WO2018006782A1 - 分离卵巢中不同种类单细胞的方法 - Google Patents

分离卵巢中不同种类单细胞的方法 Download PDF

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WO2018006782A1
WO2018006782A1 PCT/CN2017/091527 CN2017091527W WO2018006782A1 WO 2018006782 A1 WO2018006782 A1 WO 2018006782A1 CN 2017091527 W CN2017091527 W CN 2017091527W WO 2018006782 A1 WO2018006782 A1 WO 2018006782A1
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digestive juice
follicles
collagenase
cell
follicle
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French (fr)
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陈子江
马金龙
吕跃
赵涵
梁子君
王成栋
路钢
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山大生殖研发中心有限公司
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2509/10—Mechanical dissociation

Definitions

  • the invention relates to the field of cell biology.
  • the invention relates to a method of treating a mammalian early follicle to obtain a single oocyte and its granulosa cells.
  • Follicles are the basic structural and functional units of the ovary of a female mammal, and are mainly composed of an intermediate oocyte and a surrounding layer or layers of granulosa cells. According to the difference in morphology and function of follicles, it can be divided into several stages of follicles, including primordial follicles (primary follicles), primary follicles, secondary follicles, preantral follicles, sinusoid follicles and mature follicles. The size of the human follicle can be grown from the initial 30-50 ⁇ m to about 20 mm of mature follicles.
  • the mouse primordial follicle is only about 20 ⁇ m, and the diameter after ripening is about 0.5 mm.
  • the smallest diameter primordial follicles and primary follicles can be collectively referred to as early follicles.
  • the stage follicle separation method is very mature and widely used in basic research and clinical treatment.
  • there is no uniform and normative standard for the method of single early follicular separation, and the separation of two cells (ie, oocytes and their corresponding granulosa) that make up a follicle of primordial follicles and primary follicles is still in the exploratory stage. .
  • the most commonly used method is enzymatic digestion.
  • one or two enzymes are used to digest the ovarian tissue.
  • an appropriate amount of DNase can be added to prevent cell adhesion during digestion.
  • the cells can be separated by shaking or blowing the digestive juice at intervals, and the digestion is terminated by centrifugal resuspension to obtain follicles.
  • the insufficiency of the existing follicle separation method is that the separation process often requires a step of resuspending by centrifugation, which has the disadvantage of reducing the recovery efficiency of the cells, and it is easy to cause certain damage to the cells due to improper control of the action of digestive enzymes.
  • the existing method is to cut and separate the ovarian tissue after the fixed section by laser capture microdissection (LCM).
  • LCM laser capture microdissection
  • Accurate cutting takes individual cells in ovarian tissue.
  • LCM technology to separate individual cells, although very precise, must first be fixed sections of the tissue, and no active cells can be obtained.
  • the cell structure after sectioning is often incomplete and therefore has an impact on subsequent research and analysis.
  • the present invention provides, for the first time, a method for efficiently and accurately isolating early follicular tissue from a mammal to obtain an active single oocyte and its corresponding granulosa cells.
  • the present invention provides a single oocyte for obtaining early follicles of a mammal and A method of single granulocyte, characterized by comprising the steps of:
  • the ovarian tissue fragments of the mammal are digested in the first digestive juice at about 37 ° C for about 20-60 minutes, for example about 30 minutes; the first digestive juice contains collagenase I, collagenase II Or collagenase IV or a mixture thereof;
  • step (3) passing the digested solution of step (2) through a first pore size cell strainer having a pore size of about 40-100 ⁇ m (for example, about 70-80 ⁇ m), and then passing the filtrate through the second pore size. Filtered again in the cell strainer, the second pore size cell strainer has a pore size of about 8-12 ⁇ m (for example, about 8 ⁇ m); the filtrate is discarded;
  • the digestive juice mixture obtained in the step (6) is transferred to a culture solution, and single follicular cells and individual granulosa cells thereof are obtained by pipetting.
  • the method of the present invention may further comprise the step (8) of using the single oocyte obtained in the step (7) and/or its individual granulosa cells for subsequent single cell experiments, such as cell culture, nucleic acid extraction and analysis, and the like.
  • the single oocyte obtained in step (7) and/or its individual granulosa cells can be directly transferred to the culture broth for subsequent single cell experiments.
  • the early follicles of the mammal refer to primordial follicles and primary follicles. According to the difference in morphology and function of follicles, it can be divided into several periods, including primordial follicles (primary follicles), primary follicles, secondary follicles, preantral follicles, sinusoid follicles and mature follicles. Primitive follicles and primary follicles can be collectively referred to as early follicles.
  • Primordial follicles as ovarian reserve It is often in a static state, and its structure consists of an oocyte in the center and a layer of flat granular cells wrapped around it. Once activated, the primordial follicles are transformed into primary follicles consisting of an oocyte and a single layer of cubic granule cells. Mammalian primordial follicles and primary follicles are similar in volume and structure. The primordial follicles are generally 20-40 ⁇ m in diameter and consist of a flat granulosa cell surrounded by oocytes of about 20 ⁇ m in the middle.
  • the primary follicles are generally 50-80 ⁇ m in diameter and consist of a layer of cubic granule cells surrounded by oocytes of about 40 ⁇ m in the middle.
  • Statistics show that the size and structure of primordial follicles and primary follicles in mammals, especially small and medium mammals, are within the above range.
  • Small mammals generally referred to as mammals having a body length of 0.5 m or less
  • mice rats, guinea pigs, rabbits, cats, dogs, and the like.
  • Medium-sized mammals generally referred to as mammals less than 2 meters in length
  • humans monkeys, sheep, pigs, cattle, and the like.
  • step (3) the digested solution of step (2) is passed through a first pore size cell sieve, and the pore size of the first pore size cell strainer is selected to be larger than the primary follicle or primordial follicle of the mammal to be treated according to the purpose.
  • the diameter of the lens allows the target follicle to pass through the first pore cell strainer.
  • the pore size of the first pore size cell strainer is typically from about 40 to 100 ⁇ m (e.g., from about 70 to 80 ⁇ m).
  • step (1) mechanical blunt separation of the ovarian tissue of the mammal can be performed using a conventional medical device such as forceps, a needle or the like.
  • the ovarian tissue is generally divided into pieces having a size of about 1 x 10 6 to 9 x 10 6 ⁇ m 3 , that is, pieces having a length of about 100 to 300 ⁇ m.
  • the mechanical separation operation is usually carried out in a petri dish containing an anatomical solution, or directly in the first digestive juice of step (2).
  • the first digestive juice contains a mixture of collagenase I, collagenase II or collagenase IV.
  • the first digestive juice contains a mixture of Liberase and collagenase IV.
  • Liberase eg Liberase TM TL Research Grade, Sigma- , Item No. 054010020001
  • Liberase removes hybrid enzymes such as clostridium protease and trypsin and removes >98% of endotoxin and other dead cell components from the raw material.
  • Liberase is commonly used to replace traditional Collagenase I and/or Collagenase II formulations.
  • the ratio of Liberase to collagenase IV in the first digestive juice is about 1:4.
  • the first digestive juice has a Liberase of 0.02 to 0.2 mg/ml, preferably about 0.05 mg/ml, and a collagenase IV of 0.05 to 0.5 mg/ml, preferably about 0.2 mg. /ml.
  • the first digestive juice may further contain a neutral protease and/or a metalloproteinase such as thermolysin.
  • the first digestive juice may further contain DNase.
  • the second digestive juice contains trypsin, such as trypsin-EDTA solution (0.25%: 0.02%) reagent.
  • the second digestive juice contains an Accutase (eg, Cell Dissociation Reagent, ThermoFisher, Catalog No. A1110501).
  • Accutase is a widely used digestive enzyme standard preparation in the field. It has protease and collagenase activity, does not contain any animal or bacterial source components, and is often used as a replacement for trypsin. It has a mild digestion and low damage to cells. .
  • the individual follicles obtained in step (5) are directly added to the Accutase reagent for digestion.
  • the second pore size cell strainer employs a cell transwelling chamber (Transwell).
  • Transwell is a permeable cup-shaped device with a permeable polycarbonate membrane on the bottom of the cup, and the rest of the cup is made of the same material as a normal cell culture well.
  • the permeable polycarbonate membrane has micropores with pore sizes ranging from 0.4 to 12.0 [mu]m.
  • a transwell having a pore size of 8 ⁇ m was used in the method. Transwell will be resuspended in the rewell in step (4) Leave it to go to step (5).
  • step (5) may aspirate a single follicle in the resuscitation solution by using a capillary glass tube or other automated or semi-automated cell suction device or instrument. After a single follicle is aspirated, the aspirated follicle is washed in a clean medium before being transferred to the digestive juice to perform step (6).
  • the invention also provides kits for performing the aforementioned methods of obtaining early follicular single oocytes and individual granulosa cells thereof from a mammal.
  • the first digestive juice and the second digestive juice are included in the kit.
  • the components of the first digestive juice and the second digestive juice are as defined above.
  • the first digestive juice contains a mixture of collagenase I, collagenase II or collagenase IV.
  • the first digestive juice contains a mixture of Liberase and collagenase IV.
  • the ratio of Liberase to collagenase IV in the first digestive juice is about 1:4.
  • the first digestive juice may further contain a neutral protease and/or a metalloproteinase such as thermolysin.
  • the first digestive juice may further contain DNase.
  • the second digestive juice contains trypsin.
  • the second digestive juice is Accutase.
  • Figure 1 shows the separation of oocytes and granulosa cells in a single early follicle of a mouse observed under a microscope.
  • Figure 1A shows the free early follicles observed under the microscope;
  • Figure 1B shows the separated oocytes and granulosa cells observed under the microscope.
  • Figure 2 is an oocyte observed by a microscope.
  • 2A is a view of a free single oocyte of primordial follicles isolated according to the method of Example 2 observed under a microscope;
  • FIG. 2B is a free single oocyte of a primary follicle isolated according to the method of Example 2 observed under a microscope. cell.
  • FIG. 3A is an amplification curve
  • Fig. 3B is a dissolution curve.
  • FIG. 4A is a heat map based on the difference in the expression levels of different genes of cells;
  • Fig. 4B is a main component analysis.
  • Leibowitz L15 medium containing 4% fetal bovine serum (FBS) and penicillin streptomycin double antibody 100 ug/ml.
  • Digestive juice two Cell Dissociation Reagent (ThermoFisher, product number A1110501, purchase information https://www.thermofisher.com/order/catalog/product/A1110501).
  • Accutase is a widely used digestive enzyme preparation in recent years. It has protease and collagenase activity and does not contain any animal or bacterial source components. It is often used as a replacement for trypsin, which has a mild digestion and low damage to cells.
  • ⁇ MEM medium contains 10mIU/mL follicle estrogen (FSH), 3mg/mL fetal bovine serum (BSA), 1mg/mL bovine serum albumin (Fetuin: Sigma, F3385), 5mg/mL insulin, 5mg/mL transferrin, And 5 ng/ml selenium (ITS, Sigma, I3146).
  • FSH follicle estrogen
  • BSA fetal bovine serum
  • Fetuin bovine serum albumin
  • ITS 5 ng/ml selenium
  • Example 2 Separation of mouse primordial follicles from single oocytes of primary follicles and individual granulosa cells of the oocytes
  • the mouse ovary was taken out and released, and transferred to a Petri dish containing the anatomical solution for 3 times. It was then transferred to a Petri dish containing 2 ml of Digestion One.
  • the ovarian tissue was mechanically separated using a sterile needle, and the tissue was divided into pieces having a size of about 1 ⁇ 10 6 - 4 ⁇ 10 6 ⁇ m 3 under a dissecting microscope.
  • the culture dish is placed in a sterile culture cell incubator for digestion at 37 ° C for 30 minutes, and the digestive juice is blown once every 10 minutes during the digestion.
  • the follicular separation was observed in real time through a microscope during the operation, and the next step was performed when most of the follicles were in a free state and no large pieces of tissue were observed.
  • the resuspended culture contains follicles and cells between 8 ⁇ m and 70 ⁇ m in diameter:
  • mice The early follicular morphology of mice is as follows:
  • the primordial follicle (20-40 ⁇ m) consists of a flat granulosa cell surrounded by oocytes with an intermediate diameter of about 20 ⁇ m;
  • Primary follicles (50-70 ⁇ m) consist of a layer of cubic granule cells surrounded by oocytes with an intermediate diameter of about 40 ⁇ m.
  • Transwell having a pore size of 8 ⁇ m is used, and when it is placed in a culture dish, the liquid can easily pass through the filter membrane, and when suspended, the liquid can stay on the filter membrane due to surface tension.
  • the present invention utilizes this feature to pass the first digested and filtered digested solution through a Transwell filter and then recover the cells remaining in the Transwell.
  • the method of the invention has the following advantages: (1) directly filtering the culture medium containing the digestive enzyme without centrifugation; (2) using the Transwell with a pore size of 8.0 ⁇ m, the largest number of granulosa cells and membrane cells in the digestive juice can be obtained. The smaller diameter cells are directly discarded through the filter membrane, and the desired early follicles are left on the filter membrane, and then the larger diameter cells on the membrane are resuspended and absorbed by the characteristics of the membrane.
  • a single follicle was aspirated using a PicoPipet single cell separation extraction system (NEPA GENE).
  • NEPA GENE PicoPipet single cell separation extraction system
  • the early follicles and their components were manipulated according to cell morphology and size: the original follicles and their corresponding oocytes and granulosa cells were manipulated with a 30 ⁇ m S glass capillary, and the primary follicles and corresponding oocytes were manipulated with a 50 ⁇ m S glass capillary. Cells and granulosa cells.
  • the suction force should be controlled at 0.5-1.5V, and the fine-tuning amplitude should be set to 0.05V.
  • the suction should be increased by 0.5V and then removed.
  • the aspirate follicles are then washed in a clean medium, transferred to a container containing digestive solution 2 of digestive enzyme activity, and placed in a cell culture incubator for 5-10 min at 37 °C.
  • the digestive solution 2 in the method of the embodiment adopts Cell Dissociation Reagent.
  • Accutase digestive enzyme preparations have a mild and effective digestive enzyme activity that minimizes damage to cells while efficiently digesting and isolating oocytes and granulosa cells in follicles.
  • the inventors have found that the follicles are treated for a short period of time during the digestion process, for example, within 3 to 5 minutes, the damage to the cells can be minimized, thereby separating the oocytes and their corresponding granulosa cells. After being separated into single cells, it can be directly used for subsequent experiments on single cells, such as cell culture, nucleic acid extraction and amplification. Longer digestion can easily reduce the activity of oocytes and even cell degradation.
  • trypsin such as trypsin-EDTA solution (0.25%: 0.02%)
  • trypsin-EDTA solution 0.25%: 0.02%
  • the step of discontinuing digestion is required for subsequent single cell experiments, such as termination of trypsin by addition of culture medium or trypsin inhibitor.
  • the action of the cells then the cells are washed, or filtered again through a suitable cell strainer.
  • the container for taking out the digestive juice 2 is blown under a microscope (for example, using a capillary tube or a pipette, etc.) until the oocyte and its corresponding granulosa cells are separated into free single cells.
  • Example 3 Identification of single oocytes of mouse primordial follicles and primary follicles and individual granulosa cells of the oocyte
  • Example 2 From the cell morphology and size of the individual cells obtained in Example 2, individual oocytes of the mouse primordial follicles and primary follicles and individual granulosa cells of the oocytes were judged.
  • Figure 1 shows the separation of oocytes and granulosa cells in a single early follicle of a mouse observed under a microscope.
  • Figure 1A shows the free early follicles observed under the microscope;
  • Figure 1B shows the isolated oocytes and granulosa cells observed under the microscope.
  • FIG. 2A is a view of a free single oocyte of primordial follicles isolated according to the method of Example 2 observed under a microscope
  • FIG. 2B is a free single oocyte of a primary follicle isolated according to the method of Example 2 observed under a microscope. cell.
  • Example 4 Gene expression and hierarchical cluster analysis and principal component analysis of single oocytes of mouse primordial follicles and primary follicles
  • the PCR preamplification conditions are as follows:
  • Figure 3 shows the real-time PCR amplification curve and dissolution curve of the Gapdh gene of a single early oocyte.
  • the experimental results show that the obtained single oocyte can obtain good qPCR results.
  • the qPCR analysis data obtained from the single cell qPCR of the above 7 primordial follicles and 7 primary follicular oocytes were subjected to hierarchical cluster analysis and composition analysis.
  • Figure 4A is a heat map based on the difference in the expression levels of different genes in cells. It can be seen that the oocytes in the two stages have their highly expressed genes, such as ZP1 in the original follicle-derived oocytes. Oocytes are highly expressed. In addition, it can be observed that there is no significant difference in the expression of the housekeeping gene Gapdh in the two cells. After cluster analysis, it can be observed that the Gapdh status of the primordial follicle and the primary follicle are significantly different, which can be used to distinguish the primordial follicle and Primary follicles.
  • composition was analyzed for composition using the expression levels of different genes, and the results are shown in Fig. 4B. Similar to the results of the cluster analysis, it can be seen that the primordial follicles and the primary follicle oocytes are significantly different, and thus can be used to distinguish between the primordial follicles and the primary follicles.
  • oocytes primordial follicles (primary follicles), primary follicles, and obtain an active single cell.
  • Gene expression of oocytes that have lost cell activity is significantly different from gene expression of active cells.
  • the methods of the present invention enable researchers in the field to conduct further studies on individual cells. For example, oocytes can be studied in two different developmental stages by selecting genes that are specifically expressed in a certain period of time to understand their different characteristics.
  • the method of the invention operates on a single follicle level, separating the oocytes contained therein Cells and corresponding granulosa cells.
  • the method of the present invention reduces the number of manipulations of cells, thereby better protecting the integrity of the cells and enabling a greater number of early follicles to be obtained from the follicles.
  • experiments at the individual cell level are receiving increasing attention. Separation of oocytes and their corresponding granulosa cells from viable early follicles in ovarian tissue has been a problem.
  • the method of the present invention successfully solves this problem. It provides a platform basis for subsequent studies on cell activity, gene expression analysis, single cell sequencing and even proteomics from a single early oocyte and corresponding granule cell level.
  • the unit "degree” of temperature appearing in this document refers to degrees Celsius, or °C.

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Abstract

提供了用于分离哺乳动物早期卵泡获得单个卵母细胞和其单个颗粒细胞的方法。该方法能够分离哺乳动物早期卵泡获得具有活性的单个卵母细胞及其相应颗粒细胞。还提供了用于获得哺乳动物早期卵泡单个卵母细胞和其单个颗粒细胞的试剂盒。

Description

分离卵巢中不同种类单细胞的方法
本申请要求了2016年07月4日提交的、申请号为201610519416.3、发明名称为“分离卵巢中不同种类单细胞的方法”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
本发明涉及细胞生物学领域。具体的，本发明涉及处理哺乳动物早期卵泡获得单个卵母细胞和其颗粒细胞的方法。
背景技术
细胞是构成生命的基本单元，自从细胞学说被提出后，人类一直在努力寻求对细胞进行研究的方法。近年来，核酸扩增和测序技术上取得的重大突破，对单个细胞的生理和生化研究有着重要意义，使得分析单个细胞的技术方法成为了热点研究方向。因此，准确捕获单个特定种类的细胞进行研究分析也显得尤为重要。
卵泡是雌性哺乳动物卵巢的基本结构和功能单位，主要由中间的卵母细胞及周围一层或若干层颗粒细胞包裹构成。根据卵泡形态结构及功能的差异，可将其分为若干阶段的卵泡，包括原始卵泡(始基卵泡)、初级卵泡、次级卵泡、窦前卵泡、窦状卵泡及成熟卵泡。人类卵泡的直径大小可由最初的30-50μm生长到成熟卵泡20mm左右。小鼠原始卵泡只有20μm左右，成熟后直径约0.5mm。直径最小的原始卵泡及初级卵泡可统称为早期卵泡。目前，关于哺乳动物次级及其以后 阶段卵泡分离方法已经十分成熟，并广泛应用于基础研究及临床治疗当中。但对于单个早期卵泡分离的方法还没有统一和规范的标准，而对组成原始卵泡及初级卵泡的一个卵泡的两种细胞(即卵母细胞及其相应颗粒细胞)的分离方法现在还处于探索阶段。
对于卵泡的分离方法，目前最常用的是酶消化的方法，通常使用一种或两种酶对卵巢组织进行消化，在消化液中可选择添加适量DNA酶(DNase)防止消化过程中的细胞粘连，消化过程中可通过间隔的摇晃或吹打消化液来促进细胞分离，用离心重悬的方法终止消化，从而获得卵泡。现有卵泡分离方法的不足之处在于，分离过程中往往需要经过离心重悬的步骤，其缺点在于会降低细胞的回收效率，而且由于消化酶的作用控制不当，容易对细胞造成一定损伤。
而对于单个早期卵泡中卵母细胞及相应颗粒细胞的分离，现有的方法是借助激光捕获显微切割技术(Laser capture microdissection，LCM)对固定切片后的卵巢组织进行切割分离，采用此方法可以精确的切割获取卵巢组织中的单个细胞。采用LCM技术来分离单个细胞，虽然十分精确，但必须先对组织进行固定切片，而无法获取具有活性的细胞。此外，经切片处理后的细胞结构往往是不完整的，因此对于后续的研究及分析会造成一定影响。
因此本领域还需要一种更有效以及能够获得哺乳动物的具有活性的单个卵母细胞及其相应颗粒细胞的方法。
发明内容
本发明第一次提供了一种高效和准确地分离哺乳动物的早期卵泡组织，获得具有活性的单个卵母细胞及其相应颗粒细胞的方法。
本发明提供了一种获得哺乳动物的早期卵泡的单个卵母细胞和其 单个颗粒细胞的方法，其特征在于包括以下步骤：
(1)对所述哺乳动物的卵巢组织进行机械分离成为小碎块；
(2)将所述哺乳动物的卵巢组织碎块在约37℃下，在第一消化液消化约20-60分钟，例如约30分钟；所述第一消化液含有胶原蛋白酶I、胶原蛋白酶II或胶原蛋白酶IV或其混合物；
(3)将步骤(2)的消化液通过第一孔径细胞滤网，所述第一孔径细胞滤网的孔径为约40-100μm(例如为约70-80μm)，然后将滤液通过第二孔径细胞滤网中再次过滤，所述第二孔径细胞滤网的孔径为约8-12μm(例如为约8μm)；弃掉滤液；
(4)用培养液冲洗第二孔径细胞滤网内沉淀物，然后用培养液重悬沉淀；
(5)将重悬溶液中的单个卵泡吸出；
(6)将单个早期卵泡清洗后，在第二消化液中消化约3-10分钟，优选在5分钟内，所述第二消化液含有胰蛋白酶(trypsin)或
Figure PCTCN2017091527-appb-000001
(7)将步骤(6)得到的消化液混合物转移到培养液中，通过吹打分离得到单个卵泡细胞以及其单个颗粒细胞。
本发明的方法还可包括步骤(8)，将步骤(7)得到的单个卵母细胞和/或其单个颗粒细胞用于后续的单个细胞实验，例如细胞培养、核酸提取和分析等。在本发明的其中一个方面，步骤(7)得到的单个卵母细胞和/或其单个颗粒细胞可以直接转移至培养液中以用于后续的单个细胞实验。
在本发明中，所述哺乳动物的早期卵泡是指原始卵泡和初级卵泡。根据卵泡形态结构及功能的差异，可将其分为若干时期，包括原始卵泡(始基卵泡)、初级卵泡、次级卵泡、窦前卵泡、窦状卵泡及成熟卵泡。原始卵泡及初级卵泡可统称为早期卵泡。原始卵泡作为卵巢储备 往往处于静止状态，其结构由中央的一个卵母细胞及周围包裹的一层扁平颗粒细胞组成。原始卵泡一旦被激活将转变为由一个卵母细胞及单层立方形颗粒细胞组成的初级卵泡。哺乳动物的原始卵泡和初级卵泡的体积和结构较为相似。原始卵泡直径一般为20-40μm，由中间20μm左右的卵母细胞包裹一层扁平的颗粒细胞构成。初级卵泡直径一般为50-80μm，由中间40μm左右的卵母细胞包裹一层立方形颗粒细胞构成。统计数据显示，哺乳动物，特别是中小型的哺乳动物，的原始卵泡和初级卵泡的体积和结构都在上述范围。小型哺乳动物(一般指体长在0.5米以下的哺乳动物)包括小鼠、大鼠、豚鼠、兔、猫，犬等。中型哺乳动物(一般指体长在2米以下的哺乳动物)包括人、猴、羊、猪、牛等。
在步骤(3)中，将步骤(2)的消化液通过第一孔径细胞滤网，所述第一孔径细胞滤网的孔径将根据目的选择为大于待处理的哺乳动物的初级卵泡或原始卵泡的直径，由此可让目标卵泡通过第一孔径细胞滤网。第一孔径细胞滤网的孔径通常为约40-100μm(例如为约70-80μm)。
在步骤(1)中，可以采用常用的医疗器械例如镊子、针头等对所述哺乳动物的卵巢组织进行机械钝性分离。一般将卵巢组织分为大小约1×106～9×106μm3的碎块，即长度约为100～300μm的碎块。所述机械分离的操作通常在含解剖液的培养皿中进行，也可以直接在步骤(2)的第一消化液中进行。
在本发明的其中一个方面，所述第一消化液含有胶原蛋白酶I、胶原蛋白酶II或胶原蛋白酶IV的混合物。在本发明的其中又一个方面，所述第一消化液含有Liberase和胶原蛋白酶IV的混合物。Liberase(如LiberaseTM TL Research Grade，Sigma-
Figure PCTCN2017091527-appb-000002
，货号05401020001)是近 年一种广泛使用的消化酶标准制剂，其中含有高纯度的胶原蛋白酶I和胶原蛋白酶II的混合物。Liberase中去除了梭菌蛋白酶和胰蛋白酶等杂酶，并且去除了原料中＞98％的内毒素和其他死细胞成分。Liberase通常用于替换传统的Collagenase I和/或Collagenase II制剂。
在本发明的其中一个方面，所述第一消化液中Liberase和胶原蛋白酶IV的比例为约1∶4。在本发明的其中又一个方面，所述第一消化液中Liberase为0.02～0.2mg/ml，优选为约0.05mg/ml，以及胶原蛋白酶IV为0.05～0.5mg/ml，优选为约0.2mg/ml。
在本发明中，所述第一消化液还可含有中性蛋白酶和/或金属蛋白酶，如嗜热菌蛋白酶(thermolysin)。在本发明的其中又一个方面，所述第一消化液还可含有DNase。
在本发明的其中一个方面，所述第二消化液含有胰蛋白酶(trypsin)，例如胰蛋白酶-EDTA溶液(0.25％∶0.02％)试剂。
在本发明的其中另一个方面，所述第二消化液含有Accutase(如
Figure PCTCN2017091527-appb-000003
Cell Dissociation Reagent，ThermoFisher，货号A1110501)。Accutase是本领域一种广泛使用的消化酶标准制剂，具有蛋白酶和胶原酶活性，不含任何动物或细菌来源组分，常被用作胰酶的替换产品，其消化方式温和，对细胞伤害低。使用中，将步骤(5)中获得的单个卵泡直接加入到Accutase试剂中进行消化。
在本发明的其中一个方面，所述第二孔径细胞滤网采用细胞透室(Transwell)。Transwell是有通透性的杯状的装置，在杯状装置底层的一张有通透性的聚碳酸酯膜(polycarbonate membrane)，而杯状装置其余部分的材料与普通的细胞培养孔板是一样。通透性的聚碳酸酯膜带有微孔，孔径大小范围为0.4～12.0μm。本方法中采用孔径为8μm的transwell。在步骤(4)对transwell内的沉淀物进行重悬时，将transwell 悬空，以便进行步骤(5)。
在本发明的方法中，步骤(5)可以通过使用毛细玻璃管或其它自动或半自动的细胞吸取装置或器械将重悬溶液中的单个卵泡吸出。将单个卵泡吸出后，在干净培养基中将吸出的卵泡清洗后才转移至消化液二进行步骤(6)。
本发明还提供了用于实现前述从哺乳动物获得早期卵泡单个卵母细胞和其单个颗粒细胞的方法的试剂盒。所述试剂盒中包括所述第一消化液和第二消化液。所述第一消化液和第二消化液的成分如前所定义。例如，在所述试剂盒中，所述第一消化液含有胶原蛋白酶I、胶原蛋白酶II或胶原蛋白酶IV的混合物。在本发明的其中又一个方面，所述第一消化液含有Liberase和胶原蛋白酶IV的混合物。在本发明的其中又一个方面，所述第一消化液中Liberase和胶原蛋白酶IV的比例为约1∶4。在本发明中，所述第一消化液还可含有中性蛋白酶和/或金属蛋白酶，如嗜热菌蛋白酶(thermolysin)。在本发明的其中又一个方面，所述第一消化液还可含有DNase。在本发明的其中一个方面，所述第二消化液含有胰蛋白酶(trypsin)。在本发明的其中另一个方面，所述第二消化液为Accutase。
附图说明
图1为显微镜下观察到的小鼠的单个早期卵泡中卵母细胞及颗粒细胞的分离情况。图1A为显微镜下观察到的游离早期卵泡；图1B为显微镜下观察到的分离后的卵母细胞及颗粒细胞
图2为显微镜观察的卵母细胞。图2A为显微镜下观察到的根据实施例2的方法分离到的原始卵泡的游离单个卵母细胞；图2B为显微镜下观察到的根据实施例2的方法分离到的初级卵泡的游离单个卵母细胞。
图3单个早期卵母细胞Gapdh基因real-time PCR扩增曲线及溶解曲线。图3A为扩增曲线；图3B为溶解曲线。
图4小鼠原始卵泡和初级卵泡的单个卵母细胞的基因的表达情况及分层聚类分析和主成成分分析。图4A为根据细胞不同基因表达量的差异所做的热图；图4B为主成成分分析。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1 制备试验材料和准备动物
制备以下试验材料：
解剖液：Leibowitz L15培养基：包含4％胎牛血清(FBS)及青霉素链霉素双抗100ug/ml。
消化液一：αMEM培养基：包含0.05mg/ml LiberaseTM TL Research Grade(Sigma，产品编号05401020001，购买信息http://www.sigmaaldrich.com/catalog/product/roche/05401020001？lang＝zh&region＝CN)和0.2mg/ml胶原蛋白酶IV(ThermoFisher，17104-019)和0.1％DNase。
消化液二：
Figure PCTCN2017091527-appb-000004
Cell Dissociation Reagent(ThermoFisher，产品编号A1110501，购买信息https://www.thermofisher.com/order/catalog/product/A1110501)。Accutase是近年一种广泛使用的消化酶制剂，具有蛋白酶和胶原酶活性，不含任何动物或细菌来源组分。常被用作胰酶的替换产品，其消化方式温和，对细胞伤害低。
培养液：
αMEM培养基：包含10mIU/mL卵泡雌激素(FSH)，3mg/mL胎牛血清(BSA)，1mg/mL牛血清白蛋白(Fetuin：Sigma，F3385)，5mg/mL insulin，5mg/mL transferrin，and 5ng/ml selenium(ITS，Sigma，I3146)。
实验动物：
一到两周龄雌性C57小鼠
实施例2 小鼠原始卵泡和初级卵泡的单个卵母细胞以及该卵母细胞的单个颗粒细胞的分离
实验步骤：
(1)将小鼠卵巢取出并游离，转移至含解剖液的培养皿中清洗3次。然后转移至含2ml消化液一的培养皿中。
用无菌针头对卵巢组织进行机械分离，在解剖显微镜下将组织分为大小约1×106-4×106μm3的碎块。
(2)将培养皿置于无菌培养细胞培养箱中消化37℃，30分钟，消化期间每隔10分钟对消化液吹打一次。操作过程中通过显微镜实时观察卵泡分离情况，当大部分卵泡处于游离状态且观察不到大块组织存在时进行下一步操作。
(3)将消化液通过70μm细胞滤网(
Figure PCTCN2017091527-appb-000005
70μm Cell Strainer，Corning，352350)，过滤两次；然后将滤液加入孔径为8μm的Transwell(SPLInsertTM Hanging，SPL Life Sciences，35206)中再次过滤，弃掉滤液；
(4)用培养液冲洗Transwell内沉淀物三次，最后用1ml培养液重悬沉淀，重悬时将Transwell悬空于培养皿的液面上方。
重悬培养液即包含直径介于8μm-70μm之间的卵泡及细胞：
小鼠早期卵泡形态如下：
原始卵泡(20-40μm)由中间直径为约20μm左右的卵母细胞包裹一层扁平的颗粒细胞构成；
初级卵泡(50-70μm)由中间直径为约40μm左右的卵母细胞包裹一层立方形颗粒细胞构成。
本方法中采用孔径为8μm的Transwell，其在放入培养皿中时，液体可以很容易通过滤膜，而将其悬空时由于表面张力，液体可以在滤膜上面停留。本发明利用其这一特点，可将经第一次消化和过滤后的消化液再通过Transwell的滤膜，然后回收留在Transwell内的细胞。本发明的方法优点是：(一)通过滤过含有消化酶的培养基，无需离心可以直接终止消化；(二)利用8.0μm孔径的Transwell，可使消化液中数量最多的颗粒细胞及膜细胞等直径较小细胞直接通过滤膜丢弃，而将所需早期卵泡留在滤膜之上，再利用滤膜的特性，将滤膜上直径较大的细胞重悬吸出。
(5)采用PicoPipet单细胞分离提取系统(NEPA GENE)将单个卵泡吸出。操作时，依据细胞形态及大小对早期卵泡及其组分进行操作：用30μm的S玻璃毛细管操作原始卵泡及其相应卵母细胞和颗粒细胞，用50μm的S玻璃毛细管操作初级卵泡及相应卵母细胞和颗粒细胞。吸取早期卵母细胞时，吸力大小控制在0.5-1.5V为宜，微调幅度大小设定为0.05V，移取细胞时在细胞达到平衡位置后应再将吸力调大0.5V后移取。
(6)然后在干净培养基中将吸出的卵泡清洗后，转移至含有消化酶活性的消化液二的容器中，置于细胞培养箱中37℃消化5-10min。
本实施例的方法中的消化液二采用
Figure PCTCN2017091527-appb-000006
Cell  Dissociation Reagent。Accutase消化酶制剂具有温和和有效的消化酶活性，能够在有效消化和分离卵泡中的卵母细胞和颗粒细胞的同时，最大程度地减少对细胞的损害。同时，发明人发现，在消化过程中对卵泡进行较短时间的处理，例如在3～5分钟内，能够最大程度地减少对细胞的损害，由此使得卵母细胞及其相应的颗粒细胞分离成游离的单个细胞后，能够直接用于对单细胞进行后续的实验，如细胞培养、核酸提取和扩增等。时间较长的消化，容易使得卵母细胞活性降低，甚至发生细胞降解情况。
实验发现，在消化液二中采用胰蛋白酶(Trypsin)，如胰蛋白酶-EDTA溶液(0.25％∶0.02％)试剂，也能消化和分离得到卵泡中的卵母细胞和颗粒细胞。但是，在卵母细胞及其相应的颗粒细胞分离成游离的单个细胞后，需要经过中止消化的步骤才能用于后续的单个细胞实验，例如经过加入培养液或是胰酶抑制剂终止胰酶对细胞的作用，然后对细胞进行清洗，或是再次通过合适的细胞滤网过滤的过程。
实验发现，在消化液二采用其它消化酶，例如胶原蛋白酶时，不能在保持细胞活性需要的短时间内(例如半小时内，特别是在3-10分钟内)实现有效地消化和分离卵泡中的卵母细胞和颗粒细胞。
(7)消化结束后，取出消化液二的容器在显微镜下进行吹打(例如用毛细管或移液器等)，直到卵母细胞及其相应的颗粒细胞分离成游离的单个细胞为止。
(8)将单个卵母细胞和/或其单个颗粒细胞直接转移至培养液中以用于后续的单个细胞实验。
实施例3 小鼠原始卵泡和初级卵泡的单个卵母细胞以及该卵母细胞的单个颗粒细胞的鉴定
根据实施例2获得的单个细胞的细胞形态和尺寸，判断获得了小鼠原始卵泡和初级卵泡的单个卵母细胞以及该卵母细胞的单个颗粒细胞。
图1为显微镜下观察到的小鼠的单个早期卵泡中卵母细胞及颗粒细胞的分离情况。图1A为显微镜下观察到的游离早期卵泡；图1B为显微镜下观察到的分离后的卵母细胞及颗粒细胞。
如图2所示，获得了小鼠原始卵泡和初级卵泡的单个卵母细胞。图2A为显微镜下观察到的根据实施例2的方法分离到的原始卵泡的游离单个卵母细胞；图2B为显微镜下观察到的根据实施例2的方法分离到的初级卵泡的游离单个卵母细胞。
实施例4 小鼠原始卵泡和初级卵泡的单个卵母细胞的基因的表达情况及分层聚类分析和主成成分分析
1.对实施例2中获得的小鼠原始卵泡和初级卵泡的单个卵母细胞进行基因的表达分析。
实验试剂：Single Cell Lysis Kit(ThermoFisher，4458235)；SuperScript VILO cDNA Synthesis Kit(ThermoFisher，1754-050)；Platinum Taq DNA Polymerase(ThermoFisher，10966-034)；PowerUp SYBR Green Master Mix(ThermoFisher，A25777)。
实验步骤：
(1)将实施例2分离到的7个原始卵泡的卵母细胞及7个初级卵泡的卵母细胞分别转移到一个含有10μl细胞裂解液(Single Cell Lysis Kit)的PCR管中，根据产品说明书在室温下裂解5分钟(裂解后可-20℃保存)。
(2)每个PCR管中分别加入1μl终止液，室温静止2分钟。
(3)依照SuperScript VILO cDNA Synthesis Kit说明书进行单个细胞的反转录。反转录后将每管中分别加入50μl去核酸水稀释得到的cDNA。
(4)用以下特异性的引物进行预扩增，根据Platinum Taq DNA Polymerase的说明书反应体系如下：
Figure PCTCN2017091527-appb-000007
PCR预扩增条件如下：
Figure PCTCN2017091527-appb-000008
(5)扩增产物即可以进行单细胞qPCR，反应体系如下：
Figure PCTCN2017091527-appb-000009
依照SYBR说明书进行qPCR。
实验结果可以以图三为例。图三显示单个早期卵母细胞的Gapdh基因的real-time PCR扩增曲线及溶解曲线。实验结果说明获得的单个卵母细胞可以得到很好的qPCR结果。
2.对实施例2中获得的小鼠原始卵泡和初级卵泡的单个卵母细胞进行分层聚类分析和组成成分分析。
将上述在7个原始卵泡以及7个初级卵泡的卵母细胞的单细胞qPCR获得的qPCR分析数据进行分层聚类分析和组成成分分析。
其中，对qPCR得到的CT值不与内参进行标准化，而是直接对其处理，设定有效阈值并删除假阳性值、处理缺失值，最后将其转换为2为底的Log值来进行分析。将处理后的数据导入Qlucore Omics软件进行分析作图。结果如图4所示。图4A为根据细胞不同基因表达量的差异所做的热图，可以看出两种时期的卵母细胞各自存在其高表达的基因，如原始卵泡来源的卵母细胞中ZP1相对初级卵泡来源的卵母细胞呈高表达。另外，可以观察到管家基因Gapdh在两种细胞中的表达没有明显差异，再对其进行聚类分析后可以观察到原始卵泡和初级卵泡的Gapdh状况有明显区别，由此可用于区别原始卵泡和初级卵泡。
用不同基因的表达量对样本进行组成成分分析，结果如图4B所示。与聚类分析的结果类似，可以看出原始卵泡和初级卵泡的卵母细胞有明显区别，由此可用于区别原始卵泡和初级卵泡。
以上结果说明了本发明的分离卵巢组织中不同组织种类单细胞的方法可以正确的分离两种不同的早期卵母细胞，即原始卵泡(始基卵泡)、初级卵泡，并且获得其具有活性的单细胞。失去细胞活性的卵母细胞的基因表达与具有活性的细胞的基因表达是明显不同的。本发明的方法使得本领域的研究人员能够对单个细胞进行进一步的研究。例如可以通过挑选一些时期特异性表达的基因，对两种不同发育时期的卵母细胞进行研究，了解其不同的特性。
本发明的方法在单个卵泡水平上进行操作，分离其所包含的卵母细 胞及相应颗粒细胞。本发明的方法减少了对细胞的操作次数，从而更好的保护了细胞的完整性，并且能够从卵泡中获得更多数量的早期卵泡。随着单细胞技术的发展和成熟，单个细胞水平上的实验是越来越受关注。而对于卵巢组织中活的早期卵泡中卵母细胞及其相应颗粒细胞的分离一直是一个难题。而本发明的方法成功的解决了这一问题。对后续从单个早期卵母细胞及相应颗粒细胞水平上进行细胞活性研究、基因表达分析、单细胞测序甚至蛋白质组学的研究提供了平台基础。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。

Claims (15)

  1. 一种获得哺乳动物的早期卵泡的单个卵母细胞和其单个颗粒细胞的方法，其特征在于包括以下步骤：
    (1)对哺乳动物卵巢组织进行钝性分离；
    (2)将哺乳动物卵巢组织碎块在约37℃下，在第一消化液中消化约20-60分钟，例如约30分钟，所述第一消化液含有胶原蛋白酶I、胶原蛋白酶II或胶原蛋白酶IV或其混合物；
    (3)将步骤(2)得到的消化液混合物通过第一孔径细胞滤网，所述第一孔径细胞滤网的孔径为约40-100μm(例如为约70-80μm)，然后将滤液通过第二孔径细胞滤网，所述第二孔径细胞滤网的孔径为约8-12μm(例如为约8μm)；
    (4)用培养液冲洗第二孔径细胞滤网内沉淀物，然后用培养液重悬沉淀；
    (5)吸出重悬溶液中的单个早期卵泡；
    (6)将吸出的单个早期卵泡清洗后，在第二消化液中消化约3-10分钟，优选在5分钟内，所述第二消化液含有胰蛋白酶或Accutase；
    (7)将步骤(6)得到的消化液混合物转移到培养液中，通过分离得到单个卵母细胞以及其相应单个颗粒细胞。
  2. 权利要求1的方法，其中还包括以下步骤：
    (8)将单个卵母细胞和/或其单个颗粒细胞用于单个细胞实验。
  3. 权利要求1的方法，其中所述早期卵泡为原始卵泡或初级卵泡。
  4. 权利要求1的方法，其中所述第一消化液含有Liberase和胶原蛋白酶IV的混合物，优选的，其中所述第一消化液中Liberase和胶原蛋白酶IV的比例为约1∶4。
  5. 权利要求1的方法，其中所述第一消化液还含有中性蛋白酶和/或金 属蛋白酶，优选的，其中所述第一消化液还含有DNase。
  6. 权利要求1的方法，其中所述第二消化液含有Accutase。
  7. 权利要求1的方法，其中步骤(6)中，在第二消化液中消化约3-5分钟。
  8. 权利要求1的方法，其中所述第二孔径细胞滤网为细胞透室，即Transwell。
  9. 权利要求1的方法，其中所述哺乳动物为中小型哺乳动物，例如人、猴、羊、猪、牛、小鼠、大鼠、豚鼠、兔、猫，犬。
  10. 一种用于如权利要求1-9中任一项的从哺乳动物获得早期卵泡单个卵母细胞和其单个颗粒细胞的方法的试剂盒，所述试剂盒中包括所述第一消化液和第二消化液，所述第一消化液含有胶原蛋白酶I、胶原蛋白酶II或胶原蛋白酶IV或其混合物，所述第二消化液含有胰蛋白酶或Accutase。
  11. 权利要求10的试剂盒，其中所述早期卵泡为原始卵泡或初级卵泡。
  12. 权利要求10的试剂盒，其中所述第一消化液含有Liberase和胶原蛋白酶IV的混合物，优选的，其中所述第一消化液中Liberase和胶原蛋白酶IV的比例为约1∶4。
  13. 权利要求12的试剂盒，其中所述第一消化液还含有中性蛋白酶和/或金属蛋白酶，优选的，其中所述第一消化液还含有DNase。
  14. 权利要求10的试剂盒，其中所述第二消化液含有Accutase。
  15. 权利要求10的试剂盒，其中所述哺乳动物为中小型哺乳动物，例如人、猴、羊、猪、牛、小鼠、大鼠、豚鼠、兔、猫，犬。
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