WO2022120916A1 - 一种提取神经突触体的方法 - Google Patents

一种提取神经突触体的方法 Download PDF

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WO2022120916A1
WO2022120916A1 PCT/CN2020/137128 CN2020137128W WO2022120916A1 WO 2022120916 A1 WO2022120916 A1 WO 2022120916A1 CN 2020137128 W CN2020137128 W CN 2020137128W WO 2022120916 A1 WO2022120916 A1 WO 2022120916A1
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陈宇
屈雪琪
林力
许进英
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深圳先进技术研究院
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Definitions

  • the present application belongs to the technical field of molecular biology, and relates to a method for extracting neural synaptosomes.
  • Synapses are key sites for functional connections and signaling between neurons, connecting billions of neurons into neuronal and glial circuits, forming the basis for information processing and driving behavior, and in regulating human learning. , memory, and thinking styles play a key role. Structural and functional damage to synapses can lead to brain dysfunction.
  • RNA in situ hybridization technology has revealed the existence of a large amount of RNA in neuronal dendrites, axons and synapses, and the RNA is diverse, and a large number of studies have demonstrated the local translation of certain proteins at these sites.
  • RNA is diverse, and a large number of studies have demonstrated the local translation of certain proteins at these sites.
  • the characteristics and mechanisms of transcription and translation are still unclear, and a lot of research work is still needed, such as gene transcription level analysis.
  • the technology is still immature, which limits the development of related research.
  • Synaptodendrosome refers to synaptosomes and their pinched synaptic terminals and connected dendritic segments, including enriched around synaptosomes and composed of specific proteins related to synapse formation and function.
  • Subcellular structures composed of proteins are potential sources of local in situ RNA in dendrites.
  • the commonly used method for the extraction of neural synaptosomes is sucrose, Ficoll or Percoll density gradient ultracentrifugation (Rao A, Steward O.
  • RNAs Present in Synaptodendrosomes Dendritic, Glial, and Neuronal Cell Body Contribution [J]. Journal of Neurochemistry, 1993, 61(3): 835-844. Nagy A, Delgado-Escueta A V. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll) [J]. Journal of Neurochemistry, 2010, 43 (4): 1114-1123. Kanhema T, Dagestad G, et al. Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo:evidence for compartment-specific translation control[J]. Journal of Neurochemistry, 2010 , 99(5):1328-1337.).
  • RNA extracted from the obtained synaptosomes cannot meet the requirements of accurate or quantitative gene expression research experiments such as biochips, gene expression matrix analysis, high-throughput Sequencing or qRT-PCR, etc.
  • the present application provides a method for extracting synaptosomes (Synaptodendrosome, SD).
  • the method can effectively extract the synaptic body, maintains the integrity of the RNA in the synaptic body, has a short extraction time and is easy to operate.
  • the present application provides a method for extracting neural synaptosomes, and the method for extracting neural synaptosomes includes the following steps:
  • tissue homogenization buffer for grinding and filtering to obtain tissue homogenate
  • the method for extracting neural synaptosomes of the present application is controlled to operate at 2-8° C. throughout the process, which can effectively maintain the cell activity in the neural synaptosomes and avoid the degradation of RNA. Efficient and rapid acquisition of synaptosomes with reduced extraction time, resulting in synaptosomes with high activity and RNA integrity.
  • the rotation speed of the centrifugation (ie, one centrifugation) in step (2) is 800-1200 rpm, including but not limited to 900 rpm, 1000 rpm or 1100 rpm.
  • the centrifugation time of step (2) is 10-30 min, including but not limited to 12 min, 14 min, 16 min, 18 min, 20 min, 25 min, 26 min or 28 min.
  • the rotation speed of one centrifugation is controlled to be 800-1200 rpm and the time is 10-30 min, which can efficiently remove large tissues and meninges in the tissue homogenate.
  • the tissue homogenization buffer in step (1), the buffer in step (3) and the buffer in step (4) contain RNase inhibitors.
  • the concentration of the RNase inhibitor is 4-40 U/mL, including but not limited to 5 U/mL, 8 U/mL, 10 U/mL, 12 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 32U/mL, 34U/mL, 36U/mL or 38U/mL.
  • RNase inhibitors can effectively alleviate the degradation of RNA by RNases.
  • the dilution factor of step (3) is ⁇ 1.5 times, including but not limited to 2 times, 3 times, 4 times, 5 times or 6 times.
  • diluting the synaptosome layer obtained by the first centrifugation to more than 1.5 times helps to efficiently separate and purify the synaptosome during the second centrifugation.
  • the animal tissue in step (1) includes mouse brain tissue.
  • the tissue homogenization buffer (Homogenization buffer, HB) in step (1) comprises sucrose, tris hydrochloride buffer, ethylenediaminetetraacetic acid, dithiothreitol and sodium fluoride.
  • the concentration of the sucrose in the tissue homogenization buffer is 300-340 mM, including but not limited to 305 mM, 310 mM, 320 mM, 330 mM, 335 mM or 338 mM.
  • the concentration of the tris hydrochloride buffer in the tissue homogenization buffer is 5-15 mM, including but not limited to 6 mM, 8 mM, 10 mM, 12 mM or 14 mM.
  • the concentration of the EDTA in the tissue homogenization buffer is 0.5-3 mM, including but not limited to 0.6 mM, 0.8 mM, 1.0 mM, 1.5 mM, 2.5 mM or 2.8 mM.
  • the concentration of the dithiothreitol in the tissue homogenization buffer is 1-3 mM, 1.2 mM, 1.6 mM, 2 mM, 2.4 mM or 2.8 mM.
  • the concentration of the sodium fluoride in the tissue homogenization buffer is 0.1-0.5 mM, including but not limited to 0.2 mM, 0.3 mM, 0.35 mM, 0.4 mM or 0.45 mM.
  • the tissue homogenization buffer in step (1), the buffer in step (3) and the buffer in step (4) further include protease inhibition.
  • the rotational speed of the grinding in step (1) is 1000-1500 rpm, including but not limited to 1100 rpm, 1200 rpm, 1300 rpm or 1400 rpm.
  • the size of the filter in step (1) is 25-35 ⁇ m, including but not limited to 23 ⁇ m, 28 ⁇ m, 30 ⁇ m, 32 ⁇ m or 34 ⁇ m.
  • the filter of step (1) comprises a nylon filter.
  • the tissue homogenate-OptiPrep solution in step (2) needs to be mixed with tissue homogenization buffer before being added to the centrifuge tube.
  • the volume ratio of the tissue homogenate-OptiPrep solution and the tissue homogenization buffer is (1-1.5):2, including but not limited to 1.1:2, 1.2:2, 1.3:2, 1.4:2 or 1.45:2.
  • the OptiPrep solution in step (2) further includes tissue homogenization buffer (HB).
  • tissue homogenization buffer (HB) tissue homogenization buffer
  • the OptiPrep solution is formulated as shown in Table 1.
  • Density Gradient (%) OptiPrep(mL) HB(mL) 8 to 10 0.32 ⁇ 0.4 3.6 ⁇ 3.68 11.5 ⁇ 13.5 0.46 ⁇ 0.54 3.46 ⁇ 3.54 14 ⁇ 16 0.56 ⁇ 0.64 3.36 ⁇ 3.44 24 ⁇ 26 0.96 ⁇ 1.04 2.96 ⁇ 3.04
  • the neural synaptic body layer in step (2) includes the first layer of layered tissue and/or the second layer of layered tissue from top to bottom in the primary centrifugation fluid.
  • the buffer in step (3) includes phosphate buffer.
  • the volume ratio of the synaptic body layer and the buffer in step (3) is 1:(1-3), including but not limited to 1:1.5, 1:1.8, 1:2 or 1:2.5.
  • the Percoll solution in step (3) further includes tissue homogenization buffer or Dulbecco's phosphate buffer.
  • the Percoll solution is formulated as shown in Table 2.
  • the rotational speed of the centrifugation in step (3) is 2400-3600 rpm, including but not limited to 2500 rpm, 2800 rpm, 3000 rpm, 3400 rpm or 3500 rpm.
  • the centrifugation time of step (3) is 8-12 min, including but not limited to 9 min, 10 min or 11 min.
  • the neural synaptic body layer in step (4) includes the third layer of stratified tissue and/or the fourth layer of stratified tissue from top to bottom in the secondary centrifugation fluid.
  • the buffer in step (4) includes phosphate buffer.
  • the rotational speed of the centrifugation in step (4) is 480-720 rpm, including but not limited to 490 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm or 710 rpm.
  • the centrifugation time of step (4) is 2-7 min, including but not limited to 3 min, 4 min, 5 or 6 min.
  • the preparation method for extracting neural synaptosomes comprises the following steps:
  • tissue homogenization buffer a compound that at 2 ⁇ 8°C, take animal tissue and add it to tissue homogenization buffer, grind at 1000 ⁇ 1500rpm, and filter with 25 ⁇ 35 ⁇ m nylon filter to obtain tissue homogenate;
  • the method for extracting neural synaptosomes of the present application is controlled to operate at 2-8°C throughout the process, which can effectively maintain the cell activity in the neural synaptosomes and avoid the degradation of RNA.
  • the density of the centrifuge during centrifugation Gradient distribution can obtain synaptosomes efficiently and quickly, shortening the extraction time, thereby obtaining synaptosomes with higher RNA integrity;
  • the synaptosomes extracted by the method for extracting neural synaptosomes of the present application contain correct biomarker proteins, and the total RNA integrity is high, which has broad development prospects in the field of neural synaptosomes.
  • Fig. 1 is the flow chart of extracting neural synaptosomes
  • Fig. 2 is a schematic diagram of gradient centrifugation
  • FIG. 3 shows the results of protein expression of biomarkers in various layers of tissue
  • Figure 4A is a sample electrophoresis diagram
  • Figure 4B is a sample peak map.
  • the present embodiment extracts neuronal synaptosomes from mouse brain tissue.
  • the extraction process is shown in Figure 1, and the specific process includes the following steps:
  • the second step of gradient centrifugation extracts the first layer of stratified tissue (C1B1) and the second layer of stratified tissue (C1B2) from top to bottom in the centrifugation fluid, and placed them in a new 15mL
  • the centrifuge tube add 2 volumes of PBS solution containing 4U/mL of RI and 1 tablet/7mL of PIC, use step (1) isotonic Percoll to prepare gradient centrifugation solution as shown in Table 5 and add as shown in Figure 2.
  • the samples were centrifuged at 32,000 ⁇ g for 9 min at 4°C to obtain a secondary centrifuge.
  • the cellular component-specific antibodies are the astrocyte marker GFAP, the synaptic vesicle glycoprotein Synaptophysin, the neuron-specific nuclear protein NeuN, the assembly of the postsynaptic dense complex and the functionally related scaffold protein PSD95 and neuron-specific marker ⁇ III-tubulin, using Western blot (Western blot, WB) method to analyze the purity of SD separation, the results are shown in Figure 3, the results are consistent with the published articles, indicating that the SD extracted in this application is correct of.
  • Western blot Western blot
  • the total RNA in SD was extracted by Trizon method. The detailed process was carried out according to the instructions of TRIzol TM Reagent of Invitrogen Company. The total RNA in SD was obtained, and its integrity was analyzed. The total RNA of whole brain tissue (HOM) was used as a control. The analysis experiment was performed with one technical repetition, that is, two SD samples (SD1 and SD2) and two whole brain tissue samples (HOM1 and HOM2) were analyzed for total RNA integrity.
  • the SD1 and SD2 samples had 28S and 18S have two clear bands and no impurity bands, which are similar to the results of HOM1 and HOM2; in Figure 4B, the peaks of the four samples are relatively flat, the peaks in the 28S and 18S regions are obvious, and there is no significant difference in the peak areas of the peaks.
  • the RNA integrity scores (RNA integrity number, RIN) of the four samples were all above 8.5. Based on the above, it is shown that the RNA extracted from the SD sample maintains a high integrity, and the present application can efficiently extract SD and effectively protect its total RNA.
  • the method for extracting neural synaptosomes of the present application can efficiently extract neural synaptosomes, and can effectively protect the integrity of total RNA in neural synaptosomes, and has broad development in the field of neural synaptosome research. prospect.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

一种提取神经突触体的方法。所述提取神经突触体的方法包括于2～8℃，依次获取组织匀浆，并对组织匀浆进行一次密度梯度离心和二次梯度离心，获得神经突触体。所述提取神经突触体的方法，全程控制在2～8℃进行操作，能够有效保持神经突触体中细胞活度，避免了RNA的降解，通过控制离心时离心液的密度梯度分布能够高效快速地获取神经突触体，缩短了提取时间，获得的神经突触体具有较高活性和总RNA完整性。

Description

一种提取神经突触体的方法 技术领域
本申请属于分子生物学技术领域，涉及一种提取神经突触体的方法。
背景技术
突触是神经元之间功能联系和信号传导的关键部位，其将数十亿的神经元连接到神经元和神经胶质环路中，构成信息处理和驱动行为的基础，在调节人的学习、记忆以及思考方式中发挥关键作用。若突触在结构和功能上损伤，可能会导致大脑功能障碍疾病。
原位杂交技术揭示了在神经元树突、轴突和突触中存在大量的RNA，且RNA具有多样性，并且大量研究已经证明了某些蛋白在这些部位存在局部翻译。但在神经元与神经元之间，转录和翻译的特征及机制尚不清楚，仍需进行大量研究工作，如基因转录水平分析等，但目前对突触、树突及轴突部位RNA提取的技术尚不成熟，限制了相关研究的的发展。
神经突触体(Synaptodendrosome，SD)指突触小体及其夹断的突触末端和相连的树突片段，包括富集在突触小体周围并且由与突触形成和功能相关的特定的蛋白组成的亚细胞结构(特别是包含部分轴突、树突及突触前膜、突触后膜和突触链接复合体部分)，是树突中局部原位RNA的潜在来源。目前，常用的神经突触体的提取方法是蔗糖、Ficoll或Percoll密度梯度超速冷冻离心法(Rao A,Steward O.Evaluation of RNAs Present in Synaptodendrosomes:Dendritic,Glial,and Neuronal Cell Body Contribution[J].Journal of Neurochemistry,1993,61(3):835-844.Nagy A,Delgado-Escueta A V.Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material(Percoll)[J].Journal of Neurochemistry,2010,43(4):1114-1123.Kanhema T,Dagestad G,et al.Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo:evidence for compartment-specific translation control[J].Journal of Neurochemistry,2010,99(5):1328-1337.)。上述方法均是针对一般蛋白表达水平检测，会引起RNA的降解，从得到的神经突触体中提取的RNA不能满足精确或定量的基因表达研究实验如生物芯片、基因表达矩阵分析、高通量测序或qRT-PCR等。
综上所述，提供一种操作简便且可改善RNA降解的提取神经突触体的方法，从而能够从其中获得高质量的总RNA，对于突触研究领域具有重要意义。
发明内容
本申请提供了一种提取神经突触体(Synaptodendrosome，SD)的方法。所述方法能够有效提取到神经突触体，且保持了神经突触体内RNA的完整性，提取时间短，操作简便。
本申请提供一种提取神经突触体的方法，所述提取神经突触体的方法包括以下步骤：
(1)于2～8℃，将动物组织加入到组织匀浆缓冲液中进行研磨和过滤处理，得到组织匀浆；
(2)一次离心，于2～8℃，依次将密度梯度为30％～40％的组织匀浆-OptiPrep溶液、密度梯度为24％～26％的OptiPrep溶液、密度梯度为14％～16％的OptiPrep溶液、密度梯度为11.5％～13.5％的OptiPrep溶液和密度梯度为8％～10％的OptiPrep溶液加入到离心管中，离心，得到一次离心液；
(3)二次离心，于2～8℃，取一次离心液中神经突触体层，并加入到缓冲液中进行稀释，获得混合液，依次将密度梯度为20％～26％的Percoll溶液、密度梯度为14％～16％的Percoll溶液、密度梯度为9％～11％的Percoll溶液、密度梯度为5％～7％的Percoll溶液和混合液加入到离心管中，离心，得到二次离心液；以及
(4)于2～8℃，取二次离心液中神经突触体层，加入缓冲液进行稀释，离心，收集沉淀，得到所述神经突触体。
本申请的提取神经突触体的方法，全程控制在2～8℃进行操作，能够有效保持神经突触体中细胞活度，避免了RNA的降解，通过控制离心时离心液的密度梯度分布能够高效快速地获取神经突触体，缩短了提取时间，从而获得具有高活性和RNA完整性的神经突触体。
优选地，步骤(2)所述离心(即一次离心)的转速为800～1200rpm，包括但不限于900rpm、1000rpm或1100rpm。
优选地，步骤(2)所述离心的时间为10～30min，包括但不限于12min、14min、16min、18min、20min、25min、26min或28min。
本申请中，控制一次离心的转速为800～1200rpm，时间为10～30min，能够高效去除组织匀浆中较大的组织和脑膜。
优选地，步骤(1)所述组织匀浆缓冲液、步骤(3)所述缓冲液和步骤(4)所述缓冲液中含有RNA酶抑制剂。
优选地，所述RNA酶抑制剂的浓度为4～40U/mL，包括但不限于5U/mL、8U/mL、10U/mL、12U/mL、20U/mL、25U/mL、30U/mL、32U/mL、34U/mL、36U/mL或38U/mL。
本申请中，使用RNA酶抑制剂能够有效缓解RNA被RNA酶降解。
优选地，步骤(3)所述稀释的倍数为≥1.5倍，包括但不限于2倍、3倍、4倍、5倍或6倍。
本申请中，将一次离心得到的神经突触体层稀释至1.5倍以上，有助于二次离心时高效分离纯化神经突触体。
优选地，步骤(1)所述动物组织包括鼠脑组织。
优选地，步骤(1)所述组织匀浆缓冲液(Homogenization buffer，HB)包括蔗糖、三羟甲基氨基甲烷盐酸盐缓冲液、乙二胺四乙酸、二硫苏糖醇和氟化钠。
优选地，所述蔗糖在所述组织匀浆缓冲液中的浓度为300～340mM，包括但不限于305mM、310mM、320mM、330mM、335mM或338mM。
优选地，所述三羟甲基氨基甲烷盐酸盐缓冲液在所述组织匀浆缓冲液中的浓度为5～15mM，包括但不限于6mM、8mM、10mM、12mM或14mM。
优选地，所述乙二胺四乙酸在所述组织匀浆缓冲液中的浓度为0.5～3mM，包括但不限于0.6mM、0.8mM、1.0mM、1.5mM、2.5mM或2.8mM。
优选地，所述二硫苏糖醇在所述组织匀浆缓冲液中的浓度为1～3mM，1.2mM、1.6mM、2mM、2.4mM或2.8mM。
优选地，所述氟化钠在所述组织匀浆缓冲液中的浓度为0.1～0.5mM，包括但不限于0.2mM、0.3mM、0.35mM、0.4mM或0.45mM。
优选地，步骤(1)所述组织匀浆缓冲液、步骤(3)所述缓冲液和步骤(4)所述缓冲液中还包括蛋白酶抑制。
优选地，步骤(1)步骤所述研磨的转速为1000～1500rpm，包括但不限于1100rpm、1200rpm、1300rpm或1400rpm。
优选地，步骤(1)所述过滤的过滤器规格为25～35μm，包括但不限于23μm、28μm、30μm、32μm或34μm。
优选地，步骤(1)所述过滤的过滤器包括尼龙过滤器。
优选地，步骤(2)所述组织匀浆-OptiPrep溶液加入离心管前还需和组织匀浆缓冲液混合。
优选地，所述组织匀浆-OptiPrep溶液和所述组织匀浆缓冲液的体积比为(1～1.5):2，包括但不限于1.1:2、1.2:2、1.3:2、1.4:2或1.45:2。
优选地，步骤(2)所述OptiPrep溶液中还包括组织匀浆缓冲液(HB)。
优选地，按表1所示配制OptiPrep溶液。
表1
密度梯度(％) OptiPrep(mL) HB(mL)
8～10 0.32～0.4 3.6～3.68
11.5～13.5 0.46～0.54 3.46～3.54
14～16 0.56～0.64 3.36～3.44
24～26 0.96～1.04 2.96～3.04
优选地，步骤(2)所述神经突触体层包括一次离心液中由上至下的第1层分层组织和/或第2层分层组织。
优选地，步骤(3)所述缓冲液包括磷酸盐缓冲液。
优选地，步骤(3)所述神经突触体层和缓冲液的体积比为1:(1～3)，包括但不限于1:1.5、1:1.8、1:2或1:2.5。
优选地，步骤(3)所述Percoll溶液还包括组织匀浆缓冲液或杜氏磷酸盐缓冲液。
优选地，按表2所示配制Percoll溶液。
表2
密度梯度(％) Percoll(mL) HB/DPBS(mL)
5～7 0.2～0.35 3.65～3.8
9～11 0.36～0.44 3.56～3.64
14～16 0.56～0.64 3.36～3.44
20～26 0.8～1.04 2.96～3.2
优选地，步骤(3)所述离心的转速为2400～3600rpm，包括但不限于2500rpm、2800rpm、3000rpm、3400rpm或3500rpm。
优选地，步骤(3)所述离心的时间为8～12min，包括但不限于9min、10min或11min。
优选地，步骤(4)所述神经突触体层包括二次离心液中由上至下第3层分层组织和/或第4层分层组织。
优选地，步骤(4)所述缓冲液包括磷酸盐缓冲液。
优选地，步骤(4)所述离心的转速为480～720rpm，包括但不限于490rpm、500rpm、550rpm、600rpm、650rpm、700rpm或710rpm。
优选地，步骤(4)所述离心的时间为2～7min，包括但不限于3min、4min、5或6min。
作为优选的技术方案，所述提取神经突触体的制备方法包括以下步骤：
(1)于2～8℃，取动物组织加入到组织匀浆缓冲液中，于1000～1500rpm进行研磨，并使用25～35μm的尼龙过滤器进行过滤，得到组织匀浆；
(2)一次离心，于2～8℃，依次将密度梯度为30％～40％的组织匀浆-OptiPrep溶液、密度梯度为24％～26％的OptiPrep溶液、密度梯度为14％～16％的OptiPrep溶液、密度梯度为11.5％～12.5％的OptiPrep溶液和密度梯度为8％～10％的OptiPrep溶液加入到离心管中，800～1200rpm离心10～30min，得到一次离心液；
(3)二次离心，于2～8℃，取一次离心液中由上至下的第1层分层组织和/或第2层分层组织，并加入到含有4～40U/mL RNA酶抑制剂和蛋白酶抑制剂的磷酸盐缓冲液中稀释≥1.5倍，获得混合液，依次将密度梯度为20％～26％的Percoll溶液、密度梯度为14％～16％的Percoll溶液、密度梯度为9％～11％的Percoll溶液、密度梯度为5％～7％的Percoll溶液和混合液加入到离心管中，2400～3600rpm离心8～12min，得到二次离心液；以及
(4)于2～8℃，取二次离心液中由上至下第3层分层组织和/或第4层分层组织，加入含有4～40U/mL RNA酶抑制剂和蛋白酶抑制剂的磷酸盐缓冲液，480～720rpm离心2～7min，收集沉淀，得到所述神经突触体。
与现有技术相比，本申请具有以下技术效果：
(1)本申请的提取神经突触体的方法，全程控制在2～8℃进行操作，能够有效保持神经突触体中细胞活度，避免了RNA的降解，通过控制离心时离心液的密度梯度分布能够高效快速地获取神经突触体，缩短了提取时间，从而获得具有较高RNA完整性的神经突触体；
(2)本申请的提取神经突触体的方法提取得到的突触体中含有正确的生物标志物蛋白，且总RNA完整性高，在神经突触体领域中具有广阔的发展前景。
附图说明
图1为提取神经突触体流程图；
图2为梯度离心示意图；
图3为各层组织生物标志物蛋白表达结果；
图4A为样品电泳图；
图4B为样品峰图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例和附图对本申请作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本申请，而非对本申请的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例对小鼠脑组织神经突触体进行提取，提取流程如图1所示，具体过程包括以下步骤：
(1)在取组织前准备好以下试剂及材料：DEPC水；2.5M的蔗糖溶液(将蔗糖溶解在DEPC水中)；等渗Percoll溶液(1体积2.5M的蔗糖溶液加入到9体积的100％Percoll中)；组织匀浆缓冲液(Homogenization Buffer，HB)，按表3所示配制。
表3
Figure PCTCN2020137128-appb-000001
(2)实验开始前于2～8℃预冷所有器械、试剂和耗材(不包含麻醉剂和注射针头)，在通风厨中，无菌条件下进行异氟烷麻醉鼠后，断颈，解剖取脑组织(cortex)迅速放进2mL含有4U/mL的RNA酶抑制剂(RNAse Inhibitor，RI)和蛋白酶抑制剂预混液(Proteinase Inhibitor Cocktail，PIC)的HB溶液中，于1200rpm，在冰上反复上下研磨18次，直到组织研磨充分，用30μm的尼龙过滤器过滤研磨后的组织，获得组织匀浆(Homogenate lysate，HOM)。
(3)第一步梯度离心，于2～8℃，将组织匀浆与OptiPrep溶液混合成35％的混合液(35％opti-prep with homogenate)，并按表4所示配制梯度离心溶液(HB中还含有4U/mL的RI和1片/7mL的PIC)，35％opti-prep with homogenate为1.3mL，加入前与2mL HB混合，并根据图2加样，于4℃、10000×g离心10min，得到一次离心液。
表4
密度梯度(％) OptiPrep(mL) HB(mL)
9 0.36 3.64
12.5 0.5 3.5
15 0.6 3.4
25 1 3
(4)第二步梯度离心，如图1所示，抽取一次离心液中由上至下的第1层分层组织(C1B1)和第2层分层组织(C1B2)，置于新的15mL离心管中，加入2倍体积的含有4U/mL的RI和1片/7mL的PIC的PBS溶液，用步骤(1)等渗Percoll按表5所示配制梯度离心溶液并按照图2所示加样，于4℃、32000×g离心9min，得到二次离心液。
表5
密度梯度(％) Percoll(mL) HB/DPBS(mL)
6 0.24 3.76
10 0.4 3.6
15 0.6 3.4
23 0.92 3.08
(5)于2～8℃，如图1所示取二次离心液中由上至下第3层分层组织(C2B3)和第4层分层组织(C2B4)，并加入含有4～40U/mL RNA酶抑制剂和蛋白酶抑制剂的PBS溶液，于4℃、6000×g离心5min，重复2次，收集沉淀，得到所述 神经突触体(SD)，置于-80℃保存。
试验例1
本试验例根据已发表文章([1]Westmark PR,Westmark CJ,Jeevananthan A,Malter JS.Preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient.J Vis Exp.2011 Sep 17；(55):3196.[2]Kiebler MA,López-García JC,Leopold PL.Purification and characterization of rat hippocampal CA3-dendritic spines associated with mossy fiber terminals.FEBS Lett.1999 Feb 19；445(1):80-6.)，验证本次SD提取过程中，每个层带的蛋白是否和已发表文章相一致，采用细胞成分特异性抗体图1所示的C1B1、C1B2、C1B3、C1B4、C2B1、C2B2、C2B3、C2B4和HOM进行标记，细胞成分特异性抗体为星形胶质细胞标志物GFAP、神经突触小泡糖蛋白Synaptophysin、神经元特异性核蛋白NeuN、突触后致密复合体的组装和功能相关的支架蛋白PSD95和神经元特异性标志物βⅢ-tubulin，使用免疫印迹(Western blot，WB)方法进行SD分离纯度分析，结果如图3所示，结果与已发表文章相一致，说明本申请提取的SD是正确的。
试验例2
为进一步验证本申请提取的SD是否满足转录组分析要求，本试验例提取所获得的SD中的总RNA。
使用Trizon法提取SD中总RNA，详细过程按invitrogen公司的TRIzol TMReagent的使用说明进行，获得SD中总RNA，并对其进行完整性分析，以全脑组织(HOM)的总RNA为对照，分析实验进行1次技术重复，即分别对两个SD样品(SD1和SD2)和两个全脑组织样品(HOM1和HOM2)进行总RNA完整性分析，图4A中，SD1和SD2两个样品有28S和18S有两个清晰条带，并且无杂带，与HOM1和HOM2结果相近；图4B中，四个样品峰图基线比较平整，28S和18S区峰值明显，峰图峰面积无显著差异。如表6所示，四个样品的RNA完整性评分(RNA integrity number，RIN)均在8.5以上。综合上述表明SD中样品提取的RNA保持了较高的完整性，本申请可以高效的提取SD且有效保护其总RNA。
表6
样品名称 RIN
SD1 8.5
SD2 8.5
HOM1 9.4
HOM2 9.0
综上所述，本申请的提取神经突触体的方法能够高效提取神经突触体，并能有效保护神经突触体中总RNA的完整性，在神经突触体研究领域中具有广阔的发展前景。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (15)

  1. 一种提取神经突触体的方法，其包括以下步骤：
    (1)于2～8℃，将动物组织加入到组织匀浆缓冲液中进行研磨和过滤处理，得到组织匀浆；
    (2)于2～8℃，依次将密度梯度为30％～40％的组织匀浆-OptiPrep溶液、密度梯度为24％～26％的OptiPrep溶液、密度梯度为14％～16％的OptiPrep溶液、密度梯度为11.5％～13.5％的OptiPrep溶液和密度梯度为8％～10％的OptiPrep溶液加入到离心管中，离心，得到一次离心液；
    (3)于2～8℃，取一次离心液中神经突触体层，并加入到缓冲液中进行稀释，获得混合液，依次将密度梯度为20％～26％的Percoll溶液、密度梯度为14％～16％的Percoll溶液、密度梯度为9％～11％的Percoll溶液、密度梯度为5％～7％的Percoll溶液和混合液加入到离心管中，离心，得到二次离心液；以及
    (4)于2～8℃，取二次离心液中神经突触体层，加入缓冲液进行稀释，离心，收集沉淀，得到所述神经突触体。
  2. 根据权利要求1所述的方法，其中，步骤(2)所述离心的转速为800～1200rpm。
  3. 根据权利要求1或2所述的方法，其中，步骤(2)所述离心的时间为10～30min。
  4. 根据权利要求1-3中任一项所述的方法，其中，步骤(1)所述组织匀浆缓冲液、步骤(3)所述缓冲液和步骤(4)所述缓冲液中含有RNA酶抑制剂；
    任选地，所述RNA酶抑制剂的浓度为4～40U/mL。
  5. 根据权利要求1-4中任一项所述的方法，其中，步骤(3)所述稀释的倍数为≥1.5倍。
  6. 根据权利要求1-5中任一项所述的方法，其中，步骤(1)所述动物组织包括鼠脑组织。
  7. 根据权利要求1-6中任一项所述的方法，其中，步骤(1)所述组织匀浆缓冲液包括蔗糖、三羟甲基氨基甲烷盐酸盐缓冲液、乙二胺四乙酸、二硫苏糖醇和氟化钠；
    任选地，所述蔗糖在所述组织匀浆缓冲液中的浓度为300～340mM；
    任选地，所述三羟甲基氨基甲烷盐酸盐缓冲液在所述组织匀浆缓冲液中的浓度为5～15mM；
    任选地，所述乙二胺四乙酸在所述组织匀浆缓冲液中的浓度为0.5～3mM；
    任选地，所述二硫苏糖醇在所述组织匀浆缓冲液中的浓度为1～3mM；
    任选地，所述氟化钠在所述组织匀浆缓冲液中的浓度为0.1～0.5mM。
  8. 根据权利要求1至7中任一项所述的方法，其中，步骤(1)所述组织匀浆缓冲液、步骤(3)所述缓冲液和步骤(4)所述缓冲液中还包括蛋白酶抑制；
    任选地，步骤(1)步骤所述研磨的转速为1000～1500rpm；
    任选地，步骤(1)所述过滤的过滤器规格为25～35μm；
    任选地，步骤(1)所述过滤的过滤器包括尼龙过滤器。
  9. 根据权利要求1-8任一项所述的方法，其中，步骤(2)所述组织匀浆-OptiPrep溶液加入离心管前还需和组织匀浆缓冲液混合；
    任选地，所述组织匀浆-OptiPrep溶液和所述组织匀浆缓冲液的体积比为(1～1.5):2；
    任选地，步骤(2)所述OptiPrep溶液中还包括组织匀浆缓冲液。
  10. 根据权利要求1-9任一项所述的方法，其中，步骤(2)所述神经突触体层包括一次离心液中由上至下的第1层分层组织和/或第2层分层组织。
  11. 根据权利要求1-10任一项所述的方法，其中，步骤(3)所述缓冲液包括磷酸盐缓冲液；
    任选地，步骤(3)所述神经突触体层和缓冲液的体积比为1:(1～3)。
  12. 根据权利要求1-11任一项所述的方法，其中，步骤(3)所述Percoll溶液还包括组织匀浆缓冲液或杜氏磷酸盐缓冲液。
  13. 根据权利要求1-12任一项所述的方法，其中，步骤(3)所述离心的转速为2400～3600rpm；
    任选地，步骤(3)所述离心的时间为8～12min。
  14. 根据权利要求1-13任一项所述的方法，其中，步骤(4)所述神经突触体层包括二次离心液中由上至下第3层分层组织和/或第4层分层组织；
    任选地，步骤(4)所述缓冲液包括磷酸盐缓冲液；
    任选地，步骤(4)所述离心的转速为480～720rpm；
    任选地，步骤(4)所述离心的时间为2～7min。
  15. 根据权利要求1-14任一项所述的制备方法，其中，所述制备方法包括 以下步骤：
    (1)于2～8℃，取动物组织加入到组织匀浆缓冲液中，于1000～1500rpm进行研磨，并使用25～35μm的尼龙过滤器进行过滤，得到组织匀浆；
    (2)于2～8℃，依次将密度梯度为30％～40％的组织匀浆-OptiPrep溶液、密度梯度为24％～26％的OptiPrep溶液、密度梯度为14％～16％的OptiPrep溶液、密度梯度为11.5％～12.5％的OptiPrep溶液和密度梯度为8％～10％的OptiPrep溶液加入到离心管中，800～1200rpm离心10～30min，得到一次离心液；
    (3)于2～8℃，取一次离心液中由上至下的第1层分层组织和/或第2层分层组织，并加入到含有4～40U/mL RNA酶抑制剂和蛋白酶抑制剂的磷酸盐缓冲液中稀释≥1.5倍，获得混合液，依次将密度梯度为20％～26％的Percoll溶液、密度梯度为14％～16％的Percoll溶液、密度梯度为9％～11％的Percoll溶液、密度梯度为5％～7％的Percoll溶液和混合液加入到离心管中，2400～3600rpm离心8～12min，得到二次离心液；以及
    (4)于2～8℃，取二次离心液中由上至下第3层分层组织和/或第4层分层组织，加入含有4～40U/mL RNA酶抑制剂和蛋白酶抑制剂的磷酸盐缓冲液，480～720rpm离心2～7min，收集沉淀，得到所述神经突触体。
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