WO2009000213A1

WO2009000213A1 - A method for isolating aging cells and the model

Info

Publication number: WO2009000213A1
Application number: PCT/CN2008/071463
Authority: WO
Inventors: Jinqiu Zhou; Xiaohong Fu; Feilong Meng
Original assignee: Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
Priority date: 2007-06-27
Filing date: 2008-06-27
Publication date: 2008-12-31
Also published as: CN101333503B; CN101333503A

Abstract

An isolated cell population of Candida is provided, as well as the method for preparing the cell population and its uses, in which most cells of the cell population are at the same age stage. Based on the feature that candida cells have two distinct growth forms during different growth conditions, methods for preparing cell population at the same age stage on a large scale have been developed. The cell population at the same age stage may be used to study cells growth, development and the characterizations of cells at various age stages, as well as cellular aging.

Description

分离衰老细胞的方法和模型技术领域 Method and model for isolating senescent cells

本发明属于生物技术和细胞生物学领域；更具体的，本发明涉及产生同一年龄阶段细胞群的方法以及由该方法获得的细胞群。背景技术 The present invention is in the field of biotechnology and cell biology; more specifically, the present invention relates to a method of producing a cell population at a stage of the same age and a cell population obtained by the method. Background technique

衰老问题是长久以来被广泛关注和研究的热点问题之一，通常是指随年龄增长而发生的渐进的、受遗传因素影响的、复杂的形态结构与生理功能不可逆的退行性变化，主要表现为生育力的下降和死亡率的上升。衰老在进化上是相对保守的，从最高等的生物人类，到非常低等的原核生物细菌，都存在衰老这一生理过程。 The problem of aging is one of the hot issues that has been widely concerned and studied for a long time. It usually refers to the progressive, irreversible degenerative changes of morphological structure and physiological functions that occur with age. Declining fertility and rising mortality. Aging is relatively conservative in evolution. From the highest biological humans to very low prokaryotic bacteria, there is a physiological process of aging.

细胞衰老是生物体衰老的基本单位，是细胞生理和生化发生复杂变化的过程，最终可能反映在细胞的形态、结构和生理功能上。 Cell senescence is the basic unit of organism aging, a process of complex changes in cell physiology and biochemistry, which may ultimately be reflected in the morphology, structure and physiological functions of cells.

单细胞真核生物酿酒酵母 (Sacc/2aro _y«^ cerei^/ae，简称 S. cerev /ae)将细胞衰老和个体衰老联系起来，是目前被广泛应用的衰老实验模型之一。 S. Single-cell eukaryotic Saccharomyces cerevisiae (Sacc/2aro _y«^ cerei^/ae, referred to as S. cerev /ae) links cell aging with individual aging and is one of the widely used aging experimental models. S.

cerevisiae 以出芽生殖的方式产生子细胞，母细胞经过一定次数的***之后走向衰老，随之发生一系列形态结构等方面的变化 (； Jazwinski, S. M. ( 1996). Longevity, genes, and aging. Science 273, 54-59)。母细胞所能***的终极次数定义为酵母细胞的寿命 (life span)(Barton, A. A. ( 1950). Some aspects of cell division in Cerevisiae produces daughter cells in a budding manner. After a certain number of divisions, the mother cells go to senescence, followed by a series of changes in morphological structure (Jazwinski, SM (1996). Longevity, genes, and aging. Science 273 , 54-59). The ultimate number of divisions that a mother cell can divide is defined as the life span of a yeast cell (Barton, A. A. (1950). Some aspects of cell division in

saccharomyces cerevisiae. J Gen Microbiol 4, 84-86)。 Saccharomyces cerevisiae. J Gen Microbiol 4, 84-86).

然而，由于母细胞和子细胞形态上的相似性以及衰老细胞在整个细胞群体中所占比例很小，分离衰老的酵母细胞需要复杂的过程以及特殊的装置 (Park, P. U. 等 (2002 Separation of mother and daughter cells. Methods Enzymol 351, 468-477) , 获得大量衰老细胞在高通量的层面上进行研究就显得非常困难，衰老细胞的分离问题因此成为本领域研究的瓶颈和制约因素。 However, due to the morphological similarity between blast cells and daughter cells and the small proportion of senescent cells in the entire cell population, the isolation of senescent yeast cells requires complex processes and special devices (Park, PU, etc. (2002 Separation of mother and Methods. Enzymol 351, 468-477) It is very difficult to obtain a large number of senescent cells on a high-throughput level. The separation of senescent cells has become a bottleneck and constraint in this field.

因此，本领域迫切需要找到一种可方便地分离出处于同一年龄阶段 (如处于衰老阶段；)的细胞群的方法，从而制备出年龄相对统一的细胞模型，用于研究细胞的生长和衰老机制。发明内容 Therefore, there is an urgent need in the art to find a method for conveniently isolating cell populations at the same age (eg, in the aging phase) to prepare a relatively uniform cell model for studying cell growth and aging mechanisms. . Summary of the invention

本发明的目的在于提供一种分离的念珠菌细胞群，所述细胞群中大多数细胞处于同一年龄阶段。 It is an object of the present invention to provide an isolated population of Candida cells in which most of the cells are at the same age.

本发明的另一目的在于提供一种制备所述念珠菌细胞群的方法以及所述念珠菌细胞群的用途。 Another object of the present invention is to provide a method of preparing the Candida cell population and the use of the Candida cell population.

在本发明的第一方面，提供一种分离的念珠菌细胞群，所述细胞群具有以下特征： In a first aspect of the invention, there is provided an isolated population of Candida cells, said population of cells having the following characteristics:

(a) 所述细胞群中细胞数量为 10²-10^1Q个； (a) the number of cells in the cell population is 10 ² -10 ^1Q ;

(b) 所述细胞群中 80%以上细胞为念珠菌的菌丝状细胞；和 (b) more than 80% of the cells in the cell population are mycelial cells of Candida;

(c) 70%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段。 (c) More than 70% of the mycelial cells of the Candida are at the same age.

在本发明的另一优选例中，所述细胞群中， 90%以上细胞为念珠菌的菌丝状细胞。 In another preferred embodiment of the present invention, more than 90% of the cells in the cell population are mycelial cells of Candida.

在另一优选例中，所述细胞群中， 80%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段。 In another preferred embodiment, more than 80% of the mycelial cells of the Candida are in the same age group.

在另一优选例中，所述细胞群中 90%以上细胞为念珠菌的菌丝状细胞；和 /或 In another preferred embodiment, more than 90% of the cells in the cell population are mycelial cells of Candida; and/or

90%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段。 More than 90% of the mycelial cells of the Candida are at the same age.

在另一优选例中，所述细胞群中细胞数量为 10³-10⁹个；更优选的，所述细胞群中细胞数量为 10⁵-10⁸个。 In another preferred embodiment, the number of cells in the cell population is from 10 ^{3 to} 10 ⁹ ; more preferably, the number of cells in the cell population is from 10 ^{5 to} 10 ⁸ .

在另一优选例中，所述念珠菌细胞群的平均传代次数为 1-30代，更特别的为 5-25代。 In another preferred embodiment, the average number of passages of the Candida cell population is from 1 to 30 generations, more particularly from 5 to 25 passages.

在另一优选例中，所述的同一年龄阶段是指衰老阶段 (如细胞群的平均传代次数为 20代以上)。 In another preferred embodiment, the same age stage refers to the aging stage (e.g., the average number of passages of the cell population is 20 generations or more).

在另一优选例中，所述的念珠菌是白念珠菌。在本发明的第二方面，提供一种制备所述的念珠菌细胞群的方法，所述方法包括步骤： In another preferred embodiment, the Candida is Candida albicans. In a second aspect of the invention, a method of preparing the Candida cell population is provided, the method comprising the steps of:

(a) 在适合菌丝状念珠菌细胞生长的条件下，培养念珠菌细胞群，形成念珠菌菌丝状细胞群； (a) cultivating a Candida cell population under conditions suitable for the growth of mycelial cells of Candida to form a filamentous cell population of Candida;

(b) 在适合酵母状念珠菌细胞生长的条件下，培养步骤 (a)的念珠菌的菌丝状细胞群，从而形成含有念珠菌的菌丝状细胞和酵母状子细胞的混合细胞群；和 (b) cultivating the mycelial cell population of Candida in step (a) under conditions suitable for the growth of Candida albicans cells, thereby forming a mixed cell population of Candida-containing mycelial cells and yeast-like daughter cells;

(c) 从步骤 (b)获得的混合细胞群中分离出念珠菌的酵母状子细胞群，并且在适合菌丝状念珠菌细胞生长的条件下，培养该酵母状子细胞群，从而形成念珠菌的菌丝状细胞群； (c) isolating the yeast cell population of Candida from the mixed cell population obtained in step (b) and is suitable The yeast-like daughter cell population is cultured under the condition of growth of mycelial cells of Candida, thereby forming a mycelial cell population of Candida;

(d) 在适合酵母状念珠菌细胞生长的条件下，培养上一步骤所获得的念珠菌的菌丝状细胞群，从而形成含有念珠菌的菌丝状细胞和酵母状子细胞的混合细胞群； (e) 从步骤 (d)获得的混合细胞群中分离出念珠菌的菌丝状细胞群。 (d) cultivating the mycelial cell population of Candida obtained in the previous step under conditions suitable for the growth of the Candida species, thereby forming a mixed cell population containing Candida-like mycelial cells and yeast-like daughter cells; (e) separating the mycelial cell population of Candida from the mixed cell population obtained in the step (d).

在本发明的另一优选例中，该方法还包括以下步骤： In another preferred embodiment of the present invention, the method further comprises the steps of:

(f) 重复步骤 (d)和 (e)l-20次，从而获得平均传代次数为 2-30代的念珠菌的菌丝状细胞群。 (f) Repeat steps (d) and (e) l-20 times to obtain a mycelial cell population of Candida with an average passage number of 2-30 passages.

在另一优选例中，在步骤 (e)或 (f)中，还包括：分析所获得的念珠菌的菌丝状细胞群的平均传代次数。 In another preferred embodiment, in the step (e) or (f), the method further comprises: analyzing the average number of passages of the mycelial cell population of the obtained Candida.

在另一优选例中，步骤（b) 中培养时间为 0.5-5小时。 In another preferred embodiment, the incubation time in step (b) is from 0.5 to 5 hours.

在另一优选例中，步骤 (b)中培养时间为 1-4小时，进一步优选的是 2-3小时。在另一优选例中，所述的适合菌丝状念珠菌细胞生长的条件为 35-42°C，血清诱导。 In another preferred embodiment, the incubation time in the step (b) is from 1 to 4 hours, further preferably from 2 to 3 hours. In another preferred embodiment, the conditions suitable for the growth of mycelial cells of Mycelia are 35-42 ° C, and serum is induced.

在另一优选例中，所述的适合菌丝状念珠菌细胞生长的条件为 36-40°C。进一步优选的，所述的适合菌丝状念珠菌细胞生长的条件为 37-38°C。 In another preferred embodiment, the conditions suitable for the growth of mycelial cells of Mycelia are 36-40 °C. Further preferably, the conditions suitable for the growth of mycelial cells of Mycelia are 37-38 °C.

更优选的，所述的血清是小牛血清或胎牛血清。 More preferably, the serum is calf serum or fetal bovine serum.

在另一优选例中，所述的适合酵母状念珠菌细胞生长的条件为 25-32°C。 In another preferred embodiment, the conditions suitable for the growth of the Candida albicans cells are 25-32 °C.

更优选的，所述的适合酵母状念珠菌细胞生长的条件为 28-30°C。进一步优选的，所述的适合酵母状念珠菌细胞生长的条件为 30°C。 More preferably, the conditions suitable for the growth of the Candida albicans cells are 28-30 °C. Further preferably, the condition suitable for the growth of the Candida albicans cell is 30 °C.

在另一优选例中，在步骤（c) 中，通过密度梯度离心分离出念珠菌的酵母状子细胞群；或者， In another preferred embodiment, in step (c), the yeast cell population of Candida is isolated by density gradient centrifugation; or

在步骤（e) 中，通过密度梯度离心分离出念珠菌的菌丝状细胞群。 In step (e), the mycelial cell population of Candida is isolated by density gradient centrifugation.

在另一优选例中，所述的念珠菌是白念珠菌。 In another preferred embodiment, the Candida is Candida albicans.

在另一优选例中，所述念珠菌的培养基是 YPD培养基。在本发明的第三方面，提供所述的细胞群的用途，用于作为研究细胞的生长、发育、代谢、衰老的模型；或用于筛选调节细胞的生长、发育、代谢、衰老的物质。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。附图说明 In another preferred embodiment, the culture medium of Candida is YPD medium. In a third aspect of the invention, the use of the cell population is provided for use as a model for studying the growth, development, metabolism, senescence of cells; or for screening for substances that regulate the growth, development, metabolism, and senescence of cells. Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

图 1显示了白念珠菌的形态及寿命。其中： Figure 1 shows the morphology and lifespan of Candida albicans. among them:

图 1A显示了白念珠菌在不同生长条件下的形态转换。 Figure 1A shows the morphological transformation of Candida albicans under different growth conditions.

左： 30°C，酵母形态； Left: 30 ° C, yeast form;

中： 37°C，血清诱导，菌丝形态； Medium: 37 ° C, serum induction, mycelial morphology;

右：菌丝状母细胞在 30°C非诱导条件下产生酵母状子细胞。 Right: Mycelial progenitor cells produce yeast-like daughter cells under non-inducing conditions at 30 °C.

图 1B显示了酵母形态和菌丝形态的白念珠菌细胞具有相同的复制型寿命。图 2显示了在白念珠菌中大规模分离衰老细胞***的建立。其中： Figure 1B shows that Candida albicans cells in yeast morphology and mycelial morphology have the same replication life. Figure 2 shows the establishment of a large-scale separation of senescent cell systems in Candida albicans. among them:

图 2A显示了该分离***中的菌丝状母细胞主要是由 4-6个细胞串联组成的菌丝体； Figure 2A shows that the mycelial mother cells in the separation system are mainly mycelia composed of 4-6 cells in series;

图 2B显示了菌丝状细胞的分离效率接近 80%; Figure 2B shows that the separation efficiency of mycelial cells is close to 80%;

图 2C显示了随时间进程分离得到的细胞具有依次递减的复制型寿命。图中，从右至左的曲线分别代表细胞群的平均复制型寿命分别为： 22.9， 13.1， 8.4， 5.4。图 3显示了检测衰老过程中白念珠菌细胞的氧化损伤蛋白水平的结果。其中：图 3A显示了 Coomassie blue染色显示不同年龄细胞样品的总蛋白水平基本一致。其中，泳道 1、 3、 5、 7分别代表平均寿命 22.9代、 13.1代、 8.4代、 5.4 代的细胞样品加入 2,4-二硝基苯酰肼反应后的检测结果，泳道 2、 4、 6、 8分别代表平均寿命 22.9代、 13.1代、 8.4代、 5.4代的细胞样品不加 2,4-二硝基苯酰肼反应的检测结果。 Figure 2C shows that cells isolated over time have a progressively decreasing replication life. In the figure, the right-to-left curves represent the average replicated lifespan of the cell population: 22.9, 13.1, 8.4, 5.4, respectively. Figure 3 shows the results of detecting oxidative damage protein levels of Candida albicans cells during senescence. Among them: Figure 3A shows that Coomassie blue staining shows that the total protein levels of cell samples of different ages are basically the same. Among them, lanes 1, 3, 5, and 7 represent the detection results of the cell samples of the average lifespan of 22.9, 13.1, 8.4, and 5.4, respectively, after adding 2,4-dinitrophenylhydrazide, lanes 2, 4, 6, 8 respectively represent the test results of the reaction of 2,4-dinitrobenzoyl hydrazide in cell samples with an average life span of 22.9, 13.1, 8.4, and 5.4.

图 3B显示了抗 DNP抗体免疫检测细胞中氧化损伤蛋白水平， Tubulin作为内参。其中，泳道 1、 3、 5、 7分别代表平均寿命 22.9代、 13.1代、 8.4代、 5.4 代的细胞样品加入 2,4-二硝基苯酰肼反应后的检测结果，泳道 2、 4、 6、 8分别代表平均寿命 22.9代、 13.1代、 8.4代、 5.4代的细胞样品不加 2,4-二硝基苯酰肼反应的检测结果。图 4显示了碘染色法检测白念珠菌细胞中的糖原水平。其中： Figure 3B shows the level of oxidative damage protein in cells immunoreactive with anti-DNP antibodies, Tubulin as an internal reference. Among them, lanes 1, 3, 5, and 7 represent the detection results of the cell samples of the average lifespan of 22.9, 13.1, 8.4, and 5.4, respectively, after adding 2,4-dinitrophenylhydrazide, lanes 2, 4, 6, 8 respectively represent the test results of the reaction of 2,4-dinitrobenzoyl hydrazide in cell samples with an average life span of 22.9, 13.1, 8.4, and 5.4. Figure 4 shows the detection of glycogen levels in Candida albicans cells by iodine staining. among them:

图 4A显示了在白念珠菌细胞衰老过程中，随年龄的递增碘染色逐渐加深，显示细胞内糖原水平依次升高。其中， 1(平均寿命 22.9代)、 2(平均寿命 13.1代)、 3(平均寿命 8.4代；)、 4(平均寿命 5.4代）分别表示滤纸上年龄依次递增的白念珠菌细胞点区域。 Fig. 4A shows that during the aging process of Candida albicans cells, the iodine staining gradually increases with age, indicating that the intracellular glycogen levels are sequentially increased. Among them, 1 (average life of 22.9 generations), 2 (average life of 13.1 generations), 3 (average life of 8.4 generations;), 4 (average life of 5.4 generations) respectively indicate the age of Candida albicans cells on the filter paper.

图 4B显示了图 4A的定量分析。图 5显示了 Southern杂交检测白念珠菌细胞的端粒长短。基因组 DNA经酶切消化后进行琼脂糖电泳分离，与 46nt端粒特异性探针杂交。其中，泳道 M为 DNA marker; 泳道 1-8: 不同年龄的细胞提取的样品的杂交结果，其中，泳道 1， 2：平均寿命 22.9代；泳道 3， 4：平均寿命 13.1代；泳道 5， 6：平均寿命 8.4代；泳道 7， 8：平均寿命 5.4代；泳道 Y: 酵母形态的细胞样品的杂交结果；泳道 H: 菌丝形态的细胞样品的杂交结果。图 6显示了白念珠菌衰老细胞中 ERC的检测。其中： Figure 4B shows the quantitative analysis of Figure 4A. Figure 5 shows the Southern blot to detect the telomere length of Candida albicans cells. The genomic DNA was digested and subjected to agarose electrophoresis, and hybridized with a 46 nt telomere-specific probe. Wherein, lane M is a DNA marker; Lanes 1-8: results of hybridization of samples extracted from cells of different ages, wherein lanes 1, 2: average lifespan 22.9 generations; lanes 3, 4: average lifespan 13.1 generations; lanes 5, 6 : average life expectancy of 8.4 generations; lanes 7, 8: average lifespan of 5.4 passages; lanes Y: results of hybridization of cell samples in yeast form; lanes H: results of hybridization of cell samples in hyphal morphology. Figure 6 shows the detection of ERC in Candida albicans senescent cells. among them:

图 6A显示了平均寿命分别为 22.9(泳道 1)， 13.1(泳道 2)， 8.4(泳道 3)， 5.4(泳道 4)的细胞群样品的基因组 DNA进行 0.7%琼脂糖凝胶电泳，转膜后与 rDNA探针相杂交的结果。 Figure 6A shows genomic DNA of cell population samples with average lifespan of 22.9 (lane 1), 13.1 (lane 2), 8.4 (lane 3), and 5.4 (lane 4) performed on 0.7% agarose gel electrophoresis after transfer The result of hybridization with the rDNA probe.

图 6B显示了平均寿命分别为 22.9(泳道 1)， 13.1(泳道 2)， 8.4(泳道 3)， 5.4(泳道 4)的细胞群样品的基因组 DNA经酶切，电泳，转膜，与 rDNA探针杂交，洗掉信号后再与 ACT1探针杂交的结果。 Figure 6B shows the genomic DNA of the cell population samples with average lifespan of 22.9 (lane 1), 13.1 (lane 2), 8.4 (lane 3), and 5.4 (lane 4), which were digested, electrophoretically transfected, transfected, and rDNA probed. Hybridization of the needle, washing out the signal and then hybridizing with the ACT1 probe.

图 6C显示了对图 6B的定量分析。具体实施方式 Figure 6C shows the quantitative analysis of Figure 6B. detailed description

本发明人经过广泛而深入的研究和试验，基于念珠菌在不同生长条件下具有明显不同的两种生长形态 (酵母状形态和菌丝状形态)且形态上的不同并不影响其传代次数的特点，首次开发了一种可方便大量地获得处于同一年龄阶段 (如衰老阶段;)的细胞群的方法。所述处于同一年龄阶段的细胞群可用于研究细胞生长、发育及其在各个年龄阶段的特征，以及用于研究细胞的衰老，从而为生物体的生长、发育、衰老机制的研究提供了有效的途径。在此基础上完成了本发明。如本发明所用，所述的 "菌丝状"是念珠菌在高温 (通常 35-42°C)和诱导条件 (如血清诱导;)下形成的一种形态，通常由 1个或多个菌丝细胞串联组成菌丝体。 The inventors have conducted extensive and intensive research and experiments, based on the two different growth forms (yeast-like morphology and mycelial morphology) of Candida under different growth conditions, and the difference in morphology does not affect the number of passages. Characteristics, for the first time, a method for easily obtaining a large number of cell populations at the same age (such as the aging stage) has been developed. The cell population at the same age can be used to study cell growth, development and its characteristics at various ages, as well as to study the senescence of cells, thereby providing an effective study for the growth, development, and aging mechanisms of organisms. way. The present invention has been completed on this basis. As used herein, the "hyphal" is a form formed by Candida under high temperature (usually 35-42 ° C) and induction conditions (such as serum induction;), usually consisting of one or more bacteria. The silk cells are connected in series to form mycelium.

如本发明所用，所述的 "念珠菌的菌丝状细胞群"是指一群细胞，该细胞群中大多数 (；通常 80%以上，更佳地 90%以上，最佳地 95%以上)细胞是以念珠菌的菌丝状形态生长的。如本发明所用，所述的 "酵母状"是念珠菌在常规适合酵母生长的条件 (如 30 °C左右)下形成的一种菌体形态，通常呈现为圆形或椭圆形。 As used herein, the "mycelium cell group of Candida" refers to a group of cells in which the cell population is Most (typically more than 80%, more preferably more than 90%, optimally more than 95%) cells are grown in the mycelial form of Candida. As used in the present invention, the "yeast-like" is a bacterial form formed by Candida under conditions suitable for yeast growth (e.g., about 30 ° C), and is usually in the form of a circle or an ellipse.

如本发明所用，所述的 "念珠菌的酵母状细胞群"是指一群细胞，该细胞群中大多数 (；通常 80%以上，更佳地 90%以上，最佳地 95%以上)细胞是以念珠菌的酵母状形态生长的。如本发明所用，所述的 "处于同一年龄阶段"是指所述的念珠菌细胞群中大多数细胞（一般为总细胞数的 70%以上，更佳地为总细胞数的 80%以上，最佳地为总细胞数的 90%以上）处于接近的细胞年龄，也即各细胞的传代次数接近 (通常各个细胞的传代次数相差不超过 ±5代；更优选的，各个细胞的传代次数相差不超过 ±3代；最优选的，各个细胞的传代次数相差不超过 ±2代；)。如本文所用，所述的 "平均传代次数"是指细胞群中各细胞的传代次数的平均值。例如，细胞群中有 80%细胞处于传代第 10代， 8%细胞处于传代第 9代， 8%细胞处于传代第 11代， 2%处于传代第 8代， 2%处于传代第 12代，则按照平均计算，该细胞群的传代次数为传代第 10代。如本文所用，所述的 "复制型寿命 (Replicative Life Span) " 或 "复制寿命" 是指待测念珠菌细胞可产生的酵母状子细胞的个数，如果菌丝体包含大于 1个细胞，则计算每个细胞产生子细胞个数的平均值。例如，念珠菌 1只包含一个细胞，则该念珠菌的复制型寿命即是其总共产生的子细胞个数；念珠菌 2(菌丝状；)包含 3 个细胞，则该念珠菌的复制型寿命即是其总共产生的子细胞个数除以 3获得的数值。可通过常规的 Life Span Assay方法来分析念珠菌细胞的复制型寿命。通常，待测念珠菌细胞的年龄越大 (传代次数越多），其复制型寿命越低；反之，待测念珠菌细胞的年龄越小 (传代次数越少；)，其复制型寿命越高。 As used herein, "the yeast cell population of Candida" refers to a population of cells, most of which (typically more than 80%, more preferably more than 90%, optimally more than 95%) of cells. It is grown in the yeast form of Candida. As used herein, "at the same age stage" refers to the majority of cells in the Candida cell population (generally 70% or more of the total number of cells, more preferably 80% or more of the total number of cells, Optimally more than 90% of the total number of cells) at a close cell age, that is, the number of passages of each cell is close (usually the number of passages of each cell differs by no more than ±5 generations; more preferably, the number of passages of each cell differs Not more than ±3 generations; most preferably, the number of passages of each cell differs by no more than ±2 generations;). As used herein, "average passage number" refers to the average number of passages of each cell in a cell population. For example, 80% of the cells in the cell population are in passage 10, 8% are in passage 9th generation, 8% are in passage 11th generation, 2% are in passage 8th generation, and 2% are in passage 12th generation. According to the average calculation, the number of passages of the cell population was passage 10th generation. As used herein, "replicative life span" or "replication life" refers to the number of yeast-like daughter cells that can be produced by a Candida cell, and if the mycelium contains more than one cell, The average of the number of daughter cells produced per cell was calculated. For example, if Candida 1 contains only one cell, the replicating life of the Candida is the total number of daughter cells produced; Candida 2 (mycelium;) contains 3 cells, then the replication type of Candida The lifetime is the value obtained by dividing the total number of daughter cells produced by 3. The replication life of Candida cells can be analyzed by the conventional Life Span Assay method. Generally, the older the age of the Candida cells to be tested (the more the number of passages), the lower the replication type lifespan; conversely, the younger the age of the Candida cells to be tested (the fewer the number of passages;), the higher the replication life .

如本文所用，所述的 "平均复制型寿命"或 "平均复制寿命"或 "平均寿命" 是指一个念珠菌细胞群中细胞的复制型寿命的平均值。 ■ ^珠菌 As used herein, "average replication life" or "average replication life" or "average life" refers to the average of the replication life of a cell in a Candida cell population. ■ ^Cute

本发明的用于制备处于同一年龄阶段细胞群的方法是利用念珠菌 (Ο^ ^) 进行的。念珠菌属于半知菌纲的酵母菌，其具有多形态，在不同的条件下能形成酵母状形态和菌丝体形态。 The method of the present invention for preparing a cell population at the same age is carried out using Candida (Ο^^). Candida belongs to the semi-bacterial yeast, which has multiple forms and can form yeast-like morphology and mycelial morphology under different conditions.

作为本发明的一种优选方式，所述的念珠菌是白念珠菌 (； Ca«i /ito a/b/ca ，简称 C. a/&/^ms)。白念珠菌是念珠菌的一种，亦称"白色假丝酵母"，在约 30°C时以酵母形态生长，在约 37-40 °C以及某些诱导因子存在时可以形成菌丝体，但如果回复到 30°C非诱导条件培养时菌丝状的细胞又可以***产生酵母状的子细胞，但菌丝状细胞本身不会再回复到酵母状的形态。 As a preferred mode of the present invention, the Candida species is Candida albicans (; Ca«i /ito a/b/ca, abbreviated as C. a/&/^ms). Candida albicans is a type of Candida, also known as "Candida albicans", which grows in yeast at about 30 ° C and forms mycelium at about 37-40 ° C and in the presence of certain inducing factors. However, if the cells are cultured in a non-inducing condition at 30 ° C, the mycelial cells can be split to produce yeast-like daughter cells, but the mycelial cells themselves do not return to the yeast-like form.

本发明人正是利用了念珠菌的形态差异，来建立大规模有效分离衰老细胞的方法。制备处于同一年龄阶段细胞群的方法 The inventors have utilized the morphological differences of Candida to establish a method for large-scale and effective separation of senescent cells. Method for preparing cell populations at the same age

基于念珠菌在不同的培养条件下菌体形态明显不同以及该形态上的不同并不影响念珠菌的复制和传代这一特征，本发明提供了一种制备处于同一年龄阶段细胞群的方法。 Based on the fact that Candida has distinct cell morphology under different culture conditions and this morphological difference does not affect the replication and passage of Candida, the present invention provides a method of preparing cell populations at the same age.

由于念珠菌在生长过程中，不断地复制产生子代细胞，子代细胞再复制产生下一代细胞，因此在常规的念珠菌培养物中，一般含有处于各种不同年龄阶段的细胞，有的是年轻的细胞 (如传代次数为 5次以下，或如复制型寿命为 20代以上；)，有的是衰老的细胞 (如传代次数为 20次以上，或如复制型寿命为 5代以下；)。由于处于不同年龄阶段的细胞的复制速度、代谢机制、生理特征等均可能是不同的，这给研究细胞的发育和衰老机制造成了困难。因此需要分离出属于同一年龄阶段的细胞，从而便于研究人员细致地观察细胞生长的不同年龄阶段的各种特征。 Since Candida is continuously replicating to produce progeny cells during the growth process, the progeny cells are replicated to produce the next generation of cells. Therefore, in conventional Candida cultures, cells of various ages are generally contained, and some are young. Cells (such as subcultures of 5 times or less, or replication type lifespan of 20 generations or more;), some are senescent cells (such as the number of passages is more than 20 times, or if the replication type life is less than 5 generations;). Since the replication speed, metabolic mechanism, and physiological characteristics of cells at different ages may be different, this makes it difficult to study the development and aging mechanisms of cells. Therefore, it is necessary to isolate cells belonging to the same age stage, thereby facilitating the study of various characteristics of different age stages of cell growth.

通常念珠菌细胞在复制了 20-30代后成为衰老细胞，作为前提条件，为了后续获得处于同一年龄阶段的细胞群，必须在复制起始的时候各细胞也处于同一年龄阶段。因此，为了获得复制起始的时候处于同一年龄阶段的细胞，本发明人首先在适合菌丝状念珠菌细胞生长的条件下，培养念珠菌细胞群，形成菌丝状细胞群；然后，将该菌丝状细胞群在适合酵母状念珠菌细胞生长的条件下、在相对短的时间内培养（一般是 0.5-5小时，更优选的为 1-4小时，进一步优选的是 2-3小时)，形成含有菌丝状细胞和酵母状子细胞的混合细胞群；之后从该混合细胞群中分离出酵母状子细胞群，该子细胞群可作为起始的最年轻的细胞 (处于同一年龄阶段；)，用于后续制备处于同一所需年龄阶段的细胞群。 Usually, Candida cells become senescent cells after 20-30 generations of replication. As a precondition, in order to obtain subsequent cell populations at the same age, each cell must be at the same age stage at the beginning of replication. Therefore, in order to obtain cells at the same age stage at the time of initiation of replication, the inventors first cultured a Candida cell population to form a mycelial cell population under conditions suitable for the growth of mycelial cells of Mycelia; and then, The mycelial cell population is cultured in a relatively short period of time under conditions suitable for the growth of the Candida albicans cells (generally 0.5-5 hours, more preferably 1-4 hours, further preferably 2-3 hours) Forming a mixed cell population containing mycelial cells and yeast-like daughter cells; and then separating the yeast-like daughter cell population from the mixed cell population, the daughter cell population being the youngest cell at the beginning (at the same age;) For subsequent preparations in the same required Cell population at the age stage.

之后，在适合菌丝状念珠菌细胞生长的条件下，培养前述获得的作为起始的最年轻的酵母状细胞群，使之形成最年轻的菌丝状细胞群。 Thereafter, the most prolific yeast cell population obtained as described above is cultured under conditions suitable for the growth of mycelial cells of Mycelium, and the youngest mycelial cell population is formed.

之后，将前述形成的菌丝状细胞群在适合酵母状念珠菌细胞生长的条件下培养，使之产生酵母状子细胞，形成含有菌丝状细胞和酵母状子细胞的混合细胞群；然后，在子细胞达到一定浓度后，根据菌丝状细胞与酵母状细胞的形态不同，从中去除酵母状子细胞，分离出菌丝状细胞群。然后，可分析或估测所述菌丝状细胞群的细胞年龄，如细胞未达到所需年龄，则可在适合酵母状念珠菌细胞生长的条件下再培养菌丝状细胞，使之产生酵母状子细胞，在子细胞达到一定浓度后除去子细胞，分离菌丝状细胞，该步骤可重复多次进行，直至获得处于同一所需年龄阶段细胞的菌丝状细胞群。 Thereafter, the mycelial cell population formed as described above is cultured under conditions suitable for the growth of yeast-like Candida cells to produce yeast-like daughter cells, thereby forming a mixed cell population containing mycelial cells and yeast-like daughter cells; After the cells reach a certain concentration, the yeast-like daughter cells are removed from the mycelial cells and the yeast-like cells, and the mycelial cell population is isolated. Then, the cell age of the mycelial cell population can be analyzed or estimated, and if the cells do not reach the desired age, the mycelial cells can be cultured under conditions suitable for the growth of the Candida albicans cells to produce yeast. The progenitor cells, after the daughter cells reach a certain concentration, remove the daughter cells and isolate the mycelial cells. This step can be repeated multiple times until a mycelial cell population of cells at the same desired age is obtained.

由于子细胞在达到一定的浓度后会抑制菌丝状母细胞的复制，因此，在菌丝状母细胞产生的子细胞达到一定的浓度后，需要从混合培养物中分离掉子细胞，使得菌丝状母细胞能够继续产生子细胞且年龄逐渐增加。本领域人员通常可根据常规的念珠菌复制规律来确定该浓度，通常，当混合培养物的 OD_6(X)值达到 2-5时，可分离掉子细胞。 Since the daughter cells inhibit the replication of mycelial cells after reaching a certain concentration, after the daughter cells produced by the mycelial cells reach a certain concentration, it is necessary to separate the daughter cells from the mixed culture, so that the bacteria Filamentous cells can continue to produce daughter cells and gradually increase in age. Those skilled in the art can generally determine this concentration according to conventional Candida replication rules. Typically, when the mixed culture has an OD6 _(X ) value of 2-5, the daughter cells can be isolated.

本发明对适合酵母状念珠菌细胞生长的条件没有特别的限制，只要该条件是适合于酵母状念珠菌生长的，且基本上不会使酵母状念珠菌生长成菌丝状念珠菌。作为本发明的优选方式，所述的适合酵母状念珠菌细胞生长的条件为 25-32°C ; 更优选的，所述的适合菌丝状细胞生长的条件为 28-30°C ; 进一步优选的，所述的适合菌丝状细胞生长的条件为 30°C。 The present invention is not particularly limited as to the conditions suitable for the growth of Candida albicans cells, as long as the conditions are suitable for the growth of Candida albicans, and substantially no growth of Candida albicans into Mycelium. As a preferred embodiment of the present invention, the condition suitable for the growth of the Candida albicans cell is 25-32 ° C; more preferably, the condition suitable for the growth of the mycelial cell is 28-30 ° C; further preferred The conditions suitable for the growth of mycelial cells are 30 °C.

本发明对适合菌丝状念珠菌细胞生长的条件没有特别的限制，只要该条件是适合于菌丝状念珠菌生长的，且基本不会使菌丝状念珠菌生长成酵母状念珠菌。作为本发明的优选方式，所述的适合菌丝状念珠菌细胞生长的条件为 35-42°C，血清诱导（通常可调节 pH 7.0左右，起始酵母细胞浓度 10⁶/ml左右；)。更优选的，适合菌丝状细胞生长的温度条件为 36-40°C ; 进一步优选的，适合菌丝状细胞生长的温度条件为 37-38 V。 The present invention is not particularly limited as to the conditions suitable for the growth of mycelial cells of Mycelia, as long as the conditions are suitable for the growth of Mycelia of Candida, and the Mycelium of Candida is not substantially grown into Candida. As a preferred embodiment of the present invention, the conditions suitable for the growth of mycelial cells of Mycelia are 35-42 ° C, serum induction (usually adjustable pH 7.0, initial yeast cell concentration of about 10 ⁶ /ml;). More preferably, the temperature condition suitable for the growth of mycelial cells is 36-40 ° C; further preferably, the temperature condition suitable for the growth of mycelial cells is 37-38 V.

任何本领域已知的适合于培养酵母状念珠菌和菌丝状念珠菌的培养基均可用于本发明，只要该培养基能够提供念珠菌进行生长、复制等所需的足够的能量和营养。作为本发明的优选方式，念珠菌的培养基是 YPD培养基。 Any medium known in the art suitable for cultivating yeast-like Candida and Myceliophthora can be used in the present invention as long as the medium can provide sufficient energy and nutrients required for growth, replication, and the like of Candida. As a preferred mode of the present invention, the culture medium of Candida is YPD medium.

本发明对诱导酵母状念珠菌形成菌丝状念珠菌的诱导方法没有特别的限制，任何可使得酵母状念珠菌形成菌丝状念珠菌的诱导方法均是可用的，包括但不限于采用血清诱导。当需要培养菌丝状念珠菌时，可在 YPD培养基中加上诱导成分如血清等；诱导成分的加入浓度是本领域人员熟知的常识。 The present invention has no particular limitation on the method for inducing the formation of Candida albicans by the yeast Candida, any Induction methods for the formation of Mycelium of Candida albicans can be made available, including but not limited to the use of serum induction. When it is desired to culture mycelium, an inducing component such as serum or the like may be added to the YPD medium; the concentration of the inducing component to be added is a common knowledge well known to those skilled in the art.

本发明对分离酵母状念珠菌和菌丝状念珠菌的方法没有特别的限制，只要该方法能够在不影响菌株存活和特性的前提下可将两种的念珠菌进行分离。作为本发明的优选方式，所述的分离是基于酵母状念珠菌和菌丝状念珠菌形态的不同进行分离的。通常，所述的方法包括但不限于：离心法、过滤法。作为本发明的更优选方式，通过密度梯度离心分离酵母状细胞和菌丝状细胞，该方法不仅不影响菌株存活和特性，而且分离效率非常高，其优于过滤法的特点还在于不会在分离过程中造成滤器的堵塞。 The method of the present invention for isolating the yeast-like Candida and Mycelia candida is not particularly limited as long as the method can separate the two Candida species without affecting the survival and characteristics of the strain. As a preferred mode of the present invention, the separation is carried out based on the difference in the morphology of Candida albicans and Mycelia. Generally, the methods include, but are not limited to, centrifugation, filtration. As a more preferred mode of the present invention, yeast cells and mycelial cells are separated by density gradient centrifugation, which not only does not affect the survival and characteristics of the strain, but also has a very high separation efficiency, and is superior to the filtration method in that it is not The filter is clogged during the separation process.

Life Span Assay表明，白念珠菌形态上的不同并不影响其复制性寿命长短 (；见图 1B)，也即不影响白念珠菌细胞的传代次数。因此，本发明人以菌丝体为母细胞，使其不断***产生酵母状的子细胞，从而发生复制性衰老。按照上述思路，本发明人通过蔗糖密度梯度离心的方法分离菌丝状的母细胞和酵母状的子细胞，在整个复制性细胞衰老的过程中收集各个年龄阶段的菌丝状母细胞，对不同年龄的白念珠菌细胞 (包括年轻细胞;)进行研究。由于菌丝状的母细胞和酵母状的子细胞在形态上存在非常显著的差异，从而使分离效率大大提高，因此本发明的方法与现有技术人员在 S. cerev/s/ae中的分离方法相比，具有明显的优势。 Life Span Assay showed that the difference in morphology of Candida albicans did not affect its longevity of replication (see Figure 1B), ie, it did not affect the number of passages of Candida albicans cells. Therefore, the present inventors used mycelium as a mother cell to cause it to continuously divide to produce yeast-like daughter cells, thereby causing replicative senescence. According to the above idea, the present inventors separated mycelial cells and yeast-like daughter cells by sucrose density gradient centrifugation, and collected mycelial cells at various ages during the whole process of replicating cell senescence. Age of Candida albicans cells (including young cells;) were studied. Since the mycelial-like mother cells and the yeast-like daughter cells have very significant differences in morphology, so that the separation efficiency is greatly improved, the separation of the method of the present invention from those of the prior art in S. cerev/s/ae Compared with the method, it has obvious advantages.

在本发明的实施例中，本发明人根据已知的衰老细胞的特征对本发明所建立的分离方法进行了一系列实验验证。结果证明，采用本发明的方法分离得到的衰老的菌丝状母细胞确实是复制型衰老的。同时还发现了白念珠菌的细胞衰老过程不同于 S. cerevisiae的一些特性，因而对细胞衰老的机理有了更深入的理解。 In the examples of the present invention, the inventors conducted a series of experimental verifications on the separation method established by the present invention based on the characteristics of known senescent cells. As a result, it was confirmed that the senescent mycelial cells isolated by the method of the present invention were indeed replicative senescence. It has also been found that the cell senescence process of Candida albicans is different from that of S. cerevisiae, and thus has a deeper understanding of the mechanism of cell senescence.

本发明的方法操作简单易行，便于大量制备，并且可以获得处于衰老各个阶段的不同年龄的细胞，解决了大规模高通量实验细胞制备的难题，为更好更深入地研究细胞衰老的发生机制奠定了基础。 The method of the invention is simple and easy to operate, is convenient for large-scale preparation, and can obtain cells of different ages at various stages of aging, and solves the problem of large-scale high-throughput experimental cell preparation, in order to better and more deeply study the occurrence of cell senescence The mechanism laid the foundation.

本发明的方法为大规模高通量的获得同一年龄阶段细胞提供了有效的途径，从而可用于后续开展大规模的基因组学以及蛋白质组学研究，筛选出调控细胞生长、衰老过程的基因或途径，在更深的层面上进一步探索细胞生长、衰老的分子机制。处于同一年龄阶段的细胞群及其用途 The method of the present invention provides a large-scale and high-throughput method for obtaining cells of the same age, and can be used for subsequent large-scale genomics and proteomics research, screening for genes or pathways regulating cell growth and aging processes. Further explore the molecular mechanisms of cell growth and aging at a deeper level. Cell populations at the same age and their uses

本发明还提供了一种细胞群，其是由通过本发明的方法制备的处于同一年龄阶段的细胞组成。 The invention also provides a population of cells consisting of cells of the same age stage prepared by the method of the invention.

本发明所述细胞群具有以下特征： The cell population of the present invention has the following characteristics:

作为本发明的优选方式，所述细胞群中， 90%以上细胞为念珠菌的菌丝状细胞；更优选的， 95%以上细胞为念珠菌的菌丝状细胞。 In a preferred embodiment of the present invention, 90% or more of the cells in the cell population are mycelial cells of Candida; more preferably, 95% or more of the cells are mycelial cells of Candida.

作为本发明的优选方式，所述细胞群中， 80%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段；更优选的， 90%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段；进一步优选的， 95%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段。 In a preferred embodiment of the present invention, 80% or more of the mycelial cells of the Candida are in the same age stage; more preferably, more than 90% of the mycelial cells of the Candida are in the same Age stage; further preferably, more than 95% of the mycelial cells of the Candida are at the same age.

本发明的方法对于大规模地获得同一年龄阶段的细胞是特别有用的，因此，本发明所述的细胞群通常有 10²- 10^1Q个细胞 (优选的，所述细胞群中细胞数量为 10³-10⁹个；更优选的，所述细胞群中细胞数量为 10⁵-10⁸个)。然而，应理解，在得知了本发明提供的方法后，本领域人员也可能可以利用该方法制备出少于 10²或多于 10^{1 Q}个念珠菌细胞的细胞群，该细胞群也应被涵盖在本发明的范围内。 The method of the present invention is particularly useful for obtaining cells of the same age on a large scale, and therefore, the cell population of the present invention usually has 10 ² - 10 ^1Q cells (preferably, the number of cells in the cell population is 10 ³ - 10 ⁹ ; More preferably, the number of cells in the cell population is 10 ⁵ - 10 ⁸ ). However, it should be understood that after the method provided by the present invention is known, the person skilled in the art may also use the method to prepare a cell population of less than 10 ² or more than 10 ^{1 Q} Candida cells, and the cell population should also be It is intended to be covered by the scope of the invention.

本发明的方法可以制备出处于任何年龄阶段的细胞群，例如可以是平均传代次数为 3代的年轻细胞，或是平均传代次数为 20代的衰老细胞；或者，也可以是平均复制型寿命为 3代的衰老细胞，或是平均复制型寿命为 20代的年轻细胞。通常，所述念珠菌的菌丝状细胞的平均传代次数为 1-30代，更佳的为 5-25代。 The method of the present invention can prepare a cell population at any age, for example, a young cell with an average passage number of 3 passages, or an aging cell with an average passage number of 20 passages; or, the average replica life is 3rd generation of senescent cells, or younger cells with an average replication life of 20 generations. Typically, the average number of passages of the mycelial cells of Candida is from 1 to 30 generations, more preferably from 5 to 25 passages.

通常，当需要研究衰老细胞的特性时，可制备平均传代次数高于 15代，更优选的高于 20代的念珠菌细胞群。 In general, when it is desired to study the characteristics of senescent cells, a population of Candida cells having an average passage number higher than 15 passages, more preferably 20 generations, can be prepared.

本发明的处于同一年龄阶段的念珠菌细胞群可以作为一种细胞模型，用于研究细胞在生长、发育、衰老等各种阶段中的形态、结构、代谢等机制；还可用于筛选调节细胞生长、发育、代谢、衰老等机制的物质。筛选抗衰老物质 The Candida cell population at the same age of the present invention can be used as a cell model for studying the morphology, structure, metabolism and other mechanisms of cells in various stages of growth, development, aging, etc.; and can also be used for screening and regulating cell growth. Substances of development, metabolism, aging, etc. Screening for anti-aging substances

作为本发明的一种应用方式，可利用本发明的方法来制备衰老细胞群，从而用于筛选抗衰老的药物。所述的筛选方法为：将候选物质与衰老细胞接触，判断该物质对于衰老细胞的影响，如果该物质能够延缓衰老细胞的死亡或促进 (优选显著促进;)衰老细胞的代谢，则说明该物质是一种抗衰老的潜在物质。 As an application of the present invention, the method of the present invention can be used to prepare a population of senescent cells for use in screening for anti-aging drugs. The screening method is as follows: contacting the candidate substance with the senescent cell to determine the effect of the substance on the senescent cell, if the substance can delay the death or promotion of the senescent cell (preferably Promotion;) The metabolism of aging cells indicates that the substance is a potential anti-aging substance.

优选的，所述筛选方法例如包括步骤： Preferably, the screening method includes the following steps:

在测试组中，在衰老细胞的培养物中添加候选物质；检测衰老细胞的平均传代次数，并与对照组比较，其中所述的对照组是不添加所述候选物质的、与测试组衰老细胞处于同一年龄阶段的细胞。将测试组中细胞的平均传代次数与对照组中细胞的平均传代次数进行比较，如果测试组中细胞的平均传代次数在统计学上显著高于对照组 (如测试组细胞的平均传代次数比对照组高 3代；更优选的为高 4 代，进一步优选的为高 5代或更高；)，就表明该候选物是抗衰老的潜在药物。本发明的主要优点在于： In the test group, a candidate substance is added to the culture of the senescent cells; the average number of passages of the senescent cells is detected, and compared with the control group, wherein the control group is the senescent cells that are not added with the candidate substance and the test group Cells at the same age. The average number of passages of cells in the test group was compared with the average number of passages of cells in the control group, if the average number of passages of the cells in the test group was statistically significantly higher than that of the control group (eg, the average number of passages of the cells in the test group was higher than that of the control group). The group is 3 generations higher; more preferably 4 generations higher, further preferably 5 generations higher or higher;), indicating that the candidate is a potential drug for anti-aging. The main advantages of the invention are:

(1) 首次基于念珠菌在不同生长条件下具有明显不同的两种生长形态的特点，开发出制备处于同一年龄阶段 (如衰老阶段；)的细胞群的方法。 (1) For the first time, based on the characteristics of Candida having two distinct growth patterns under different growth conditions, a method for preparing a cell population at the same age (e.g., aging stage) was developed.

(2) 所述处于同一年龄阶段的细胞群可用于研究细胞生长、发育及其在各个年龄阶段的特征，以及用于研究细胞的衰老，从而为生物体的生长、发育、衰老机制的研究提供了极其有效的途径。 (2) The cell population at the same age can be used to study cell growth, development and its characteristics at various ages, as well as to study the senescence of cells, thereby providing research for the growth, development and aging mechanisms of organisms. An extremely effective way.

(3) 采用本发明的方法，可方便地大量制备年龄接近的衰老细胞，从而为研究和筛选抗衰老的药物提供了极其有效的途径。 (3) By adopting the method of the present invention, it is convenient to mass-produce aging cells of close age, thereby providing an extremely effective way for researching and screening anti-aging drugs.

(4) 本发明的方法操作简单易行，特别有利于高通量地制备处于同一年龄阶段的细胞群，克服了以往本领域技术人员难以有效地分离衰老细胞和年轻细胞的技术缺陷。下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如 Sambrook等人，分子克隆：实验室指南 (New York: Cold Spring Harbor Laboratory Press , 1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。 (4) The method of the present invention is simple and easy to operate, and is particularly advantageous for high-throughput preparation of cell populations at the same age stage, overcoming technical defects that are difficult for those skilled in the art to effectively separate senescent cells and young cells. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。实施例 1 处于同一年龄阶段的细胞的分离方法 1. 菌株及培养条件 Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1 Method for isolating cells at the same age 1. Strain and culture conditions

实验所用菌株为白念珠菌 (OwiftWa 野生型菌株 SC5314 (购自 The strain used in the experiment was Candida albicans (OwiftWa wild type strain SC5314 (purchased from

ATCC) 。 ATCC).

酵母状细胞培养条件为 30°C， YPD培养基。在该培养条件下，白念珠菌的生长状态如图 1A左图所示。 Yeast cell culture conditions were 30 ° C, YPD medium. Under the culture conditions, the growth state of Candida albicans is shown in the left panel of Fig. 1A.

菌丝状细胞培养条件为 37°C， YPD+15%小牛血清。在该培养条件下，白念珠菌的生长状态如图 1A中图所示。当将白念珠菌菌丝状细胞从该培养条件回复到培养条件为 30°C， YPD培养基继续培养时，其可产生酵母状的子细胞如图 1A 右图所示。 The mycelial cell culture conditions were 37 ° C, YPD + 15% calf serum. Under the culture conditions, the growth state of Candida albicans is shown in the figure in Fig. 1A. When the Candida albicans mycelial cells were returned from the culture condition to a culture condition of 30 ° C, and the YPD medium was further cultured, it produced yeast-like daughter cells as shown in the right panel of Fig. 1A.

YPD培养基的配方如下：蛋白胨 2%，酵母抽提物 1%，葡萄糖 2%，配制在蒸馏水中。 The formulation of YPD medium was as follows: peptone 2%, yeast extract 1%, glucose 2%, formulated in distilled water.

2. 分离衰老细胞的方法 2. Method for isolating senescent cells

① . 取新鲜白念珠菌野生型菌株 SC5314单克隆接种至 YPD液体培养基，于 30°C过夜培养，该培养物中包含各种年龄阶段的酵母状细胞； 1. The wild type Candida albicans strain SC5314 was inoculated into YPD liquid medium and cultured overnight at 30 ° C, and the culture contained yeast cells of various ages;

② . 用新鲜 YPD稀释过夜培养物至 OD_6Q()约为 0.1，加 15%小牛血清于 37°C 诱导 ₄_5小时，得到各种年龄阶段的菌丝状细胞； 2. Dilute the overnight culture with fresh YPD to an OD _{6Q () of} about 0.1, and add 15% calf serum at 37 ° C for _{4 -} 5 hours to obtain mycelial cells of various ages;

③ . 将菌丝状细胞收集并转移至新鲜 YPD液体培养基中，30°C培养 2.5小时，使菌丝状母细胞在一个相对短的时间内产生酵母状子细胞，以 0-30%不连续蔗糖密度梯度于 lOOOrpm离心 3min收集上层水相中的酵母状子细胞，该子细胞被认为是最年轻的子细胞； 3. The mycelial cells were collected and transferred to fresh YPD liquid medium and cultured at 30 ° C for 2.5 hours to cause the mycelial cells to produce yeast-like daughter cells in a relatively short period of time, with 0-30% discontinuity. The sucrose density gradient was centrifuged at 1000 rpm for 3 min to collect the yeast-like daughter cells in the upper aqueous phase, which was considered to be the youngest daughter cell;

④ . 以 2中的条件诱导菌丝状细胞，得到作为起始的母细胞； 4. Inducing mycelial cells under the conditions of 2, and obtaining the starting mother cells;

⑤ . 换用新鲜 YPD液体培养基于 30°C连续培养菌丝状母细胞，根据需要在不同时间以 0-30%不连续蔗糖密度梯度离心，去掉上层酵母状子细胞，收集下层菌丝状母细胞，并换用新鲜 YPD培养液继续培养，直至得到最衰老的母细胞。在连续培养过程中，当培养物的 OD₆oo值达到 2-5时，都需进行细胞分离，去掉培养物中大量的子细胞并换新鲜培养液以保证母细胞的正常生长状态。 5. Transfer the mycelial mother cells continuously at 30 °C with fresh YPD liquid medium, centrifuge at 0-30% discontinuous sucrose density gradient at different times, remove the upper layer of yeast-like daughter cells, and collect the lower mycelial cells. And continue to culture with fresh YPD medium until the most senescent mother cells are obtained. In the continuous culture process, when the OD ₆ oo value of the culture reaches 2-5, cell separation is required, and a large number of daughter cells in the culture are removed and fresh culture medium is exchanged to ensure the normal growth state of the mother cells.

显微观察显示，菌丝状母细胞主要由 4-6个细胞串联而成，如图 2A。菌丝状细胞的分离效率接近 80%，如图 2B，考虑到每个菌丝状母细胞端部的酵母状母细胞约占 10%以上，该方法分离衰老细胞的效率可达 90%以上，已经相当理想。本发明人将平均每个菌丝细胞所产生的酵母状子细胞的个数定义为该菌丝状母细胞的复制型寿命（Replicative Life Span) 。如果菌丝体包含大于 1个菌丝细胞，则计算每个菌丝细胞产生子细胞个数的平均值。 Microscopic observation showed that mycelial cells were mainly composed of 4-6 cells in series, as shown in Fig. 2A. The isolation efficiency of mycelial cells is close to 80%. As shown in Fig. 2B, considering that the yeast-like mother cells at the end of each mycelial mother cell account for more than 10%, the efficiency of separating senescent cells by the method can reach more than 90%. Already quite ideal. The inventors defined the number of yeast-like daughter cells produced by each hyphal cell as the mycelial cell Replicative Life Span. If the mycelium contains more than one hyphal cell, the average number of daughter cells per hyphal cell is calculated.

复制型寿命检测实验（Life Span Assay) 的操作如下： The operation of the Life Span Assay is as follows:

从需要进行复制型寿命检测的细胞群体中随机选取细胞放置于 YPD平板上，通过显微操作仪将 50-80个细胞有序的排布到平板上的固定位置。在 30°C培养使细胞进行 1-2次***，去掉子细胞并计数，重复该步骤，直至所有的母细胞不再 ***。夜间将平板置于 4°C。对于只包含一个菌丝细胞的菌丝体，可以以子细胞的个数来代表***代数；对于包含多于一个菌丝细胞的菌丝体，则计算每个细胞产生子细胞个数的平均值，即以获得的总的子细胞数除以细胞个数作为***代数。统计数据并由 Wilcoxon rank-sum检验确定是否有统计学意义， P值小于 0.05表示两组数据之间存在显著差异。 Cells were randomly selected from cell populations requiring replication-type life testing and placed on YPD plates, and 50-80 cells were arranged in a fixed position on the plate by a micromanipulator. The cells were cultured at 30 ° C for 1-2 divisions, the daughter cells were removed and counted, and the procedure was repeated until all the mother cells no longer split. Place the plate at 4 °C at night. For mycelium containing only one mycelial cell, the number of daughter cells can be used to represent the split algebra; for mycelium containing more than one mycelial cell, the average number of daughter cells per cell is calculated. That is, the total number of daughter cells obtained is divided by the number of cells as a split algebra. The statistical data was determined by the Wilcoxon rank-sum test to determine whether it was statistically significant. A P value of less than 0.05 indicates a significant difference between the two sets of data.

3. 分离处于各个年龄阶段的细胞群 3. Isolation of cell populations at all ages

除了上述制备衰老细胞以外，可通过减少菌丝状母细胞的复制时间和复制代数，来获得所需各个年龄阶段的细胞。因此，根据前述制备衰老细胞类似的方法，本发明人还在细胞复制的不同时间点分别获取细胞群，获得 4种处于各个年龄阶段的菌丝体细胞群 (其中一种细胞群是最年轻的细胞；)。 In addition to the above-described preparation of senescent cells, cells of various age stages can be obtained by reducing the replication time and replication number of mycelial cells. Therefore, according to the aforementioned method for preparing senescent cells, the inventors also separately obtained cell populations at different time points of cell replication, and obtained four mycelial cell populations at various ages (one of which is the youngest). cell;).

分别对前述获得的 4种处于各个年龄阶段的细胞群进行 Life Span Assay实验，结果见图 2C。由结果表明，本发明人分离得到的不同年龄阶段的细胞具有不同的复制型寿命，从起始最年轻细胞算起，平均复制型寿命分别是 22.9(最年轻的细胞，平均传代次数约为 1代；)， 13.1(平均传代次数约为 10.8代；)， 8.4(平均传代次数约为 15.5代;)， 5.4(平均传代次数约为 18.5代；)代，且每一细胞群中的细胞大多处于同一年龄阶段，确实反映了白念珠菌细胞复制性衰老的进程。实施例 2. 氧化损伤蛋白在白念珠菌衰老过程中不断积累 The Life Span Assay experiments were performed on the above-mentioned four cell populations at various ages, and the results are shown in Figure 2C. From the results, it was revealed that the cells of different ages isolated by the present inventors have different replication lifespans, and the average replicative lifespan is 22.9 from the youngest cells initiating (the youngest cells, the average number of passages is about 1). Generation;), 13.1 (average passage number is about 10.8 generations;), 8.4 (average passage number is about 15.5 generations;), 5.4 (average passage times is about 18.5 generations;) generation, and most of the cells in each cell population At the same age, it does reflect the process of replicative senescence of Candida albicans cells. Example 2. Oxidative damage protein accumulates during the senescence process of Candida albicans

衰老的细胞对某些压力如氧化压力的抗性会大大下降，所以衰老细胞中积累的氧化损伤的蛋白应该也较年轻细胞要多。被氧化修饰的蛋白分子中会被不可逆的引入羰基基团，本发明人利用羰基基团的化学特性，结合免疫检测的方法对分离得到的不同年龄的白念珠菌细胞中氧化损伤蛋白的水平进行了对比分析。 The resistance of senescent cells to certain stresses, such as oxidative stress, is greatly reduced, so oxidatively damaged proteins accumulated in senescent cells should also be more abundant than younger cells. The oxidatively modified protein molecule is irreversibly introduced into the carbonyl group. The present inventors utilized the chemical properties of the carbonyl group in combination with immunoassay to measure the levels of oxidative damage proteins in Candida albicans cells of different ages. Comparative analysis.

本发明人分别取平均寿命为 22.9, 13.1, 8.4, 5.4代的白念珠菌细胞群，进行氧化损伤蛋白检测实验。氧化损伤蛋白检测实验如下： The inventors took the Candida albicans cell population with an average lifespan of 22.9, 13.1, 8.4, and 5.4, respectively, and performed an oxidative damage protein detection experiment. The oxidative damage protein detection experiments are as follows:

用常规的玻璃珠方法裂解细胞抽提总蛋白， Bradford定量蛋白浓度，将各样品总蛋白浓度调整至相同量 (<10 μ₈/μ1) ₀ 取 1倍体积样品与 1倍体积 12%SDS混合，力 Π 2倍体积 2,4-二硝基苯酰肼 (2,4-Dinitrophenylhydrazine， 20mM，溶于 10% 三氟乙酸)反应 (Le vine等 (1994)， Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233, 346-357)，室温， 15-30min。力口入 1.5倍体积 2M Tris/30% 甘油 /19% 巯基乙醇中和。进行 SDS-PAGE电泳， Coomassie blue染色；及利用抗 2,4-二硝基苯酚 (2,4-dinitrophenyl)抗体 (购自 ZYMED公司）进行 Western Blot检测，出现条带且条带越深，表示氧化损伤的蛋白水平越高。 The cells were extracted by conventional glass bead method, Bradford was used to quantify the protein concentration, and the total protein concentration of each sample was adjusted to the same amount (<10 μ ₈ /μ1). ₀ 1 volume sample was mixed with 1 volume 12% SDS. , 2 times the volume of 2,4-dinitrophenylhydrazine (20 mM, dissolved in 10% trifluoroacetic acid) (Le vine et al (1994), Carbonyl assays for determination of oxidatively modified proteins Methods Enzymol 233, 346-357), room temperature, 15-30 min. The mouth was neutralized with 1.5 volumes of 2M Tris/30% glycerol/19% mercaptoethanol. SDS-PAGE electrophoresis, Coomassie blue staining; and Western Blot detection using anti-2,4-dinitrophenyl (2,4-dinitrophenyl) antibody (purchased from ZYMED), the band appeared and the band deeper, indicating The higher the level of protein that is oxidatively damaged.

结果显示，随着年龄增加，细胞中氧化损伤的蛋白水平明显升高，见图 3A 和图 3B所示。这说明氧化损伤的蛋白在衰老的细胞中不断积累。结合在 S. The results showed that the level of oxidative damage in the cells increased significantly with age, as shown in Figures 3A and 3B. This indicates that oxidatively damaged proteins accumulate in senescent cells. Combined in S.

cerei^/ae中的研究成果，本发明人认为：衰老细胞中氧化损伤蛋白的积累主要是抗氧化压力能力的下降所引起，而非细胞衰老过程中活性氧的形成增多所导致。实施例 3. 糖原水平伴随白念珠菌细胞衰老进程不断升高 The results of the research in cerei^/ae, the present inventors believe that: The accumulation of oxidative damage proteins in senescent cells is mainly caused by a decrease in the ability to resist oxidation, rather than an increase in the formation of reactive oxygen species during cellular aging. Example 3. Glycogen levels are accompanied by an increase in the aging process of Candida albicans cells

衰老的细胞是代谢不旺盛的细胞，因此某些代谢产物会在衰老细胞中积累。酵母中细胞衰老的过程伴随着糖原异生以及能量储存，因此衰老细胞中糖原水平较高 (Lin, S. S.等 (2001)， Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem 276, 36000-36007.)。基因组学研究表明，糖原合成的基因在衰老细胞中表达上调 (； Lesur, I.等 (2004)， The transcriptome of prematurely aging yeast cells is similar to that of Aging cells are cells that are not metabolized, so certain metabolites accumulate in senescent cells. The process of cell senescence in yeast is accompanied by gluconeogenesis and energy storage, so the level of glycogen in senescent cells is higher (Lin, SS et al. (2001), Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem 276, 36000-36007.). Genomics studies have shown that glycogen synthesis genes are upregulated in senescent cells (; Lesur, I. et al. (2004), The transcriptome of prematurely aging yeast cells is similar to that of

telomerase-deficient cells. Mol Biol Cell 15, 1297- 1312.)。在高等生物中，糖原积累不但在生理性衰老过程中发生，还在一些病理性条件下出现。 Telomerase-deficient cells. Mol Biol Cell 15, 1297- 1312.). In higher organisms, glycogen accumulation occurs not only during physiological aging, but also under some pathological conditions.

本发明人取平均寿命分别为 22.9, 13.1, 8.4, 5.4代的白念珠菌细胞群，进行糖原水平检测实验。 The inventors took the Candida albicans cell population with an average lifespan of 22.9, 13.1, 8.4, and 5.4, respectively, and tested the glycogen level.

糖原水平检测实验如下： The glycogen level test was as follows:

用血球计数板对各组细胞样品进行细胞计数，取等量的各组细胞样品点在 GF/C 滤纸 (；购自 Whatman公司）上，于 37°C用碘蒸气熏蒸 2-3min，拍照并用 Cell counts were performed on each group of cell samples using a hemocytometer. An equal amount of each cell sample was spotted on GF/C filter paper (purchased from Whatman) and fumigation with iodine vapor for 2-3 min at 37 ° C.

Bio-Rad Quantity-One program (购自 Bio-Rad公司）定量分析，结果见图 4A和图 5B 所示。 The Bio-Rad Quantity-One program (purchased from Bio-Rad) was quantitatively analyzed, and the results are shown in Fig. 4A and Fig. 5B.

本发明人的实验结果表明，在白念珠菌衰老过程中糖原水平发生了显著升高。糖原的积累可能反映了衰老细胞中某种程度的糖代谢紊乱和能量翻转。实施例 4. 白念珠菌细胞衰老过程中端粒长度保持稳定 The experimental results of the present inventors showed that the level of glycogen increased significantly during the senescence of Candida albicans. High. The accumulation of glycogen may reflect some degree of glucose metabolism disorder and energy turnover in senescent cells. Example 4. Telomere length remained stable during cell senescence of Candida albicans

端粒是真核生物染色体末端由重复的 DNA序列及其结合蛋白所组成的核蛋白复合物，端粒可以维持染色体的稳定性以及帮助 DNA完整复制的进行。端粒酶是一个特化的逆转录酶，起着延伸端粒的作用。在野生型 S. cerei^/ae细胞中，端粒酶是有活性的，因此端粒并不会随着细胞***即复制型细胞衰老的进行而缩短。为了回答在白念珠菌中是否也存在同样的机制，本发明人检测了不同年龄的白念珠菌细胞端粒的长短。 Telomeres are nuclear protein complexes composed of repetitive DNA sequences and their binding proteins at the ends of eukaryotic chromosomes. Telomeres maintain chromosome stability and help complete DNA replication. Telomerase is a specialized reverse transcriptase that acts to extend telomeres. In wild-type S. cerei^/ae cells, telomerase is active, so telomeres do not shrink with cell division, ie, replication-type cell senescence. In order to answer whether the same mechanism exists in Candida albicans, the inventors examined the length of telomeres of Candida albicans cells of different ages.

本发明人取平均寿命分别为 22.9, 13. 1, 8.4, 5.4代的白念珠菌细胞群，进行端粒长短检测实验。 The inventors took the Candida albicans cell population with an average lifespan of 22.9, 13. 1, 8.4, and 5.4, respectively, and tested the telomere length.

端粒长短检测实验如下： The telomere length test is as follows:

用常规的玻璃珠法抽提各组细胞的基因组 DNA， Nlalll和 AM酶切后进行 0.9%琼脂糖凝胶电泳，转移至 Hybond-N+ 膜 (Amersham Biosciences) , 与包含两个白念珠菌端粒重复序列的 5 '末端 ³²P标记的寡核苷酸探针 The genomic DNA of each group of cells was extracted by conventional glass bead method, and Nlalll and AM were digested and subjected to 0.9% agarose gel electrophoresis, transferred to Hybond-N+ membrane (Amersham Biosciences), and containing two Candida albicans telomeres. Repeated sequence 5' end ³² P labeled oligonucleotide probe

(S '-gaa-gtt-aga-cat-ccg-tac-acc-aag-aag-tta-gac-atc-cgt-aca-cca-a-S^jS^T Southern杂交 (Singh, S. M.等 (2002)， Analysis of Telomerase in Candida albicans: Potential Role in Telomere End Protection. Eukaryot Cell 1, 967-977)。 (S '-gaa-gtt-aga-cat-ccg-tac-acc-aag-aag-tta-gac-atc-cgt-aca-cca-aS^jS^T Southern hybridization (Singh, SM et al. (2002), Analysis of Telomerase in Candida albicans: Potential Role in Telomere End Protection. Eukaryot Cell 1, 967-977).

结果见图 5，由结果可见，白念珠菌在整个生命周期中端粒维持在一个稳定的水平。实施例 5. 染色体外核糖体 DNA环的积累不是白念珠菌衰老的主要原因在 S. cerevisiae中，核糖体 DNA是位于第 12染色体上的一段 100-200个串联重复的大约 9. 1kb的序列。 ERC理论认为，相邻的核糖体 DNA重复序列之间的同源重组导致产生 ERC， ERC可以自我复制并且随着细胞***不均等的分配积累在母细胞中，从而导致细胞衰老。 ERC的积累既是导致酵母细胞衰老的原因，同时也是细胞衰老的标志（ Sinclair, D. A.和 Guarente, L. (1997). Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033- 1042.)。 The results are shown in Figure 5. From the results, it can be seen that Candida albicans maintains a stable level throughout the life cycle. Example 5. Accumulation of extrachromosomal ribosomal DNA loops is not a major cause of Candida albicans senescence In S. cerevisiae, ribosomal DNA is a sequence of about 9.1 kb in a section of 100-200 tandem repeats on chromosome 12. . According to ERC theory, homologous recombination between adjacent ribosomal DNA repeats results in the production of ERC, which can self-replicate and accumulate in the mother cells as the cell division is unevenly distributed, leading to cell senescence. Accumulation of ERC is both a cause of senescence in yeast cells and a hallmark of cellular senescence (Sinclair, D. A. and Guarente, L. (1997). Extrachromosomal rDNA circles - a cause of aging in yeast. Cell 91, 1033- 1042.).

本发明人在白念珠菌中对该理论进行了验证。取平均寿命分别为 22.9, 13. 1, 8.4, 5.4代的白念珠菌细胞群进行检测。 The inventors verified this theory in Candida albicans. The Candida albicans cell population with average lifespan of 22.9, 13. 1, 8.4, and 5.4 generations was tested.

采用的染色体外核糖体 DNA环（extrachromosomal rDNA circle, ERC) 检测实验如下： Exochromosomal DNA strand (ERC) detection The experiment is as follows:

用 Zymolyase裂解法抽提基因组 DNA， 0.7%琼脂糖凝胶电泳， lV/cm， 40hr_; 转移至 Hybond-N+膜，用 ³²P标记的白念珠菌 18S rDNA的 1.7kb PCR产物 (常规方法制备）进行 Southern杂交检测。为了检测 rDNA的拷贝数，基因组 DNA用 EcoRV和 Sail酶切， 0.9%琼脂糖凝胶电泳，转膜然后与 rDNA探针 (；常规方法制备）杂交。将膜上杂交的探针洗掉，重新与 l. lkb的 C Cr (ACTl) 探针 (常规方法制备；)杂交，对 rDNA杂交信号进行标准化。用 Image Quant软件 (Amersham Biosciences) 对 rDNA拷贝数进行量化处理，最年轻细胞的 rDNA拷贝数定为 55/ 细胞 (Jones, T.等 (2004)， The diploid genome sequence of Candida albicans. Proc Natl Acad Sci U S A 101, 7329-7334) 。 Genomic DNA was extracted by Zymolyase lysis method, 0.7% agarose gel electrophoresis, lV/cm, 40 hr _; transferred to Hybond-N+ membrane, 1.7 kb PCR product of ³² P-labeled Candida albicans 18S rDNA (prepared by conventional method) Southern hybridization assays were performed. To detect the copy number of rDNA, genomic DNA was digested with EcoRV and Sail, electrophoresed on a 0.9% agarose gel, and then transfected with an rDNA probe (prepared by a conventional method). The probe hybridized on the membrane was washed away and re-hybridized with a C. lkb C Cr (ACT1) probe (prepared by conventional methods) to normalize the rDNA hybridization signal. The rDNA copy number was quantified using Image Quant software (Amersham Biosciences), and the rDNA copy number of the youngest cells was determined to be 55/cell (Jones, T. et al. (2004), The diploid genome sequence of Candida albicans. Proc Natl Acad Sci USA 101, 7329-7334).

结果与以往的研究截然不同，不但没有检测到 ERC的存在 (图 6A；)，整个基因组的 rDNA水平也未发现有明显变化 (；图 6B和图 6C；)。联系前人研究结果不难发现， ERC目前也只在 S. cerev /ae中被证明是存在的，因此并不是一种普遍存在的导致细胞衰老的因素。 The results were quite different from previous studies. Not only did the presence of ERC not be detected (Fig. 6A;), but no significant changes were observed in the rDNA levels of the entire genome (Fig. 6B and Fig. 6C;). It is not difficult to find out the results of previous studies. ERC is currently only found in S. cerev /ae, so it is not a ubiquitous factor leading to cell aging.

并且，本发明人还发现，单拷贝 S/R2基因的敲除可以导致白念珠菌寿命缩短，说明 S/R2对白念珠菌寿命的正调控可能不是通过抑制 ERC的产生实现的。这些结果表明酵母细胞衰老过程中还存在着其它 ERC非依赖性的途径， ERC的积累并不是导致白念珠菌衰老的原因。实施例 6. 筛选抗衰老药物的方法 Moreover, the inventors have also found that knockout of the single copy S/R2 gene can lead to a shortened lifespan of Candida albicans, indicating that the positive regulation of S/R2 on the lifespan of Candida albicans may not be achieved by inhibiting the production of ERC. These results indicate that there are other ERC-independent pathways in the senescence of yeast cells, and the accumulation of ERC is not the cause of senescence of Candida albicans. Example 6. Method for screening anti-aging drugs

细胞模型：实施例 1制备的衰老细胞。 Cell model: The senescent cells prepared in Example 1.

测试组：添加候选物的实施例 1制备的衰老细胞。 Test group: The senescent cells prepared in Example 1 in which the candidate was added.

对照组：未添加候选物的实施例 1制备的衰老细胞 (与测试组衰老细胞处于同一年龄阶段)。所述的筛选方法包括以下步骤： Control group: The senescent cells prepared in Example 1 in which no candidate was added (at the same age as the test group senescent cells). The screening method includes the following steps:

在测试组中，在实施例 1制备的衰老细胞的培养物中添加候选物，观察细胞的平均传代次数。 In the test group, candidates were added to the culture of the senescent cells prepared in Example 1, and the average number of passages of the cells was observed.

将前述测试组中细胞的平均传代次数与对照组中细胞的平均传代次数进行比较，如果测试组中细胞的平均传代次数在统计学上显著高于对照组 (例如测试组的平均传代次数比对照组高出 3-5代；)，就表明该候选物是抗衰老的潜在药物。在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 The average number of passages of cells in the aforementioned test group was compared with the average number of passages of cells in the control group, if the average number of passages of cells in the test group was statistically significantly higher than that of the control group (for example, the average number of passages of the test group was higher than that of the control group). The group is 3-5 generations higher;), indicating that the candidate is a potential anti-aging drug. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

权利要求 Rights request

1. 一种分离的念珠菌细胞群，其特征在于，所述细胞群具有以下特征： An isolated population of Candida cells, characterized in that said population of cells has the following characteristics:

2. 如权利要求 1 所述的念珠菌细胞群，其特征在于，所述细胞群中， 90%以上细胞为念珠菌的菌丝状细胞。 The Candida cell population according to claim 1, wherein 90% or more of the cell population is a mycelial cell of Candida.

3. 如权利要求 1 所述的念珠菌细胞群，其特征在于，所述细胞群中， 80%以上的所述念珠菌的菌丝状细胞处于同一年龄阶段。 The Candida cell population according to claim 1, wherein more than 80% of the mycelial cells of the Candida are in the same age stage.

4. 一种制备权利要求 1所述的念珠菌细胞群的方法，其特征在于，所述方法包括步骤： A method of producing a Candida cell population according to claim 1, wherein the method comprises the steps of:

(a) 在适合菌丝状念珠菌细胞生长的条件下，培养念珠菌细胞群，形成念珠菌菌丝状细胞群； (a) cultivating a Candida cell population to form a Candida mycelium cell population under conditions suitable for the growth of mycelial cells of Candida;

(c) 从步骤 (b)获得的混合细胞群中分离出念珠菌的酵母状子细胞群，并且在适合菌丝状念珠菌细胞生长的条件下，培养该酵母状子细胞群，从而形成念珠菌的菌丝状细胞群； (c) isolating the yeast cell population of Candida from the mixed cell population obtained in the step (b), and cultivating the yeast cell population under conditions suitable for the growth of the Mycelium cell, thereby forming Candida Mycelial cell population;

(d) 在适合酵母状念珠菌细胞生长的条件下，培养上一步骤所获得的念珠菌的菌丝状细胞群，从而形成含有念珠菌的菌丝状细胞和酵母状子细胞的混合细胞群；和 (d) cultivating the mycelial cell population of Candida obtained in the previous step under conditions suitable for the growth of the Candida species; thereby forming a mixed cell population containing Candida-like mycelial cells and yeast-like daughter cells; with

(e) 从步骤 (d)获得的混合细胞群中分离出念珠菌的菌丝状细胞群。 (e) Segregating the mycelial cell population of Candida from the mixed cell population obtained in the step (d).

5. 如权利要求 4所述的方法，其特征在于，该方法还包括以下步骤： 5. The method according to claim 4, wherein the method further comprises the following steps:

6. 如权利要求 4所述的方法，其特征在于，步骤（； b) 中培养时间为 0.5-5小时。 6. The method according to claim 4, wherein the incubation time in step (; b) is 0.5-5 hours.

7. 如权利要求 4所述的方法，其特征在于，所述的适合菌丝状念珠菌细胞生长的条件为 35-42 °C，血清诱导。 The method according to claim 4, wherein the condition suitable for growth of Mycelial cells of Mycelia is 35-42 ° C, and serum is induced.

8. 如权利要求 4所述的方法，其特征在于，所述的适合酵母状念珠菌细胞生长的条件为 25-32°C。 The method according to claim 4, wherein the condition suitable for the growth of the Candida albicans cell is 25-32 °C.

9. 如权利要求 4所述的方法，其特征在于，在步骤（； c) 中，通过密度梯度离心分离出念珠菌的酵母状子细胞群；或者， 9. The method according to claim 4, wherein in step (; c), centrifugation by density gradient a population of yeast-like cells that are isolated from Candida; or,

10. 权利要求 1所述的细胞群的用途，其特征在于，用于作为研究细胞的生长、发育、代谢、衰老的模型；或用于筛选调节细胞的生长、发育、代谢、衰老的物质。 The use of the cell population according to claim 1, which is used as a model for studying growth, development, metabolism, and senescence of cells; or for screening for substances regulating growth, development, metabolism, and senescence of cells.