CN107974463B - Slc6a11 gene and application of protein thereof - Google Patents
Slc6a11 gene and application of protein thereof Download PDFInfo
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Abstract
The invention provides an application of Slc6a11 gene and protein thereof, in particular to a preparation method of a pain tolerance animal model of a non-human mammal, which comprises the following steps: (1) Providing a non-human mammal embryonic stem cell, and inactivating the Slc6a11 gene in the cell to obtain an embryonic stem cell with the Slc6a11 gene inactivated; (2) Preparing a chimeric blastocyst by using the embryonic stem cell inactivated by the Slc6a11 gene and a wild type blastocyst; (3) And preparing a pain tolerance animal model by using the chimeric blastocyst. The pain tolerance animal model provided by the invention is insensitive to pain stimulus, can be used for related research of pain sense, and can be used for screening and testing specific drugs.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an application of an Slc6a11 gene and a protein thereof.
Background
Gamma-Aminobutyric acid (GABA) is the most important inhibitory amino acid neurotransmitter in the central nervous system of mammals, can mediate inhibitory synaptic transmission, thereby preventing neuronal hyperexcitation in the brain and participating in important processes such as control of nervous excitation, information processing, cognition, memory, learning and the like.
Neurons capable of releasing GABA are called gabaergic neurons. GABAergic neurons are present in all cortical layers, with a high proportion between layer IV and II to III, accounting for approximately 20-30% of all cortical neurons. GABA stored in the vesicles at the axon terminals of gabaergic neurons is released by exocytosis and binds to GABA receptors located in postsynaptic membranes. In mature brain tissue, GABA receptors (GABA) A Receptor and GABA B Receptor) is activated by GABA, and then a chloride ion channel is opened, so that chloride ions flow into nerve cells, membrane potential hyperpolarization is caused, and the inhibitory effect of the nerve cells is exerted. The concentration of extracellular GABA determines the extent to which GABA receptors are activated, and therefore maintaining low levels of extracellular GABA concentrations is critical for reducing or terminating inhibitory synaptic transmission of GABA energy and maintaining normal neural signaling. In the mammalian nervous system, this maintenance of low extracellular GABA concentrations is accomplished by re-uptake of GABA by cells. Reuptake of GABA is caused by specific Na having high affinity + /Cl - Ion dependent GABA Transporters (GATs). The GABA transport protein can effectively control the retention time and concentration of GABA in synaptic cleft, thereby accurately regulating GABA energy signal conduction, maintaining normal transmitter level of the central nervous system and ensuring the correctness of GABA signal transmission.
Currently, the research on GABA in the art is insufficient, and further research on the mechanism and function thereof is urgently needed.
Disclosure of Invention
The invention aims to provide an animal model of non-human mammal pain tolerance.
In a first aspect of the invention, there is provided a method of preparing an animal model of pain tolerance in a non-human mammal, the method comprising the steps of:
(a) Providing a cell of a non-human mammal, and inactivating the Slc6a11 gene in the cell to obtain a cell with the Slc6a11 gene inactivated;
(b) And (b) preparing a pain tolerance animal model with the Slc6a11 gene inactivated by using the cells with the Slc6a11 gene inactivated obtained in the step (a).
In another preferred embodiment, the cells are embryonic stem cells.
In another preferred example, the step (b) further comprises the steps of:
(b1) Preparing a chimeric blastocyst by using the cell with the inactivated Slc6a11 gene and a wild type blastocyst;
(b2) And preparing an animal model with pain tolerance by using the chimeric blastocyst.
In another preferred embodiment, the step (b 2) further comprises the steps of:
(i) Culturing said chimeric blastocyst to develop into a chimeric non-human mammal;
(ii) Mating the chimeric non-human mammal with a wild non-human mammal for breeding, and screening in the offspring to obtain a heterozygote non-human mammal with an inactivated Slc6a11 gene;
(iii) And (3) mutually mating and breeding the heterozygote non-human mammals, and screening the heterozygote non-human mammals with the Slc6a11 gene inactivated in the later generation to obtain the homozygote non-human mammals, namely the pain tolerance animal model.
In another preferred embodiment, the Slc6a11 gene of a part of the cells in said chimeric blastocyst is inactivated.
In another preferred embodiment, both the heterozygote non-human mammal and the homozygote non-human mammal can normally breed.
In another preferred embodiment, the animal model of pain tolerance is characterized by pain tolerance as compared to a wild-type control animal.
In another preferred embodiment, the pain tolerance comprises a reduced sensitivity to thermal, mechanical, inflammatory and/or neuropathic pain.
In another preferred embodiment, said reduced sensitivity is an increased response time to thermal, mechanical, inflammatory and/or neuropathic pain.
In another preferred embodiment, the non-human mammal is a rodent or primate, preferably including a mouse, rat, rabbit, monkey.
In another preferred example, said inactivating the Slc6a11 gene comprises gene knockout, gene disruption or gene insertion.
In another preferred example, the gene inactivation comprises that the Slc6a11 gene is not expressed, or that no active Slc6a11 protein is expressed.
In another preferred example, the Slc6a11 gene inactivation is systemic Slc6a11 gene inactivation.
In another preferred example, in step (a), one or more exons in the Slc6a11 gene are deleted or interrupted and replaced with a selection marker using a DNA homologous recombination technique, resulting in a non-human mammalian cell in which the Slc6a11 gene is inactivated.
In another preferred example, exons 1-3 of the Slc6a11 gene are deleted and replaced with a selection marker.
In another preferred embodiment, the selection marker is selected from the group consisting of: neo gene, TK gene, or combinations thereof.
In a second aspect of the invention, there is provided the use of an animal model prepared by a method according to the first aspect of the invention to study the hyperalgesia.
In a third aspect of the invention, there is provided the use of an animal model prepared by a method according to the first aspect of the invention to prepare an animal model that is pain insensitive.
In a fourth aspect of the invention there is provided the use of an animal model prepared by a method according to the first aspect of the invention to screen for or identify substances (therapeutic agents) which alleviate pain sensation.
In a fifth aspect of the invention, there is provided a method of screening for or identifying potential therapeutic agents that alleviate pain sensation, comprising the steps of:
(a) Administering a test compound to an animal model prepared by a method according to the first aspect of the invention in the presence of the test compound in a test group, and measuring the response time T1 of the animal model to a pain stimulus in the test group; in the control group, the control compound (including vehicle) is applied to the animal model prepared by the method of the first aspect of the invention in the absence of the test compound, and the response time T2 of the animal model to the same pain stimulus in the test group is measured;
(b) Comparing the response time T1 and the response time T2 measured in the previous step to determine whether the test compound is a potential therapeutic agent for alleviating pain;
wherein a response time T1 that is significantly greater than a response time T2 indicates that the test compound is a potential therapeutic agent for alleviating pain sensation.
In another preferred embodiment, the pain stimulus is selected from the group consisting of: thermal pain stimuli, mechanical pain stimuli, inflammatory pain stimuli, neuropathic pain stimuli, or combinations thereof.
In another preferred embodiment, the expression "significantly greater than" means that the ratio R.gtoreq.1/R.gtoreq.1.2, preferably R.gtoreq.1.5, more preferably R.gtoreq.2 of reaction time T1/reaction time T2.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In a sixth aspect of the invention, there is provided a method of screening for or identifying potential therapeutic agents that alleviate pain sensation, comprising the steps of:
(a) In the test group, the test compound is administered to a wild-type non-human mammal or cell in a resting state, and the level V1 of expression of the Slc6a11 gene or protein, or protein transport activity, is detected; in a control group, a control compound is administered to a wild-type non-human mammal or cell, and the level V2 of expression of the Slc6a11 gene or protein, or protein transport activity, is detected;
(b) Comparing the level V1 and the level V2 detected in the previous step to determine whether the test compound is a potential therapeutic agent for alleviating pain;
wherein, if level 1 is significantly lower than level V2, it is indicative that the test compound is a potential therapeutic agent for alleviating pain sensation.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In a seventh aspect of the invention, there is provided an animal model of pain tolerance in a non-human mammal, the model being prepared by a method according to the first aspect of the invention.
In another preferred example, the Slc6a11 gene of the animal model is inactivated.
In an eighth aspect of the invention, there is provided use of an inhibitor of Slc6a11 in the preparation of a formulation or composition for alleviating pain.
In another preferred example, the Slc6a11 inhibitor inhibits the expression of the Slc6a11 gene, or inhibits the expression or activity of the Slc6a11 protein.
In another preferred embodiment, the Slc6a11 inhibitor comprises MicroRNA, siRNA, shRNA, or a combination thereof.
In another preferred embodiment, the Slc6a11 inhibitor comprises an antibody.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.
Drawings
FIG. 1 shows a schematic diagram of mGAT4 (Slc 6a 11) knockout mouse construction and genotype identification.
FIG. 1A shows a schematic diagram of the targeting strategy for mGAT4 (Slc 6a 11) knockout mouse construction.
FIG. 1B shows mGAT4 +/- And (5) carrying out genotype identification on selfing progeny of the mice. Different banding results correspond to different genotype mice: the single 750bp band was Wild Type (WT) (+/+); the single 411bp band is an mGAT4 gene knockout homozygote (-/-); double bands are heterozygotes (+/-).
FIG. 2 shows the tissue expression profile of the mGAT4 gene. (n.gtoreq.4)
FIG. 2A shows the expression of SLc6a11 gene mRNA in cortical, cardiac, liver, spleen, lung, kidney, small intestine, stomach, testis, salivary gland, thymus, lymph node tissues.
FIG. 2B shows the expression of SLc6a11 gene mRNA in cortex, cerebellum, olfactory bulb, hippocampus, striatum, thalamus, hypothalamus, medulla oblongata, spinal cord, pituitary tissue.
FIG. 3 shows the expression of mGAT4 protein in brain tissue of SLc6a11 knockout mice detected by Western blot method.
FIG. 4 shows the delayed response of mGAT4 knockout mice to thermal pain stimuli. (n.gtoreq.11)
FIG. 4A shows tail-flick latency between mGAT4 knockout mice and wild-type mice in tail-flick experiments.
FIG. 4B shows paw licking or jump latency in the hotplate experiments in the mGAT4 knockout mice versus the wild type mice.
FIG. 5 shows the delayed response of mGAT4 knockout mice to mechanical pain stimulation. (n.gtoreq.11)
FIG. 5A shows response latency between mGAT4 knockout mice and wild-type mice in plantar mechanical stimulation experiments.
FIG. 5B shows the mechanical force thresholds for the mGAT4 knockout mice and wild-type mice to produce a pain response.
FIG. 5C shows the behavioral assessment of mechanical pain response scoring of mGAT4 knockout mice versus wild type mice. Higher scores indicate more intense pain sensation.
FIG. 6 shows the expression of mGAT4 knockout mice in formalin-induced pain tests. (n.gtoreq.11)
Figure 6A shows the response time to pain for mGAT4 knockout mice versus wild-type mice at different time periods after formalin injection.
Figure 6B shows the total time to the onset of a pain response in mGAT4 knockout mice versus wild type mice in the first (left) and second (right) phase after formalin injection.
FIG. 7 is a graph showing the response of mGAT4 knockout mice to pain stimuli after sciatic nerve segment ligation. (n = 10)
FIG. 7A shows the response delay time to mechanical stimulation of mGAT4 knockout mice versus wild-type mice before and after sciatic nerve segment ligation.
FIG. 7B shows the response thresholds to mechanical stimulation of mGAT4 knockout and wild-type mice before and after ligation of sciatic nerve segments.
FIG. 7C shows the delay time of the response to plantar thermal stimulation before and after sciatic nerve portion ligation in mGAT4 knockout mice and wild-type mice.
Detailed Description
The inventor has extensively and deeply studied and established an animal model of pain tolerance, which is a mouse or other non-human mammal with the Slc6a11 gene knocked out or inactivated. The animal model is an effective pain tolerance animal model, can be used for pain related research, and can be used for screening and testing specific medicines. The present invention has been completed on the basis of this finding.
Specifically, the invention describes a method for establishing a mGAT4 gene (Slc 6a 11) deleted mouse model by utilizing a gene knockout technology, discloses related identification results, researches on the pain sense reaction of the mouse model, and finds that the mice lacking mGAT4 are insensitive to pain sense stimulation compared with normal mice, which indicates that the mouse model can be used for the related research of pain sense, and the mGAT4 is involved in the formation of pain sense. The discovery of the invention also clearly shows that GAT-3 (mGAT-4) can be used as the action target of small molecule drugs, and can be used for screening and developing inhibitors or agonists aiming at the activity of the protein and can be used for developing drugs related to treatment of pain abnormity.
Slc6a11 gene and protein thereof
Human GABA transporters were found to have 4 subtypes, designated: GAT-1, GAT-2, GAT-3, BGT-1. They are composed of 4 independent genes: slc6a1, slc6a13, slc6a11 and Slc6a12 code. Corresponding homologous genes were found in mice and they were named mGAT1, mGAT3, mGAT4 and mGAT2. The mouse 4 transporter subtype genes are all located on chromosome 6, with Slc6a1 adjacent to Slc6a11 and Slc6a12 adjacent to Slc6a 13. GAT-1 (mGAT 1) is currently being studied more and has been found to play an important role in numerous nervous system functions, including participation in processes such as learning and memory, mood regulation, addictive mechanisms, nociception, etc., but the function of GAT-3 (mGAT 4) encoded by Slc6a11 is still unclear. 627aa (SEQ ID NO: 1, protein sequence: ENSMUSP00000032451, www.ensembl.org) of mouse mGAT4 protein, and 632aa (SEQ ID NO: 2, protein sequence: ENSP00000254488, www.ensembl.org) of human GAT-3 protein in full length, wherein the homology of the two proteins is 94%; 6538 for the Gene ID of the human SLC6A11 Gene and 243616 for the Gene Slc6a11 of the mouse.
Inactivation of genes
Many methods are available for the study of genes of unknown function, such as inactivation of the gene to be studied, analysis of the resulting genetically modified phenotypic change, and subsequent acquisition of functional information about the gene. Another advantage of this approach is that it can correlate gene function with disease, thus obtaining both gene function and disease information and animal models of disease that the gene can treat as a potential drug or drug target. The method of gene inactivation may be accomplished by means of gene deletion, gene disruption or gene insertion. Among them, gene knockout technology is a very powerful means for studying the function of human genes in the whole.
Animal model
In the present invention, a very effective non-human mammal model for studying pain sensation is provided.
In the present invention, examples of non-human mammals include (but are not limited to): mouse, rat, rabbit, monkey, etc., more preferably rat and mouse.
As used herein, the term "inactivation of the Slc6a11 gene" includes the case where one or both of the Slc6a11 genes are inactivated, i.e. includes the inactivation of the Slc6a11 gene heterozygously and homozygously. For example, a mouse in which the Slc6a11 gene is inactivated can be a heterozygous or homozygous mouse.
In the present invention, a non-human mammal (e.g., a mouse) in which the Slc6a11 gene is inactivated can be prepared by gene deletion or introduction of a foreign gene (or fragment) to inactivate the Slc6a11 gene. In the art, techniques for inactivating a target gene by gene knockout or introduction of a foreign gene are known, and these conventional techniques can be used in the present invention.
In another preferred embodiment of the invention, the inactivation of the Slc6a11 gene is achieved by gene knockout.
In another preferred embodiment of the invention, the inactivation of the Slc6a11 gene is achieved by inserting a foreign gene (or fragment) into the Slc6a11 gene.
In one embodiment of the invention, a construct containing an exogenous insert can be constructed that contains homology arms homologous to flanking sequences flanking the insertion site of the target gene (Slc 6a 11) such that the exogenous insert (or gene) can be inserted into the Slc6a11 genomic sequence (particularly the exon region) via homologous recombination at high frequency, resulting in a frameshift, premature termination, or knock-out of the mouse Slc6a11 gene, resulting in the deletion or inactivation of Slc6a 11.
The homozygous or heterozygous mouse obtained by the method of the invention can be fertile and normally develop. The inactivated Slc6a11 gene can be inherited to progeny mice on a mendelian basis.
In a preferred embodiment, the invention provides a homozygous mouse model animal lacking the Slc6a11 gene.
Drug candidate or therapeutic agent
In the present invention, there is also provided a method for screening a candidate drug or therapeutic agent for treating abnormal pain using the animal model of the present invention.
In the present invention, a drug candidate or therapeutic agent refers to a substance known to have a certain pharmacological activity or being tested, which may have a certain pharmacological activity, including but not limited to nucleic acids, proteins, chemically synthesized small or large molecular compounds, cells, and the like. The candidate drug or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, or intradermally.
The main advantages of the invention include:
(a) The homozygous or heterozygous mouse obtained by the method of the invention can be fertile and normally develop. The inactivated Slc6a11 gene can be inherited to progeny mice in a Mendelian manner
(b) The mouse model is insensitive to pain stimulation, can be used for related research of pain, and can be used for screening and testing specific drugs.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
Construction of mGAT-4 gene knockout mouse model
The mGAT-4 knockout mouse is constructed by adopting a mouse embryonic stem cell gene homologous recombination mode, and figure 1 is a targeting vector, a homologous recombination targeting schematic diagram and a gene identification map of a constructed mouse model. Wherein the region of exons 1 to 3 of Slc6a11 in the mouse genome is replaced by the selection gene neo, resulting in disruption of the Slc6a11 gene. The gene targeting is carried out according to the conventional operation, the targeting vector DNA is introduced into a mouse embryonic stem cell (ES) by an electroporation method, cultured on a feeder layer cell consisting of mouse embryonic fibroblasts treated by mitomycin, clone screening is carried out under the action of G418 drugs, PCR identification is carried out on the obtained clones, one PCR primer is respectively designed on the outer side of a homologous recombination arm, the other PCR primer is respectively designed on a screening gene neo gene sequence, PCR products with expected lengths are obtained by PCR on both sides, and the obtained clones are positive clones if the PCR products are correctly verified by sequencing. After cloning and amplifying the positive ES cells, injecting a certain amount of ES cells into a blastocyst cavity of a mouse by using a microinjection method to obtain a chimeric blastocyst, transplanting the chimeric blastocyst into an oviduct of a pseudopregnant mouse to make the pseudopregnant mouse implanted and developed to produce a chimeric mouse, breeding the chimeric mouse and a wild-type mouse to obtain a heterozygote mouse with target gene deletion, and mating the heterozygote mouse to obtain a homozygote mouse with the target gene deletion.
According to the mGAT4 (Slc 6a 11) gene knockout targeting strategy (FIG. 1A), two pairs of primers were designed to identify wild-type and mGAT4 knockout homozygous mice, respectively.
Genotype identification primer sequences:
slc6a11-ko-f:5’-GGTGGCCTGGGCATCGTGT-3’；(SEQ ID NO.:3)
slc6a11-ko-r:5’-TGAGACGTGCTACTTCCATTTG-3’；(SEQ ID NO.:4)
slc6a11-wt-f:5’-GCAGCGTCTGTCACCTGCGT-3’；(SEQ ID NO.:5)
slc6a11-wt-r:5’-TGGGACCCCTCGCCACTCTG-3’。(SEQ ID NO.:6)
the PCR procedure was as follows: 94 ℃/4min;94 ℃/45s,60 ℃/30s,72 ℃/40s,35 cycles; 72 ℃/10min;4 ℃/hold.
mGAT4 +/- Mice were selfed and the resulting progeny contained three genotypes: WT, mGAT4 +/- And mGAT4 -/- . Carrying out electrophoresis on a PCR product of a wild type which is not subjected to homologous recombination to obtain an electrophoresis band of 750 bp; after the mGAT4 gene is knocked out, the size of a PCR product is 411bp, and the genotype identification result (shown in figure 1B) is shown. The number of the three genotype mice basically accords with Mendel's law after statistics, which shows that the deletion of the mGAT4 gene can not cause homozygote death. And the homozygote mouse can mate with a homozygote mouse or a wild type mouse respectively to obtain mouse offspring with normal quantity, and the genotype ratio accords with the Mendel's law. Indicating that the fertility of the mGAT4 knockout mice is not influenced.
The results are shown in FIG. 2 and FIG. 3, in which FIG. 2 shows the tissue expression profile of the mGAT4 gene, and FIG. 3 shows the expression of mGAT4 protein in brain tissue of Slc6a11 knockout mice. RT-PCR and WesternBlot methods are used for further proving that corresponding mRNA and protein can not be detected in a mouse body after mGAT4 gene knockout, and the success of establishment of mGAT4 gene knockout mice is proved.
Example 2
Decreased pain sensitivity in mGAT4 knockout mice
GABA is involved in the regulation of pain sensation, and mGAT4 is an important protein for GABA uptake outside cells of the central nervous system and plays an important role in regulating the extracellular concentration of GABA, but whether mGAT4 is involved in the regulation of pain sensation is not clear. The change of response threshold of mGAT4 knockout mice to acute injury stimulation is researched by utilizing thermal stimulation (tail flick and hot plate experiments) and mechanical stimulation (plantar mechanical pain sensation experiments); the sensitivity degree of the mGAT4 knockout mouse to inflammatory pain is researched by using a formalin experiment; study of mGAT4 in neuropathic pain Using a mouse partial sciatic nerve ligation modelThe function of (1). C57/BL6 background mGAT4 Gene knockout mGAT4 +/- Selfing heterozygote mice to obtain mGAT4 Wild Type (WT) and heterozygote (mGAT 4) +/- ) And homozygote (mGAT 4) -/- ) A mouse. Hot plate experiments selected from 8-10 weeks old female Wild Type (WT) and homozygote (mGAT 4) -/- ) Mice, other behavioral experiments selected from 8-10 weeks old male Wild Type (WT) and homozygous (mGAT 4) -/- ) A mouse. Hereinafter, mGAT4 knockout mice will be referred to as homozygote mice (mGAT 4) -/- ). As a result, it was found that in all of the above models, mGAT4 knockout mice exhibited pain tolerance, low sensitivity to thermal pain, mechanical pain, inflammatory pain, and neuropathic pain. Therefore, the mGAT4 knockout mouse can be used as an important animal model for researching the pain sense process, and the mGAT4 gene deletion causes the reduction of the mouse pain sense, so the mGAT4 and the human homologous gene GAT-3 thereof can be used as an important target for screening analgesic drugs.
The experimental methods and results will be described in detail below.
Tail flick test Tail-flick test:
the infrared heat source stimulation tail flick experiment is utilized to detect the response of the mouse to the acute thermal pain. The mouse is carefully placed in a fixed black cylinder support, and the tail of the mouse is completely exposed and can move freely. Mice were given a 3 minute pre-acclimation to avoid an overstimulation response from the procedure. Before testing, the mouse tail is lightly touched, so that the 1/3 position of the tail end naturally adheres to the surface of the infrared heat source and keeps static. Starting timing while starting the heat source, and automatically ending timing when the Tail of the mouse swings due to heat pain and leaves the surface of the heat source, and recording the delay time (Tail-flash latency) of the Tail swing of the mouse. The i.r. (infrared) light source intensity (i.r. intensity) was 30U, and the maximum observation time was set to 15 seconds to avoid tissue damage.
As shown in FIG. 4A, the tail flick response latency time of the mGAT4 knockout mouse was 5.06. + -. 0.83sec, which was significantly prolonged (p < 0.05) compared with the response time of 2.97. + -. 0.26sec in the littermate wild-type mouse.
Hot plate experiment Hot plate test:
mice were tested for response to acute thermal pain using a hotplate experiment. The experimental procedures were improved on a literature basis. One hour before the test of the official experiment, the test mouse was placed in a cylinder on a hot plate set at 25 ℃, the top cover was closed, and the test mouse was subjected to an adaptive operation for 1 minute. After one hour, the test was performed. At this point the hotplate temperature was set to 52.0 + -0.2 deg.C, the mouse was gently placed into the transparent cylinder on the hotplate and the time was started. The behavior of the mouse was closely observed, and the time when the mouse licked the hind paw, shaken continuously or swiped the hind paw, or jumped was recorded as the delay time of the mouse on the hot plate (hotspot interaction latency). To avoid tissue damage, the maximum observation time was set to 60 seconds.
As a result, as shown in FIG. 4B, the response latency of the mGAT4 knockout mouse was not significantly prolonged as compared with that of the wild-type mouse.
Plantar thermal pain test Radiation heat plate cast:
and (3) stimulating the soles of the mice by using an infrared heat source, and detecting the response of the mice to acute thermal pain. The experimental procedures were improved on a literature basis. The mouse was carefully placed in a stationary black cylinder holder so that the hind limb of the mouse was exposed intact and free to move. Mice were given a 3 minute pre-acclimation to avoid an overstimulation response from the procedure. The mouse hindpaw portion was naturally attached to the infrared heat source surface and held stationary. Starting timing while starting the heat source, and automatically ending timing when the mouse back palm swings due to heat pain and leaves the surface of the heat source, and recording as the delay time of the heat pain of the mouse sole (Paw with wasl latency). The intensity of the thermal stimulation was 30U, and the maximum observation time was set to 30 seconds to avoid tissue damage.
The results are shown in FIG. 7C.
Plantar Mechanical pain test Mechanical plantar test:
the response of mice to mechanical pain stimuli was measured using a Dynamic Plantar tactile meter (DPA). The specific method comprises the following steps: the mouse to be tested is placed in the plastic square grids 30 minutes in advance, the mouse can move freely, the bottom of each square grid is a blank platform of a wire mesh, the mechanical foot-contracting reflection threshold value is measured when the mouse is quite, and the mechanical stimulation intensity is in g. During measurement, after a power supply of a machine is turned on, a test probe (a blunt needle) is aligned to the middle part of the palm part of the hind paw of the mouse, and the following two measurement methods are adopted:
1) The pressure of the mouse hindpaw is gradually increased by the measuring probe, and the amplification is 0.5g/sec. Once the mouse fails to tolerate a slight withdrawal or lifting of the hind paw, the probe drops and the test is completed, at which point the instrument displays the pressure applied to the ball of the foot (Force). Each mouse was measured 5 times, each time at least 30 seconds apart, and the mean value was taken as a quantitative indicator of the mechanical paw withdrawal threshold, setting the maximum value to 6.5g.
2) The pressure of the mouse hindpaw is set as a fixed value (5.0 g in a natural state, 4.0g after the sciatic nerve part is ligated) by the test probe, the force application time is continuously prolonged until the mouse can not tolerate the pressure and the hindpaw slightly leaves or the foot is withdrawn or lifted, the probe immediately falls, and the test is finished. At this time, the instrument showed the response delay time of the mouse to the pressure (Latency).
Each mouse was measured 5 times with intervals of at least 30 seconds, and the mean value was taken as a quantitative indicator of the mechanical paw withdrawal threshold, setting the maximum value at 30 seconds. The behavioral movements of the hind paws of the mice were scored at the same time as the test. The scoring criteria are as follows: no obvious reaction was observed in the hind paw, and the natural state was recorded as 0 min. Pinching or lifting of the hind paw occurred, and was scored as 1 point. The behavior of licking the hindpaw was scored as 2 points. Scores of the behavior of the challenge probe appeared to be 3. The average of 5 measurements was taken as the response score (Reaction score) of the hind paw of the mouse to mechanical pain.
As shown in FIG. 5, the lag time of pain induced by mechanical force stimulation in the hind paw of the mGAT4 knockout mouse was 5.34. + -. 1.011sec; response time was 2.47 ± 0.60sec in littermate wild-type mice under the same conditions, as shown in fig. 5A, significantly shorter than mGAT4 knockout mice (p = 0.029). Meanwhile, the mGAT4 gene knockout increases the response force threshold of the mouse to pain caused by mechanical force. The response thresholds for the mGAT4 knockout and littermate wild-type mice were 4.3 ± 0.21g and 3.62 ± 0.23g, respectively, with the former being significantly higher (p = 0.035) (fig. 5B). In addition, the mice were scored for their behavioral intensity in response to mechanical force stimuli, with higher scores indicating a more intense response to pain and more sensitive pain perception. The statistical results are shown in fig. 5C, with a wild-type mouse score of 0.67 ± 0.23, significantly higher than the mGAT4 knockout mouse 0.077 ± 0.044 (p = 0.012). It was also shown that mGAT4 knockout attenuated the severity of the mice's response to pain from mechanical stimulation. All three results demonstrate that mGAT4 knock-out reduces the sensitivity of mice to mechanical pain stimuli.
Formalin-induced pain test Formalin test
Formalin can cause a persistent inflammatory pain response. Microinjection was performed by injecting 20. Mu.l of 2% formalin solution (prepared in physiological saline) subcutaneously in the right hind paw plantar region. The behavior of the mice after formalin injection was continuously observed. The time for the mouse hindpaw to tremble, scrunch, or lick the paw was recorded within 5 minutes each.
As a result, as shown in FIG. 6, the mGAT4 knockout reduced the time required for mice to lick the formalin-injected hindpaw and shake or curl the formalin-injected hindpaw, and the pain caused by the formalin solution was relieved to some extent. The response time of the mice to pain in the first time Phase and the second time Phase (Phase I and Phase II) is counted respectively, and the response time of the mice with the wild type and the mGAT4 knockout genotypes in the first time Phase is equivalent to 64.86 +/-9.59 sec and 49.93 +/-4.26 sec respectively, and has no statistical difference; whereas the two genotypes showed more significant differences only in the second phase, the response time of the mGAT4 knockout mice was 31.32 ± 5.97sec, a more significant reduction (p = 0.06) than that of the littermate wild-type mice 69.21 ± 21.44sec, fig. 6B. The response times for the two genotypes were substantially coincident in the first time phase, as shown in FIG. 6A. In the second time phase, the response time per time unit of mGAT4 knockout mice to formalin pain was lower than littermate wild type mice, specifically 9.87 ± 2.33sec at 35 minutes post formalin injection, significantly less than 23.84 ± 6.49sec for littermate wild type (p = 0.035). Therefore, the mGAT4 gene deletion has certain analgesic effect on persistent inflammatory pain.
Partial sciatic nerve ligation model (Partial scientific, PSNL)
Injecting pentobarbital sodium (80 mg/kg) into abdominal cavity of mouse, after anesthetizing the mouse, removing hair at buttocks, fixing at 38 deg.C for prone position on a constant temperature operating table, sterilizing the operation position with iodine cotton ball from inside to outside spirally, sterilizing with alcohol cotton ball once in the same method, transversely cutting a 2cm incision at pelvis bone with a surgical knife, inactivating muscle at 0.5cm position below pelvis bone, exposing sciatic nerve, ligating 1/2 sciatic nerve with 6-0 suture with needle, and suturing muscle and skin layer by layer with 6-0 suture with needle. Wound was disinfected spirally from inside to outside with iodine cotton ball. Mice were placed on a 38 ℃ rewarming blanket until awakened. After reviving, the animals were returned to clean cages for rearing (3 animals/cage).
The results are shown in fig. 7, where the sole response to mechanical stimulation was again examined on day 7 after surgery. As shown in fig. 7A and 7B, compared to before operation, wild-type mice showed stronger response to pain caused by mechanical force due to the generation of neuropathic pain after operation, and the response delay time of hind paw to mechanical force stimulation was greatly shortened from 8.55 ± 1.79sec at background to 3.25 ± 0.99sec (p = 0.0084), and the mechanical force threshold background value of 3.19 ± 0.25g for generating pain response was significantly reduced to 2.43 ± 0.24g (p = 0.011). The sciatic nerve ligation operation successfully causes sciatic nerve injury of the mice, induces neuropathic pain of the mice, and causes hypersensitivity response of the mice to pain caused by mechanical stimulation. However, the same nerve injury did not induce hypersensitivity to mechanical stimulation in the mGAT4 knockout mice. The response delay time of mGAT4 knockout mice after nerve injury was 9.36. + -. 2.40sec, and the mechanical force threshold was 4.17. + -. 0.43g, all of which were maintained at the same level as before ligation operation (p-values were 0.23 and 0.67, respectively). The mGAT4 gene knockout relieves the allodynia of mice caused by nerve injury, and the mGAT4 deletion has analgesic effect on the nerve pain. In the plantar thermal pain test, the response delay time of the mouse hind paws to the thermal pain stimulus was examined before sciatic nerve ligation, 7 days after surgery, and 60 days after surgery, respectively, as shown in fig. 7C. Similar to the results of mechanical pain, the response delay time of the pelma of the wild type mice to thermal pain was abruptly reduced from the pre-operative 6.91 ± 0.87sec to 2.72 ± 0.33sec (p = 0.0007) 7 days after sciatic nerve ligation, while the post-operative response time of 5.2615 ± 0.5783sec of the mGAT4 knockout mice was still maintained at the pre-operative level of 6.58 ± 1.10sec (p = 0.28). The response of two genotype mice to thermal pain at 7 days after operation is remarkably different (p = 0.0029), further indicating that the mGAT4 gene knockout has relieving effect on allodynia caused by nerve injury.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (11)
1. A method for preparing a pain tolerance mouse model, comprising the steps of:
(a) Providing mouse cells, and screening the cellsSlc6a11Inactivating the gene to obtainSlc6a11A cell in which the gene is inactivated;
(b) Using the product obtained in step (a)Slc6a11Gene inactivated cells, preparedSlc6a11A pain tolerance mouse model with inactivated genes,
wherein saidSlc6a11The amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 1.
2. The method of claim 1, wherein said cell is an embryonic stem cell.
3. The method of claim 1, wherein said step (b) further comprises the steps of:
(b1) By using saidSlc6a11Preparing a chimeric blastocyst from the gene-inactivated cell and a wild-type blastocyst;
(b2) And (3) preparing a pain tolerance mouse model by using the chimeric blastocyst.
4. The method of claim 3, wherein said step (b 2) further comprises the steps of:
(i) Culturing the chimeric blastocyst to develop into a chimeric mouse;
(ii) Mating and breeding the chimera mouse and wild mouse, and screening in the offspringSlc6a11A genetically inactivated heterozygous mouse;
(iii) The heterozygote mice are mutually mated and bred, and are screened in the offspringSlc6a11And (3) obtaining a homozygote mouse with the inactivated gene, namely the pain tolerance mouse model.
5. A method according to claim 1, wherein the pain tolerant mouse model is characterized by pain tolerance comprising reduced sensitivity to thermal, mechanical, inflammatory and/or neuropathic pain, as compared to a wild-type control mouse.
6. The method of claim 1, wherein in step (a), the DNA is homologous recombinationSlc6a11One or more exons in the gene are deleted or disrupted and replaced with a selectable marker to obtainSlc6a11Mouse cells with inactivated genes.
7. The method of claim 6, wherein the step of converting the signal into a signal comprises converting the signal into a signal having a frequency that is different from the frequency of the signalSlc6a11Exons 1-3 of the gene are deleted and replaced with a selectable marker.
8. Use of a mouse model prepared by the method of claim 1 for preparing a pain insensitive mouse model.
9. Use of a mouse model prepared by the method of claim 1 to screen or identify pain-alleviating substances.
10. A method of screening for or identifying potential therapeutic agents that alleviate pain sensation comprising the steps of:
(a) Administering a test compound to a mouse model prepared by the method of claim 1 in the presence of the test compound in a test group, and measuring the response time T1 of the mouse model to a pain stimulus in the test group; administering a control compound to a mouse model prepared by the method of claim 1 in the absence of the test compound in a control group, and measuring the response time T2 of the mouse model to the same pain stimulus in the control group;
(b) Comparing the response time T1 and the response time T2 measured in the previous step to determine whether the test compound is a potential therapeutic agent for alleviating pain.
11. Use of an Slc6a11 inhibitor for preparing a preparation for constructing a mouse model with pain tolerance, wherein the Slc6a11 inhibitor inhibitsSlc6a11Gene expression, or inhibition of expression of Slc6a11 protein,
wherein saidSlc6a11The amino acid sequence of the gene-encoded protein is shown as SEQ ID NO. 1, and the amino acid sequence of the Slc6a11 protein is shown as SEQ ID NO. 1.
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