缺氧预适应小鼠脑匀浆液对离体细胞缺氧耐受性的影响及其可能有效组分的初步研究

发布时间：2018-06-23 12:49

本文选题：急性重复缺氧 + 模型　；参考：《第三军医大学》2008年博士论文

【摘要】： 缺氧时,机体会出现各种代偿反应以对抗缺氧的影响。机体这种与缺氧做斗争的能力,即缺氧的耐受力显然是有一定限度的。近年来的研究表明,机体的这种对缺氧的耐受性可以通过缺氧预适应等措施得到加强。缺氧预适应是指机体经短暂时间或亚致死量的缺氧后,对后续的更长时间或更严重缺氧性损伤产生明显耐受的能力。其机制除了与缺氧引发的由缺氧诱导因子-1等转录因子介导的各种适应代偿性反应蛋白表达改变有关外,组织、细胞的能量代谢产物,特别是ATP的分解代谢产物等在缺氧预适应中也起重要作用。脑是对缺氧最敏感的器官,是影响机体缺氧耐受能力的主要限制因素。因此中枢神经系统的机能或代谢变化可能是机体缺氧耐受形成的主要机制。研究表明,缺氧时脑组织的物质变化不仅对于机体缺氧耐受能力的建立具有重要作用,而且缺氧预适应小鼠的脑匀浆液也能提高动物甚至离体细胞对缺氧的耐受能力。积极寻找并研究急性重复缺氧预适应小鼠脑匀浆液中的可提高机体缺氧耐受能力的生物活性物质具有重要的理论和实际意义。本研究通过观察不同实验条件对急性重复缺氧预适应小鼠模型的影响,建立稳定可靠、效果显著的小鼠缺氧预适应模型。由于动物对缺氧的耐受性个体差异较大,影响因素较多,因此为便于控制实验条件和探索增强缺氧耐受性的物质特性,本研究采用离体细胞观察急性重复缺氧预适应小鼠脑匀浆液对离体细胞缺氧耐受性的影响。在确定急性重复缺氧预适应小鼠脑匀浆液对不同离体培养细胞(未用NGF诱导的PC12细胞、HepG2细胞)缺氧耐受性具有双相效应的基础上,以NGF诱导分化的PC12细胞作为观察对象,探索急性重复缺氧预适应小鼠脑匀浆液对分化PC12细胞只起保护作用、无损伤作用的最适浓度,并进一步将该提取液分成蛋白部分和非蛋白部分,探讨其中存在的增强细胞缺氧耐受性的可能组分。实验分三部分进行。一、方法第一部分急性重复缺氧预适应小鼠模型的复制 (1).使小鼠一次性缺氧致死,以观察小鼠缺氧致死前的变化规律。 (2).将小鼠连续4次重复缺氧,以观察小鼠每次缺氧耐受时间的变化。 (3).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是呼吸频率约40次/min或出现第1次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。 (4).将小鼠分成2组,均经过连续4次缺氧,2组小鼠每次结束缺氧的标准分别是第一次喘呼吸或末次喘呼吸,以观察每次不同时间缺氧对小鼠缺氧耐受性的影响。 (5).将小鼠分成4组,均经过连续4次缺氧,但4组小鼠连续4次缺氧的环境温度分别是:9-11℃、13-15℃、17-19℃、21-23℃,以观察不同环境温度对小鼠缺氧耐受性的影响。 (6).将小鼠分成2组,均经连续4次缺氧,2次缺氧间的换瓶操作时间分别是5-15s或20-30s,以观察不同复氧时间对小鼠缺氧耐受性的影响。 (7).将经过连续4次缺氧的小鼠恢复常氧, 30分钟后再放回缺氧瓶缺氧1次,以观察小鼠复氧30分钟后缺氧耐受性的变化。第二部分缺氧预适应小鼠脑匀浆液对未用NGF诱导分化的PC12细胞、HepG2细胞以及NGF诱导分化的PC12细胞缺氧耐受性的影响 1.以不同浓度的小鼠脑匀浆液分别作用于未用NGF诱导的PC12细胞,观察缺氧24h、48h、72h时PC12细胞的活力、LDH透出率、早期凋亡率、晚期凋亡率的变化规律,以确定小鼠脑匀浆液对细胞缺氧耐受性的影响。 2.以不同浓度的小鼠脑匀浆液分别作用于HepG2细胞,观察缺氧24h、48h、72h缺氧时HepG2细胞的活力、早期凋亡率、晚期凋亡率的变化规律,以了解小鼠脑匀浆液对非神经元模型细胞的影响。 3.根据上述第二部分实验的结果,仅以较低浓度范围小鼠脑匀浆液分别作用于NGF诱导分化的PC12细胞,观察缺氧24h、48h、72h时分化PC12细胞的活力、LDH透出率,晚期凋亡率的变化规律,以探索缺氧预适应小鼠脑匀浆液对缺氧、NGF诱导分化的PC12细胞只起保护作用、没有损伤作用的最适浓度。第三部分:缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨 (1).以高氯酸除蛋白法制备缺氧预适应小鼠脑匀浆去蛋白液,HPLC法测定其腺苷含量。用小鼠脑匀浆去蛋白液作用于NGF诱导分化PC12细胞,以确定去蛋白液对缺氧的分化PC12细胞有无保护作用;以小鼠脑匀浆去蛋白液合并腺苷A1或A2A受体阻断剂作用于分化PC12细胞,以确定小鼠脑匀浆去蛋白液的作用是否与腺苷有关。 (2).透析法除去缺氧预适应小鼠脑匀浆液中小分子物质,并观察透析保留液对缺氧的分化PC12细胞有无保护作用;分别用RT-PCR、Western blot方法检测缺氧预适应小鼠脑组织中VEGF mRNA、VEGF的蛋白水平是否增加;用重组VEGF纯品作用于分化PC12细胞,以确定透析保留液对分化PC12细胞的保护作用是否与VEGF有关。二、结果与讨论通过上述3部分的工作获得以下结果 (一)影响急性重复缺氧模型的因素 1.小鼠经4次急性重复缺氧后,每次缺氧耐受时间显著增加。这一结果提示,小鼠在每次缺氧后,均在体内产生了增强缺氧耐受性的物质,并且各次缺氧后缺氧耐受的物质有积累效应。 2.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠呼吸频率在40次/min时换瓶比较,可显著提高小鼠每次耐受时间。这是因为小鼠在呼吸频率为40次/min时的缺氧时间比出现喘呼吸时的缺氧时间短,缺氧程度尚未达到缺氧耐受形成的程度,因此对缺氧的耐受性也不强,因为缺氧预适应的形成需要足够程度的缺氧。 3.在小鼠出现第一次喘呼吸时将小鼠换入下一个缺氧瓶与小鼠出现末次喘呼吸时换瓶比较,缺氧耐受时间无显著差异。其原因是,不同小鼠喘呼吸次数不同,有的小鼠喘呼吸3-5次即死亡,有的小鼠喘呼吸却可达30次以上,因此小鼠的个体差异较大,使组间无显著差异;并且由于末次喘呼吸后小鼠随即死亡,因此末次喘呼吸难以判断,以其作为结束缺氧标准导致小鼠死亡率增加。 4.随着环境温度的增加,小鼠的缺氧耐受时间显著降低。这可能是因为低温可减少小鼠体内有害物质的产生、降低代谢,有利于缺氧预适应的形成。 5.每次对小鼠换瓶操作时间长于15秒会显著降低小鼠的缺氧耐受时间。其原因可能是复氧时间太长,因为复氧时间短暂有利于预适应的形成。由于经过重复4次缺氧的小鼠恢复常氧30分钟后,其缺氧耐受性还可以部分保留,说明小鼠4次缺氧耐受性的完全消失需要至少30分钟以上的时间,因此赋予预适应小鼠缺氧耐受性的物质,可能部分对机体恢复常氧供应较为敏感,但部分能维持较长时间。 (二)缺氧预适应小鼠脑匀浆液对未用NGF诱导的PC12细胞、HepG2细胞、NGF诱导分化的PC12细胞缺氧耐受性的影响 1.缺氧预适应小鼠脑匀浆液对缺氧、未用NGF诱导的PC12细胞、HepG2细胞缺氧耐受性的影响是:缺氧预适应小鼠脑匀浆液可显著提高细胞对缺氧的耐受性。在缺氧初期(24h),较高浓度的保护作用显著强于低浓度的保护作用,这提示缺氧预适应小鼠脑组织中产生了某种或某些可以增强细胞缺氧耐受性的物质或使脑组织中抗缺氧成分表达增加。随着缺氧时间延长,保护作用下降,高浓度的下降更快。至缺氧72h,高浓度提取液的缺氧损伤作用显著高于低浓度组,该结果提示,缺氧预适应小鼠脑组织中可能产生了半衰期较短的抗缺氧物质及在高浓度时才显示作用的、作用时间持久的促缺氧细胞损伤的物质,即提示缺氧预适应有着双相效应,缺氧预适应小鼠脑匀浆的作用存在量效关系和时间效应。但是由于本研究的目的是为了寻找缺氧预适应小鼠脑匀浆液中增强缺氧耐受性的物质,因此实验中应避免损伤性物质的影响。为此,在后续缺氧预适应小鼠脑匀浆液对分化PC12细胞的作用研究中,以蛋白浓度对提取液进行定量,并降低实验中所用提取液的浓度范围,以期找到提取液对NGF诱导分化的PC12细胞只起保护作用,无损伤作用(即只增强NGF诱导分化的PC12细胞缺氧耐受性,对分化PC12细胞无损伤作用)的最适浓度,为进一步研究缺氧预适应小鼠脑匀浆液的保护效应及保护性物质奠定基础。 2.经较低浓度的缺氧预适应小鼠脑匀浆液对NGF诱导分化的PC12细胞的作用实验发现,蛋白终浓度为100.0μg/mL的缺氧预适应小鼠脑匀浆液与同浓度正常小鼠脑匀浆液比较,可显著增强NGF诱导分化的PC12细胞的缺氧耐受性,并且此浓度的缺氧预适应小鼠脑匀浆液对分化PC12细胞未显示出损伤作用,为增强分化PC12细胞缺氧耐受性的适合浓度。 (三)缺氧预适应小鼠脑匀浆液不同组分对NGF诱导分化的PC12细胞缺氧耐受性的影响及其可能有效成分的初步探讨 1.向离体培养的NGF诱导分化的PC12细胞中加入不同浓度的腺苷,随着腺苷浓度增加,细胞的缺氧耐受性增高,10.0μmol/L的腺苷对细胞的保护作用可维持24h。急性4次重复缺氧可使小鼠脑组织腺苷含量上升。缺氧预适应小鼠脑匀浆去蛋白液只在缺氧的24h内显著增加分化PC12细胞的缺氧耐受性,腺苷A2A受体阻断剂可阻断其保护作用。正常小鼠脑匀浆去蛋白液对分化PC12细胞无保护作用。因此,缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。 2.在缺氧24h和48h,缺氧预适应小鼠脑匀浆透析保留液与未经透析的缺氧预适应小鼠脑匀浆液对分化PC12细胞均有保护作用,且经两种孔径(3.5kD、7.0kD)透析袋透析的缺氧预适应小鼠脑匀浆透析保留液和未经透析的缺氧预适应小鼠脑匀浆液对缺氧细胞的保护作用无显著差异,说明缺氧预适应小鼠脑组织中起保护作用的主要是分子量大于7.0kD的蛋白成分。急性重复缺氧预适应小鼠脑组织中VEGF表达虽增加,但应用重组VEGF直接作用于分化的PC12细胞发现,不同浓度的VEGF对NGF诱导分化的、不同缺氧时间的PC12细胞均无保护作用。这一结果提示:缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用。由于缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用时间持久,提示蛋白质组学方法可能是研究其中抗缺氧成分的有效方法,因此本研究结果为今后进一步研究缺氧预适应小鼠脑匀浆液中抗缺氧物质提示了方向。三、结论 1.赋予重复缺氧小鼠缺氧耐受性的物质,部分对机体恢复常氧供应较为敏感,部分则能持续较长时间; 2.缺氧预适应小鼠脑匀浆液在浓度较高时对离体缺氧培养细胞的作用表现为双相性--缺氧早期,脑匀浆液对缺氧细胞具有保护效应,延长缺氧时间,其损伤作用逐渐显现,这种双相作用存在量效关系和时间效应;其只起保护作用的浓度是在较低浓度; 3.缺氧预适应小鼠脑匀浆液可显著提高不同来源离体培养细胞的缺氧耐受性,提示对缺氧细胞具有普遍的保护作用; 4.缺氧预适应小鼠脑匀浆液的蛋白组分抗缺氧作用较为持久,其中VEGF表达量虽增高,但并不参与NGF诱导分化的PC12细胞的抗缺氧作用; 5.缺氧预适应小鼠脑匀浆液的非蛋白组分的抗缺氧作用时间较短,腺苷可能是其中一个重要的活性物质。
[Abstract]:In the case of hypoxia, the body has a variety of compensatory responses to the effect of anoxia. The body's ability to combat hypoxia, the tolerance of hypoxia, is obviously limited. In recent years, the body's tolerance to hypoxia can be strengthened through hypoxia preconditioning. Hypoxic preconditioning refers to the body. After a short or sublethal dose of hypoxia, the ability to tolerate further longer or more severe anoxic damage is associated with a variety of adaptable reactive protein expressions mediated by hypoxia inducible factor -1 and other adaptable reactive protein expressions, tissue, and cell energy metabolites, especially A TP catabolic products also play an important role in hypoxic preconditioning.
Brain is the most sensitive organ to hypoxia, which is the main limiting factor of the body's tolerance to hypoxia. Therefore, the changes in the function or metabolism of the central nervous system may be the main mechanism for the formation of the body's hypoxia tolerance. The hypoxic preconditioning of the brain homogenate of mice can also improve the tolerance to hypoxia in animals and even isolated cells. It is of great theoretical and practical significance to actively seek and study bioactive substances in the brain homogenate of acute repeated hypoxia preconditioning mice, which can improve the anoxic tolerance of the body. A stable, reliable and effective model of hypoxic preconditioning in mice was established with a stable and reliable model of hypoxic preconditioning in mice. In order to facilitate the control of the experimental conditions and to explore the material characteristics of enhancing the hypoxia tolerance, this study adopted the view of isolated cells. The effects of acute repeated hypoxia preconditioning on hypoxia tolerance of isolated cells in mice were investigated. On the basis of determining the biphasic effect of acute hypoxia preconditioning mouse brain homogenate on the hypoxia tolerance of different cultured cells (without NGF induced PC12 cells, HepG2 cells), the differentiation of PC12 cells induced by NGF was used as a view. To explore the protective effect of acute hypoxic preconditioning on the differentiated PC12 cells, the optimal concentration of no damage was found, and the extract was further divided into protein and non protein parts, and the possible components of the enhanced cell hypoxia tolerance were discussed. The experiment was divided into three parts.
First, method
Part one replication of a mouse model of acute repeated hypoxia preconditioning
(1) let the mice die in a single dose of hypoxia to observe the change rule of mice before hypoxia.
(2) to observe the change of hypoxia tolerance time in mice by repeated 4 times of anoxia.
(3). The mice were divided into 2 groups, all after 4 consecutive anoxia. The standard of the 2 mice at each end of the hypoxia was about 40 times /min or first breath, in order to observe the effect of hypoxia on the hypoxia tolerance of mice at each time.
(4) the mice were divided into 2 groups, all after 4 consecutive anoxia. The standard of the 2 mice at the end of the hypoxia was the first breath or the last breath, to observe the effect of anoxia on the hypoxia tolerance of mice at each time.
(5) the mice were divided into 4 groups after 4 consecutive anoxia, but the ambient temperature of the 4 groups of mice for 4 consecutive anoxia were 9-11, 13-15, 17-19, 21-23, respectively, to observe the effects of different ambient temperatures on the hypoxia tolerance of mice.
(6). The mice were divided into 2 groups, all after 4 consecutive anoxia, and the operation time of the 2 anoxia was 5-15s or 20-30s, respectively, to observe the effect of different reoxygenation time on the hypoxia tolerance of mice.
(7) restore the normoxic mice after 4 consecutive anoxia, and then return to anoxic bottle for 1 times after 30 minutes to observe the change of hypoxia tolerance after 30 minutes of reoxygenation.
Second part of the hypoxic preconditioned mouse brain homogenate effect on the hypoxia tolerance of PC12 cells, HepG2 cells and NGF induced PC12 cells without NGF induced differentiation
1. the mouse brain homogenate of different concentrations was used to act on PC12 cells which were not induced by NGF, and the activity of PC12 cells, LDH permeability, early apoptosis rate and late apoptosis rate were observed at 24h, 48h and 72h, in order to determine the effect of mouse brain homogenate on cell hypoxia tolerance.
2. HepG2 cells were used in different concentrations of mouse brain homogenate to observe the activity of HepG2 cells, the early apoptosis rate and the late apoptosis rate of the hypoxia 24h, 48h and 72h, in order to understand the effect of the mouse brain homogenate on the non neuron model cells.
3. according to the results of the second parts of the experiment above, only the mouse brain homogenate in the lower concentration range was used to induce the differentiation of PC12 cells in NGF, and the activity of PC12 cells, LDH permeability and late apoptosis rate were observed at 24h, 48h and 72h, in order to explore the hypoxia preconditioning mouse brain homogenate solution to hypoxia and NGF induced differentiation of PC12. The cells only play a protective role, without the optimum concentration of injury.
The third part: the effect of different components of the hypoxic preconditioned mouse brain homogenate on the hypoxia tolerance induced by NGF induced PC12 cells and the preliminary study of its possible effective components
(1) the content of adenosine was determined by HPLC method with hypoxic preconditioning in the brain homogenate of mice with perchloric acid deproteinization. The mouse brain homogenate deprotein was used to induce the differentiation of PC12 cells by NGF to determine the protective effect of deproteinized solution on the anoxic differentiated PC12 cells, and the inhibition of adenosine A1 or A2A receptor blocker in the rat brain homogenate deproteinization solution. Disrupting agent acts on differentiated PC12 cells to determine whether the effect of mouse brain homogenate deproteinization is related to adenosine.
(2). The dialysis method was used to remove the small molecular substances in the mouse brain homogenate, and to observe the protective effect of the dialysis retention solution on the anoxic differentiated PC12 cells. RT-PCR and Western blot were used to detect VEGF mRNA in the brain tissue of the hypoxic preconditioned mice, and the protein level of VEGF increased, and the recombinant VEGF was used to differentiate PC12 fine. To determine whether the protective effect of dialysis fluid on differentiated PC12 cells is related to VEGF.
Two, results and discussion
The following results are obtained through the work of the above 3 parts
(1) factors affecting the model of acute anoxia
In 1. mice, after 4 acute anoxia, the tolerance time of hypoxia increased significantly each time. This result suggested that the mice produced an anoxic tolerance substance in the body after each anoxic, and the anoxic tolerance substance had accumulated effect after each anoxic.
2. during the first breath of mice, the mice were changed into the next hypoxic bottle and the breathing frequency of the mice at the 40 /min, which could significantly improve the mice tolerance time. This is because the hypoxia time of the mice at the respiratory rate of 40 times /min is shorter than that of the asthmatic respiration, and the degree of hypoxia has not reached the anoxic tolerance. The degree of formation, therefore, is also not strong enough to tolerate hypoxia, because the formation of hypoxic preconditioning requires adequate hypoxia.
3. during the first breath of mice, the mice were changed into the next hypoxic bottle and the last breath of the mice. There was no significant difference in the time of hypoxia tolerance. The reason was that the number of asthma in different mice was different, some of the mice were breathing 3-5 times or more than 30 times. There was no significant difference between the groups, and the mice died immediately after the last wheezing breathing, so the last wheezing respiration was difficult to judge, and the death rate of mice was increased as a standard to end the hypoxia.
4. with the increase of ambient temperature, the hypoxia tolerance time of mice decreased significantly. This may be because the low temperature can reduce the production of harmful substances in the mice, reduce metabolism, and be beneficial to the formation of hypoxic preconditioning.
5. the hypoxia tolerance time of mice was significantly reduced each time the operation time of the mice was longer than 15 seconds. The reason may be that the reoxygenation time was too long, because the time of reoxygenation was short for the formation of preconditioning. The hypoxia tolerance of mice after repeated 4 times of hypoxia was partly retained after 30 minutes, indicating that the mice were deficient in 4 times. The complete disappearance of oxygen tolerance requires at least 30 minutes or more, so the substance that endows the hypoxic tolerance of the preconditioned mice may be partly sensitive to the recovery of the oxygen supply in the body, but partly for a long time.
(two) hypoxic preconditioning mice brain homogenate had no effect on the hypoxia tolerance of PC12 cells, HepG2 cells and NGF induced PC12 cells induced by NGF.
1. hypoxic preconditioning mice brain homogenate on hypoxia, without NGF induced PC12 cells, the effect of hypoxia tolerance in HepG2 cells is that hypoxia preconditioning mouse brain homogenate can significantly improve the tolerance to hypoxia. At the early stage of hypoxia (24h), the higher concentration of protective effect is stronger than low concentration of protective effect, which suggests hypoxic preconditioning. In the brain tissue of mice, some or some substances that can enhance the anoxic tolerance of the cells or increase the expression of anti anoxic components in the brain tissue. With the prolonged anoxia time, the protective effect drops and the high concentration decreases faster. To hypoxia 72h, the hypoxia injury in high concentration extract is significantly higher than the low concentration group, which suggests hypoxia. Preconditioning mice may produce a short half-life of anti hypoxia substance and its effect at high concentration. The substance that has a lasting effect on hypoxia cell injury suggests that anoxic preconditioning has a biphasic effect. The effect of hypoxia preconditioning on the brain homogenate of the mice remains in the dose effect relationship and time effect. The aim of the study is to find the substance of hypoxia tolerance in the hypoxic preconditioning mouse's brain homogenate, so we should avoid the effect of damaging substances in the experiment. Therefore, in the study of the effect of subsequent hypoxia preconditioning on the differentiation of PC12 cells from the mouse brain homogenate, the concentration of protein was used to quantify the extract, and the experiment was reduced. The concentration range of the solution is used to find the protective effect of the extract on the PC12 cells induced by NGF, and the optimum concentration of no damage (that is, only enhancing the NGF induced PC12 cells' anoxic tolerance, and the no damage effect on the differentiated PC12 cells), is to further study the protective effects and protective effects of the hypoxic preconditioned mouse brain homogenate. Quality lays the foundation.
2. the effect of hypoxic preconditioning on NGF induced PC12 cells induced by hypoxic hypoxia in mice showed that the hypoxia preconditioned mouse brain homogenate with the final concentration of 100 g/mL and the same concentration of normal mice could significantly enhance the hypoxia tolerance of PC12 cells induced by NGF, and the deficiency of this concentration was deficient. Oxygen preconditioning mice brain homogenate did not show any damage to differentiated PC12 cells, and was suitable for enhancing the tolerance of differentiated PC12 cells to hypoxia.
(three) the effects of different components of hypoxic preconditioning mice brain homogenate on hypoxia tolerance of NGF induced PC12 cells and possible effective components
1. different concentrations of adenosine was added to the differentiated PC12 cells induced by NGF in vitro. With the increase of adenosine concentration, the hypoxia tolerance of the cells increased. The protective effect of adenosine at 10 mu mol/L could maintain 24h. acute 4 repeated hypoxia to increase the content of adenosine in the brain tissue of mice. Hypoxia preconditioning in the brain homogenate deproteinized solution only The hypoxia tolerance significantly increased the anoxic tolerance of differentiated PC12 cells, and adenosine A2A receptor blocker blocked its protective effect. The normal mouse brain homogenate deprotein solution had no protective effect on the differentiated PC12 cells. Therefore, the anti oxygen deficiency time of the non protein components in the brain homogenate of the hypoxic mice was shorter, and adenosine might be one of them. An important active substance.
2. in hypoxic preconditioning mice, the hypoxic preconditioning of dialysis fluid and non dialysis was performed in hypoxia 24h and 48h.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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