WO2019080667A1 - 一种共济失调动物模型的构建方法以及应用 - Google Patents

一种共济失调动物模型的构建方法以及应用

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WO2019080667A1
WO2019080667A1 PCT/CN2018/106132 CN2018106132W WO2019080667A1 WO 2019080667 A1 WO2019080667 A1 WO 2019080667A1 CN 2018106132 W CN2018106132 W CN 2018106132W WO 2019080667 A1 WO2019080667 A1 WO 2019080667A1
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ataxia
animal model
candidate agent
tmem30a
phenomenon
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PCT/CN2018/106132
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English (en)
French (fr)
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朱献军
鲁芳
杨业明
张琳
李姝锦
杨牧
张姗姗
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四川省人民医院
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Priority to AU2018357499A priority Critical patent/AU2018357499A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the present disclosure relates to the field of medical engineering technology, and in particular to a method and application for constructing an ataxia animal model.
  • Ataxia is a pathological condition in which patients cannot maintain precise gait in a certain form and perform precise movements. Any lesion involving cerebellar afferent or efferent pathways may cause ataxia. Most of them are caused by genetic factors, so they are collectively called Hereditary ataxia (HA). HA is a group of hereditary degenerative diseases characterized by chronic progressive cerebellar ataxia; the genetic background, ataxia, and cerebellar damage-related pathological changes passed down from generation to generation are three characteristics. The classification of hereditary ataxia is still unclear, and there are more than 60 reports reported so far, but there is no uniform and accepted classification method.
  • Cerebellar ataxia is the main type of hereditary ataxia, which is a large class of neurodegenerative diseases with obvious genetic heterogeneity.
  • the main symptoms are gait instability, limb weakness, and cerebellar symptoms are prominent features, accompanied by Cognitive impairment.
  • the cerebellar ataxia has a variety of genetic models, including autosomal dominant, recessive inheritance, X-linked inheritance and other genetic models, as well as some sporadic cases. At present, more than 30 kinds of cerebellar ataxia subtypes have been discovered, accounting for 10% to 15% of hereditary diseases of the nervous system.
  • cerebellar ataxia The clinical symptoms of cerebellar ataxia are complex and staggered, and even the same family can exhibit high heterogeneity. And its clinical involvement is extensive, there is a large overlap between the clinical phenotypes of each type, and clinical classification is very difficult, so the final diagnosis of cerebellar ataxia must rely on genetic testing.
  • the pathogenic genes are cumbersome and complicated, which brings great difficulties for diagnosis.
  • there is currently no specific effective treatment for cerebellar ataxia in clinical practice the main reason is the lack of detailed pathogenesis research. In the future, the diagnosis and treatment of cerebellar ataxia will depend on the current research on the discovery of pathogenic genes and the pathogenesis.
  • the purpose of the present disclosure is to include, but is not limited to, a method for constructing an ataxia animal model, which can construct a cerebellar ataxia animal model specifically knocking out the Tmem30a gene in cerebellar Purkinje cells, in order to study its The function of the cerebellum Purkinje cells.
  • Another object of the present disclosure is to provide, but is not limited to, to provide an ataxia animal model obtained by the above construction method.
  • Another object of the present disclosure is to include, but is not limited to, the use of an ataxia animal model obtained by the above construction method in the study of ataxia.
  • the sequence of interest is the Tmem30a gene.
  • the method for constructing an ataxia animal model can construct an animal model of cerebellar ataxia by specifically knocking out the Tmem30a gene sequence on the genome of the cerebellar Purkinje cell of the target animal.
  • Typical features of cerebellar ataxia gait instability, cerebellar atrophy, progressive apoptosis of Purkinje cells.
  • This model can be used for the study of cerebellar ataxia, providing a basis for the discovery of pathogenic genes and the exploration of pathogenic mechanisms of cerebellar ataxia.
  • 1 is a Temm30a specific model construction route and a knockout mouse genotype identification map of the cerebellar Purkinje cell provided by the embodiments of the present disclosure.
  • tdTomato is a transgenic red fluorescent protein tomato reporter gene, and has a terminator sequence in front of the initial codon of the red fluorescent protein;
  • the LoxP sites are arranged in the same direction at both ends, and the terminator will be deleted when the Cre enzyme is expressed, the red fluorescent protein can be expressed, and the Cre positive cells are labeled only red).
  • Figure 3 is an immunofluorescence staining (IHC) provided by an embodiment of the present disclosure, indicating that Pcp2-Cre is specifically expressed in mouse cerebellar Purkinje cells (in the figure: GCL: Granular cell layer, granule cell layer; PCL: Purkinje cell layer) , Purkinje cell layer; ML: Molecular layer, molecular layer; Calbindin is a marker protein expressed in cerebellar Purkinje cells).
  • IHC immunofluorescence staining
  • WB Western blot
  • Fig. 5 is a photograph showing the hindlimb contracture of the Purkinje cell-specific knockout Tmem30a KO mouse when lifting the tail according to the embodiment of the present disclosure (in the figure: 6mo means 6 months old).
  • Figure 6 is a photograph of cerebellar atrophy of Tmem30a KO mice provided in an embodiment of the present disclosure (in the figure: 10 mo means 10 months old).
  • Figure 7 is a staining result of paraffin sections provided by an embodiment of the present disclosure.
  • Figure 8 is a progressive reduction of cerebellar Purkinje cells in Tmem30a KO mice according to an embodiment of the present disclosure.
  • FIG. 9 is an immunohistochemical staining map of endoplasmic reticulum stress marker proteins Chop and PDI in Tmem30a KO cerebellum according to an embodiment of the present disclosure;
  • A is a medium endoplasmic reticulum stress marker protein C in Tmem30a KO cerebellar slice
  • EBP homologous protein CHOP
  • the arrow indicates the cells with endoplasmic reticulum stress, it can be seen that the expression of CHOP protein in Tmem30a KO cerebellum is significantly increased.
  • B is the endoplasmic reticulum stress marker in Tmem30a KO cerebellar slice.
  • PDI protein Protein Disulfide Isomerase
  • PC Purkinje cell
  • P4-ATPase is an enzyme with phospholipid inversion activity on the cell membrane, so it is also called phospholipid invertase, which is very important for maintaining the asymmetric distribution of phospholipids on both sides of the membrane.
  • Membrane microenvironment is stable, membrane protein function, cell vesicle transport, cell polarity, apoptosis and so on.
  • P4-ATPase Some of these phospholipids are concentrated on the cytoplasmic side, such as PE and PS, and this asymmetric distribution depends on the activity of the P4-ATPase on the membrane.
  • the mammalian genome encodes 14 different P4-ATPases, and defects in P4-ATPase can lead to a variety of diseases, suggesting that their function in vivo is critical.
  • the Cdc50 family (Cdc50a, b, c, also known as Tmem30a, b, c) is capable of interacting with the P4-ATPase and is required for proper folding and subcellular localization of the P4-ATPase and is therefore considered to be P4- The beta subunit of ATPase. Since there are 14 kinds of P4-ATPase in animals, they are redundant in the tissues and cross- influence, so it is difficult to study their functions.
  • Tmem30a is the best choice.
  • the disclosure of the present disclosure provides a basis for further study of the pathogenic mechanism leading to cerebellar ataxia by constructing a mouse model specifically knocking out Tmem30a in the cerebellar Purkinje cell.
  • an embodiment of the present disclosure provides a method for constructing an ataxia animal model, which knocks out a target sequence on a genome in a cerebellar Purkinje cell of a target animal;
  • the sequence of interest is the Tmem30a gene.
  • the sequence of interest is an exon sequence on the Tmem30a gene.
  • the Tmem30a gene has four exons (exon 1 exon, exon 2, exon 3, exon 4), therefore, one or several exons are knocked out arbitrarily. Sequences can inactivate the Tmem30a gene for knockout purposes.
  • the target animal is selected from any one of the group consisting of a mouse, a rat, a dog, a monkey, and a donkey.
  • the target animal is a mouse and the exon sequence is an exon 3 sequence.
  • the exon 1 exon sequence, the exon 2 exon sequence or the exon 4 exon sequence may be selected, or a combination thereof may be used, which belongs to the present disclosure.
  • sequence of interest is knocked out using the Cre-loxP knockout technique.
  • the Tmem30a gene conditional knockout homozygous mouse can also be directly mated with the transgenic Pcp2-Cre gene mouse to obtain a cerebellar Purkinje cell conditional knockout Tmem30a gene mouse.
  • Tmem30a gene conditional knockout homozygous mouse can be obtained by referring to Chinese Patent Application No. 2017103803265, entitled "Construction Method and Application of Mouse Model of Islet ⁇ Cell Conditional Knockout Tmem30a Gene”.
  • the embodiments of the present disclosure also provide an application of the ataxia animal model obtained by the above-described construction method of the ataxia animal model in the study of ataxia.
  • the study is a human ataxia pathogenesis study or pathogenic mechanism study.
  • the present disclosure provides the use of an ataxia animal model obtained by the above-described method of constructing an ataxia animal model for screening a medicament for preventing or treating an ataxia disease.
  • the applying comprises: applying a candidate agent to the ataxia animal model
  • the candidate agent is indicated as a drug for preventing or treating an ataxia disease:
  • Phenomenon (1) The ataxia animal model to which the candidate agent was applied showed that the gait was normal or more stable, or that the hind limb did not collapse when the tail was lifted, compared to the ataxia animal model to which the candidate agent was not applied. Or a degree of contraction that is lower than an ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (2) Compared to the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the cerebellum did not show progressive atrophy or atrophy with age. An ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (3) Compared to the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the Purkinje cells in the cerebellum did not decrease with age. The phenomenon or the reduction of Purkinje cells in the cerebellum is lower than that of the ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (4) Compared with the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the endoplasmic reticulum stress marker protein CHOP expression was lower than the candidate was not applied. An anemia model of the agent.
  • the present disclosure provides an ataxia animal model in which the Tmem30a gene on the genome of the cerebellar Purkinje cells is knocked out.
  • the animal model is a mouse, a rat, a dog, a monkey or a donkey.
  • the animal model is a mouse, and exon 3 of the Tmem30a gene on the genome in the cerebellar Purkinje cell of the animal model is knocked out.
  • the present disclosure provides the use of an ataxia animal model as described above in an ataxia study.
  • the study is a human ataxia pathogenesis study or pathogenic mechanism study.
  • the present disclosure provides a method of screening for a medicament for preventing or treating an ataxia disease comprising: applying a candidate agent to the above-described ataxia animal model.
  • Phenomenon (1) The ataxia animal model to which the candidate agent was applied showed that the gait was normal or more stable, or that the hind limb did not collapse when the tail was lifted, compared to the ataxia animal model to which the candidate agent was not applied. Or a degree of contraction that is lower than an ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (2) Compared to the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the cerebellum did not show progressive atrophy or atrophy with age. An ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (3) Compared to the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the Purkinje cells in the cerebellum did not decrease with age. The phenomenon or the reduction of Purkinje cells in the cerebellum is lower than that of the ataxia animal model to which the candidate agent is not applied;
  • Phenomenon (4) Compared with the ataxia animal model to which the candidate agent was not applied, the ataxia animal model to which the candidate agent was applied showed that the endoplasmic reticulum stress marker protein CHOP expression was lower than the candidate was not applied. An anemia model of the agent.
  • a mouse as a target animal is taken as an example, and a method for constructing an ataxia animal model provided by the present disclosure will be described, specifically as follows.
  • the Tmem30a gene (exon 3 exon) conditional knockout homozygous is obtained by referring to the construction method of the mouse model of the conditional knockout Tmem30a gene with the application number of 2017103803265, and the Chinese patent application method. mouse;
  • Tmem30a gene conditional knockout homozygous mice were mated with transgenic Pcp2-Cre gene mice to obtain cerebellar Purkinje cell conditional knockout Tmem30a gene mice (Fig. 1A).
  • the transgenic Pcp2-Cre gene mice were mated with the Tmem30a gene conditional knockout homozygous mice, and half of the offspring of the offspring carried both Pcp2-Cre and Tmem30a conditional knockouts.
  • This animal was mated with a Tmem30a gene conditional knockout homozygous mouse to obtain a cerebellar Purkinje cell-specific knockout Tmem30a mutant animal (expressed as Tmem30a KO).
  • Cre enzyme in the Pcp2-Cre gene Purkinje cells enables the conditional knockout of the Tmem30a gene in the cerebellar Purkinje cells.
  • FIG. 1 The results in Figure 1 show (in the figure: WT represents wild-type mice, Tmem30a +/- , Pcp2-Cre represents the conditional knockout of the CME Purkinje cells, Tmem30a heterozygous mice, Tmem30 -/- , Pcp2-Cre represents the cerebellum Purkinje cells conditionally knocked out the Tmem30a gene homozygous mouse, referred to herein as Tmem30a KO), and obtained a conditional knockout Tmem30a gene homozygous mouse of the cerebellum Purkinje cell.
  • this example constructed the tdTomato reporter gene ( Figure 2).
  • the STOP gene cassette with loxP recombination sites on both sides prevents the expression of the downstream red fluorescent protein tdTomato.
  • Cre is present, the STOP gene cassette is removed in the tissue specifically expressed by Cre, resulting in the expression of tdTomato, and thus the expression position and expression efficiency of Cre recombinase can be determined by observing the fluorescence generated by tdTomato expression.
  • Cre was specifically expressed in cerebellar Purkinje cells in Tmem30a KO animals (as shown in Figure 3).
  • the label in Fig. 3 means: GCL: Granular cell layer, granule cell layer; PCL: Purkinje cell layer, Purkinje cell layer; ML: Molecular layer, molecular layer).
  • the eyeballs were quickly taken and placed in 4% PFA. After fixing for 15 minutes on ice, a hole was cut on the cornea and then fixed on ice. After 2 h, the PBS buffer was washed 3 times, then the eyeball was dehydrated in a 30% sucrose solution for 2 h, then the cornea and crystals were cut off under a dissecting microscope, OCT was embedded and quickly frozen in a -80 ° C refrigerator. After about 10 minutes, the OCT-embedded eyeballs were taken out and placed in a frozen slicer at -25 ° C for about 30 min to be sliced. The slice thickness was 12 ⁇ m.
  • the higher quality film is placed in an oven at 37 ° C for 30 min, then the immunohistochemical pen is circled in the place with retinal tissue, washed three times with PBS to remove OCT, and then 5% of NDS (containing 0.25% Triton) The cells were blocked for 2 h, and the primary antibody was incubated at 4 ° C overnight. The next day, after PBS was washed twice, the corresponding fluorescent secondary antibody was incubated, then washed twice with PBS, mounted, and observed.
  • TMEM30A protein in Tmem30a KO mice was verified by western blot (WB).
  • the membrane was washed 3 times for 10 minutes each time with 1 ⁇ TBST, and the protein was detected by Thermo's ELC luminescence kit.
  • the instrument used was Bio-Rad's chemiluminescent gel imaging system.
  • a mouse model Tmem30a KO specifically knocking out Tmem30a in cerebellar Purkinje cells can be provided.
  • Tmem30a KO knockout mice had symptoms of cerebellar ataxia. Specifically, the gait is unstable, and the tail hind limb contracture is lifted (Fig. 5). In Fig. 5, 6mo means 6 months old.
  • Fig. 6 The cerebellum of the knockout mice was dissected and it was found that the knockout mice progressively atrophied with the age (Fig. 6, in which: WT refers to the wild type control, and KO refers to the Purkinje cell knockout animal; 10mo refers to 10 months of age; Cross-sectional area of cerebellum refers to cerebellar longitudinal section area), and the cerebellar longitudinal section is only 50% of WT mice at 10 months of age.
  • the cerebellum of the mice of different ages was subjected to paraffin sectioning, and H&E staining was performed on the sections, and it was found that the cerebellar molecular layer (ML) was gradually thinned and the granule cells were decreased (Fig. 7).
  • WT refers to a wild type control
  • KO refers to a Tmem30a Purkinje cell knockout animal
  • P42 means 42 days old
  • 5mo means 5 months old
  • 12mo means 12 months old.
  • Calbindin refers to calcium binding protein, which is a Purkinje cell marker protein
  • DAPI refers to 4',6-diamidino-2-phenylindole
  • P16 means 16 days old
  • P25 means 25 days old
  • P30 means 30 days old
  • P42 means 42 days old.
  • Tmem30a knockout causes endoplasmic reticulum stress.
  • cells undergo endoplasmic reticulum stress they activate apoptosis-related pathways, which ultimately lead to apoptosis.
  • the embodiment of the present disclosure constructs a mouse model for specifically knocking out the Tmem30a gene in the cerebellar Purkinje cell, which shows typical characteristics of cerebellar ataxia: gait instability, cerebellar atrophy, Purkin Wild cells undergo progressive apoptosis and the like.
  • This model can be used for the study of cerebellar ataxia.
  • the construction method of the ataxia animal model provided by the present disclosure can construct a cerebellar ataxia animal model in which islet ⁇ cells specifically knock out Tmem30a in cerebellar Purkinje cells, and the model exhibits typical ataxia.
  • the mouse model can be used in the field of ataxia research or screening for drugs for treating or preventing ataxia, and provides a model basis for further understanding the pathogenesis of ataxia and screening for ataxia drugs.

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Abstract

提供了一种共济失调动物模型的构建方法，其包括敲除目标动物的小脑浦肯野细胞中的基因组上的目的序列；所述目的序列为Tmem30a基因。还提供了该方法构建的小脑共济失调的动物模型以及利用该动物模型筛选用于预防或治疗共济失调疾病的药物的方法。

Description

一种共济失调动物模型的构建方法以及应用
本申请要求于2017年10月26日提交中国专利局的申请号为201711012105.9、名称为“一种共济失调动物模型的构建方法以及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及医学工程技术领域，具体而言，涉及一种共济失调动物模型的构建方法以及应用。
背景技术
共济失调(Ataxia)是患者不能按一定的形式维持精细步态、完成精确动作的一种病理状态，任何涉及小脑传入或传出途径的病变都可能导致共济失调。其中多数由遗传因素所致,故统称为遗传性共济失调(Hereditary ataxia，HA)。HA是一组以慢性进行性小脑性共济失调为特征的遗传变性病；世代相传的遗传背景、共济失调表现及小脑损害为主的病理改变是三大特征。遗传性共济失调分类尚不明晰，至今报道的已有60多种，但尚无统一和公认的分类方法。根据其病发部位可分为四种类型：①深感觉障碍共济失调；②小脑共济失调；③前庭迷路共济失调；④大脑型共济失调。小脑共济失调是遗传性共济失调主要类型，是—大类具有明显遗传异质性的神经系统变性疾病，主要症状为步态不稳，肢体无力，以小脑症状为突出特点，并伴有认知障碍。小脑共济失调遗传模式多样，包括常染色体显性、隐性遗传，X-连锁遗传等多种遗传模式，还包括一些散发病例。目前已发现了30余种小脑共济失调亚型，约占神经系统遗传性疾病的10％～15％。
小脑共济失调的临床症状复杂、交错重叠，即使同一家族也可表现高度异质性。并且其临床累及面广，各型之间临床表型存在较大的重叠，临床分型非常困难，因此最终诊断小脑共济失调必须依靠基因检测。但因其分型众多，致病基因繁冗复杂，为诊断带来极大困难。此外，目前临床上尚无针对小脑共济失调具体有效的治疗方法，其主要原因就是缺乏详细的发病机理研究。而未来对于小脑共济失调的诊断和治疗还要依赖于目前对其致病基因的发现和致病机理的研究。
而目前，缺乏相关的可用于共济失调研究的动物模型。
鉴于此，特提出本公开。
公开内容
本公开的目的在于包括但不限于提供一种共济失调动物模型的构建方法，该构建方法可以构建在小脑浦肯野细胞中特异敲除Tmem30a基因的小脑共济失调动物模型，以便研究其在小脑浦肯野细胞中的功能。
本公开的另一目的在于包括但不限于提供一种由上述构建方法得到的共济失调动物模型。
本公开的另一目的在于包括但不限于提供一种由上述构建方法得到的共济失调动物模型在共济失调研究中的应用。
本公开是这样实现的：
一种共济失调动物模型的构建方法，敲除目标动物的小脑浦肯野细胞中的基因组上的目的序列；
所述目的序列为Tmem30a基因。
由上述的共济失调动物模型的构建方法所得到的共济失调动物模型在共济失调研究中的应用。
由上述的共济失调动物模型的构建方法所得到的共济失调动物模型在筛选用于预防或治疗共济失调疾病药物中的应用。
一种共济失调动物模型，该动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因被敲除。
本公开具有以下有益效果：
本公开提供的共济失调动物模型的构建方法，首次通过特异性敲除目标动物的小脑浦肯野细胞中的基因组上的Tmem30a基因序列，可以构建出小脑共济失调的动物模型，该动物模型表现出小脑共济失调的典型特征：步态不稳，小脑萎缩，浦肯野细胞进行性凋亡等。该模型可用于小脑共济失调的研究，为小脑共济失调的致病基因的发现和致病机理的探索提供基础。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例提供的小脑浦肯野细胞Tmem30a特异模型构建路线和敲除小鼠基因型鉴定图。
图2为本公开实施例提供的小脑浦肯野细胞中TdTomato报告基因构建示意图(图中：tdTomato是一个转基因红色荧光蛋白Tomato报告基因，在红色荧光蛋白的初始密码子前面有终止子序列；终止子两端带有同向排列的LoxP位点，在有Cre酶表达的时候终止子会被删除，红色荧光蛋白可以表达，Cre阳性细胞被标记惟红色)。
图3为本公开实施例提供的免疫荧光染色(Immunohistochemistry，IHC)表明Pcp2-Cre特异表达于小鼠小脑浦肯野细胞(图中：GCL:Granular cell layer，颗粒细胞层；PCL：Purkinje cell layer，浦肯野细胞层；ML：Molecular layer，分子层；Calbindin是表达于小脑浦肯野细胞中的标记蛋白)。
图4为本公开实施例提供的免疫印迹(Western blot，WB)法验证Tmem30a蛋白在敲除小鼠小脑中表达量降低(图中：WT:Wildtype，野生型对照；KO：Knockout，基因敲除突变；Cerebrum：大脑；Cerebellum：小脑)。
图5为本公开实施例提供的浦肯野细胞特异敲除Tmem30a KO小鼠提起尾部时后肢蜷缩的照片图(图中：6mo是指6个月龄)。
图6为本公开实施例提供的Tmem30a KO小鼠的小脑萎缩照片图(图中：10mo是指10个月龄)。
图7为本公开实施例提供的石蜡切片染色结果。
图8为本公开实施例提供的Tmem30a KO小鼠小脑浦肯野细胞进行性减少。
图9为本公开实施例提供的Tmem30a KO小脑中内质网应激标志蛋白Chop和PDI的免疫组化染色图；(图中A是Tmem30a KO小脑切片中中内质网应激标志蛋白C/EBP homologous protein(CHOP)表达情况，箭头指示出现内质网应激的细胞，可以看出Tmem30a KO小脑中CHOP蛋白表达细胞明显增加。图中B是Tmem30a KO小脑切片中中内质网应激标志蛋白Protein Disulfide Isomerase(PDI)表达情况，箭头指示出现内质网应激的细胞，可以看出Tmem30a KO小脑中PDI表达细胞明显增加。图中C是A，B表达水平的量化)。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开实施例的共济失调动物模型的构建方法以及应用进行具体说明。
小脑共济失调的典型神经病理学改变是小脑萎缩，浦肯野细胞的退行性病变和丢失。浦肯野细胞(Purkinje cell，PC)是从小脑皮质发出的唯一能够传出冲动的神经元。PC轴突穿过颗粒层和白质到达深部小脑核团，PC在运动协调中起着重要的作用。
ATP8A2(一种P4-ATP酶)的基因突变能够导致小脑功能异常，患者表现出小脑共济失调的症状，表明P4-ATP酶的活性与小脑共济失调息息相关。P4-ATP酶是细胞膜上一种具有磷脂内翻活性的酶，故又称为磷脂内翻酶，它对于维持膜两侧的磷脂不对称分布非常重要，膜两侧磷脂不对称分布对于维持质膜微环境稳定，膜蛋白功能发挥，细胞囊泡转运，细胞极性，细胞凋亡等至关重要。其中一些磷脂分布集中在细胞质一侧，例如PE和PS，这种不对称分布就取决于膜上P4-ATP酶的活性。哺乳动物基因组编码14种不同的P4-ATP酶，P4-ATP酶的缺陷可导致多种疾病，说明其在体内的功能至关重要。
Cdc50家族(Cdc50a，b，c，也称为Tmem30a，b，c)能够和P4-ATP酶相互作用，并且对于P4-ATP酶的正确折叠和亚细胞定位是必须的，因此被认为是P4-ATP酶的β亚基。由于P4-ATP酶在动物体内有14种，在组织中功能冗余，相互交叉影响，不易研究其功能。
而其β亚基只有三个，并且绝大多数P4-ATP酶与Tmem30a相互作用，因此想要研究P4-ATP酶的功能，阐明其导致小脑共济失调的致病机理，从其β亚基Tmem30a入手是最佳选择。
综上分析，本公开的公开人通过构建在小脑浦肯野细胞特异敲除Tmem30a的小鼠模型，从而为深入研究其导致小脑共济失调的致病机理提供基础。
一方面，本公开实施例提供了一种共济失调动物模型的构建方法，敲除目标动物的小脑浦肯野细胞中的基因组上的目的序列；
所述目的序列为Tmem30a基因。
进一步地，在本公开的一些实施方案中，所述目的序列为Tmem30a基因上的外显子序列。
Tmem30a基因具有4个外显子(分别是第1号外显子、第2号外显子、第3号外显子、第4号外显子)，因此，任意敲除其中的一个或几个外显子序列，均可使Tmem30a基因失活，实现敲除目的。
进一步地，在本公开的一些实施方案中，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
进一步地，在本公开的一些实施方案中，所述目标动物为小鼠，所述外显子序列为第3号外显子序列。
当然，在其他的实施例中，也可以选择敲除第1号外显子序列、第2号外显子序列或第4号外显子序列，或者是它们之间的组合均可，其均属于本公开的保护范围。
进一步地，在本公开的一些实施方案中，采用Cre-loxP敲除技术敲除所述目的序列。
通常来说，实现基因敲除的技术手段有很多，例如CRISPR/Cas9技术。因此，在其他的实施例中采用CRISPR/Cas9技术或其他的技术手段敲除Tmem30a基因，也属于本公开的保护范围。
当然，在本公开的一些实施方案中，也可以直接将Tmem30a基因条件性敲除纯合子小鼠与转Pcp2-Cre基因小鼠交配，得到小脑浦肯野细胞条件性敲除Tmem30a基因小鼠。
需要说明的是，Tmem30a基因条件性敲除纯合子小鼠的获取可参考申请号为2017103803265、名称为“胰岛β细胞条件性敲除Tmem30a基因小鼠模型的构建方法及应用”的中国专利申请。
另一方面，本公开实施例还提供了由上述的共济失调动物模型的构建方法所得到的共济失调动物模型在共济失调研究中的应用。
进一步地，在本公开的一些实施方案中，所述研究为人类共济失调发病机理研究或致病机制研究。
再一方面，本公开提供了由上述的共济失调动物模型的构建方法所得到的共济失调动物模型在筛选用于预防或治疗共济失调疾病药物中的应用。
进一步地，在本公开的一些实施方案中，所述应用包括：向所述共济失调动物模型施加候选试剂；
如果施加所述候选试剂后，检测到所述共济失调动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗共济失调疾病的药物：
现象(1)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：步态正常或更稳定，或者提起尾部时其后肢未出现蜷缩或蜷缩程度低于未施加该候选试剂的共济失调动物模型；
现象(2)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑未出现进行性萎缩或萎缩程度低 于未施加该候选试剂的共济失调动物模型；
现象(3)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑中的浦肯野细胞未出现减少的现象或小脑中浦肯野细胞减少的幅度低于未施加该候选试剂的共济失调动物模型；
现象(4)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：内质网应激标志蛋白CHOP表达量低于未施加该候选试剂的共济失调动物模型。
再一方面，本公开提供了一种共济失调动物模型，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因被敲除。
进一步地，在本公开的一些实施方案中，所述动物模型为小鼠、大鼠、狗、猴或猿。
进一步地，在本公开的一些实施方案中，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因的第1号外显子、第2号外显子、第3号外显子和第4号外显子中的任意一个外显子被敲除或任意多个外显子同时被敲除。
进一步地，在本公开的一些实施方案中，所述动物模型为小鼠，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因的第3号外显子被敲除。
再一方面，本公开提供了如上所述的共济失调动物模型在共济失调研究中的应用。
进一步地，在本公开的一些实施方案中，所述研究为人类共济失调发病机理研究或致病机制研究。
再一方面，本公开提供了一种筛选用于预防或治疗共济失调疾病药物中的方法，其包括：向上述共济失调动物模型施加候选试剂。
进一步地，在本公开的一些实施方案中，如果施加所述候选试剂后，检测到所述共济失调动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗共济失调疾病的药物：
现象(1)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：步态正常或更稳定，或者提起尾部时其后肢未出现蜷缩或蜷缩程度低于未施加该候选试剂的共济失调动物模型；
现象(2)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑未出现进行性萎缩或萎缩程度低于未施加该候选试剂的共济失调动物模型；
现象(3)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑中的浦肯野细胞未出现减少的 现象或小脑中浦肯野细胞减少的幅度低于未施加该候选试剂的共济失调动物模型；
现象(4)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：内质网应激标志蛋白CHOP表达量低于未施加该候选试剂的共济失调动物模型。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例以小鼠作为目标动物为例，对本公开提供的共济失调动物模型的构建方法进行说明，具体如下。
1.参照申请号为2017103803265、名称为胰岛β细胞条件性敲除Tmem30a基因小鼠模型的构建方法及应用的中国专利申请的方法得到Tmem30a基因(第3号外显子)条件性敲除纯合子小鼠；
2.将Tmem30a基因条件性敲除纯合子小鼠与转Pcp2-Cre基因小鼠交配，得到小脑浦肯野细胞条件性敲除Tmem30a基因小鼠(图1A)。如图1A所示意，转Pcp2-Cre基因小鼠和Tmem30a基因条件性敲除纯合子小鼠交配，后代有一半的动物同时携带Pcp2-Cre和Tmem30a条件性敲除。此动物和Tmem30a基因条件性敲除纯合子小鼠交配，即可得到小脑浦肯野细胞特异敲除Tmem30a突变动物(以Tmem30a KO表示)。
Pcp2-Cre基因浦肯野细胞中表达Cre酶，实现在小脑浦肯野细胞条件性敲除Tmem30a基因的效果。
3.用引物F2：5’-TGGTTAGGAACACATAGAACAAA-3’；和R2：5’-CTAAGGGGCTGGTATGGGAAT-3’鉴定Tmem30a条件性敲除小鼠；用引物CreF：5’-ATTTGCCTGCATTACCGGTC-3’；Cre-R:5’-ATCAACTGGTTCTTTTCGG-3’鉴定Cre基因型，结果如图1B所示。图1结果显示(图中：WT代表野生型小鼠，Tmem30a +/-,Pcp2-Cre代表小脑浦肯野细胞条件性敲除Tmem30a基因杂合子小鼠，Tmem30 -/-,Pcp2-Cre代表小脑浦肯野细胞条件性敲除Tmem30a基因纯合子小鼠，在此简称Tmem30a KO)，得到了小脑浦肯野细胞条件性敲除Tmem30a基因纯合子小鼠。
实施例2
为了确定Cre介导的loxP位点重组效率，本实施例构建了tdTomato报告基因(图2)。在Cre缺失时，两侧带有loxP重组位点的STOP基因盒能够防止下游的 红色荧光蛋白tdTomato的表达。当Cre存在时，在Cre特异表达的组织中STOP基因盒被移除，导致tdTomato的表达，因此能够通过观察tdTomato表达产生的荧光来确定Cre重组酶的表达位置和表达效率。使用免疫组织化学方法，观察到Tmem30a KO动物中Cre在小脑浦肯野细胞特异表达(如图3所示)。图3中标示是指：GCL:Granular cell layer，颗粒细胞层；PCL：Purkinje cell layer，浦肯野细胞层；ML：Molecular layer，分子层)。
免疫组织化学方法：
小鼠断颈处死后，快速取眼球，并放入4％的PFA中，冰上固定15min后，在角膜上剪一个口子，然后继续冰上固定。2h后，PBS缓冲液冲洗3遍，然后将眼球置于30％蔗糖溶液中脱水2h，然后解剖镜下剪去角膜及晶体，OCT包埋并迅速置于-80℃冰箱冷冻。大约10min后，取出OCT包埋的眼球，置于冰冻切片机-25℃平衡约30min后即可切片。切片厚度为12μm。
切片完成后，选取质量较高的片子于37℃烘箱放置30min，然后免疫组化笔在有视网膜组织的地方画圈，PBS洗三遍以去除OCT，然后5％的NDS(含有0.25％Triton)封闭通透2h，孵育一抗，4℃过夜。第二天，PBS清洗两遍后，孵育相应的荧光二抗，然后再用PBS清洗两遍，封片，观察。
实施例3
通过免疫印迹(western blot，WB)验证Tmem30a KO小鼠中的TMEM30A蛋白表达情况。
免疫印迹方法：
①分别分离野生型和突变型小鼠视网膜组织，置于1.5ml离心管，并加入200μl蛋白裂解液RIPA；
②超声破碎视网膜组织后，在冰上裂解20min；
③4℃，16000g离心10min后，取上清转移至另一干净离心管，加入50μl的蛋白上样液，混匀后95℃加热5min；
④待样本冷却后，分别取20μl，160V电压进行聚丙烯酰胺凝胶电泳(SDS-PAGE)以分离蛋白；
⑤SDS-PAGE结束后，根据需要，裁剪适当大小的硝酸纤维素膜，按顺序铺上滤纸、胶、硝酸纤维素膜及滤纸，并赶去气泡，转膜槽放入冰水浴中，采用恒流0.28A的电流进行转膜，转膜2h；
⑥转膜完毕后，纯水冲洗硝酸纤维素膜一遍，晾干并标记。然后用8％的脱脂牛奶封闭2h；
⑦封闭完成后，加入一定量的按一定比例(按照抗体使用说明书)稀释于封闭液的一抗，4℃孵育过夜。
⑧回收一抗，1×TBST缓冲液洗膜4次，每次10min，根据一抗来源，选择合适二抗，用1×TBST稀释辣根过氧化氢酶(HRP)标记的二抗，室温于摇床上孵育2h
⑨二抗孵育结束后，用1×TBST洗膜3次，每次10min，用Thermo的ELC发光试剂盒检测蛋白，所用仪器为Bio-Rad的化学发光凝胶成像系统。
结果见图4，图中WT代表野生型小鼠，KO代表Tmem30a KO小鼠。Cerebrum是指大脑组织；Cerebellum是指小脑组织。免疫印迹实验证明了在Tmem30a KO小鼠小脑浦肯野细胞中，Tmem30a蛋白被特异敲除(图4)。由于小脑中还有颗粒细胞、胶质细胞，在这两种细胞中Tmem30a未被敲除，所以在小脑中Tmem30a的表达量只是部分降低。
根据上述结果可以提供出在小脑浦肯野细胞中特异敲除Tmem30a的小鼠模型Tmem30a KO。
实施例4
利用构建的Tmem30a基因敲除小鼠模型进行小脑共济失调研究
1Tmem30a KO小鼠出现小脑共济失调症状
对Tmem30a KO行为观察以及后肢紧抱实验(Hind-limb clasping test)表明Tmem30a KO敲除小鼠有小脑共济失调的症状。具体表现为步态不稳，提起尾部后肢蜷缩(图5)。图5中6mo是指6个月龄。
2Tmem30a KO小鼠小脑进行性萎缩
对敲除小鼠的小脑进行了解剖，发现敲除小鼠随着年龄的增长小脑进行性萎缩(图6，图中：WT是指野生型对照，KO是指浦肯野细胞敲除动物；10mo是指10月龄；Cross-sectional area of cerebellum是指小脑纵切面面积)，在10月龄时小脑纵切面积只有WT小鼠的50％。并对不同年龄段小鼠的小脑进行石蜡切片，对切片做H&E染色，发现小脑分子层(ML)逐渐变薄，颗粒细胞减少(图7)。图7中WT是指野生型对照，KO是指Tmem30a浦肯野细胞敲除动物；P42是指42天龄，5mo是指5月龄，12mo是指12月龄。
3Tmem30a KO小鼠小脑浦肯野细胞进行性减少
对敲除小鼠的小脑进行了冰冻切片、免疫组织化学染色分析，对浦肯野细胞标志蛋白Calbindin-D28K染色，发现KO小鼠随着年龄增长，小脑中浦肯野细胞逐渐减少，在出生后42天(P42)时敲除小鼠中浦肯野细胞几乎全部死亡(图8)。图8中：Calbindin是指钙结合蛋白，是浦肯野细胞标志蛋白；DAPI是指4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole)，是一种可以较强结合DNA的荧光染料，常用于荧光显微镜观测。P16是指16天龄，P25是指25天龄,P30是指30天龄,P42是指42天龄。
4Tmem30a KO小鼠小脑浦肯野细胞出现内质网应激(ER stress)
为探明浦肯野细胞死亡原因，对敲除小鼠的小脑冰冻切片做免疫组织化学染色分析。由于Tmem30a与胞内囊泡转运相关，因此它的缺失可能会引起胞内分泌小泡转运紊乱，导致内质网应激。因此我们对小脑冰冻切片进行内质网应激标志蛋白CHOP和PDI染色，发现在出生后20天时，Tmem30a KO小鼠浦肯野细胞CHOP和PDI的表达量明显增加(图9)。图9中标示是指：GCL：Granular cell layer，颗粒细胞层；PCL：Purkinje cell layer，浦肯野细胞层；ML：Molecular layer，分子层)；P20是指20天龄。与野生型对照比较，Tmem30a KO小脑组织中内质网应激蛋白标志CHOP表达增加了近6倍，PDI增加了近4倍(图9C)。Tmem30a敲除后会导致内质网应激。细胞出现内质网应激后会激活细胞凋亡相关通路，最终导致细胞凋亡。
综上所述，本公开实施例首次构建了在小脑浦肯野细胞特异敲除Tmem30a基因的小鼠模型，该模型表现出小脑共济失调的典型特征：步态不稳，小脑萎缩，浦肯野细胞进行性凋亡等。该模型可用于小脑共济失调的研究。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性：本公开提供的共济失调动物模型的构建方法可以构建出胰岛β细胞在小脑浦肯野细胞中特异敲除Tmem30a的小脑共济失调动物模型，该模型表现出典型的共济失调特征，该小鼠模型可以用于共济失调研究或用于筛选出治疗或预防共济失调的药物等领域，为进一步了解共济失调的发病机制、筛选共济失调药物提供了模型基础。

Claims (17)

  1. 一种共济失调动物模型的构建方法，其特征在于，敲除目标动物的小脑浦肯野细胞中的基因组上的目的序列；
    所述目的序列为Tmem30a基因。
  2. 根据权利要求1所述的共济失调动物模型的构建方法，其特征在于，所述目的序列为Tmem30a基因上的外显子序列。
  3. 根据权利要求2所述的共济失调动物模型的构建方法，其特征在于，所述目标动物选自小鼠、大鼠、狗、猴以及猿中的任意一种。
  4. 根据权利要求3所述的共济失调动物模型的构建方法，其特征在于，所述目标动物为小鼠，所述外显子序列为第3号外显子序列。
  5. 根据权利要求4所述的共济失调动物模型的构建方法，其特征在于，其包括：将Tmem30a基因条件性敲除纯合子小鼠与转Pcp2-Cre基因小鼠交配，得到小脑浦肯野细胞条件性敲除Tmem30a基因小鼠。
  6. 由权利要求1-5中任一项所述的共济失调动物模型的构建方法所得到的共济失调动物模型在共济失调研究中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述研究为人类共济失调发病机理研究或致病机制研究。
  8. 由权利要求1-5中任一项所述的共济失调动物模型的构建方法所得到的共济失调动物模型在筛选用于预防或治疗共济失调疾病药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述应用包括：向所述共济失调动物模型施加候选试剂；
    如果施加所述候选试剂后，检测到所述共济失调动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗共济失调疾病的药物：
    现象(1)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：步态正常或更稳定，或者提起尾部时其后肢未出现蜷缩或蜷缩程度低于未施加该候选试剂的共济失调动物模型；
    现象(2)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑未出现进行性萎缩或萎缩程度低于未施加该候选试剂的共济失调动物模型；
    现象(3)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑中的浦肯野细胞未出现减少的 现象或小脑中浦肯野细胞减少的幅度低于未施加该候选试剂的共济失调动物模型；
    现象(4)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：内质网应激标志蛋白CHOP表达量低于未施加该候选试剂的共济失调动物模型。
  10. 一种共济失调动物模型，其特征在于，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因被敲除。
  11. 根据权利要求10所述的共济失调动物模型，其特征在于，所述动物模型为小鼠、大鼠、狗、猴或猿。
  12. 根据权利要求10或11所述的共济失调动物模型，其特征在于，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因的第1号外显子、第2号外显子、第3号外显子和第4号外显子中的任意一个外显子被敲除或任意多个外显子同时被敲除。
  13. 根据权利要求10-12任一项所述的共济失调动物模型，其特征在于，所述动物模型为小鼠，所述动物模型的小脑浦肯野细胞中的基因组上的Tmem30a基因的第3号外显子被敲除。
  14. 权利要求10-12任一项所述的共济失调动物模型在共济失调研究中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述研究为人类共济失调发病机理研究或致病机制研究。
  16. 一种筛选用于预防或治疗共济失调疾病药物中的方法，其特征在于，其包括：向所述共济失调动物模型施加候选试剂。
  17. 根据权利要求16所述的方法，其特征在于，如果施加所述候选试剂后，检测到所述共济失调动物模型出现以下现象中的一个或一个以上，则指示该候选试剂可以作为用于预防或治疗共济失调疾病的药物：
    现象(1)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：步态正常或更稳定，或者提起尾部时其后肢未出现蜷缩或蜷缩程度低于未施加该候选试剂的共济失调动物模型；
    现象(2)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑未出现进行性萎缩或萎缩程度低于未施加该候选试剂的共济失调动物模型；
    现象(3)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：随着年龄的增长，小脑中的浦肯野细胞未出现减少的现象或小脑中浦肯野细胞减少的幅度低于未施加该候选试剂的共济失调动物模型；
    现象(4)：相较于未施加该候选试剂的共济失调动物模型，施加了该候选试剂的共济失调动物模型表现出：内质网应激标志蛋白CHOP表达量低于未施加该候选试剂的共济失调动物模型。
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