WO2019091034A1 - 一种提取动物组织线粒体的方法 - Google Patents

一种提取动物组织线粒体的方法 Download PDF

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WO2019091034A1
WO2019091034A1 PCT/CN2018/079790 CN2018079790W WO2019091034A1 WO 2019091034 A1 WO2019091034 A1 WO 2019091034A1 CN 2018079790 W CN2018079790 W CN 2018079790W WO 2019091034 A1 WO2019091034 A1 WO 2019091034A1
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mitochondria
suspension
animal
tissue
animal tissue
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马颖
马铎
阮雯莉
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广州资生生物科技有限公司
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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  • the invention belongs to the field of biotechnology, and in particular relates to a method for breaking a cell membrane of an animal tissue and a method for extracting mitochondria.
  • Mitochondria are important organelles in animal tissue cells and are found in most living cells. Their main function is to provide the energy needed for the metabolism of various substances in cells. Because of this, the study of the structure, function and physicochemical properties of mitochondrial inner and outer membranes, respiratory chain enzymes and mitochondrial DNA has become an important topic in cell biology research. In addition, mitochondria are increasingly being used as targets for the treatment of systemic functional diseases. For example, Wang Shuai et al. found that a variety of mitochondria-targeted drugs can effectively alleviate myocardial ischemia-reperfusion injury (practical cardio-pulmonary vascular disease). Journal, 2017, 25(3)). Extraction of active mitochondria is the basis of mitochondrial research. Since mitochondria are abundantly present in metabolically active cells, such as animal heart, liver, kidney and other cells, a large number of mitochondria are extracted from these tissue cells.
  • the conventional mitochondrial extraction kit method is costly, and the extracted mitochondria concentration is unstable, low in efficiency, and low in viability.
  • the sucrose density gradient centrifugation method belongs to the conventional method, and the extraction method is low in cost, but it is time consuming and laborious, and requires an ultra-low temperature centrifuge, and the equipment and equipment conditions are high. Both of the above methods need to extract cells from animal tissues first, and after culturing, expanding and culturing to obtain a sufficient number of cells, the mitochondria can be extracted, which is time consuming and laborious, and the number of extracted mitochondria is limited.
  • the present invention provides a method for extracting mitochondria of animal tissues, which is used for solving the problem that the existing method for extracting animal tissue mitochondria is time-consuming and laborious, and the number of extracted mitochondria is limited.
  • a method for extracting mitochondria from animal tissues comprising:
  • the isolated animal tissue has an ex vivo time of 0.5 to 1 h.
  • the frozen tissue block has a temperature after thawing of -15 to 20 °C.
  • the method further includes:
  • the isolated animal tissue is sterilized.
  • the method further comprises:
  • the isolated animal tissue is washed.
  • the suspension is centrifuged at 6000 to 6500 rpm for 1 to 1.2 hours, and further includes:
  • the suspension was placed at 4-8 ° C for 12 h.
  • the suspension further comprises:
  • the suspension is sterilized.
  • the method further comprises:
  • the sheet is subjected to a sterilization treatment.
  • the present invention provides a method for disrupting an animal tissue cell membrane, comprising: a) dicing an isolated animal tissue into small pieces and freezing at -75 to -85 ° C for 1.5 to 2 hours to obtain a frozen tissue block; b) thawing the frozen tissue block and cutting into a slice having a thickness smaller than the diameter of the animal cell; c) adding the slice to distilled water to prepare a suspension, and centrifuging the suspension at 6000 to 6500 rpm for 1 to 1.2 hours to obtain a supernatant. d) The supernatant is centrifuged at 12000 to 13000 rpm for 1 to 1.2 hours, and then precipitated to obtain mitochondria; wherein the thickness of the sheet is larger than the diameter of the animal mitochondria.
  • the method for extracting mitochondria of the present invention comprises two steps of centrifugation, which is simple and efficient, has few steps, and has a large number of extracted mitochondria, and the whole process is low in the extraction process, and the extracted mitochondria has high activity.
  • the invention provides a method for extracting mitochondria of animal tissues, which is used for solving the problem that the existing method for extracting animal tissue mitochondria is time-consuming and laborious, and the number of extracted mitochondria is limited.
  • the umbilical cord tissue of the mammalian sheep after delivery is collected and placed in 4 °C physiological saline for storage.
  • the umbilical cord tissue is sent to a GMP-certified sterile laboratory for distilled water for 7 minutes, and 75% alcohol is disinfected. After that, the umbilical cord tissue is collected.
  • the umbilical tissue tissue was sterilized with 75% alcohol and centrifuged at 3000 rpm for 30 min to remove the supernatant to remove the alcohol.
  • the suspension was shaken to make a suspension. After that, the suspension was placed in a refrigerator at 4 ° C for 1 h and then centrifuged at 6000 rpm. After centrifugation at °C for 1 h, the supernatant was obtained, and the supernatant was centrifuged at 12000 rpm at 4 ° C for 1 h, and then the precipitate was taken to obtain umbilical cord tissue mitochondria.
  • the fetal brain is obtained within 24 hours before the delivery of the mammalian sheep, and the brain tissue is taken. After 30 minutes, the isolated brain tissue was transferred to a biological laboratory for cleaning. The cleaning steps were as follows:
  • the brain tissue was cut into small pieces in a sterile operation table and placed in a tissue cassette of a tissue microtome, and frozen at -85 ° C for 2 h to obtain a frozen brain tissue block, and the frozen brain tissue block was thawed -15 to -20 ° C.
  • the brain tissue block was then cut into 2 ⁇ m brain tissue slices with a tissue microtome to ensure complete destruction of the brain tissue membrane.
  • the brain tissue slices were stored in 50 ml of 75% alcohol in a 200 ml small container. Each 100 g was collected and placed in a biofilter to remove alcohol. The filtered flakes were placed in a 500 ml glass container and immersed in 400 ml of sterile distilled water. The suspension was shaken, and then the suspension was placed in a refrigerator at 4 ° C for 12 h. The suspension was first centrifuged at 1500 rpm for 10 min. In this operation, the intact cell and nucleus with the highest sedimentation coefficient (Svedberg unit) formed particles, and the other cell components had a relatively low precipitation rate and therefore remained in the supernatant. Can't be separated.
  • Svedberg unit sedimentation coefficient
  • Mitochondrial extraction was performed using a mitochondrial extraction kit (Pierce, USA), 2 g of brain tissue was taken out, and the tissue was washed once with PBS, then cut into pieces in an ice bath dish, and 20 ml of mitochondrial separation reagent A was added to the pre-cooled glass.
  • the homogenizer was homogenized 10 times at 600 rpm, centrifuged at 800 rpm for 5 min at 4 ° C, and the supernatant was transferred to another centrifuge tube and centrifuged at 14600 rpm for 10 min. After carefully removing the supernatant, the precipitate is taken as the separated mitochondria.
  • the mitochondria were suspended with an appropriate amount of mitochondrial detection solution for subsequent experiments. The above operations were all carried out at 4 ° C and completed within 1 h.
  • membrane potential measuring medium containing 225 mM sucrose, 8 mM Tris-HCl, 13 mM K 2 HPO 4 , 10 mM KH 2 PO 4 , 5 mM MgCl 2 , 20 mM KCl, 5 ⁇ M rotenone, and 5 mM succinic acid
  • the membrane potentials at three time points of 0.5 h, 1.5 h and 2.5 h after extraction of mitochondria were determined by two methods.
  • the experimental results are shown in Table 1. At three time points of 0.5, 1.5 and 2.5 h, the mitochondrial membrane potential extracted by the extraction method of the present invention was higher than that of the mitochondrial membrane potential extracted by the kit method, and the difference was statistically significant (P ⁇ 0.05). The mitochondria extracted by the extraction method of the present invention has higher intrinsic membrane potential value after extraction than the mitochondria extracted by the kit method, and the difference is statistically significant. The mitochondrial membrane extracted by the extraction method of the invention has good functional activity.
  • Quantitative detection of mitochondrial protein was performed using a BCA (bicinchoninic acid) protein quantification kit (Pierce, USA).

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Abstract

提供一种提取动物组织线粒体的方法，包括：a）将离体动物组织切成小块后在-75～-85℃下冷冻1.5～2h，得到冷冻组织块；b）将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片；c）将所述薄片加入蒸馏水制成悬液，将所述悬液6000～6500rpm离心1～1.2h，得到上清液；d）将所述上清液12000～13000rpm离心1～1.2h后取沉淀，得到线粒体；其中，所述薄片的厚度大于动物线粒体的直径。

Description

一种提取动物组织线粒体的方法
本申请要求于2017年11月13日提交中国专利局、申请号为2017111151070、发明名称为“一种提取动物组织线粒体的方法”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
本发明属于生物技术领域，尤其涉及一种动物组织细胞膜的破碎方法和提取线粒体的方法。
背景技术
线粒体是动物组织细胞中重要的一种细胞器，存在于绝大多数活细胞中,它的主要功能是提供细胞内各种物质代谢所需要的能量。正因为此，研究线粒体内外膜、呼吸链酶及线粒体DNA等成分的结构、功能及理化性质已成为细胞生物学研究中的重要课题。另外，线粒体越来越多被作为系统功能性疾病治疗药物的靶点进行研究，如汪帅等人研究发现多种线粒体靶向药物可有效减轻心肌缺血再灌注损伤(实用心脑肺血管病杂志，2017，25(3))。提取有活性的线粒体是线粒体研究的基础。由于线粒体大量存在于代谢旺盛的细胞中，如动物的心肌、肝、肾等细胞，大量制备线粒体就是从这些组织细胞中提取。
目前，线粒体的提取方法很多,不同实验室根据实际情况往往采用不同的方法。选择线粒体提取方法的考虑因素主要有线粒体提取方法是否能减少提取过程中胞质的污染、提取得到的线粒体活性是否强、提取方法是否方便快捷等。常规的线粒体提取试剂盒方法成本高，提取的线粒体浓度不稳定、效率低、活率不高。蔗糖密度梯度离心法属于传统方法，该提取方法成本低，但是费时费力，而且需要超速低温离心机，仪器设备条件要求高。以上两种方法均需要先从动物组织中提取细胞，经过传代、扩增培养得到足够数量的细胞后才可以进行线粒体的提取，费时费力，并且提取的线粒体数量有限。
发明内容
有鉴于此，本发明提供了一种提取动物组织线粒体的方法，用于解决现有提取动物组织线粒体方法费时费力、提取到的线粒体数量有限的问题。
本发明的具体技术方案如下：
一种提取动物组织线粒体的方法，包括：
a)将离体动物组织切成小块后在-75～-85℃下冷冻1.5～2h，得到冷冻组织块；
b)将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片；
c)将所述薄片加入蒸馏水制成悬液，将所述悬液6000～6500rpm离心1～1.2h，得到上清液；
d)将所述上清液12000～13000rpm离心1～1.2h后取沉淀，得到线粒体；其中，所述薄片的厚度大于动物线粒体的直径。
优选的，所述离体动物组织的离体时间为0.5～1h。
优选的，所述冷冻组织块解冻后的温度为-15～20℃。
进一步的，所述步骤a)之前还包括：
将所述离体动物组织进行灭菌处理。
进一步的，将所述离体动物组织进行灭菌处理之前还包括：
将所述离体动物组织进行清洗。
进一步的，将所述薄片加入蒸馏水制成悬液之后，将所述悬液6000～6500rpm离心1～1.2h之前，还包括：
将所述悬液在4～8℃下放置12h。
进一步的，将所述悬液在4～8℃下放置12h之前还包括：
将所述悬液进行灭菌处理。
进一步的，将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片之后，将所述薄片加入蒸馏水制成悬液之前，还包括：
将所述薄片进行消毒处理。
综上所述，本发明提供了一种动物组织细胞膜的破碎方法，包括：a)将离体动物组织切成小块后在-75～-85℃下冷冻1.5～2h，得到冷冻组织块； b)将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片；c)将所述薄片加入蒸馏水制成悬液，将所述悬液6000～6500rpm离心1～1.2h，得到上清液；d)将所述上清液12000～13000rpm离心1～1.2h后取沉淀，得到线粒体；其中，所述薄片的厚度大于动物线粒体的直径。本发明提取线粒体的方法将薄片进行两步离心，简单高效、步骤少，提取得到的线粒体数量大，提取过程中全程低温，提取到的线粒体活性高。
具体实施方式
本发明提供了一种提取动物组织线粒体的方法，用于解决现有提取动物组织线粒体方法费时费力、提取到的线粒体数量有限的问题。
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
采集哺乳动物绵羊分娩后的脐带组织后放入4℃生理盐水中保存备用，30min内将脐带组织送入GMP认证的无菌实验室内进行蒸馏水清洗、75％酒精消毒处理，之后，将脐带组织切成2.0cm×2.0cm×(0.3～0.4)cm小块放入组织切片机盒后在-85℃冰箱中冷冻2h，得到冷冻脐带组织块，之后将组织切片机和冷冻脐带组织块解冻到-15～-20℃后，在组织切片机中将脐带组织块切成2μm的脐带组织薄片，以保证脐带组织细胞膜完全破碎。
将脐带组织薄片用75％酒精消毒、3000rpm离心30min除去取上清液以除去酒精、放入蒸馏水中振荡制成悬液，之后，将悬液放入4℃冰箱冷藏1h后用离心机6000rpm 4℃离心1h，得到上清液，再将上清液12000rpm 4℃离心1h后取沉淀，得到脐带组织线粒体。
实施例2
在哺乳动物绵羊分娩前24h内得到胎儿的大脑器官，取大脑组织。30min将离体大脑组织转移至生物实验室进行清洗，清洗步骤具体为：
1)用过滤自来水不间断冲刷大脑组织2min；
2)在容器内用无菌蒸馏水清洗大脑组织2min；
3)将大脑组织放入75％酒精中浸泡5min；
4)把大脑组织放入无菌蒸馏水容器内浸泡3min；
5)重复步骤2)至步骤4)一次。
之后，在无菌操作台将大脑组织切成小块放入组织切片机的组织盒内，在-85℃下冷冻2h，得到冷冻大脑组织块，将冷冻大脑组织块解冻-15～-20℃后用组织切片机将大脑组织块切成2μm的脑组织薄片，以保证大脑组织细胞膜完全破碎。
将脑组织薄片保存在200ml小容器盛放的50ml 75％酒精内，每采集到100g即放入生物滤器去除酒精，过滤后的薄片放入500ml的玻璃容器内并浸泡在400ml的无菌蒸馏水中振荡制成悬液，之后，将悬液放置在4℃的冰箱12h。先将悬液以1500rpm离心10min，此操作中，具有最高沉降系数值(Svedberg unit)的完整的细胞和细胞核形成颗粒，其它的细胞成分沉淀速率相对低很多，因此将继续被保留在上清液中无法被分离。将离心后得到的上清液转移到另一个无菌的离心管中，然后开始第二次离心步骤。第二次离心采用6000rpm高速离心力离心20min，此操作可分离沉淀出沉降系数值(Svedberg unit)比细胞核低的线粒体，收集沉淀即得到线粒体。其中，该实施例使用的容器、工具均经过无菌消毒。
实施例3试剂盒法提取线粒体
使用线粒体提取试剂盒(美国Pierce公司)进行线粒体提取，取出2g大脑组织，用PBS洗涤组织1次，然后在冰浴的平皿中剪成碎块，加入20ml线粒体分离试剂A，于预冷的玻璃匀浆器中600rpm匀浆10次，4℃下800rpm离心5min，将上清液转移到另一离心管后，14600rpm离心10min。小心去除上清后，取沉淀即为分离得到的线粒体。用适量的线粒 体检测液悬浮线粒体，进行后续实验。以上操作均在4℃进行，1h内完成。
实施例4线粒体膜功能活性的测定
在比色杯中加入2ml膜电位测定介质(膜电位测定介质含225mM蔗糖、8mM Tris-HCl、13mM K 2HPO 4、10mM KH 2PO 4、5mM MgCl 2、20mM KCl、5μM鱼藤酮和5mM琥珀酸钠，pH＝7.4)，加入5μl 0.1mM罗丹明123，充分混匀后，在激发波长520nm、发射波长525nm下，测定基础荧光值F1，测定后将液体倒入试管中，加入100μl线粒体悬液，25℃孵育15min后，12000g离心10min，取上清，测定荧光值F2。膜电位的大小用每毫克线粒体能引起荧光值的变化来表示ψm＝(F1－F2)/线粒体蛋白含量(mg)。分别测定两种方法提取线粒体后0.5h、1.5h和2.5h共三个时间点的膜电位。
实验结果如表1所示，在0.5、1.5和2.5h共三个时间点上，本发明提取方法提取的线粒体膜电位高于试剂盒法提取的线粒体膜电位，差异具有统计学意义(P<0.05)。本发明提取方法提取的线粒体在提取后的2.5h内膜电位值高于试剂盒法提取的线粒体，差异具有统计学意义，本发明提取方法提取的线粒体膜功能活性良好。
表1不同提取方法得到的线粒体的线粒体膜电位结果
Figure PCTCN2018079790-appb-000001
实施例5线粒体蛋白定量检测
线粒体蛋白定量检测采用BCA(bicinchoninic acid)蛋白定量试剂盒(美国Pierce公司)。
按照BCA蛋白定量试剂盒说明书的步骤进行操作。配制蛋白标准品并绘制标准曲线图。取试剂盒中的A液和B液按50：1的比例混匀，在酶标板中加入待测样品10μl，再加入200μl BCA孵育液，混匀后，37℃气浴30min。于酶标仪562nm波长下，按照BCA蛋白定量试剂盒说明书的步骤进行测定操作。配制蛋白标准品并绘制标准曲线图。取试剂盒各孔的吸光度值，再计算出样品的蛋白浓度。通过BCA方法定量线粒体蛋白，结果如表2所示，结果表明同样组织量下，应用本发明提取方法提取的线粒体蛋白浓度高于试剂盒法，差异有统计学意义(P<0.05)。
表2不同提取方法得到的线粒体浓度结果
Figure PCTCN2018079790-appb-000002
以上对本发明所提供的动物组织细胞膜的破碎方法和提取线粒体的方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (8)

  1. 一种提取动物组织线粒体的方法，其特征在于，包括：
    a)将离体动物组织切成小块后在-75～-85℃下冷冻1.5～2h，得到冷冻组织块；
    b)将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片；
    c)将所述薄片加入蒸馏水制成悬液，将所述悬液6000～6500rpm离心1～1.2h，得到上清液；
    d)将所述上清液12000～13000rpm离心1～1.2h后取沉淀，得到线粒体；其中，所述薄片的厚度大于动物线粒体的直径。
  2. 根据权利要求1所述的提取动物组织线粒体的方法，其特征在于，所述离体动物组织的离体时间为0.5～1h。
  3. 根据权利要求1所述的提取动物组织线粒体的方法，其特征在于，所述冷冻组织块解冻后的温度为-15～20℃。
  4. 根据权利要求1所述的提取动物组织线粒体的方法，其特征在于，将离体动物组织切成小块后在-75～-85℃下冷冻1.5～2h，得到冷冻组织块之前，还包括：
    将所述离体动物组织进行灭菌处理。
  5. 根据权利要求4所述的提取动物组织线粒体的方法，其特征在于，将所述离体动物组织进行灭菌处理之前还包括：
    将所述离体动物组织进行清洗。
  6. 根据权利要求1所述的提取动物组织线粒体的方法，其特征在于，将所述薄片加入蒸馏水制成悬液之后，将所述悬液6000～6500rpm离心1～1.2h之前，还包括：
    将所述悬液在4～8℃下放置12h。
  7. 根据权利要求6所述的提取动物组织线粒体的方法，其特征在于，将所述悬液在4～8℃下放置12h之前还包括：
    将所述悬液进行灭菌处理。
  8. 根据权利要求7所述的提取动物组织线粒体的方法，其特征在于，将所述冷冻组织块解冻后切成厚度小于动物细胞直径的薄片之后，将所述薄片加入蒸馏水制成悬液之前，还包括：将所述薄片进行消毒处理。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101717772A (zh) * 2009-12-18 2010-06-02 聊城大学 一种动物线粒体dna的提取及纯化方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101717772A (zh) * 2009-12-18 2010-06-02 聊城大学 一种动物线粒体dna的提取及纯化方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DEVALL, M. ET AL.: "A Comparison of Mitochondrial DNA Isolation Methods in Frozen Post- Mortem Human Brain Tissue--Applications for Studies of Mitochondrial Genetics in Brain Disorders", BIOTECHNIQUES, vol. 59, no. 4, 1 October 2015 (2015-10-01), pages 241-2 - 244-6, XP055608278, ISSN: 1940-9818, DOI: 10.2144/000114343 *
XIA, YULING ET AL.: "The Principle and Method for Extracting Mitochondrial DNA from Animal", NEWSLETTER OF SERICULTURAL SCIENCE, vol. 22, no. 3, 30 September 2002 (2002-09-30), ISSN: 1006-0561 *
ZANG, YINGAN ET AL.: "Method for Extracting Mitochondria from Animal Tissues", CHINESE JOURNAL OF VETERINARY MEDICINE, vol. 42, no. 2, 28 February 2006 (2006-02-28), ISSN: 0529-6005 *

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