant homozygous mice in forced swimming experiments.
  • Figure 13 shows a morphological comparison of Glce mutant homozygous mice with C57 mice.
  • the left picture the upper is C57 female mice, the lower is Glce mutant homozygous female mice;
  • the right picture the upper is C57 male mice, the lower is Glce mutant homozygous male mice.
  • Figure 14 shows the comparison of body weight between Glce mutant homozygous mice and C57 mice.
  • Figure 15 shows the comparison of the body fat wet weight of Glce mutant homozygous mice and C57 mice.
  • Figure 16 shows the comparison of the wet weight of the peripheral gonadal adipose tissue in Glce mutant homozygous mice and C57 mice.
  • Figure 17 shows the comparison of wet weight of bilateral perirenal adipose tissue in Glce mutant homozygous mice and C57 mice.
  • Figure 18 shows the comparison of the wet weight of visceral adipose tissue in Glce mutant homozygous mice and C57 mice.
  • Figure 19 shows the comparison of wet weight of bilateral inguinal adipose tissue in Glce mutant homozygous mice and C57 mice.
  • Figure 20 shows a stroke behavioral evaluation experiment.
  • Figure 21 shows the detection of brain tissue infarction in stroke mice by TTC staining.
  • Figure 22 shows the spontaneous activity, time and speed of Glce mutant homozygous mice.
  • Figure 23 shows the travel, time and velocity of Glce mutant homozygous mice in the central region of the open field.
  • Figure 24 shows the time of the open arms, central region, and closed arms of the Glce mutant homozygous mice in an elevated plus maze experiment.
  • the inventors have conducted extensive and intensive research to establish a genetically stable, phenotypically stable neuropsychiatric disease model, which is a mouse or other non-human mammal whose Glce gene has been knocked out or inactivated.
  • the animal model of the present invention is an effective animal model of neuropsychiatric diseases and can be used for studying neuropsychiatric diseases such as schizophrenia, bipolar disorder, depression, anxiety, phobia, autism spectrum disorder, stroke and Alzheimer's disease. And can be used for screening and testing of specific drugs.
  • an animal model obtained by knocking out or inactivating the Glce gene can also be used for studying diseases such as stroke, obesity, and anxiety.
  • the present invention has been completed on this basis.
  • Heparan sulfate is a polysaccharide widely present on the cell surface and cytoplasmic matrix. As a negatively charged linear macromolecule, many cytokines, growth factors, chemokines and interleukins can specifically bind to it, and then in the embryo. Play a role in physiological processes such as development, cell growth, inflammatory response, coagulation, tumor metastasis, and viral infection.
  • Glucuronic acid C5 isomerase (Glce) is a key enzyme in the synthesis of glycosaminoglycan proteoglycans. It can heterogeneize D-glucuronic acid in sugar chains to L-iduronic acid 2 , greatly increases the complexity of heparan sulfate, and provide more flexibility to the sugar chain, L- iduronic acid of heparan sulfate is essential to identify a large number of site 3 protein molecule.
  • Glce mRNA and protein expression levels was significantly downregulated or completely lost 4.
  • overexpression of Glce in breast and lung cancer cell lines inhibits proliferation of small cell lung cancer and breast cancer cells, suggesting that Glce may be a potential tumor suppressor gene 5,6 .
  • the Glce gene is located on chromosome 9 of the mouse genome and has a full length of 618 (EnsemblGene ID: ENSMUSG00000032252, Genebank accession number: 93683).
  • the Glce genome sequence consists of 4 introns and 5 exons, Glce gene-expressing protein has 3 transcripts, transcript 1 has 3 exons, 2 introns, and transcript 2 has 5 explicit The transcript 3 has two exons and one intron.
  • the Glce gene of other species such as humans can also be found in the literature or public databases such as EnsemblGene and Genebank.
  • Glce also includes variant forms of the various naturally occurring Glce genes. Representative examples include a nucleotide sequence encoding a Glce protein identical to the wild type due to degeneracy of the codon, and a nucleotide sequence encoding a conservative variant polypeptide of the wild type Glce protein. Furthermore, for other mammals other than mice, the term refers to homologs of the Glce gene in the mammal. For example, for humans, the term refers to human Glce (the cDNA homology of the known mouse Glce gene to the human Glce gene is 91.4%, and the amino acid sequence has a homology of 97.4%).
  • the inactivating agent of Glce includes all inactivation or partial inactivation.
  • the inactivating agent of the Glce protein of the present invention includes (a) an inhibitor, and examples of the inhibitor include, but are not limited to, a small molecule compound, an antibody, an antisense nucleic acid, a miRNA, an siRNA, or a combination thereof; a knockout agent for the Glce gene.
  • Neuropsychiatric disorders are a class of neurodevelopmental disorders caused by disorders of the nervous system, or more precisely pathological changes in the brain's neural circuits, including autism spectrum disorders, schizophrenia, depression, anxiety, phobia , epilepsy, etc. Neurodevelopmental disorders such as neuronal dysplasia, improper synaptic connections, and misconnections in the neural circuits can lead to severe neuropsychiatric disorders. Depression is a typical neuropsychiatric disorder. Studies have shown that 7-10 , abnormal growth of neurons and abnormal dendritic development lead to depression.
  • hippocampus is a brain region closely related to learning, memory and emotional and emotional regulation. Functionally, the occurrence of adult neonatal neurons in hippocampal dentate gyrus plays an important role in maintaining the plasticity of neuronal networks. At the same time, hippocampus The dentate gyrus is also the most vulnerable brain area in the early stages of Alzheimer's disease. It is also often associated with hippocampal function abnormalities in patients with mental illness such as schizophrenia and depression. More and more evidence suggests that adult hippocampal dentate gyrus of newborn neurons (adult neurogenesis) the reduction may be one of schizophrenia and depression and mental illness important reason for the onset of dementia 11.
  • diseases associated with the reduction of adult neonatal neurons include, but are not limited to, mental disorders such as schizophrenia, bipolar disorder, depression, anxiety, phobia, autism spectrum disorders, neurodegenerative diseases such as Alzheimer's disease.
  • Parkinson's disease, stroke, etc. preferably, includes schizophrenia, bipolar disorder, depression, anxiety, phobia, autism spectrum disorder, stroke, and/or Alzheimer's disease.
  • a number of methods can be used, such as inactivating the gene to be studied, analyzing the resulting phenotypic changes of the genetic modification, and obtaining functional information of the gene.
  • Another advantage of this research method is that it can associate gene function with disease, so that when the gene function is obtained, the disease information and disease animal model that the gene can be treated as a potential drug or drug target can be obtained.
  • Methods of gene inactivation can be accomplished by gene knockout, gene disruption, or gene insertion. Among them, gene knockout technology is a very powerful means to study the function of human genes in the whole.
  • a very effective non-human mammalian model of neuropsychiatric disorders is provided.
  • non-human mammals include, but are not limited to, mice, rats, rabbits, monkeys, and the like, and more preferably rats and mice.
  • Glce gene inactivation includes the case where one or both Glce genes are inactivated, ie, including the Glce gene, heterozygously and homozygously inactivated.
  • a mouse inactivated by the Glce gene can be a heterozygous or homozygous mouse.
  • a non-human mammal e.g., a mouse
  • the Glce gene inactivated
  • a foreign gene or fragment
  • Techniques for inactivating target genes by gene knockout or transfer into foreign genes are known in the art, and these conventional techniques can be used in the present invention.
  • inactivation of the Glce gene is achieved by gene knockout.
  • the inactivation of the Glce gene is achieved by insertion of a foreign gene (or fragment) into the Glce gene.
  • a construct comprising an exogenous insert comprising a homology arm homologous to a flanking sequence flanking the insertion site of a target gene (Glce), thereby allowing High frequency insertion of exogenous inserts (or genes) into the Glce genome sequence (especially exons) by homologous recombination Region), resulting in frameshift, early termination, or knockout of the mouse Glce gene, resulting in deletion or inactivation of the Glce gene.
  • Glce target gene
  • the homozygous or heterozygous mouse obtained by the method of the invention is fertile.
  • the inactivated Glce gene can be inherited to the offspring mice by Mendelian law.
  • the invention provides a homozygous mouse model animal lacking the Glce gene.
  • a drug candidate or a therapeutic agent refers to a substance which is known to have a certain pharmacological activity or is being tested and which may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, sugars, and small chemical synthesis. Molecular or macromolecular compounds, cells, and the like.
  • the drug substance or therapeutic agent can be administered orally, intravenously, intraperitoneally, subcutaneously, spinally, or directly into the brain.
  • the present invention can better simulate clinically primary depression.
  • the neuropsychiatric disease model of the present invention is genetically stable and phenotypically stable.
  • a homozygous or heterozygous animal model obtained by the method of the invention is fertile.
  • Transgenic heterozygous mice have reproductive capacity, and the inactivated Glce gene can be inherited to the offspring mice by Mendelian law.
  • the animal model of neuropsychiatric disease of the present invention exhibits various neurological and psychiatric disease-like symptoms, and thus can be widely used for drug screening and testing of neuropsychiatric diseases, including schizophrenia, bipolar disorder, depression, Anxiety disorders, phobias, autism spectrum disorders, stroke and Alzheimer's disease.
  • the present invention discloses for the first time that an animal model obtained by knocking out or inactivating a Glce gene can also be used for studying diseases such as stroke, obesity, and anxiety.
  • Example 1 Obtaining a homozygous mouse of the Glce gene mutation carrying Cre recombinase
  • the Glce mutant gene sequence was first constructed (Fig. 1).
  • the Glce gene knockout targeting vector sequence was designed as shown in Figure 1.
  • the Loxp/Loxp allele was inserted into the exon 3 of the Glce gene, and the neo gene was inserted at the 3' end.
  • the 5' end arm was 3125 bp and the 3' end arm was 3718 bp.
  • Figure 2 is a plasmid map of the Glce knockout targeting vector. 1. Obtain a homologous fragment of the gene of interest (Glce), and clone the DNA fragment into a plasmid vector; 2.
  • the gene loci are shown in the following table:
  • the gene position labeling number is based on "10kb Up and Down of Glce gene”.
  • FIG. 3 shows the restriction enzyme digestion of the Glce gene knockout targeting vector plasmid DNA using a 1 Kb DNA ladder.
  • Linearization of the targeting vector 100 ⁇ g of Glce-CKO plasmid DNA (purchased from Biovector NTCC) was linearized with NotI (enzyme dosage: 150 U), the enzyme digestion system was 150 ⁇ l, and digested overnight at 37 ° C, after treatment with an equal volume of phenol chloroform and chloroform, Precipitate with absolute ethanol and resuspend in 100 ⁇ l sterile PBS for use.
  • ES cell targeting was derived from embryonic stem cells of 129S V /E V strain male mice (purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences), linearized DNA amount: 35 ⁇ g, electrorotator model: Bio-Rad Gene Pulser (Cat. No.165-2105), electroporation conditions: voltage 240v, capacitance 500 ⁇ F, actual energization time 10.5ms, actual voltage 256v, clone screening conditions: 300 ⁇ g/ml G418 and 2 ⁇ M GanC for 8 days. A total of 96 copies of resistant clones and DNA samples were obtained.
  • the P1 primer is located outside the 5'arm, and the P2 primer is located in the neo recombination region, 8.2 kb from the extra-arm primer.
  • the P4 primer is located outside the 3'arm, and the P3 primer is located in the neo recombination region, 4.7 kb from the extra-arm primer.
  • P4 and P3 primer sequences :
  • P4 GAGAGGCTTGGAGGCGGTGCTGATCTT (gene site: 29603-29629) (SEQ ID NO.: 3)
  • P3 GATATACTATGCCGATGATTAATTGTC (SEQ ID NO.: 4)
  • ES cell cloning identification results PCR identified 96 drug-resistant ES cell clones, of which 19 ES clones developed double-arm homologous recombination. The PCR product was further confirmed by DNA sequencing.
  • Figure 4 shows the results of electrophoresis of the 5'-end PCR product of ES cloned genomic DNA.
  • Figure 5 shows the results of electrophoresis of the 3'-end PCR product of ES cloned genomic DNA.
  • Microinjection blastocyst source C57BL/6J mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) have superovulation and naturally develop into the blastocyst stage. 60 embryos were injected and transplanted into the recipient uterus of three pseudopregnant mice. The recipients were hybrids of C57BL/6J ( ⁇ ) and CBA ( ⁇ ) (purchased from Shanghai Slack Laboratory Animal Co., Ltd.). Mice with a chimeric rate greater than 50% were selected from the born mice and raised to adulthood. They were mated with C57BL/6J female mice, and the gray mice of the offspring were identified by PCR using the extracted tail genomic DNA (identification strategy is the same as above). As shown in Figure 6, two-arm positive F1 mice (Loxp/+) were obtained.
  • F1 generation mice were raised to adulthood, and NSE-Cre tool mice (purchased from Shanghai Slack experimental animals) The company was crossed to obtain F2 generation Loxp/+Cre mice.
  • the F2 generation mice were raised to adulthood, and the males and females in the F2 generation were mated together, and the F3 generation mutation homozygotes (Loxp/Loxp Cre) were obtained according to the Mendelian law: the mutant heterozygote (Loxp/+Cre): Wild type (+/ The ratio of +Cre) is approximately 1:2:1.
  • the tail genomic DNA was extracted for PCR identification (the identification strategy is the same as above), and the results are shown in FIG. 7 . Mutant homozygous mice (Loxp/Loxp Cre) were used for subsequent animal behavioral experiments.
  • the Glce gene-mutated homozygous mice carrying the Cre recombinase were placed in a dark experimental chamber (110 mm X 110 mm X 330 mm), and the mice were tested for spontaneous activity for 5 min. The activity of the mice was photographed by infrared imaging. The mouse trajectory and activity time were analyzed using video tracking software and analysis software of Shanghai Jiliang Software Technology Co., Ltd.
  • mice were placed in a bright open experiment box (500 mm X 500 mm X 590 mm) and tested for 5 min.
  • the activity of the mice was photographed by infrared imaging.
  • the mouse trajectory and activity time were analyzed using video tracking software and analysis software of Shanghai Jiliang Software Technology Co., Ltd.
  • adolescent Glce mutant homozygous mice were in the market The distance, activity time, and activity rate of the side-region activities were significantly reduced (Fig. 11). The results showed that the activity of the young Glce mutant homozygous mice was significantly reduced compared with the C57BL/6 normal mice.
  • mice were placed in a water tank of 12 cm in diameter and 25 cm in height (water temperature 21-22 ° C). The mice were forced to swim in water with low water temperature. The mice were tested for activity for 6 min, and the mice were recorded for the next 4 min. Time. The activity of the mice was photographed by camera. The activity time of the mice was analyzed. The study found that the adolescent Glce mutant homozygous mice had a significant increase in immobility time in forced swimming experiments compared to adolescent C57BL/6 normal mice ( Figure 12).
  • adolescent Glce mutant homozygous mice had a certain degree of depression compared with C57BL/6 normal mice. There was no significant difference between the female adolescent Glce mutant homozygous mice and the male adolescent Glce mutant homozygous mice. In addition, adolescent Glce mutant homozygous mice with a certain degree of depression also produce a certain degree of anxiety.
  • the birth rate of Glce mutant homozygous mice is in accordance with Mendel's law, and the body weight of the mice is tested. The results are shown in FIG. The results showed that the morphology of adult Glce neuron-specific knockout mice was more obese than that of adult C57BL/6 normal mice.
  • Glce mutant homozygous mice were weighed on an electronic balance to study the difference in body weight between adult Glce mutant homozygous mice and adult C57BL/6 normal mice.
  • Glce mutant homozygous mice were dissected and adipose tissue including peripheral adipose tissue, bilateral perirenal adipose tissue and bilateral inguinal adipose tissue were weighed on an electronic balance to study adult Glce mutant homozygous mice and adulthood. Difference in wet tissue wet weight of C57BL/6 normal mice.
  • the average body fat wet weight of adult C57BL/6 normal mice was 2.21g, and the adult Glce mutant homozygous mice (including male and female) The body fat wet weight averaged 8.01 g, which was statistically significant (P ⁇ 0.01) compared with adult C57BL/6 normal mice.
  • the Glce mutant homozygous mice were dissected and the adipose tissue around the gonads was weighed on an electronic balance to study the difference in wet weight between the bilateral gonadal adipose tissue in adult Glce mutant homozygous mice and adult C57BL/6 normal mice. .
  • the bilateral gonads of adult C57BL/6 normal mice (including male and female)
  • the average wet weight of surrounding adipose tissue was 1.24g
  • the average wet weight of bilateral adipose tissue in adult Glce mutant homozygous mice was 2.98g, which was statistically significant compared with adult C57BL/6 normal mice. (P ⁇ 0.01).
  • Glce mutant homozygous mice were dissected and bilateral renal peripheral adipose tissue was weighed on an electronic balance to study the difference in wet weight of bilateral renal adipose tissue between adult Glce mutant homozygous mice and adult C57BL/6 normal mice. .
  • the wet weight of bilateral perirenal adipose tissue was significantly increased in adult Glce mutant homozygous mice compared to adult C57BL/6 normal mice (Fig. 17).
  • the Glce mutant homozygous mice were dissected and the bilateral adipose tissue and bilateral perirenal adipose tissue were weighed on an electronic balance.
  • the total weight of visceral adipose tissue was bilateral adipose tissue and bilateral nephropathy.
  • the sum of the wet weight of adipose tissue was studied to study the difference in wet weight of visceral adipose tissue between adult Glce mutant homozygous mice and adult C57BL/6 normal mice.
  • the wet weight of visceral adipose tissue was significantly increased in adult Glce mutant homozygous mice compared to adult C57BL/6 normal mice (Fig. 18).
  • the Glce mutant homozygous mice were dissected and the bilateral inguinal adipose tissue was weighed on an electronic balance.
  • the wet weight of subcutaneous adipose tissue was the sum of the wet weight of the bilateral inguinal adipose tissue.
  • the adult Glce mutant homozygous mice and adult C57BL were studied. /6 The difference in wet weight of subcutaneous adipose tissue in normal mice.
  • Glce gene mutant homozygous mice with Cre recombinase were housed in a clean feeding environment.
  • the mouse animal model developed stroke-like symptoms at the age of aging (about 1.5 years).
  • a series of stroke behavioral experiments were used to evaluate the stroke-like symptoms and severity of the mouse model.
  • the experimental method was based on a series of systematic behavioral experiments on the severity of stroke symptoms published in Stroke, 2001 (Chen J, et al.Stroke.2001), including sports test, sensory test, balance ability test, body reflex test, abnormal activity test, etc., specifically for flat test, tail test, visual tactile test, prophetic test, balance beam test Behavioral tests of mice, auricular reflexes, sacral reflexes, convulsions, convulsions, convulsions, and dystonia were scored.
  • Tail test Lift the tail of the mouse and observe the movement of the limbs of the mouse. If the forelimbs are bent inward and the claws are grasped, if the forelimbs of the mice are bent inward and the paws are grasped, the score is 1 point, otherwise the score is 0; The hind limbs are bent inward and the claws are grasped. If the hind limbs of the mouse are bent inward and the paws are grasped and scored 1 point, otherwise score 0 points; if the head is raised within 30 seconds, the angle with the vertical axis is greater than 10 degrees, if the mouse is within 30 seconds The head is raised and scored 1 point with the vertical axis angle greater than 10 degrees, otherwise scored 0 points.
  • Ontology test grasp the mouse body, let the hind limbs of the mouse move freely, place the forelimb of the mouse on the edge of the table or the edge of the cage, and hang the hind limbs. Use the forceps to clip the hind leg muscles of the mouse and observe whether the hind limbs of the mouse are Can quickly retract. If the hind limbs of the mice cannot be retracted quickly, score 1 point, otherwise score 0 points.
  • the time on the balance beam is greater than 40 seconds. If the mouse tries to balance on the balance beam but eventually falls, the time on the balance beam is greater than 40 seconds and scores 4 points.
  • the time on the balance beam is less than 20 seconds. If the mouse does not try to balance on the balance beam or hold the balance beam, the time on the balance beam. Score less than 20 seconds and score 6 points.
  • Auricular reflex The cotton ear can be used to stimulate the ear canal of the mouse to observe whether the mouse has a taro reaction. If there is a description of the auricle reflex, the mouse has no auricle reflex and scores 1 point.
  • (2) sputum reflex use a cotton swab to stimulate the iris of the mouse, and observe whether the mouse has a reaction to close the eyelid. If there is a description that the mouse has a sacral reflex, if there is no indication that the mouse has no reflex, score 1 point.
  • Shocking reflex Make a big noise, such as the water bottle falling, observe whether the mouse has a shocked reaction, if there is a shocking reflex, if there is no indication that the mouse does not have a shocking reflection, Score 1 point.
  • mice (4) Observe whether the mice have convulsions, spasms, dystonia and other phenomena. If the mouse has convulsions, spasms, dystonia, etc., score 1 point.
  • the mouse in Fig. 20 cannot score 1 point in a straight line test, score 1 point around the injury side, and score 1 point on the injury side; in the tailing test, the forelimb bends inward and the paw grasps 1 point. The hind limbs are bent inward and the paws are grasped and scored 1 point. The head is lifted within 30 seconds and the angle is greater than 10 degrees with the vertical axis score of 1 point.
  • the forelimbs can quickly extend forward and open the claws in time to accurately grasp Score 0 points on the edge of the table or on the edge of the cage; the hind limbs can quickly retract the score by 0 in the propensity test; in the balance beam test, the mouse tries to balance on the balance beam but eventually falls, and the time on the balance beam is greater than 40 seconds.
  • the mice had an auricle reflex score of 0; a score of 0 with a closed reflex; a score of 0 with a stun response; and a score of 1 for a convulsion in the mouse.
  • TTC 2,3,5-triphenyltetrazolium chloride staining Infarction.
  • the operation steps are: take the brain directly after anesthesia, and freeze in a -20 degree refrigerator for about 5-10 minutes, which is convenient for slicing.
  • Slice Cut one slice every 1 mm. The sections were placed in TTC at a conventional concentration of 2%. Cover with tin foil paper, put it into a 37-degree incubator for 15-30min, and turn the brain slices from time to time to make the brain slices evenly contact the staining solution. It was then fixed with 4% paraformaldehyde for 30 min. Take a photo.
  • TTC is a fat-soluble, light-sensitive complex that can be used to stain for ischemic infarction in mammalian tissues. It is a proton acceptor of the pyridine-nucleoside structural enzyme system in the respiratory chain, which reacts with dehydrogenase in normal tissues and is red, while the activity of dehydrogenase in the ischemic tissue decreases and cannot be reacted, so no change occurs. pale.
  • Fig. 21 The results show that after TTC staining, the normal brain tissue of the stroke mice was red, and the infarcted brain tissue was pale. Infarction was observed in the olfactory bulb, prefrontal cortex, corpus callosum, hippocampus, striatum, amygdala, hypothalamus, temporal lobe, cerebellum, pons, and medulla.
  • Glce gene homozygous mice carrying the Cre recombinase (hereinafter referred to as Glce mutant homozygous mice) were placed in a clean feeding environment. Anxiety-like symptoms in mouse animal models were evaluated in adolescent mice (>3 months old) through a series of animal behavioral experiments including autonomic activities, open field experiments, elevated plus maze, and the like.
  • mice were placed in a dark experimental chamber (110 mm X 110 mm X 330 mm) and tested for spontaneous activity of the mice for 5 min.
  • the activity of the mice was photographed by infrared imaging.
  • the mouse trajectory and activity time were analyzed using video tracking software and analysis software of Shanghai Jiliang Software Technology Co., Ltd.
  • mice were placed in a bright open experiment box (500 mm X 500 mm X 590 mm) and tested for 5 min.
  • the activity of the mice was photographed by infrared imaging.
  • the mouse trajectory and activity time were analyzed using video tracking software and analysis software of Shanghai Jiliang Software Technology Co., Ltd.
  • mice were placed in an elevated cross maze with a single arm length of 30 cm, a width of 6 cm, a closed arm single arm length of 30 cm, a width of 6 cm, and a height of 14.5 cm. The height was about 50 cm from the ground, and the mouse was photographed for 5 minutes in the elevated cross maze. Activity situation in the middle. Analysis of mouse activity found that compared with adolescent C57BL/6 normal mice, adolescent Glce mutant homozygous mice significantly reduced the time of open arms in the elevated plus maze test, and the time spent in the closed arm was significant. There was no significant difference in the time spent in the central area (Figure 24).
  • Example 10 Drug Verification Drug Screening Platform for Treating Neuropsychiatric Diseases (such as Depression)
  • the model animal mouse constructed in Example 1 was injected with the current clinical treatment of neuropsychiatric drugs such as fluoxetine or timosaponin, and then the model animals were subjected to spontaneous activity, open field experiment, forced swimming test. The behavioral indicators in the assessment are evaluated.
  • neuropsychiatric drugs such as fluoxetine or timosaponin
  • Example 11 screening drug candidates using a drug screening platform for treating neuropsychiatric diseases such as depression
  • the present embodiment it is planned to inject a therapeutic animal for neuropsychiatric diseases by the model animal mouse constructed in Example 1, and then evaluate the behavioral indexes of the model animal mice in the spontaneous activity, the open field experiment, and the forced swimming test. .
  • neuropsychiatric diseases can also be referred to the above methods, and corresponding behavioral indicators are used for drug candidate screening.

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Abstract

一种非人哺乳动物的神经精神疾病动物模型的制备方法，该方法包括以下步骤：(a)提供非人哺乳动物的细胞，将所述细胞中的Glce基因失活，得到Glce基因失活的非人哺乳动物细胞；(b)利用步骤(a)中得到的Glce基因失活的细胞，制备得到Glce基因失活的神经精神疾病动物模型。所述动物模型是一种有效的神经精神疾病动物模型，可用于研究精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和老年痴呆症等神经精神疾病，并可以用于特定药物的筛选和测试试验。

Description

一种非人哺乳动物神经精神疾病动物模型的建立方法及其用途 技术领域
本发明涉及生物技术领域，具体地，涉及一种非人哺乳动物神经精神疾病动物模型的建立方法及其用途。
背景技术
抑郁症又称抑郁障碍，是一种典型的神经精神疾病，临床表现主要有显著而持久的心境低落、动力缺失、行为绝望等核心症状。抑郁症因其具有高发病率、高复发率和高致残率的特点，已经成为最常见和最致残的神经精神疾病之一。
目前抑郁症的致病机理尚未研究清楚，缺乏安全有效的治疗手段，因此深入探讨其发病机理是治疗抑郁症的重要基础。小鼠疾病模型在研究人类疾病的致病机理和药物筛选中起到了非常重要的作用。在探究人类的认知功能、神经退行性疾病、神经精神疾病等方面，小鼠模型具有巨大的优势。
基因敲除是上世纪80年代发展起来的一项复杂的分子生物学技术，其基于小鼠胚胎干细胞DNA同源重组原理，也称为“基因打靶”技术。其后利用这项技术构建了数千种基因突变小鼠，这些基因工程小鼠不仅为生物科学和医学的研究带来了突破，也在新药研发中发挥了至关重要的作用。
目前已有的小鼠抑郁症模型的建立方法大多要经过外源性药物或其它物理的、化学的方法处理，甚至是通过手术手段，不能完全模拟人类的原发性抑郁症，并且存在如手术复杂、实际操作较困难，造模时间较长，效果不稳定，个体差异较大等缺点。
因此，本领域迫切需要开发一种可以较好地模拟临床上原发性的抑郁症，个体之间不存在手术操作或者药物剂量导致的差异，其遗传背景高度一致，可以作为研究抑郁症的发病机理和新药筛选的非人哺乳动物的神经精神疾病的动物模型。
发明内容
本发明的目的在于提供一种可以较好地模拟临床上原发性的抑郁症，个体之间不存在手术操作或者药物剂量导致的差异，其遗传背景高度一致，可以作 为研究抑郁症的发病机理和新药筛选的非人哺乳动物的神经精神疾病的动物模型。
本发明的第一方面提供了一种非人哺乳动物的神经精神疾病动物模型的制备方法，包括以下步骤：
(a)提供非人哺乳动物的细胞，将所述细胞中的葡萄糖醛酸C5异构酶(Glucuronyl C5-epimerase,Glce)基因失活，得到Glce基因失活的非人哺乳动物细胞；
(b)利用步骤(a)中得到的Glce基因失活的细胞，制备得到Glce基因失活的神经精神疾病动物模型。
在另一优选例中，在步骤(a)中，还包括如下步骤：
(a1)利用DNA同源重组技术，将所述Glce基因中的外显子3至外显子5中一个或多个外显子剔除或中断，并用筛选标记替换，得到Glce基因失活的非人哺乳动物细胞。
在另一优选例中，在步骤(b)中，还包括如下步骤：
(b1)利用步骤(a)中得到的Glce基因失活的非人哺乳动物细胞制备得到嵌合非人哺乳动物；
(b2)将步骤(b1)中得到的嵌合非人哺乳动物和正常野生型非人哺乳动物交配繁育，在后代中筛选获得Glce基因失活的杂合子非人哺乳动物；
(b3)通过将步骤(b2)中得到的杂合子非人哺乳动物相互交配获得Glce基因失活的纯合子非人哺乳动物，从而得到Glce基因失活的非人哺乳动物的动物模型。
在另一优选例中，在步骤(b3)中，还包括步骤(b4)：将Glce基因失活的纯合子非人哺乳动物与同一物种的神经元特异性敲除工具非人哺乳动物进行杂交，从而获得神经元特异性的Glce基因失活的非人哺乳动物的动物模型。
在另一优选例中，所述将Glce基因失活包括基因剔除、基因中断或基因插入。
在另一优选例中，所述基因失活包括Glce基因不表达，或表达没有活性的Glce蛋白。
在另一优选例中，所述Glce基因失活是通过缺失或敲除Glce的外显子3而失活。
在另一优选例中，所述的Glce基因失活是神经元特异性的Glce基因失活。
在另一优选例中，所述非人哺乳动物为啮齿动物或灵长目动物，较佳地包括小鼠、大鼠、兔和/或猴。
在另一优选例中，所述非人哺乳动物是小鼠，并且在步骤(b4)中把GlceLoxp/Loxp小鼠与工具鼠NSE(neuron-specific enolase)-Cre交配，即得到在神经元细胞特异性Glce基因敲除小鼠简称cKO小鼠(即神经元特异性Glce失活小鼠)。
在另一优选例中，所述筛选标记选自下组：抗性基因、荧光蛋白基因、或其组合。
在另一优选例中，所述筛选标记包括neo基因。
在另一优选例中，所述步骤(b)中得到的Glce基因失活的非人哺乳动物的动物模型中，与野生型对照动物相比，具有以下一个或多个特征：
(t1)旷场活动水平降低；
(t2)探索新异环境的欲望降低；
(t3)抑郁样行为增加；
(t4)抑郁程度增加；
(t5)焦虑样行为增加；
(t6)焦虑程度增加；
(t7)恐惧样行为增加；
(t8)恐惧程度增加；
(t9)认知障碍增加；
(t10)中风的发生率增加；
(t11)形态的肥胖程度增加；
(t12)肥胖症状的发生率增加；
(t13)脂肪组织湿重增加。
在另一优选例中，所述旷场活动水平选自下组：旷场活动的路程、活动时间、活动速度、或其组合。
在另一优选例中，所述抑郁样行为选自下组：在旷场实验中探索中央区域的时间减少，表现出探索新异环境的欲望降低、在强迫游泳实验中的不动时间增加，表现出行为绝望、或其组合。
在另一优选例中，所述脂肪组织湿重选自下组：内脏脂肪组织湿重、皮下脂肪组织湿重、或其组合。
在另一优选例中，所述内脏脂肪组织湿重选自下组：双侧性腺周围脂肪组织湿重、双侧肾周脂肪组织湿重、或其组合。
在另一优选例中，所述皮下脂肪组织湿重包括双侧腹股沟脂肪组织湿重。
本发明第二方面提供了一种本发明第一方面所述方法制备的非人哺乳动物模型的用途，将该模型用作研究神经精神疾病的动物模型。
在另一优选例中，所述神经精神疾病包括：精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和/或老年痴呆症。
本发明第三方面提供了一种本发明第一方面所述方法制备的非人哺乳动物模型的用途，其中，将该模型用于筛选或鉴定可减轻或治疗神经精神疾病的物质(治疗剂)。
在另一优选例中，所述的神经精神疾病是新生神经元的神经突发生减少相关疾病。
在另一优选例中，所述的新生神经元的神经突发生减少相关疾病包括精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和/或老年痴呆症。
本发明第四方面提供了一种筛选或鉴定治疗或缓解神经精神疾病的潜在治疗剂的方法，包括以下步骤：
(a)在测试组中，在测试化合物的存在下，将测试化合物施用于本发明第一方面所述方法制备的非人哺乳动物模型，对测试组的所述动物模型的行为进行行为学分析；并且在不施用所述测试化合物且其他条件相同的对照组中，对对照组的所述动物模型的行为进行行为学分析；
(b)对测试组和对照组动物模型的行为进行比较，其中，与对照组相比，如果施用了测试化合物的动物模型中表征神经精神疾病行为得到改善，则表明该测试化合物可作为神经精神疾病的潜在治疗剂。
在另一优选例中，所述的行为学分析包括：自主活动、旷场实验、强迫游泳实验、高架十字迷宫实验、条件恐惧实验、Y迷宫实验、新物体识别实验、水迷宫实验、或其组合。
在另一优选例中，所述的方法是非诊断性和非治疗性的。
在另一优选例中，所述方法包括步骤(c)，将步骤(b)筛选或鉴定的潜在治疗剂施用于本发明第一方面所述方法制备的非人哺乳动物模型，从而测定其对所述动物模型的行为的影响。
在另一优选例中，所述改善是在统计学上具有显著性意义的改善。
本发明第五方面提供了一种非人哺乳动物模型，用本发明第一方面所述方法制备。
在另一优选例中，对于Glce基因失活而言，所述的非人哺乳动物模型是杂合的或纯合的。
在另一优选例中，所述的Glce基因失活是神经元特异性的Glce基因失活。
本发明第六方面提供了一种细胞的用途，所述细胞中的葡萄糖醛酸C5异构酶(Glucuronyl C5-epimerase,Glce)基因失活或下调，用于制备构建非人哺乳动物的神经精神疾病动物模型的生物制剂。
在另一优选例中，所述生物制剂为液态制剂。
本发明第七方面提供了一种Glce基因或其蛋白的失活剂的用途，用于制备构建非人哺乳动物的神经精神疾病动物模型的制剂。
在另一优选例中，所述失活剂包括抑制剂。
在另一优选例中，所述失活剂选自下组：抗体、小分子化合物、核酸、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Glce基因突变序列设计策略。
图2显示了Glce基因剔除打靶载体质粒图谱。
图3显示了Glce基因剔除打靶载体质粒DNA的酶切鉴定结果。
图4显示了ES克隆基因组DNA的5’端PCR产物的电泳结果。
图5显示了ES克隆基因组DNA的3’端PCR产物的电泳结果。
图6显示了Glce转基因小鼠(Loxp/+)序列突变位点PCR结果。
图7显示了Glce突变纯合子(Loxp/Loxp Cre)、突变杂合子(Loxp/+Cre)和Wild type(+/+Cre)的序列突变位点PCR结果。
图8显示了Glce突变纯合子小鼠的自发活动路程、时间及速度。
图9显示了Glce突变纯合子小鼠在旷场实验中的活动总路程、时间及速度。
图10显示了Glce突变纯合子小鼠在旷场中央区域的活动路程、时间及速度。
图11显示了Glce突变纯合子小鼠在旷场周边区域的活动路程、时间及速度。
图12显示了Glce突变纯合子小鼠在强迫游泳实验中的不动时间。
图13显示了Glce突变纯合子小鼠与C57小鼠的形态对比。其中，左图：上为C57雌性小鼠，下为Glce突变纯合子雌性小鼠；右图：上为C57雄性小鼠，下为Glce突变纯合子雄性小鼠。
图14显示了Glce突变纯合子小鼠与C57小鼠的体重对比情况。
图15显示了Glce突变纯合子小鼠与C57小鼠的体脂湿重对比情况。
图16显示了Glce突变纯合子小鼠与C57小鼠的双侧性腺周围脂肪组织湿重对比情况。
图17显示了Glce突变纯合子小鼠与C57小鼠的双侧肾周脂肪组织湿重对比情况。
图18显示了Glce突变纯合子小鼠与C57小鼠的内脏脂肪组织湿重对比情况。
图19显示了Glce突变纯合子小鼠与C57小鼠的双侧腹股沟脂肪组织湿重对比情况。
图20显示了中风行为学评价实验。
图21显示了TTC染色检测中风小鼠脑组织梗塞情况。
图22显示了Glce突变纯合子小鼠的自发活动路程、时间及速度。
图23显示了Glce突变纯合子小鼠在旷场中央区域的活动路程、时间及速度。
图24显示了Glce突变纯合子小鼠在高架十字迷宫实验中分别探究开放臂、中央区域和封闭臂的时间。
具体实施方式
本发明人经过广泛而深入的研究，建立了一种遗传稳定、表型稳定的神经精神疾病模型，它是Glce基因被剔除或失活的小鼠或其他非人哺乳动物。本发明的动物模型是一种有效的神经精神疾病动物模型，可用于研究精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和老年痴呆症等神经精神疾病，并可以用于特定药物的筛选和测试试验。
此外，本发明还意外地发现，剔除或失活Glce基因所得到的动物模型还可以同时用于研究中风、肥胖、焦虑等疾病。在此基础上完成了本发明。
Glce基因
硫酸乙酰肝素是在细胞表面和细胞质基质中广泛存在的一种多糖，作为一个带负电的线性大分子，许多细胞因子、生长因子、趋化因子和白介素能与之特异性地结合，进而在胚胎发育、细胞生长、炎症反应、凝血、肿瘤转移和病毒侵染等生理过程中发挥作用1。
葡萄糖醛酸C5异构酶(Glce)是硫酸乙酰肝素蛋白聚糖糖链合成过程中的一个关键酶，它能将糖链中的D-葡萄糖醛酸异构为L-艾杜糖醛酸2，大大提高了硫酸乙酰肝素的复杂度，并为糖链提供了更多的柔性，L-艾杜糖醛酸是硫酸乙酰肝素识别众多蛋白分子的一个必不可缺的位点3。
在对21例正常乳腺组织和74例乳腺肿瘤组织的研究发现，82％-84％的人乳 腺肿瘤组织中Glce在mRNA水平和蛋白水平的表达是显著下调或者完全丧失的4。此外，在乳腺癌和肺癌细胞系中过表达Glce能抑制小细胞肺癌和乳腺癌细胞的增殖，这提示我们Glce可能是一个潜在的抑癌基因5,6。
Glce基因位于小鼠基因组9号染色体上，全长618(EnsemblGene ID:ENSMUSG00000032252，Genebank登录号:93683)。Glce基因组序列包括4个内含子和5个外显子，Glce基因表达蛋白有3个转录本，转录本1具有3个外显子、2个内含子，转录本2具有5个外显子、4个内含子，转录本3具有2个外显子、1个内含子。这些序列信息可参见文献或EnsemblGene、Genebank等公共数据库。
人类等其他物种的Glce基因也可参见文献或EnsemblGene、Genebank等公共数据库。
应理解，术语“Glce”还包括各种天然存在的Glce基因的变异形式。代表性的例子包括：因密码子的简并性而编码与野生型相同的Glce蛋白的核苷酸序列，编码野生型Glce蛋白的保守性变异多肽的核苷酸序列。此外，对于小鼠之外的其他哺乳动物时，该术语指Glce基因在该哺乳动物中的同系物。例如对于人而言，该术语指人的Glce(已知小鼠Glce基因与人类Glce基因的cDNA同源度为91.4％，氨基酸序列的同源度为97.4％)。
Glce基因或其蛋白的失活剂
在本发明中，所述Glce的失活剂包括全部失活或部分失活。
本发明的Glce蛋白的失活剂包括(a)抑制剂，所述抑制剂的例子包括(但并不限于)：小分子化合物、抗体、反义核酸、miRNA、siRNA、或其组合；(b)Glce基因的敲除剂。
神经精神疾病及新生神经元发生(neurogenesis)减少相关疾病
神经精神疾病是一类由于神经系统失调导致的神经发育障碍性疾病，或者更准确地说是大脑神经环路的病理改变，包括孤独症谱系障碍、精神分裂症、抑郁症、焦虑症、恐惧症、癫痫等。神经元发育异常、突触连接修饰不当以及神经环路的错误连接等神经发育障碍均会导致严重的神经精神疾病发生。抑郁症是一种典型的神经精神疾病。研究表明7-10，神经元的生长异常、树突发育异常导致抑郁症的发生。
近年的研究表明，海马是与学习、记忆以及情绪情感调控密切相关的脑区，从功能上看，海马齿状回成年新生神经元发生对维持神经元网络的可塑性具有重要的作用，同时，海马齿状回也是老年痴呆症早期阶段最容易受损的脑区，在精神分裂症与抑郁症等精神疾病病人中也常伴有海马功能的异常。越来越多的证据提示海马齿状回区成年新生神经元发生(adult neurogenesis)的减少可能是精神分裂症与抑郁症等精神疾病及老年痴呆症发病的重要原因之一11。
在本发明中，成年新生神经元发生减少相关疾病包括但不限于精神疾病如精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍，神经退行性疾病如老年痴呆症、帕金森症、中风等，优选地，包括精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和/或老年痴呆症。
基因失活
对于功能未知基因的研究可采用许多方法，例如使有待研究的基因失活，分析所得的遗传修饰的表型变化，进而获得该基因的功能信息。这一研究方法的另一优点是可以将基因功能和疾病进行关联，从而在获得基因功能的同时也能获得该基因作为潜在药物或者药物靶点所能治疗的疾病信息和疾病动物模型。基因失活的方法可通过基因剔除、基因中断或基因插入的方式来完成。其中，基因剔除技术是研究人类基因在整体中的功能的非常强有力的手段。
动物模型
在本发明中，提供了一种非常有效的神经精神疾病的非人哺乳动物模型。
在本发明中，非人哺乳动物的例子包括(但并不限于)：小鼠、大鼠、兔、猴等，更佳地是大鼠和小鼠。
如本文所用，术语“Glce基因失活”包括一个或两个Glce基因被失活的情况，即包括Glce基因杂合地和纯合地失活。例如，Glce基因失活的小鼠可以是杂合或纯合的小鼠。
在本发明中，可基因剔除或转入外源基因(或片段)而使Glce基因失活等方法制备Glce基因失活的非人哺乳动物(如小鼠)。在本领域中，通过基因剔除或转入外源基因而使靶基因失活的技术是已知的，这些常规技术都可用于本发明。
在本发明的另一优选例中，Glce基因的失活是通过基因剔除实现的。
在本发明的另一优选例中，Glce基因的失活是通过Glce基因中插入外源基因(或片段)而实现的。
在本发明的一具体实例中，可构建一含有外源插入片段的构建物，该构建物含有与靶基因(Glce)的插入位点的两侧的侧翼序列同源的同源臂，从而可以通过同源重组高频地将外源插入片段(或基因)插入至Glce基因组序列(尤其是外显子 区域)，造成小鼠Glce基因的移码、提前终止、或敲除，从而导致Glce基因缺失或失活。
用本发明方法获得的纯合或杂合的小鼠可育。失活的Glce基因可以孟德尔规律遗传给后代小鼠。
在一优选例中，本发明提供了一种缺失Glce基因的纯合小鼠模型动物。
候选药物或治疗剂
在本发明中，还提供了一种利用本发明的动物模型，筛选治疗神经精神疾病的候选药物或治疗剂的方法。
在本发明中，候选药物或治疗剂是指已知具有某种药理学活性或正在被检测的可能具有某种药理学活性的物质，包括但不限于核酸、蛋白、糖类、化学合成的小分子或大分子化合物、细胞等。候选药物或治疗剂的给药方式可以是口服、静脉注射、腹腔注射、皮下注射、椎管给药或直接脑内注射。
本发明的主要优点包括：
(1)本发明可以较好地模拟临床上原发性的抑郁症。
(2)个体之间不存在手术操作或者药物剂量导致的差异，其遗传背景高度一致。
(3)可以作为研究抑郁症的发病机理和新药筛选的有力工具。
(4)本发明神经精神疾病模型的遗传稳定、表型稳定。
(5)用本发明方法获得的纯合或杂合的动物模型可育。转基因杂合小鼠具有生殖能力，失活的Glce基因可以孟德尔规律遗传给后代小鼠。
(6)本发明的神经精神疾病动物模型表现出多种神经和精神疾病样的症状，因此可以广泛用于神经精神类疾病的药物筛选和测试，包括精神分裂症、躁郁症、抑郁症、焦虑症、恐惧症、孤独症谱系障碍、中风和老年痴呆症等。
(7)本发明首次揭示了剔除或失活Glce基因所得到的动物模型还可以同时用于研究中风、肥胖、焦虑等疾病。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，实施例所用的材料均为市售产品。
实施例1获得携带Cre重组酶的Glce基因突变纯合子小鼠
首先构建Glce突变基因序列(图1)。如图1所示设计Glce基因剔除打靶载体序列，将Loxp/Loxp等位基因插入到Glce基因3号外显子两侧，在3’端插入neo基因，5’端臂3125bp，3’端臂3718bp。图2为Glce基因剔除打靶载体质粒图谱。1.获得目的基因(Glce)的同源片段，将此DNA片段克隆到质粒载体中；2.从重组质粒中切除目的基因的大部分同源DNA序列，只留部分序列在线性质粒载体的两端；3.将neo基因克隆到带有目的基因同源顺序的线性质粒中，使之位于残留目的基因同源顺序的中间；4.在目的基因同源顺序的外侧线性化重组质粒载体，将hsv－tk基因克隆到此线性载体中。
基因位点如下表所示：
Figure PCTCN2017098535-appb-000001
注：基因位置标注号根据“10kb Up and Down of Glce gene”。
图3为Glce基因剔除打靶载体质粒DNA的酶切鉴定，使用1Kb DNA ladder。对打靶载体进行线性化：100μg Glce-CKO质粒DNA(购自Biovector NTCC)用NotI(酶用量：150U)线性化，酶切体系为150μl，37℃消化过夜，等体积酚氯仿、氯仿处理后，无水乙醇沉淀，100μl无菌PBS重悬备用。ES细胞打靶：ES细胞SCR012来源于129SV/EV品系雄性小鼠(购自中科院上海实验动物中心)的胚胎干细胞，线性化DNA量：35μg，电转仪型号：Bio－Rad Gene Pulser(Cat.No.165-2105)，电穿孔条件：电压240v，电容500μF,实际通电时间10.5ms，实际电压256v，克隆筛选条件：300μg/ml G418和2μM GanC筛选8天。挑取抗性克隆和提供DNA样本共96份。
阳性ES细胞基因组鉴定方法：
1.5’arm PCR鉴定
P1引物位于5’arm外，P2引物位于neo重组区域，距离臂外引物8.2kb。
P1和P2引物序列:
P1:GGCATTTGCACTCACATACACAACCCA(gene site:15824-15850)(SEQ ID NO.:1)
P2:GTGCCACTCCCACTGTCCTTTCC(SEQ ID NO.:2)
PCR反应体系(ul)：
Figure PCTCN2017098535-appb-000002
PCR反应条件：
Figure PCTCN2017098535-appb-000003
PCR仪：Eppendorf AG 22331 Hamburg
试剂：TaKaRa La Taq宝生物工程(大连)有限公司(Cat:DRR002B)
分子量Marker:MBI GeneRuler 1kb DNA Ladder(晶美生物，Cat：SM0311)
2.3’arm PCR鉴定
P4引物位于3’arm外，P3引物位于neo重组区域，距离臂外引物4.7kb。P4和P3引物序列:
P4:GAGAGGCTTGGAGGCGGTGCTGATCTT(gene site:29603-29629)(SEQ ID NO.:3)
P3:GATATACTATGCCGATGATTAATTGTC(SEQ ID NO.:4)
PCR反应体系(ul)：
Figure PCTCN2017098535-appb-000004
PCR反应条件：
Figure PCTCN2017098535-appb-000005
PCR仪：Eppendorf AG 22331 Hamburg
试剂：TaKaRa La Taq宝生物工程(大连)有限公司(Cat:DRR002B)
分子量Marker:MBI GeneRuler 1kb DNA Ladder(晶美生物，Cat：SM0311)
ES细胞克隆鉴定结果PCR鉴定药物抗性ES细胞克隆96个，其中19个ES克隆发生双臂同源重组。PCR产物经DNA序列测定进一步证实。图4显示了ES克隆基因组DNA的5’端PCR产物的电泳结果。图5显示了ES克隆基因组DNA的3’端PCR产物的电泳结果。
阳性ES克隆囊胚注射：
显微注射用囊胚来源：C57BL/6J小鼠(购自上海斯莱克实验动物有限公司)超数排卵,自然受孕体内发育至囊胚阶段。注射60枚胚胎，注射后移植入3只假孕小鼠受体子宫，受体为C57BL/6J(♂)与CBA(♀)(购自上海斯莱克实验动物有限公司)的杂交一代。从出生的小鼠中挑选嵌合率大于50％的小鼠饲养至成年，与C57BL/6J雌性小鼠进行交配，后代灰色小鼠经提取尾基因组DNA进行PCR鉴定(鉴定策略同上)，结果如图6所示，获得双臂阳性F1代小鼠(Loxp/+)。
将F1代小鼠饲养至成年，与NSE-Cre工具鼠(购自上海斯莱克实验动物有 限公司)杂交，获得F2代Loxp/+Cre小鼠。将F2代小鼠饲养至成年，F2代内雌雄相互交配，按照孟德尔规律遗传，获得F3代突变纯合子(Loxp/Loxp Cre)：突变杂合子(Loxp/+Cre)：Wild type(+/+Cre)的比例约为1：2：1。经提取尾基因组DNA进行PCR鉴定(鉴定策略同上)，结果如图7所示。将突变纯合子小鼠(Loxp/Loxp Cre)用于后续动物行为学实验。
实施例2Glce突变纯合子小鼠的抑郁行为学分析
2.1自主活动
将携带Cre重组酶的Glce基因突变纯合子小鼠放置于黑暗实验箱内(110mm X 110mm X 330mm)，测试小鼠的自发活动5min。通过红外摄像拍摄小鼠的活动情况。使用上海吉量软件科技有限公司的视频跟踪软件和分析软件对小鼠活动轨迹以及活动时间进行分析。
研究显示，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠的自发活动的总路程、活动时间以及活动速度并没有显著差异(图8)。
结果表明，青年期Glce突变纯合子小鼠天生的习性与C57BL/6正常小鼠相比较并没有显著差异。
2.2旷场实验
将小鼠放置于明亮空旷实验箱内(500mm X 500mm X 590mm)，测试小鼠的探索活动5min。通过红外摄像拍摄小鼠的活动情况。使用上海吉量软件科技有限公司的视频跟踪软件和分析软件对小鼠活动轨迹以及活动时间进行分析。
结果显示，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在旷场实验中活动的总路程、活动时间以及活动速度均显著减小(图9)。结果表明，青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较活动显著减少。
与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在旷场中央区域活动的路程、活动时间以及活动速度均显著减小(图10)，结果表明，青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较探索新异环境的活动显著减少。
与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在旷场周 边区域活动的路程、活动时间以及活动速度均显著减小(图11)，结果表明青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较活动显著减少。
总的来说，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠的活动显著减少，并且探索新异环境的活动显著减少，表明青年期Glce突变纯合子小鼠有一定程度的抑郁。
2.3强迫游泳
将小鼠放置于直径12cm、高度25cm的水缸中(水温21-22℃)，小鼠被强迫在水温较低的水中游泳，测试小鼠的活动6min，记录小鼠在后4min中的不动时间。通过摄像拍摄小鼠的活动情况。对小鼠活动时间进行分析。研究发现，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在强迫游泳实验中的不动时间显著增加(图12)。
结果表明，青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较有一定程度的抑郁。雌性青年期Glce突变纯合子小鼠与雄性青年期Glce突变纯合子小鼠相比较无显著差异。除此之外，产生一定程度抑郁的青年期Glce突变纯合子小鼠同时也产生一定程度的焦虑。
实施例3Glce突变纯合子小鼠的形态学分析
Glce突变纯合子小鼠的出生比例符合孟德尔定律，通过检测小鼠的体重，结果如图13所示。结果表明，与成年C57BL/6正常小鼠相比较，成年Glce神经元特异敲除小鼠的形态更加肥胖。
实施例4Glce突变纯合子小鼠的体重分析
将Glce突变纯合子小鼠置于电子天平上称重，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的体重差异。
研究发现(图14)，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠肥胖症状的发生率更高，并且体重与成年C57BL/6正常小鼠相比较具有统计学显著差异。
结果显示，以超过成年C57BL/6正常小鼠体重平均值的20％以上的标准认为 是肥胖，雌性成年C57BL/6正常小鼠的体重平均为28.7g，雌性成年Glce突变纯合子小鼠的体重平均为34.6g，雌性成年Glce突变纯合子小鼠肥胖症状的发生率约为50％，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的体重平均为32.2g，雄性成年Glce突变纯合子小鼠的体重平均为39.5g，雄性成年Glce突变纯合子小鼠肥胖症状的发生率约为66％，与雄性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的体重平均为30.7g，成年Glce突变纯合子小鼠(包含雌雄)的体重平均为37.8g，成年Glce突变纯合子小鼠肥胖症状的发生率总计约为60％，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠肥胖症状的发生率更高。
实施例5Glce突变纯合子小鼠的体脂分析
将Glce突变纯合子小鼠解剖后取脂肪组织包括双侧性腺周围脂肪组织、双侧肾周脂肪组织、双侧腹股沟脂肪组织置于电子天平上称重，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的脂肪组织湿重差异。
研究发现(图15)，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的体脂湿重增加，具有统计学显著差异。
结果显示，雌性成年C57BL/6正常小鼠的体脂湿重平均为0.94g，雌性成年Glce突变纯合子小鼠的体脂湿重平均为8.50g，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的体脂湿重平均为2.98g，雄性成年Glce突变纯合子小鼠的体脂湿重平均为7.52g，与雄性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的体脂湿重平均为2.21g，成年Glce突变纯合子小鼠(包含雌雄)的体脂湿重平均为8.01g，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的体脂湿重增加。
实施例6Glce突变纯合子小鼠的脂肪组织分析
6.1Glce突变纯合子小鼠的双侧性腺周围脂肪组织分析
将Glce突变纯合子小鼠解剖后取双侧性腺周围脂肪组织置于电子天平上称重，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的双侧性腺周围脂肪组织湿重差异。
与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧性腺周围脂肪组织湿重显著增加(图16)。
结果显示，雌性成年C57BL/6正常小鼠的双侧性腺周围脂肪组织湿重平均为0.54g，雌性成年Glce突变纯合子小鼠的双侧性腺周围脂肪组织湿重平均为3.10g，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的双侧性腺周围脂肪组织湿重平均为1.66g，雄性成年Glce突变纯合子小鼠的双侧性腺周围脂肪组织湿重平均为2.85g，与雄性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的双侧性腺周围脂肪组织湿重平均为1.24g，成年Glce突变纯合子小鼠(包含雌雄)的双侧性腺周围脂肪组织湿重平均为2.98g，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧性腺周围脂肪组织湿重显著增加。
6.2Glce突变纯合子小鼠的双侧肾周脂肪组织分析
将Glce突变纯合子小鼠解剖后取双侧肾周脂肪组织置于电子天平上称重，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的双侧肾周脂肪组织湿重差异。
与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧肾周脂肪组织湿重显著增加(图17)。
结果显示，雌性成年C57BL/6正常小鼠的双侧肾周脂肪组织湿重平均为0.36g，雌性成年Glce突变纯合子小鼠的双侧肾周脂肪组织湿重平均为2.06g，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的双侧肾周脂肪组织湿重平均为1.18g，雄性成年Glce突变纯合子小鼠的双侧肾周脂肪组织湿重平均为1.61g，与雄性成年C57BL/6正常小鼠相比较具有 统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的双侧肾周脂肪组织湿重平均为0.87g，成年Glce突变纯合子小鼠(包含雌雄)的双侧肾周脂肪组织湿重平均为1.83g，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧肾周脂肪组织湿重显著增加。
6.3Glce突变纯合子小鼠的内脏脂肪组织分析
将Glce突变纯合子小鼠解剖后取双侧性腺周围脂肪组织和双侧肾周脂肪组织置于电子天平上称重，内脏脂肪组织湿重总重为双侧性腺周围脂肪组织和双侧肾周脂肪组织湿重之和，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的内脏脂肪组织湿重差异。
与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的内脏脂肪组织湿重显著增加(图18)。
结果显示，雌性成年C57BL/6正常小鼠的内脏脂肪组织湿重平均为0.90g，雌性成年Glce突变纯合子小鼠的内脏脂肪组织湿重平均为5.16g，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的内脏脂肪组织湿重平均为2.84g，雄性成年Glce突变纯合子小鼠的内脏脂肪组织湿重平均为4.45g，与雄性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的内脏脂肪组织湿重平均为2.11g，成年Glce突变纯合子小鼠(包含雌雄)的内脏脂肪组织湿重平均为4.81g，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的内脏脂肪组织湿重显著增加。
6.4Glce突变纯合子小鼠的皮下脂肪组织(如双侧腹股沟脂肪组织)分析
将Glce突变纯合子小鼠解剖后取双侧腹股沟脂肪组织置于电子天平上称重，皮下脂肪组织湿重为双侧腹股沟脂肪组织湿重之和，研究成年Glce突变纯合子小鼠与成年C57BL/6正常小鼠的皮下脂肪组织湿重差异。
与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧腹股沟 脂肪组织(代表皮下脂肪组织)湿重显著增加(图19)。
结果显示，雌性成年C57BL/6正常小鼠的双侧腹股沟脂肪组织湿重平均为0.04g，雌性成年Glce突变纯合子小鼠的双侧腹股沟脂肪组织湿重平均为3.34g，与雌性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；雄性成年C57BL/6正常小鼠的双侧腹股沟脂肪组织湿重平均为0.13g，雄性成年Glce突变纯合子小鼠的双侧腹股沟脂肪组织湿重平均为3.07g，与雄性成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)；成年C57BL/6正常小鼠(包含雌雄)的双侧腹股沟脂肪组织湿重平均为0.10g，成年Glce突变纯合子小鼠(包含雌雄)的双侧腹股沟脂肪组织湿重平均为3.20g，与成年C57BL/6正常小鼠相比较具有统计学显著差异(P<0.01)。
上述结果充分表明，与成年C57BL/6正常小鼠相比较，成年Glce突变纯合子小鼠的双侧腹股沟脂肪组织(代表皮下脂肪组织)湿重显著增加。
实施例7Glce突变纯合子小鼠的中风行为学分析
将带Cre重组酶的Glce基因突变纯合子小鼠置于清洁饲养环境饲养。该小鼠动物模型在进入老年期(约1.5年)发生中风样症状。通过一系列中风行为学实验评价小鼠动物模型的中风样症状及严重程度，实验方法参考2001年发表于Stroke上的评价大鼠中风症状严重程度的一系列系统的行为学实验(Chen J,et al.Stroke.2001)，包括运动测试、感觉测试、平衡能力测试、本体的反射测试、异常的活动能力测试等，具体为平放测试、提尾测试、视觉触觉测试、本体觉测试、平衡木测试、耳廓反射、睑闭反射、惊跳反射、抽搐、痉挛、肌张力障碍等行为测试，对小鼠的行为进行评分。
Glce基因突变纯合子小鼠组40只有10只在老年时期发生中风，中风发生率为25％；对照组C57BL/6小鼠组40只小鼠有2只在老年时期发生中风，中风发生率为5％，结果表明，Glce基因突变纯合子小鼠与C57BL/6小鼠相比较更易发生中风。
7.1运动测试。
(1)平放测试：将小鼠放置于平地上，观察小鼠是否能够正常行走，正常行走得分0分；是否能够直行，不能直行得分1分，否则得分0分；是否围绕损伤侧转圈，围绕损伤侧转圈得分1分，否则得分0分；是否向损伤侧倾倒， 向损伤侧倾倒得分1分，否则得分0分。
(2)提尾测试：提起小鼠的尾巴，观察小鼠肢体活动的情况，是否前肢向内弯曲、爪子抓紧，若小鼠前肢向内弯曲、爪子抓紧得分1分，否则得分0分；是否后肢向内弯曲、爪子抓紧，若小鼠后肢向内弯曲、爪子抓紧得分1分，否则得分0分；是否30秒内头部抬起、与垂直轴角度大于10度，若小鼠30秒内头部抬起、与垂直轴角度大于10度得分1分，否则得分0分。
7.2感觉测试。
(1)视觉触觉测试：把握住小鼠身体，让小鼠前肢能够自由活动，使小鼠面向台子边缘或者笼子边缘，将小鼠快速靠近台子边缘或者笼子边缘，观察小鼠前肢是否能够快速向前伸、并且及时张开爪子准确抓住台子边缘或者笼子边缘。若小鼠前肢不能快速向前伸、并且及时张开爪子准确抓住台子边缘或者笼子边缘得分1分，否则得分0分。
(2)本体觉测试：把握住小鼠身体，让小鼠后肢能够自由活动，将小鼠前肢放置于台子边缘或者笼子边缘、后肢悬空，使用镊子夹小鼠后肢大腿肌肉，观察小鼠后肢是否能够快速回缩。若小鼠后肢不能快速回缩得分1分，否则得分0分。
7.3平衡能力测试。
将小鼠放置于平衡木一端，观察小鼠在平衡木上自由活动的情况，主要包括以下7种情况：(1)是否能够保持身体平衡、在平衡木上自由行走，若小鼠能够保持身体平衡、在平衡木上自由行走得分0分。
(2)是否抓住平衡木边缘，若小鼠抓住平衡木边缘得分1分。
(3)抱住平衡木，但是有一后肢掉落，若小鼠抱住平衡木，但是有一后肢掉落得分2分。
(4)抱住平衡木，但是有两后肢掉落，或者在平衡木上旋转，并且在平衡木上的时间大于60秒，若小鼠抱住平衡木，但是有两后肢掉落，或者在平衡木上旋转，并且在平衡木上的时间大于60秒得分3分。
(5)尝试在平衡木上保持平衡但是最终掉落，在平衡木上的时间大于40秒，若小鼠尝试在平衡木上保持平衡但是最终掉落，在平衡木上的时间大于40秒得分4分。
(6)尝试在平衡木上保持平衡但是最终掉落，在平衡木上的时间大于20秒，若小鼠尝试在平衡木上保持平衡但是最终掉落，在平衡木上的时间大于20秒得分5分。
(7)没有尝试在平衡木上保持平衡或者抱紧平衡木的欲望，在平衡木上的时间小于20秒掉落，若小鼠没有尝试在平衡木上保持平衡或者抱紧平衡木的欲望，在平衡木上的时间小于20秒掉落得分6分。
7.4本体的反射测试及异常的活动能力测试。
(1)耳廓反射：使用棉签刺激小鼠耳道，观察小鼠是否有甩头反应，若有说明小鼠具有耳廓反射，若无说明小鼠没有耳廓反射、得分1分。
(2)睑闭反射：使用棉签刺激小鼠虹膜，观察小鼠是否有闭上眼睑的反应，若有说明小鼠具有睑闭反射，若无说明小鼠没有睑闭反射、得分1分。
(3)惊跳反射：制造一个大的噪声，比如水瓶掉落，观察小鼠是否有受惊吓跳起来的反应，若有说明小鼠具有惊跳反射，若无说明小鼠没有惊跳反射、得分1分。
(4)观察小鼠是否出现抽搐、痉挛、肌张力障碍等现象。若小鼠出现抽搐、痉挛、肌张力障碍等现象，得分1分。
计算每一项相加的总得分，总得分1-6分为轻度损伤，总得分7-12分为中度损伤，总得分13-18分为重度损伤。
例如，图20中的小鼠在平放测试中不能直行得分1分，围绕损伤侧转圈得分1分，向损伤侧倾倒得分1分；在提尾测试中前肢向内弯曲、爪子抓紧得分1分，后肢向内弯曲、爪子抓紧得分1分，30秒内头部抬起、与垂直轴角度大于10度得分1分；在视觉触觉测试中前肢能快速向前伸、并且及时张开爪子准确抓住台子边缘或者笼子边缘得分0分；在本体觉测试中后肢能快速回缩得分0分；在平衡木测试中小鼠尝试在平衡木上保持平衡但是最终掉落，在平衡木上的时间大于40秒得分4分；小鼠具有耳廓反射得分0分；具有睑闭反射得分0分；具有惊跳反射得分0分；小鼠出现抽搐得分1分。该小鼠的总得分为：1+1+1+1+1+1+0+0+4+0+0+0+1＝11分，评价为中度中风。
实施例8Glce突变纯合子小鼠的脑组织梗塞分析
通过TTC(2，3，5—氯化三苯基四氮唑)染色方法检测中风小鼠脑组织的 梗塞情况。操作步骤是：麻醉后直接取脑，-20度冰箱中速冻5-10分钟左右，便于切片。切片：每隔1mm切一片。将切片置于TTC中，常规浓度为2％。用锡箔纸盖住后，放入37度温箱15-30min，不时翻动脑片，使脑片均匀接触到染色液。然后使用4％多聚甲醛固定30min。拍照。
TTC是脂溶性光敏感复合物，可用来染色检测哺乳动物组织的缺血梗塞。它是呼吸链中吡啶-核苷结构酶系统的质子受体，与正常组织中的脱氢酶反应而呈红色，而缺血组织内脱氢酶活性下降，不能反应，故不会产生变化呈苍白。
结果如图21所示，结果显示，经过TTC染色之后，中风小鼠的正常脑组织呈红色，而梗塞的脑组织呈苍白。观察到嗅球、前额叶、胼胝体、海马组织、纹状体、杏仁核、下丘脑、颞叶、小脑、脑桥、延髓等均有梗塞现象。
实施例9Glce突变纯合子小鼠的焦虑行为学分析
将携带Cre重组酶的Glce基因突变纯合子小鼠(以下简称Glce突变纯合子小鼠)置于清洁饲养环境饲养。在小鼠青年期(>3月龄)通过一系列动物行为学实验包括自主活动、旷场实验、高架十字迷宫等实验评价小鼠动物模型的焦虑症样症状。
9.1自主活动
将小鼠放置于黑暗实验箱内(110mm X 110mm X 330mm)，测试小鼠的自发活动5min。通过红外摄像拍摄小鼠的活动情况。使用上海吉量软件科技有限公司的视频跟踪软件和分析软件对小鼠活动轨迹以及活动时间进行分析。
研究发现，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠的自发活动的总路程、活动时间以及活动速度并没有显著差异(图22)。
结果表明，青年期Glce突变纯合子小鼠天生的习性与C57BL/6正常小鼠相比较并没有显著差异。
9.2旷场实验
将小鼠放置于明亮空旷实验箱内(500mm X 500mm X 590mm)，测试小鼠的探索活动5min。通过红外摄像拍摄小鼠的活动情况。使用上海吉量软件科技有限公司的视频跟踪软件和分析软件对小鼠活动轨迹以及活动时间进行分析。
研究发现，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在旷场中央区域活动的路程、活动时间以及活动速度均显著减小(图23)。
结果表明，青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较探索 新异环境的活动显著减少，并产生更强烈的焦虑情绪。
9.3高架十字迷宫实验
将小鼠放置于开放臂单臂长30cm、宽6cm、封闭臂单臂长30cm、宽6cm、高14.5cm的高架十字迷宫中，离地面高度约50cm，通过摄像拍摄小鼠5min内在高架十字迷宫中的活动情况。对小鼠的活动进行分析发现，与青年期C57BL/6正常小鼠相比较，青年期Glce突变纯合子小鼠在高架十字迷宫实验中探究开放臂的时间显著减少，停留在封闭臂的时间显著增加，在中央区域停留的时间没有显著差异(图24)。
结果表明，青年期Glce突变纯合子小鼠与C57BL/6正常小鼠相比较，焦虑程度明显增加。雄性青年期Glce突变纯合子小鼠与雌性青年期Glce突变纯合子小鼠相比较焦虑情绪更明显。除此之外，产生一定程度焦虑的青年期Glce突变纯合子小鼠同时也产生一定程度的抑郁。
实施例10用治疗神经精神疾病(如抑郁症)的药物验证药物筛选平台
在本实施例中，给实施例1构建的模型动物小鼠注射当前临床治疗神经精神疾病的药物氟西汀或知母皂苷，随即对模型动物小鼠在自发活动、旷场实验、强迫游泳实验中的行为学指标进行评估。
结果表明，药物氟西汀或知母皂苷增加模型动物小鼠的自发活动，增加在旷场实验中探索中央区域的时间，减少在强迫游泳实验中的不动时间，说明氟西汀或知母皂苷能够减轻抑郁。
实施例11利用治疗神经精神疾病(如抑郁症)药物筛选平台筛选候选药物
在本实施例中，计划通过给实施例1构建的模型动物小鼠注射神经精神疾病的治疗药物，随即对模型动物小鼠在自发活动、旷场实验、强迫游泳实验中的行为学指标进行评估。
通过与给安慰剂的模型动物小鼠比较自发活动、旷场实验、强迫游泳实验中的行为学指标的差异，能够改善行为学指标的候选药物，即为该神经精神疾病的潜在治疗药物。
其它神经精神类疾病也可以参照上述方法，采用相应的行为学指标进行候选药物筛选。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献：
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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.
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Claims (10)

  1. 一种非人哺乳动物的神经精神疾病动物模型的制备方法，其特征在于，包括以下步骤：
    (a)提供非人哺乳动物的细胞，将所述细胞中的葡萄糖醛酸C5异构酶(Glucuronyl C5-epimerase,Glce)基因失活，得到Glce基因失活的非人哺乳动物细胞；
    (b)利用步骤(a)中得到的Glce基因失活的细胞，制备得到Glce基因失活的神经精神疾病动物模型。
  2. 如权利要求1所述的方法，其特征在于，在步骤(a)中，还包括如下步骤：
    (a1)利用DNA同源重组技术，将所述Glce基因中的外显子3至外显子5中一个或多个外显子剔除或中断，并用筛选标记替换，得到Glce基因失活的非人哺乳动物细胞。
  3. 如权利要求1所述的方法，其特征在于，在步骤(b)中，还包括如下步骤：
    (b1)利用步骤(a)中得到的Glce基因失活的非人哺乳动物细胞制备得到嵌合非人哺乳动物；
    (b2)将步骤(b1)中得到的嵌合非人哺乳动物和正常野生型非人哺乳动物交配繁育，在后代中筛选获得Glce基因失活的杂合子非人哺乳动物；
    (b3)通过将步骤(b2)中得到的杂合子非人哺乳动物相互交配获得Glce基因失活的纯合子非人哺乳动物，从而得到Glce基因失活的非人哺乳动物的动物模型。
  4. 如权利要求3所述的方法，其特征在于，在步骤(b3)中，还包括步骤(b4)：将Glce基因失活的纯合子非人哺乳动物与同一物种的神经元特异性敲除工具非人哺乳动物进行杂交，从而获得神经元特异性的Glce基因失活的非人哺乳动物的动物模型。
  5. 如权利要求1所述的方法，其特征在于，所述步骤(b)中得到的Glce基因失活的非人哺乳动物的动物模型中，与野生型对照动物相比，具有选自下组的一个或多个特征：
    (t1)旷场活动水平降低；
    (t2)探索新异环境的欲望降低；
    (t3)抑郁样行为增加；
    (t4)抑郁程度增加；
    (t5)焦虑样行为增加；
    (t6)焦虑程度增加；
    (t7)恐惧样行为增加；
    (t8)恐惧程度增加；
    (t9)认知障碍增加；
    (t10)中风的发生率增加；
    (t11)形态的肥胖程度增加；
    (t12)肥胖症状的发生率增加；
    (t13)脂肪组织湿重增加。
  6. 一种权利要求1所述方法制备的非人哺乳动物模型的用途，其特征在于，将该模型用作研究神经精神疾病的动物模型。
  7. 一种权利要求1所述方法制备的非人哺乳动物模型的用途，其中，将该模型用于筛选或鉴定可减轻或治疗神经精神疾病的物质(治疗剂)。
  8. 一种筛选或鉴定治疗或缓解神经精神疾病的潜在治疗剂的方法，其特征在于，包括以下步骤：
    (a)在测试组中，在测试化合物的存在下，将测试化合物施用于权利要求1所述方法制备的非人哺乳动物模型，对测试组的所述动物模型的行为进行行为学分析；并且在不施用所述测试化合物且其他条件相同的对照组中，对对照组的所述动物模型的行为进行行为学分析；
    (b)对测试组和对照组动物模型的行为进行比较，其中，与对照组相比，如果施用了测试化合物的动物模型中表征神经精神疾病行为得到改善，则表明该测试化合物可作为神经精神疾病的潜在治疗剂。
  9. 一种细胞的用途，其特征在于，所述细胞中的葡萄糖醛酸C5异构酶(Glucuronyl C5-epimerase,Glce)基因失活或下调，用于制备构建非人哺乳动物的神经精神疾病动物模型的生物制剂。
  10. 一种Glce基因或其蛋白的失活剂的用途，其特征在于，用于制备构建非人哺乳动物的神经精神疾病动物模型的制剂。
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