CN116098123A - Application of Per2 gene in preparing diphasic disorder animal model - Google Patents

Application of Per2 gene in preparing diphasic disorder animal model Download PDF

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CN116098123A
CN116098123A CN202211437055.XA CN202211437055A CN116098123A CN 116098123 A CN116098123 A CN 116098123A CN 202211437055 A CN202211437055 A CN 202211437055A CN 116098123 A CN116098123 A CN 116098123A
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per2
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bipolar disorder
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王新玲
陈炜
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Shandong University
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Abstract

The invention belongs to the field of genes and application thereof, and relates to application of Per2 genes in preparing a bipolar disorder animal model, wherein the Per2 genes belong to clock genes and are also called periodical genes, and the inventor utilizes related viral vectors to knock down and overexpress the Per2 genes respectively, so that the inventor finds that the dual functions of depression and mania emotion are influenced by regulating and controlling the expression level of the Per2 genes, and then the mania-like behavior phenotype and the depression-like behavior phenotype of rats can be induced, so that a corresponding animal model is obtained, the limitation that the traditional bipolar disorder animal model can only simulate one phenotype is broken, and the model is an ideal model for respectively researching two different phenotypes of the bipolar disorder, and can be widely applied to pathogenesis of the bipolar disorder and research and development of new drugs.

Description

Application of Per2 gene in preparing diphasic disorder animal model
Technical Field
The invention belongs to the field of genes and application thereof, and relates to application of a Per2 gene in preparing a bipolar disorder animal model.
Background
Bipolar disorder is a severe mood disorder, often misdiagnosed as depression, due to the alternating occurrence of two symptom groups, manic or hypomanic and depressive, in the clinical manifestations. The bottleneck existing in the basic research field of the current diphasic disorder is as follows: that is, the existing internationally recognized animal models of bipolar disorder (including the sodium-potassium-atpase inhibitor ouabain model, amphetamine model, mitochondrial DNA accumulation mutation model, etc.) can only simulate the manic state of bipolar disorder, and cannot fully simulate the manic/depressive two phenotypes of the disease. The animal model limited by the bipolar disorder can not simulate mania/depression by one mechanism, and severely restricts the research of the pathogenesis of the bipolar disorder and the development of new therapeutic drugs. Therefore, the invention of the two phenotype-switched bipolar disorder animal model has a great pushing effect on the pathogenesis of bipolar disorder and the development of new drugs.
The clock gene Period (Per) is also called a Period gene, and belongs to one of core clock genes. Including three subtypes Per1, per2 and Per 3. Among them, there are many reports of Per1 and Per2 genes involved in affective disorders. Clinical studies have found that peripheral blood Per2 gene level reduction in bipolar disorder patients is associated with manic symptoms and can be reversed by lithium salt treatment. However, whether the Per2 gene is involved in the depressive episode of bipolar disorder patients has not been reported yet. Therefore, the relation between the gene and the bipolar disorder can be revealed, and further, the bipolar disorder animal model is obtained, a better platform is provided for the treatment and research of the bipolar disorder, and the method becomes one of the important scientific problems to be solved in the field.
Disclosure of Invention
Aiming at the situation in the prior art, the inventor provides application of Per2 gene in preparing a bipolar disorder animal model, wherein the Per2 gene belongs to a clock gene, which is also called a periodic gene, and the inventor uses related viral vectors to knock down and overexpress the Per2 gene respectively, and finds that the dual effects of depression and mania emotion are influenced by regulating and controlling the expression level of the Per2 gene, so that a mania-like behavior phenotype and a depression-like behavior phenotype of a rat can be induced, and a corresponding animal model is obtained, the limitation that the prior bipolar disorder animal model can only simulate one phenotype is broken, and the model is an ideal model for respectively researching two different phenotypes of the bipolar disorder, and can be widely applied to development of a pathogenesis of the bipolar disorder and a new drug.
The specific principle of the invention is as follows:
the inventors have found for the first time that the Per2 gene is also involved in the depressive episode of bipolar disorder, namely: per2 gene mediates both mania and depression-the occurrence of both phenotypes. Based on the role of Per2 gene in mania and depression occurrence, the inventor applies the gene to the preparation of animal models, and finally obtains new animal models of bipolar disorder: knocking down the expression of the hippocampal CA1 brain region Per2 gene by adopting adeno-associated virus (AAV-shPer 2) can induce the manic behavior phenotype of rats; overexpression of the hippocampal CA1 brain region Per2 gene by lentivirus (LV-Per 2) induces a rat depressive-like behavioral phenotype. The inventor utilizes knock-down and over-expression technology to regulate the expression level of Per2 gene, can more completely present two opposite phenotypes of the bipolar disorder, and the process only involves the intervention of one gene, thereby avoiding the interference of two factors, better embodying the pathophysiology process of a disease and breaking through the difficult problem of an international animal model of the bipolar disorder.
The specific technical scheme of the invention is as follows:
the application of Per2 gene in preparing animal model with diphasic disorder includes the following steps:
respectively constructing adeno-associated virus vector AAV-shPer2 with knocked-down Per2 gene expression, wherein the nucleotide sequence is shown as SEQ ID NO.1, and constructing lentiviral vector LV-Per2 with over-expressed Per2 gene, and the nucleotide sequence is shown as SEQ ID NO. 2.
After the virus vector is obtained, a manic behavior phenotype animal model can be prepared, and the specific steps are as follows:
microinjection of AAV-shPer2 viral vector 1ul in SD rat hippocampal CA1 brain region, wherein the concentration of virus liquid is 4×10 11 GC/mL, knocking down the expression of the clock gene Per2, performing a behavioral test 21 days after brain microinjection, and finding that the immobility time of rats in the experimental group in a forced swimming experiment is obviously shortened; in the open field experiment, the residence time of the central area is obviously prolonged, and the total distance of activities is obviously increased; in the elevated plus maze test, the open arm residence time was significantly increased, i.e., the manic-like behavioral phenotype.
The preparation method of the depression-like behavioral phenotype animal model comprises the following specific steps:
microinjection into the CA1 brain region of the hippocampus of SD ratsLV-Per2 viral vector 1ul, wherein the viral titer is 4X 10 8 TU/ml, over-expressing Per2, performing behavioral tests 12 days after brain microinjection, and finding that rats in the experimental group are shown to have significantly prolonged immobility time in forced swimming experiments; in the sugar water test, the sugar water preference ratio is obviously reduced; in the open field experiment, the residence time in the middle central area is obviously shortened, and the number of activities and the total distance of activities are obviously reduced; in the elevated plus maze test, the open arm residence time was significantly reduced, i.e., the depression-like behavioral phenotype.
It can be seen that the Per2 gene is knocked down in the CA1 brain region of the rat hippocampus, and the manic behavior phenotype of the rat can be induced; the overexpression of Per2 gene in the CA1 brain region of rat hippocampus can induce the behavior phenotype of rat depression, and the technical method simulates the manic state and depression state of patients with bipolar disorder, thus providing an animal model with complete phenotype for deep research on pathogenesis of bipolar disorder and development of new therapeutic drugs.
As described in the background art, in the existing animal model of bipolar disorder, the conventional animal model can only simulate one state of bipolar disorder, and cannot sufficiently show two phenotype switching of the disease, which is not beneficial to sufficiently reveal the pathogenesis thereof. To solve this problem, and to further reveal the pathogenesis of bipolar disorder, the inventors for the first time utilized the dual role of Per2 gene expression level in affecting depression and manic emotion in the art, knockdown and over-express Per2 gene in the CA1 brain region of rat hippocampus, respectively, to induce manic-like behavior phenotype and depression-like behavior phenotype of rat; compared with other existing models, the model has the following advantages:
first, the model has a full phenotype, which simulates both the manic phenotype of bipolar disorder and the depressive phenotype of bipolar disorder. This is the first international realization of an animal model that mimics both the bipolar disorder mania and depressive phenotypes, with innovations in theory and methodology that will have a significant impact on the art.
Secondly, the model innovatively finds that the Per2 gene can cause mania and depression and a specific brain region, namely a hippocampal CA1 brain region, where the gene plays a role. This model involves the intervention of only one gene, the Per2 gene. This accords with the pathophysiological process of a disease, and avoids the interference of two factors.
Thirdly, the Per2 gene is knocked down and overexpressed in the CA1 brain of the sea horse respectively to induce mania and depression phenotypes, so as to simulate mania attacks and depression attacks of patients with bipolar disorder, and the model is favorable for researching pathogenesis of bipolar disorder, is an ideal model for researching two different phenotypes of bipolar disorder respectively, and can be widely applied to the pathogenesis of bipolar disorder and research and development of new drugs.
Drawings
FIG. 1 is a schematic diagram showing a construction process of an adeno-associated viral vector AAV-shPer2,
FIG. 2 is a schematic diagram showing the construction process of lentiviral vector LV-Per 2;
FIG. 3 is a graph showing the manic-like behavior of the rats in example 1
A is an experimental flow; b shows brain region microinjection sites by immunofluorescence technique; c is the PER2 protein knockdown result of the CA1 brain region of the Hippocampus; d is a sugar water preference test result; e is a forced swimming test result; f is an overhead plus maze experiment result, which comprises the stay time of an open arm, the times of entering the open arm, the times of opening an arm probe, and the total moving distance; g is the experimental result of open field, including the total distance of the activity and the residence time in the central zone.
SPT, forced swimming; OPT, open field experiment; EPM, overhead plus maze experiment; FST, forced swimming test. Data normalization test, consistent with normal distribution, using group t test, p <0.05, p <0.01, p <0.001.
FIG. 4 is a diagram showing the behavior of rats in example 2
A is an experimental flow; b shows the microinjection LV site in brain region by immunofluorescence technique; c is the over-expression result of PER2 protein in CA1 brain region of Hippocampus; d is a sugar water preference test result; e is a forced swimming test result; f is an overhead plus maze experimental result, including the times of entering the open arm and the residence time in the open arm; g is the experimental result of open field, including the total distance of the activity and the residence time in the central zone.
SPT, forced swimming; OPT, open field experiment; EPM, overhead plus maze experiment; FST, forced swimming test. Data normalization test, which is consistent with normal distribution, using group t test, p <0.05, with statistical difference.
Detailed Description
The above summary of the invention is further described in detail below with reference to the accompanying drawings. It is to be understood that these examples are for the purpose of illustrating the invention only and are not intended to limit the scope of the invention. The specific techniques employed in the examples are all conventional in the art, and the biological materials employed are all known biological materials obtained by the inventors from normal pathways and legal sources during the course of the study, and the inventors listed the following related techniques: however, other specific techniques not included are known and will not be described in detail.
The method comprises the steps of respectively constructing adeno-associated virus vector AAV-shPer2 with knockdown Per2 gene expression, constructing lentiviral vector LV-Per2 with the nucleotide sequence shown in SEQ ID NO.1 and over-expressing Per2 gene, constructing both vectors by the prior art, specifically synthesizing by gene company, and only illustrating the construction process of adeno-associated virus vector AAV-shPer2 shown in figure 1 and the construction process of lentiviral vector LV-Per2 shown in figure 2. In addition, other viral vectors with knockdown and over-expression of Per2 gene may be used in the specific application of the present invention, and the inventors will not be described in detail herein.
After obtaining the above virus vectors, the inventors provided specific preparation methods for animal models as follows:
example 1
Microinjection of AAV-shPer2 viral vector 1ul in SD rat hippocampal CA1 brain region, wherein the concentration of virus liquid is 4×10 11 GC/mL, knocking down the expression of the clock gene Per2, detecting the virus transfection condition and knocking down efficiency by adopting Western immunoblotting and immunofluorescence technology after 21 days of operation, and the result is shown in figures 3b-c; and performing emotion related behavioural tests of the rats, including a sugar water preference test, a forced swimming test, an elevated plus maze test and an open field test, and observing whether manic behaviors of the rats occur or not (the experimental flow is shown in figure 3 a). The method comprises the following specific steps:
male SD (Sprague Dawley) rats purchased from experimental animal breeding limited, license number: SCXK (Lu) 2020 0022, weight 220-240g when purchased. The animals are adaptively bred for 7 days, the animal house is kept at constant temperature and humidity (the temperature is 21-25 ℃ and the humidity is 40-70%), and the circadian rhythm is 12h/12h (8:00 lights are turned on and 20:00 lights are turned off).
Animals were fed adaptively for 7 days before the start of the experiment, with free water intake and feeding. The rats were anesthetized for 5 minutes with isoflurane gas induction, the head hair of the rats was shaved, the prone position was fixed on a rat brain stereotactic apparatus, the head of the rats was fixed with ear sticks, and simultaneously anesthesia was maintained with isoflurane gas oral-nasal mask, with the bregmas on the same horizontal plane. The surgical area was sterilized with iodophor and 75% alcohol and cut along the midline at the top of the cranium, ranging from the inner canthus to the root of the ear. According to the Paxinos and Watson rat brain stereotactic maps, the coordinates of the hippocampal CA1 brain region are: A/P, -4.3mm, M/L, + -2.0mm, D/V, -2.0mm.
Brain region microinjection: the needle tube and the soft tube of the microinjector are washed by deionized water and alcohol and dried. The syringe and the hose are filled with physiological saline, and the air bubbles in the tube are discharged. A small section of bubbles is left before the virus is sucked so as to prevent the virus from being mixed with normal saline. The needle of the microinjection needle is inserted into the sleeve of the brain region of the rat, and the microinjection of the virus is stopped before the air bubble enters the needle so as to prevent the air bubble from entering the brain. The injection rate was 0.2 μl/min and virus was pumped for 5 minutes and left for 3 minutes to allow sufficient spread of the virus throughout the target brain region. Wherein the control group is injected with the control virus AAV-scramble, and the experimental group is injected with the AAV-shPer2. After the injection is completed, the scalp is sutured. The rats recovered one week after the operation, were kept in a single cage, and 20 ten thousand units of penicillin were injected daily 3 days before the operation to prevent infection.
21 days after operation, performing behavioral tests including a sugar water preference test, an open field test, an overhead plus maze test and a forced swimming test: the sugar preference test showed that the two groups of sugar preference ratios did not differ statistically (p >0.05, fig. 3 d); in the forced swimming test, the group t test shows that the immobility time of the experimental group is significantly shortened compared with the control group (t=2.175, p <0.05, fig. 3 e), i.e. depression-like behavior or manic-like behavior; in the overhead plus maze experiment, the residence time of rats in the experimental group on the open arm is remarkably prolonged (t=3.502, p <0.01, fig. 3 f), the number of times of the probes on the open arm is remarkably increased (t=2.795, p <0.01, fig. 3 f), and the total distance of activities is remarkably increased (t=2.053, p <0.05, fig. 3 f). In open field experiments, the total range of activity was very significantly increased in the experimental group compared to the control group (t=4.369, p <0.001, fig. 3 g), the residence time was very significantly increased in the central zone (t=3.584, p <0.01, fig. 3 g), which was manifested as increased activity, decreased anxiety-like behavior and increased exploratory behavior, i.e. manic-like behavior.
Mice were sacrificed and dissected, rat brain tissue was collected, hippocampal CA1 brain region proteins were extracted, and western immunoblotting experiments were performed to detect AAV virus knockdown Per2 effects; the method comprises the steps of perfusing a rat, fixing the tissue by using 4% paraformaldehyde, taking the brain tissue of the rat, soaking the brain tissue in the 4% paraformaldehyde for fixing, soaking the brain tissue for 24 hours, dehydrating by using 30% sucrose solution, draining and freezing the dehydrated brain tissue, slicing in a frozen slicer, rinsing, mounting, rinsing, drying in the shade, sealing and the like, and detecting under a fluorescence microscope. Immunofluorescence results show the effect of AAV virus transfection and expression sites in the CA1 brain region (FIG. 3b, left panel shows the layer of the rat brain stereotactic map CA1 brain region, the low-power mirror in the figure, right panel shows the localization of the virus transfected brain region. Western immunoblotting experiments show the effect of PER2 protein knockdown in the CA1 brain region, and the experimental group is significantly lower than the control group (t=2.494, p <0.05, FIG. 3 c).
In conclusion, the AAV-shPer2 virus is microinjected into the CA1 brain region of the Hippocampus to induce mania-like behavior of the rat, and the mania-like behavior phenotype animal model of the rat can be prepared by using the method.
Example 2
The preparation method of the depression-like behavioral phenotype animal model comprises the following specific steps:
microinjection LV surgery was performed on the hippocampal CA1 brain region of SD rats, control group was injected with control virus LV-Con, and experimental group was injected with LV-Per2 virus. The virus transfection condition and the knocking-down efficiency are detected 12 days after the operation, and the emotion-related behavioural test of the rat is carried out, including a sugar water preference test, a forced swimming test, an elevated plus maze test and an open field experiment, so as to observe whether the rat has depression-like behaviors or not (the experimental flow is shown in figure 4 a).
The steps of rat feeding and brain microinjection are the same as those of example 1, and the inventors do not repeat the description.
12 days after operation, immunofluorescence results show transfection effect and expression sites of the LV virus in the CA1 brain region (fig. 4b, left is a low-power mirror, right is a high-power mirror), western immunoblotting experiments show overexpression results of the PER2 protein in the CA1 brain region, and the experimental group is remarkably improved compared with the control group (t=3.059, p <0.05, fig. 4 c).
And performing behavioral tests, including a sugar water preference test, an open field test, an overhead plus maze test and a forced swimming test. The sugar water preference test shows that the sugar water preference ratio of the rats in the experimental group is significantly reduced compared with that of the rats in the control group (t=2.523, p <0.05, fig. 4 d); in the forced swimming test, the immobility time of the experimental group is significantly prolonged compared with the control group (t=2.275, p <0.05, fig. 4 e), and the experimental group shows a depression-like behavior; in the overhead plus maze experiment, the residence time of the rats in the experimental group on the arm was significantly shortened (t=2.423, p <0.05, fig. 4 f) compared with the rats in the control group, and the number of times of entering the arm was not significantly changed (p >0.05, fig. 4 f). In open field experiments, the experimental group showed significantly reduced residence time in the central zone (t=2.997, p <0.05, fig. 4 g) compared to the control group, and no significant change in total course of activity (p >0.05, fig. 4 g) was manifested as anxiety-like behavior.
In conclusion, the micro-injection of the LV-Per2 virus into the CA1 brain region of the hippocampus induces the depression-like behavior of the rat, and the method can be used for preparing a phenotype animal model of the depression-like behavior of the rat.

Claims (3)

  1. The application of Per2 gene in preparing animal model of diphasic disorder is characterized in that: when the diphasic disturbance animal model is prepared, an adeno-associated virus vector AAV-shPer2 with knocked-down Per2 gene expression is adopted, and the nucleotide sequence is shown as SEQ ID NO. 1; a slow virus vector LV-Per2 for over-expressing the Per2 gene has a nucleotide sequence shown as SEQ ID NO. 2.
  2. 2. Use of the Per2 gene according to claim 1 for the preparation of an animal model of bipolar disorder, characterized in that: preparing a manic behavior phenotype animal model, which comprises the following specific steps:
    microinjection of AAV-shPer2 viral vector 1ul in SD rat hippocampal CA1 brain region, wherein the concentration of virus liquid is 4×10 11 GC/mL, knockdown of the expression of clock gene Per2.
  3. 3. Use of the Per2 gene according to claim 1 for the preparation of an animal model of bipolar disorder, characterized in that: preparing a depression-like behavioral phenotype animal model, which comprises the following specific steps:
    microinjection of LV-Per2 viral vector 1ul in the hippocampal CA1 brain region of SD rats, wherein the viral titer was 4X 10 8 TU/mL, over-express Per2.
CN202211437055.XA 2022-11-16 2022-11-16 Application of Per2 gene in preparing diphasic disorder animal model Pending CN116098123A (en)

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Citations (4)

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