CN114255823A - Construction method and application of retinal degenerative disease model - Google Patents
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Abstract
The invention discloses a construction method and application of a retinal degenerative disease model, and relates to the technical field of medical engineering. The model is constructed by a method that Tmem184b gene in the genome of the non-human target animal is not expressed or the expression is inhibited through a genetic engineering technology. Therefore, the non-human target animal body can show the characteristics related to the retinal degeneration, and on the basis, the animal body can be used as an animal model of the retinal degeneration so as to provide a good model research basis for researching the disease mechanism and drug screening of the retinal degeneration. The invention provides a new non-human animal model for the research of retinal degenerative disease and the screening of drugs or therapies for treating the retinal degenerative disease, and enriches the model for the research of retinal degenerative disease to a great extent.
Description
Technical Field
The invention relates to the technical field of medical engineering, in particular to a construction method and application of a retinal degenerative disease model.
Background
Retinal degeneration is a serious disease that can lead to irreversible visual impairment and even blindness, and is typically characterized by the loss of highly differentiated cells in the neural retina, such as photoreceptors or the Retinal pigment epithelium. Photoreceptor damage is an important group of these diseases, and is the decrease in retinal function, irreversible impairment of vision, and even blindness due to abnormal death of photoreceptor cells. Currently, there are few animal models for studying retinal degeneration.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a construction method of a retinal degenerative disease model and application thereof to solve the technical problems.
The invention is realized by the following steps:
the invention provides a method for constructing a retinal degenerative disease model, which comprises the following steps: tmem184b gene in the genome of the non-human target animal was rendered non-expressed or expression inhibited by genetic engineering techniques.
The Chinese name of Tmem184b gene is transmembrane protein184b, and the Tmem184b gene of human is located at 22q13.1 locus of chromosome 22 of human, contains 13 exons, and the transcript of the gene can form 14 alternative spliceosomes of a, b, X1, X2, X3 … X12 and the like through alternative splicing. This gene was sequenced by the human genome project in 1999 and was published in Nature J PMID:10591208 after human chromosome 22 was sequenced by Dunham et al. The homologous gene to Tmem184b exists in 318 known species and has been shown to be well conserved in both humans and in a variety of model organisms. Tmem184b is expressed in each tissue, especially with high levels of expression in brain, fat and placenta. The Tmem184b gene encodes a membrane protein with 7-fold transmembrane, related function studies are few, and only a few omics studies relate to possible pathways and correlation analysis with cancer. The Tmem184b gene is still under initial research, and the specific action mechanism of the Tmem184b gene in mice and other mammals is not clear, so that the development and application of the Tmem184b gene are limited.
The inventor finds that the Tmem184b gene in the genome of the non-human target animal is not expressed or the expression of the Tmem184b gene is inhibited, so that the non-human target animal body can show the characteristics related to the retinal degeneration, and based on the characteristics, the animal body can be used as an animal model of the retinal degeneration so as to provide a good model research basis for researching the disease mechanism and drug screening of the retinal degeneration. The invention provides a new non-human animal model for the research of retinal degenerative disease and the screening of drugs or therapies for treating the retinal degenerative disease, and enriches the model for the research of retinal degenerative disease to a great extent.
The above expression suppression includes, but is not limited to: the expression level of the Tmem184b gene is slightly reduced or the expression level is significantly reduced compared to the expression level in a normal target animal body.
In a preferred embodiment of the invention, all or a portion of the exons of the Tmem184b gene in the genome of the non-human target animal are not expressed or expression is inhibited by genetic engineering techniques.
It is to be noted that the number of exons of the Tmem184b gene varies from non-human target animal genome to non-human target animal genome, and it is within the scope of the present invention to select all exons or a portion of exons as desired for non-expression or for inhibition of expression.
The number of partial exons may be one, two, three or more. It is within the scope of the present invention to knock out a sequence of either type (partial or full length) so long as the expression of the Tmem184b gene is silenced in the cells of the animal being knocked out such that the animal exhibits characteristics corresponding to retinal degeneration.
In a preferred embodiment of the invention, the non-human target animal is a non-human target animal having a homologous gene at Tmem184 b;
the non-human target animal is mammal, and can be mouse, rat, rabbit, cow, dog, pig, horse, sheep, monkey or ape; preferably, the non-human target animal is a mouse.
In a preferred embodiment of the invention, at least one of exons 1 to 17 of the Tmem184b gene in the genome of the non-human target animal is rendered non-expressible or inhibited. Alternatively, exons 4-7, 4-10 are not expressed or expression is inhibited.
In a preferred embodiment of the invention, when the non-human target animal is a mouse, the exon 4-6 sequences of Tmem184b gene in the genome of the non-human target animal are rendered non-expressed or expression is inhibited. The mouse Tmem184b gene (Transmembrane protein184b, MGI:2445179) is located on mouse chromosome 15 79244884 and 79287503bp, and has a total length of 46.62kb, and the cDNA has a total length of 3431bp and contains 17 exons.
When other non-mouse mammals are used as disease models, those skilled in the art can easily obtain a gene homologous to the mouse Tmem184b gene, and knock out the homologous gene to obtain a retinal degenerative disease model.
In a preferred embodiment of the present invention, the genetic engineering technique is: any one or a combination of techniques of gene editing, gene knockout, and RNA interference.
In a preferred embodiment of the present invention, the gene editing technology is selected from at least one of ZFN technology, TALEN technology, CRISPR/Cas9 technology, and DNA homologous recombination technology;
the gene knockout technology is selected from complete gene knockout technology or conditional gene knockout technology.
In a preferred embodiment of the invention, the construction method comprises mating non-human target animals with Tmem184b gene knockout to obtain a Tmem184b gene knockout homozygous retinal degenerative disease model.
Tmem184b gene knock-out mice were purchased from seiki biotechnology limited, strain number: c57BL/6N-Tmem184bem1cyagen。
The knockout strategy is to knock out Tmem184b gene by using CRISPR/Cas9 technology. However, in other embodiments, gene knock-out is readily accomplished using gene editing techniques conventional in the art, provided that the gene to be knocked-out is defined. Therefore, in other embodiments, the Tmem184b gene is knocked out by using CRISPR/Cas9 technology or other technical means, which also belongs to the protection scope of the invention.
The invention also provides application of the retinal degenerative disease model obtained by the construction method of the retinal degenerative disease model in research of the retinal degenerative disease, and the research aims at diagnosis or treatment of non-diseases.
The non-human animal model obtained by the construction method has the characteristics of typical retinal degenerative diseases, has very wide application prospects, and provides a basis for deeply understanding and researching the retinal degenerative diseases by being used for researching the pathogenesis and the pathogenesis of the retinal degenerative diseases. Or screening a drug for preventing or treating a retinal degenerative disease, evaluating the efficacy or prognosis of the drug, or the like.
The invention also provides application of the retinal degenerative disease model obtained by the construction method of the retinal degenerative disease model in early molecular screening of medicines or in screening of medicines for targeted treatment of the retinal degenerative disease.
In a preferred embodiment of the invention, the degenerative retinal disease is characterized by: loss of highly differentiated cells such as photoreceptors or retinal pigment epithelium (retinal pigment epithelium) in the neurogenic retina.
In an alternative embodiment, the above-mentioned retinal degenerative disease manifests as at least one of the following symptoms:
thinning of retinal structures;
and retinal rod cell dysplasia.
Such thinning of retinal structures includes, but is not limited to: the outer core layer becomes thinner or a significant thinning occurs. Retinal rod dysplasia includes, but is not limited to: rod extracellular segment shortening; loss of cone cells, impaired visual function detected by ERG, and the like.
The present invention also provides a method for culturing a retinal degenerative disease model, which comprises: the retinal degenerative disease models obtained by the above-described construction method were mated with each other.
After obtaining a retinal degenerative disease model by the above-described construction method for the first time, in order to obtain a larger number of retinal degenerative disease models, those skilled in the art will easily think that a larger number of retinal degenerative disease models are obtained by performing a breeding method of mating the retinal degenerative disease models with each other, and such a method of breeding a retinal degenerative disease model also falls within the scope of the present invention.
The invention has the following beneficial effects:
the invention discovers that the Tmem184b gene in the genome of the non-human target animal is not expressed or the expression of the Tmem184b gene is inhibited, so that the non-human target animal body can show relevant characteristics of retinal degeneration, and based on the expression, the animal body can be used as an animal model of the retinal degeneration so as to provide a good model research basis for researching a disease mechanism and drug screening of the retinal degeneration. The invention provides a new non-human animal model for the research of retinal degenerative disease and the screening of drugs or therapies for treating the retinal degenerative disease, and enriches the model for the research of retinal degenerative disease to a great extent.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the gene knockout of TMEM184b gene knockout mouse;
FIG. 2 is an electrophoretogram for genotyping TMEM184b gene knock-out mice; KO represents a homozygous knockout; het represents heterozygosity; WT means a wild type;
FIG. 3 is a graph showing HE staining (top) and statistics (bottom) of the thickness of an ectonuclear layer of a six-month-old TMEM184b gene knock-out mouse eyeball paraffin section;
FIG. 4 is a waveform and amplitude statistics of six months old TMEM184b gene knock-out mouse ERG;
FIG. 5 is a graph showing immunofluorescence staining results of six-month-old TMEM184b gene knock-out mouse retina cryosection PRPH2 and Na/K ATPase antibody;
FIG. 6 is a graph showing the results of immunofluorescence staining of cone Arrestin antibody combined with staining of fluorescently labeled Peanut agglutinin (PNA) in a six-month-old TMEM184b gene knock-out mouse retina frozen section.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. m.j. goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), Handbook of Experimental Immunology (Handbook of Experimental Immunology) (ed. D.M.Weir and C.C.Black well), Gene Transfer Vectors for Mammalian Cells (ed. J.M.Miller and M.P.Calos) (ed. J.M.and M.P.Calos) (ed. 1987), Methods in Current Generation (Current Protocols in Molecular Biology) (ed. F.M.Ausubel.et al, 1987), PCR, Polymerase Chain Reaction (ed. PCR: The Polymerase Chain Reaction) (ed. Mullis et al, 1994), and Methods in Current Immunology (ed. J.1991).
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for constructing a retinal degenerative disease model. Referring to FIG. 1, the 4 th to 6 th exons of Tmem184b gene are knocked out by CRISPR/Csa9 technology. The obtained Tmem184b gene knockout heterozygote mice are mutually mated and bred to obtain Tmem184b gene knockout homozygote mice which can be used as a disease model of retinal degenerative disease.
Tmem184b gene knock-out mice were purchased from seiki biotechnology limited, strain number: c57BL/6N-Tmem184bem1cyagen. Purchasing web site
https://www.cyagen.com/cn/zh-cn/sperm-bank-live/223693。
Experimental example 1
In this experimental example, a Tmem184b knock-out homozygote mouse was identified for the retinal degenerative disease model constructed in example 1.
The method comprises the following steps:
1) cutting a little tissue sample from the tail tip of the Tmem184b knockout mouse, and placing the tissue sample into a clean 1.5ml centrifuge tube;
2) add 100. mu.l lysis buffer (40mM NaOH, 0.2mM EDTA solution) to the centrifuge tube and heat for 1h at 100 ℃ in a metal bath;
3) the centrifuge tube was removed, cooled to room temperature, 100. mu.l of a neutralizing solution (40mM Tris-HCl, pH5.5) was added thereto, and after centrifugation at 10000g for 2min, the supernatant was used for mouse genotyping.
4) And (3) PCR amplification: the PCR reaction system was configured as follows
The primer sequences are as follows:
tmem184b 5' Primer-F sequence (SEQ ID NO. 1):
5’-CTGACCTTCATCCCTGCAGT-3’；
tmem184b 3' Primer-R1 sequence (SEQ ID NO. 2):
5’-GCTGTGGACCATGGCTCTA-3’；
tmem184b 3' Primer-R2 sequence (SEQ ID NO. 3):
5’-AGGCTGACGGAGATGTTGTA-3’；
the amplification procedure was as follows:
preheating: 95 ℃ for 5 min; denaturation: 95 ℃ for 30s, annealing: 60 ℃, 30s, extension: 72 ℃, 30s, cycle: 25 times; 72 ℃ for 5 min; storing at 4 ℃.
5) Gel electrophoresis:
10ul of PCR product was placed in wells and electrophoresed in 1% agarose gel at 120V constant pressure for 15min.
The PCR results are shown in FIG. 2, in which WT represents the wild-type control, and the band size is 550 bp; het represents heterozygote, and has two bands which are respectively 550bp and 350 bp; KO represents homozygote, and has a single band with a size of 350 bp. According to the results of fig. 2, it is shown that the identification method can effectively identify the mouse genotype, and a mouse (homozygote) with a band indicated by KO is selected as a retinal degenerative disease model for subsequent experiments.
Experimental example 2
In this example, paraffin sections of retinas were taken and H & E staining was performed.
The retinas of the mice with the KO homozygous retinal degenerative disease determined in experimental example 1 and the wild type at 6 months of age were subjected to paraffin sectioning and staining by hematoxylin-eosin staining (H & E staining method), respectively, as follows:
1) quickly taking eyeball tissues of the mouse, and placing the eyeball tissues in a stationary liquid for fixation for 24 hours;
2) embedding in paraffin, and slicing to obtain slices with a thickness of 4 μm;
3) the slices were dewaxed conventionally with xylene, washed with multi-stage ethanol to water: xylene (I)5min → xylene (II) 5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing 2 min;
4) hematoxylin staining for 5 minutes and washing with tap water;
5) ethanol hydrochloride is differentiated for 30 seconds;
6) soaking in tap water for 15 minutes;
7) placing in eosin solution for 2 minutes.
8) Conventional dehydration, transparency, mounting: 95% ethanol (I)1min → 95% ethanol (II) 1min → 100% ethanol (I)1min → 100% ethanol (II) 1min → xylenecarbonate (3: 1)1min → xylene (I)1min → xylene (II) 1min → neutral resin sealing.
9) Take pictures under microscope.
Results as shown in figure 3, statistics of the outer nuclear layer thickness were found at 6 months to show that the outer nuclear layer of the retina had begun to thin in KO mice compared to WT mice, indicating photoreceptor cell death.
Experimental example 3
In this experimental example, ERG visual acuity test was performed on Tmem184b knockout mice (homozygote identified in experimental example 1) at 6 months of age, and the control group was WT.
1) Dark adaptation animals should adapt dark overnight, and the environment should be absolutely free of light;
2) anesthesia the next day: weighing, and injecting in an abdominal cavity; deep anesthesia is suitable;
3) animal fixation and mydriasis: after completion of anesthesia, mice were taped in front of the animal test platform under dark red light illumination: the mouse needs to be ensured to lie prone, namely, the height of two eyes is consistent relative to the stimulating port of the flash stimulator, the eyes are fully exposed, and the mydriatic agent is dripped.
4) Electrode installation: preheating a electroretinography instrument (Roland Visual Electrophysiology System, Roland Consult. Heidelberger. Germany), implanting a needle type grounding electrode under the root of the tail end of the mouse, and terminating the other end of the needle type grounding electrode with a grounding interface of an amplifier; the double-ended needle electrode is inserted into the nape skin (approximately in the middle of the ears) and simultaneously connected with the negative interfaces of the two channels; the gold ring electrode was clamped to the electrode holder of the animal experiment platform and carefully angled to slightly contact the central apex of the cornea. One channel anode is connected with the right eye, and the two channel anode is connected with the left eye. The needle tube is used for dripping physiological saline on the eyes, so that the contact effect of the gold ring electrode and the cornea is improved. The two gold ring electrodes are ensured to contact the same position of the central positive end of the cornea of two eyes in the same angle and mode.
5) And after the oscillography signal is recorded to confirm that the oscillography signal is correct, the dark red light is turned off. An ERG detection with a dark adapted intensity of 0.003cd/s · m2 can be recorded first in an attempt to confirm the quality of the signal: if a large difference from the expected amplitude occurs in the amplitudes of both eyes, it is recommended to check the mounting position of the gold ring electrode again. Then the signal of dark adapted light intensity of 0.03/0.3/3.0/10.0cd/sm2 is recorded in turn, after which the system will automatically turn on the background light.
Results as shown in figure 4, at 6 months, KO mice had a significant decrease in both a-wave and b-wave under dark and light adaptation conditions compared to WT mice, indicating that Tmem184b gene knock-out resulted in impaired vision in the mice (figure 4).
Experimental example 4
In this example, an immunostaining experiment of frozen retinal sections was performed.
The Tmem184b gene knockout mouse constructed in example 1 at 6 months of age was taken, and after the neck was cut off, the eyeball was quickly taken and placed in 4% PFA, and after fixation on ice for 15min, the mouth was cut on the cornea, and then fixation on ice was continued. After 2h, the eyes were rinsed 3 times with PBS buffer, dehydrated in 30% sucrose solution for 2h, dissected under a lens to remove the cornea and crystals, OCT embedded and rapidly frozen in liquid nitrogen. After about 10min, the OCT embedded eyeball is taken out and placed in a freezing microtome to be balanced at-25 ℃ for about 30min, and then the section can be obtained. The slice thickness was 12 μm.
After the slicing is completed, selecting the high-quality slices, placing the slices in an oven at 37 ℃ for 30min, then drawing circles on the places with the retinal tissues by an immunohistochemical pen, washing the places with PBS for three times to remove OCT, then sealing and permeating 5% NDS (containing 0.25% Triton) for 2h, incubating primary antibodies (selecting corresponding antibodies according to different targets), and standing overnight at 4 ℃. On the following day, after three washes with PBS, the corresponding fluorescent secondary antibody was incubated, then washed three more times with PBS, mounted, and observed.
The results are shown in fig. 5, and it can be seen that, at the age of 6 months in mice, after staining PRPH2 and Na/K atpase antibody by retinal frozen tissue sections, Tmem184b knockout mice have shortened outer nuclear layer of retina and rod outer segment compared to wild type mice, indicating that Tmem184b knockout results in knockout mouse retinal rod cell dysplasia.
Experimental example 5
In this example, a retinal cryosection immunostaining experiment (immunization with cone Arrestin antibody) was performed.
Frozen sections of the retina of experiment example 4 were taken and cone cell changes were detected by cone Arrestin antibody immunofluorescence staining in combination with peanut lectin staining.
The results are shown in fig. 6, and the number of cone cells in retinas of six-month-old knockout mice is dramatically reduced compared with the number of cone cells in wild-type controls, which indicates that Tmem184b gene knockout results in loss of cone cells in knockout mice retinas and impaired vision.
In summary, the present invention is exemplified by a mouse, which shows characteristics of retinal degenerative diseases after knockout of the Tmem184b gene. For example: the retina structure becomes thin, especially the outer nuclear layer is obviously thinned; rod cell dysplasia and short outer segment in photoreceptor cell; cone loss, impaired visual function as detected by ERG, etc. Therefore, the Tmem184b knockout mouse can be used as a disease model of retinal degeneration.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Sichuan academy of medical sciences, Sichuan academy of people
<120> construction method and application of retinal degenerative disease model
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
gctgtggacc atggctcta 19
<210> 3
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<212> DNA
<213> Artificial sequence
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Claims (10)
1. A method for constructing a retinal degenerative disease model is characterized by comprising the following steps: tmem184b gene in the genome of the non-human target animal was rendered non-expressed or expression inhibited by genetic engineering techniques.
2. The method of claim 1, wherein all or a part of exons of Tmem184b gene in the genome of the non-human target animal are not expressed or expression of the exons is inhibited by genetic engineering techniques.
3. The method of constructing a model for a retinal degenerative disease according to claim 1 or 2, wherein the non-human target animal is a non-human target animal having a Tmem184b homologous gene;
preferably, the non-human target animal is a mouse, rat, rabbit, cow, dog, pig, horse, sheep, monkey, or ape; preferably, the non-human target animal is a mouse.
4. The method of claim 3, wherein at least one exon of exons 1 to 17 of Tmem184b gene in the genome of the non-human target animal is not expressed or is inhibited from being expressed;
preferably, when the non-human target animal is a mouse, the exon 4-6 sequences of the Tmem184b gene in the genome of the non-human target animal are rendered non-expressed or expression is inhibited.
5. The method for constructing a model of a retinal degenerative disease according to claim 1 or 2, wherein the genetic engineering technique is: any one or a combination of techniques of gene editing, gene knockout, and RNA interference.
6. The method for constructing an animal model of retinal vascular disease according to claim 5, wherein the gene editing technique is at least one selected from the group consisting of ZFN technique, TALEN technique, CRISPR/Cas9 technique, and DNA homologous recombination technique;
the gene knockout technology is selected from complete gene knockout technology or conditional gene knockout technology.
7. The method of claim 5, wherein the method comprises mating Tmem184b knockout non-human target animals to obtain a Tmem184b knockout homozygous retinal degenerative disease model.
8. Use of a retinal degenerative disease model constructed by the method for constructing a retinal degenerative disease model according to any one of claims 1 to 7 for the study of retinal degenerative diseases for the purpose of diagnosis or treatment of non-diseases.
9. The use of the retinal degenerative disease model constructed by the method for constructing a retinal degenerative disease model according to any one of claims 1 to 7 in the early molecular screening of drugs or in the screening of drugs for the targeted treatment of retinal degenerative diseases.
10. The use of claim 9, wherein the retinal degenerative disease is manifested by at least one of the following symptoms:
thinning of retinal structures;
and retinal rod cell dysplasia.
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| CN109666743A (en) * | 2019-01-31 | 2019-04-23 | 上海市长宁区妇幼保健院 | A kind of cervical carcinoma molecular marker and its application |
| WO2021151059A1 (en) * | 2020-01-24 | 2021-07-29 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Compounds and methods for treating or reducing pruritus |
| CN113621649A (en) * | 2021-09-14 | 2021-11-09 | 商丘市第一人民医院 | Construction method and application of retinal pigment degeneration disease model |
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| CN109666743A (en) * | 2019-01-31 | 2019-04-23 | 上海市长宁区妇幼保健院 | A kind of cervical carcinoma molecular marker and its application |
| WO2021151059A1 (en) * | 2020-01-24 | 2021-07-29 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Compounds and methods for treating or reducing pruritus |
| CN113621649A (en) * | 2021-09-14 | 2021-11-09 | 商丘市第一人民医院 | Construction method and application of retinal pigment degeneration disease model |
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| Title |
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| MARTHA R. C. BHATTACHARYA等: ""TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance"", 《THE JOURNAL OF NEUROSCIENCE》, vol. 36, no. 17, 27 April 2016 (2016-04-27), pages 4681 - 4689 * |
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