CALCINEURIN-MEF2C信号途径参与内质网应激诱导的大鼠心肌细胞肥大

发布时间：2018-04-23 09:44

本文选题：心肌肥大 + 内质网应激　；参考：《山东大学》2010年博士论文

【摘要】： 心肌肥大是心肌细胞对多种病理因素的代偿性反应,但慢性心肌肥大参与了扩张性心肌病、心力衰竭、猝死等多种心血管疾病的发生、发展过程,严重危及人类生命；因此阐明心肌肥大的发病机制对于防治心肌肥大和心力衰竭具有重要的临床意义。内质网(endoplasmic reticulum, ER)是调节细胞内钙稳态和膜型／分泌型蛋白质合成、折叠的重要细胞器,对维持心肌细胞钙和功能蛋白质稳态具有重要作用。ER对应激刺激非常敏感,多种因素如钙紊乱、蛋白合成增加、缺血、缺氧等因素均可触发ER应激。适当的ER应激诱导ER分子伴侣如葡萄糖调节蛋白(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)等表达上调,增强ER钙调节和处理未折叠蛋白的能力,具有细胞保护作用；但是,持续或严重的ER应激可触发ER凋亡信号通路,促进ER重要的促凋亡蛋白C/EBP homologous protein (CHOP)表达增加和caspase-12剪切活化,导致细胞凋亡和组织损伤。蛋白激酶R样ER激酶(protein kinase R-like ER kinase, PERK)是重要的ER跨膜蛋白,ER应激时GRP78与PERK的解离激活PERK,诱导激活型转录因子4 (activating transcription factor 4, ATF4)转录及其下游靶分子CHOP表达,促进细胞凋亡。多种证据表明ER应激与神经退行性变、糖尿病性心肌病和缺血／再灌注损伤等多种疾病密切相关。研究发现心肌肥大时ER应激分子CRT、GRP78、GRP94等表达显著上调,CHOP介导的ER应激凋亡通路参与了心力衰竭过程中的细胞凋亡,提示心肌肥大过程中存在ER应激反应,但ER应激在心肌肥大发生、发展中的变化及作用目前并不清楚。心肌细胞内Ca2+水平升高参与了心肌肥大的发生,是触发心肌肥大反应的基本信号。钙调神经磷酸酶(calcineurin, CaN)是一种受细胞浆Ca2+及钙调素(calmodulin, CaM)调控的蛋白丝氨酸／苏氨酸磷酸酶,由催化亚基CnA和调节亚基CnB组成,在T细胞活化、心肌肥大、细胞周期调控等多种生物学过程中发挥关键性的调控作用。心肌细胞增强因子2c (myocyte enhancer factor 2c, MEF2c)是MEF2c家族中发现的第一个参与心脏发育的分子,并被CaN激活,但是CaN-MEF2c信号途径在ER应激诱导心肌肥大中的作用目前尚未报道。本工作首先在大鼠腹主动脉狭窄致高血压、心肌肥大模型上,探讨ER应激在心肌肥大发生、发展过程中的作用；其次采用ER应激诱导剂毒胡萝卜素(thapsigargin, TG,抑制ER钙泵继而排空ER内Ca2+)和衣霉素(tunicamycin, TM,抑制ER内蛋白质N-端糖基化)作用于原代培养乳鼠心肌细胞,观察TG和TM对心肌细胞肥大和ER应激的影响,并探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。主要实验方法和结果如下： 1 ER应激参与大鼠腹主动脉狭窄致高血压心肌肥大发生、发展过程本部分实验旨在观察ER应激反应在腹主动脉狭窄致高血压大鼠心肌肥大发生、发展过程中的作用。健康雄性Wistar大鼠85只,随机分为模型组(n=45)和假手术组(n=40),模型组行腹主动脉狭窄术,假手术组仅分离腹主动脉不行狭窄术,分别于术后1 d、3 d、7 d、14 d、28 d时观察各组血流动力学变化,测定全心重／体重比(whole heart weight/body weight, HW/BW)和左心室重／体重比(left ventricular weight/body weight, LVW/BW),双向电泳-质谱分析技术检测术后28 d心肌组织蛋白质表达谱的变化,RT-PCR技术检测左心室心肌组织ER应激分子GRP78、CRT和CHOP等mRNA表达变化,Western blot分析α-肌动蛋白(α-actin)、GRP78. CRT、CHOP,以及凋亡相关蛋白Bax和Bcl-2等表达变化。结果发现腹主动脉狭窄诱导大鼠心肌肥大,与假手术组比较,术后7d模型组大鼠血压升高,心功能代偿性增加,HW/BW和LVW/BW显著增加。狭窄术后28 d心肌肥大标志性蛋白肌球蛋白轻链表达上调,而心脏a-actin前体和α-肌球蛋白重链表达下调,Western blot证实模型组心肌组织a-actin表达较假手术组显著增加。其次发现模型组CRT mRNA表达于术后1d即发生显著上调,较假手术组增加136%(P0.01)；GRP78表达于术后7d显著增加,其高表达均持续至实验结束。长期ER应激触发CHOP凋亡途径,模型组大鼠心肌组织CHOP和促凋亡蛋白Bax表达均于术后14d显著增加,而抗凋亡蛋白Bcl-2表达降低。上述结果提示腹主动脉狭窄早期即可触发ER应激,诱导ER分子伴侣表达增加,ER应激反应可能参与了腹主动脉狭窄致大鼠高血压、心肌肥大过程。CHOP介导的ER应激相关凋亡途径可能参与了心肌肥大及失代偿的调节,决定肥大心肌失代偿的进程。2 ER应激诱导剂TG和TM诱导原代培养心肌细胞肥大和显著ER应激上述实验发现ER应激参与了腹主动脉狭窄致大鼠心肌肥大的发生、发展过程,为证实ER应激独立诱导心肌肥大,本部分实验在原代培养乳鼠心肌细胞模型上,采用不同浓度TG(1、2.5、5、10、50、70、100 nmol／L)处理原代培养心肌细胞48 h或50 nmol／LTG分别处理心肌细胞12、24、36、48、60、72 h；同时采用不同浓度TM(1、10、100 ng／m1)分别处理心肌细胞48、72、96 h；10-7mmol／L血管紧张素(angiotensin, Ang)Ⅱ处理心肌细胞48 h作为阳性对照。采用培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性和细胞凋亡率检测反映心肌细胞损伤变化,RT-PCR技术观察心肌细胞肥大标志性基因心房钠尿肽(atrial natriuretic peptide, ANP)和脑钠肽(brain natriuretic peptide, BNP) mRNA表达,3H-亮氨酸([3H]-Leucine)掺入技术检测心肌细胞蛋白质合成速率,F-actin染色技术观测心肌细胞骨架改变,同时分析心肌细胞表面积变化。此外采用RT-PCR技术观测ER应激分子CRT、GRP78、PERK、ATF4和CHOP mRNA表达,Western blot技术观测CRT、GRP78、CHOP、Bax和Bel-2蛋白水平改变,同时采用ER特异性荧光染料Dapoxyl观察ER形态变化,免疫荧光技术观察CRT荧光改变。结果发现,ER应激诱导剂TG和TM以时间和剂量依赖性方式诱导心肌细胞损伤,培养基LDH活性和细胞凋亡率显著增加；同时心肌细胞显著肥大,表现为TG和TM以时间和剂量依赖性方式诱导ANP和BNP mRNA表达、蛋白合成速率和细胞表面积增加；F-actin染色表明TG和TM诱导心肌细胞骨架荧光强度显著增强、应力纤维增加。而且发现50 nmol／L TG作用48 h和10 ng／ml TM作用72 h心肌细胞显著肥大,细胞损伤较轻,是诱导心肌细胞肥大的较适条件。此外,TG以剂量和时间依赖性方式诱导培养心肌细胞显著ER应激,ER应激分子CRT、GRP78表达显著增加；值得注意的是PERK和ATF4 mRNA表达于TG作用24-48 h显著增加,而在60-72 h时显著降低。严重ER应激触发细胞凋亡途径,TG以剂量和时间依赖性方式促进ER凋亡蛋白CHOP表达增加和Bcl-2/Bax比值显著降低。心肌细胞ER染色显示ER形态显著扩张,荧光颗粒分布不均、浓集并出现空泡。免疫荧光显示TG作用后CRT荧光强度增强且向核周浓集。10 ng／ml TM处理72 h心肌细胞也证实上述ER应激改变。上述结果提示ER应激诱导剂TG和TM诱导培养心肌细胞显著肥大和ER应激,而且CHOP凋亡途径激活参与了ER应激诱导的心肌细胞肥大。3 CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程 ER是细胞内重要的Ca2+处理器,细胞内Ca2+升高是触发心肌细胞肥大的基本信号,而CaN可直接受细胞内Ca2+调控,本部分实验旨在探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。不同浓度TG(1、2.5、5、10、50、70、100 nrnol／L)处理原代培养心肌细胞48 h或50 nmol／L TG分别处理心肌细胞12、24、36、48、60、72 h,同时采用10ng／ml TM处理心肌细胞72 h。其次为观察CaN活性抑制后心肌细胞肥大和ER应激变化,在50 nmol／L TG作用48 h诱导心肌细胞肥大模型上,采用CaN活性抑制剂环孢素A(CsA,5μmol/L)预处理心肌细胞10 min后继而培养基内加入50nmol/LTG作用48 h。采用Fluo-3AM染色检测心肌细胞内游离Ca2+水平,超微量Ca2+-ATP酶活性测试盒检测肌浆网／内质网钙-ATPase(sarco／ER Ca2+-ATPase,SERCA)活性,同时采用底物发色法检测心肌细胞CaN活性,采用细胞凋亡率和培养基LDH活性检测反映CaN活性抑制后细胞损伤情况,采用ANP和BNP mRNA表达、蛋白合成速率和细胞表面积检测评价CaN活性抑制后心肌细胞肥大变化。Western blot技术检测心肌细胞SERCA,受磷蛋白(phospholamban, PLB)、MEF2c、p-MEF2c以及CaN活性抑制后ER应激分子CRT、GRP78、CHOP、Bax和Bcl-2等蛋白表达,免疫荧光技术检测MEF2c荧光改变。结果发现ER应激诱导剂TG以剂量和时间依赖性方式诱导细胞内游离Ca2+水平升高,同时SERCA活性和表达降低、PLB表达增加；此外心肌细胞CaN活性和MEF2c/p-MEF2c蛋白表达显著增加。TM作用心肌细胞也证实ER应激诱导心肌细胞内游离Ca2+水平增加和SERCA活性降低,同时CaN活性升高。免疫荧光显示正常心肌细胞MEF2c主要分布在细胞浆,TG作用后MEF2c向核内转位。CsA显著抑制TG诱导的心肌细胞CaN活性升高,同时阻断TG诱导的心肌细胞肥大,表现为CsA显著阻断TG诱导的心肌细胞ANP、BNP mRNA表达、蛋白质合成速率和细胞表面积增加。免疫荧光发现TG诱导的心肌细胞MEF-2c表达和核转位均显著被CsA抑制。心肌细胞肥大抑制后细胞损伤显著增加,细胞凋亡率和培养基LDH活性升高。此外发现CaN活性抑制没有阻断TG诱导的CRT、GRP78和CHOP表达增加。上述结果提示SERCA活性降低和PLB表达增加可能参与了心肌细胞内游离Ca2+增加,Ca2+可能通过激活CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程。心肌细胞肥大可能是ER应激损伤的代偿性反应,CaN-MEF2c途径阻断显著抑制ER应激诱导的心肌细胞肥大,导致细胞损伤增加,至少部分是通过CHOP介导的ER应激凋亡途径实现的。通过上述分析,得出如下实验结论：ER应激不仅参与腹主动脉狭窄致高血压大鼠心肌肥大的发生、发展过程,而且独立诱导培养乳鼠心肌细胞肥大发生,CaN-MEF2c信号途径参与了ER应激诱导的心肌细胞肥大发生、发展过程,CaN活性抑制显著阻断ER应激诱导的心肌细胞肥大,同时细胞损伤显著增加。提示ER应激是心肌肥大的发病学因素之一,为心肌肥大、心力衰竭的临床治疗提供了新的靶点。
[Abstract]:Myocardial hypertrophy is a compensatory response to a variety of pathological factors, but chronic myocardial hypertrophy is involved in the occurrence and development of many kinds of cardiovascular diseases, such as dilated cardiomyopathy, heart failure, sudden death and so on, and seriously endangers human life. Therefore, it is important to clarify the pathogenesis of myocardial hypertrophy for the prevention and treatment of cardiac hypertrophy and heart failure. The clinical significance.
Endoplasmic reticulum (ER) is an important organelle that regulates intracellular calcium homeostasis and membrane / secretory protein synthesis and folds. It plays an important role in maintaining calcium and functional protein homeostasis in cardiac myocytes..ER is very sensitive to stress stimulation. Many factors such as calcium disorder, protein synthesis, ischemia, and hypoxia can be touched. ER stress. Appropriate ER stress induces the up regulation of ER molecular chaperones such as glucose-regulated proteins (GRPs), calreticulin (calreticulin, CRT) and so on, enhancing the ability of ER calcium regulation and processing unfolded proteins to protect the cells. However, the persistent or severe ER stress triggers the ER apoptotic signaling pathway. The important apoptotic protein C/EBP homologous protein (CHOP) expression increases and caspase-12 shear activation, leading to cell apoptosis and tissue damage. Protein kinase R like ER kinase (protein kinase R-like ER) is an important transmembrane protein. Vating transcription factor 4 (ATF4) transcripts and its downstream target molecule CHOP expression promotes cell apoptosis.
A variety of evidence suggests that ER stress is closely related to neurodegenerative changes, diabetic cardiomyopathy and ischemia / reperfusion injury. The expression of ER stress molecules CRT, GRP78, GRP94 is significantly up-regulated during myocardial hypertrophy, and CHOP mediated ER stress apoptosis pathway participates in apoptosis during cardiac failure and suggests myocardial hypertrophy. There is a ER stress reaction in the process, but the changes and roles of ER stress in cardiac hypertrophy are not clear. The increase of Ca2+ levels in cardiac myocytes is the basic signal to trigger cardiac hypertrophy. Calcineurin (CaN) is a kind of cytoplasmic Ca2+ and calmodulin (CALMODUL). In, CaM) regulated protein serine / threonine phosphatase, composed of catalytic subunit CnA and regulatory subunit CnB, plays a key role in many biological processes, such as T cell activation, cardiac hypertrophy, cell cycle regulation, and so on. Cardiomyocyte enhancement factor 2C (myocyte enhancer factor 2C, MEF2c) is the first found in the MEF2c family. A molecule that participates in cardiac development and is activated by CaN, but the role of CaN-MEF2c signaling pathway in ER stress induced cardiac hypertrophy has not been reported.
In this work, we first discussed the role of ER stress in the development and development of cardiac hypertrophy in hypertensive rat abdominal aortic stenosis and myocardial hypertrophy, followed by ER stress inducer (thapsigargin, TG, inhibition of ER calcium pump and emptying ER Ca2+) and ycomycin (tunicamycin, TM, N- terminal sugar in ER). The effects of TG and TM on cardiomyocyte hypertrophy and ER stress were observed and the effect of CaN-MEF2c signal pathway on the hypertrophy of cardiomyocytes induced by ER stress was investigated. The main experimental methods and results were as follows:
1 ER stress participates in the occurrence and development of hypertensive cardiac hypertrophy induced by abdominal aortic stenosis in rats.
This part of the experiment was designed to observe the role of ER stress reaction in the development of hypertrophy of myocardium in hypertensive rats induced by abdominal aorta stenosis. 85 healthy male Wistar rats were randomly divided into model group (n=45) and sham operation group (n=40), the model group underwent abdominal aorta stenosis, and the sham operation group was only separated from abdominal aorta stenosis. After 1 D, 3 D, 7 d, 14 d, and 28 d, the hemodynamic changes were observed in each group. The changes of the total cardiac weight / weight ratio (whole heart weight/body weight, HW/BW) and the left ventricular weight / weight ratio (left ventricular) were measured, and the changes in the protein expression profiles of 28 cardiac muscle tissues were detected by two dimensional electrophoresis mass spectrometry. The changes in the expression of ER stress molecules, such as GRP78, CRT and CHOP, were detected in left ventricular myocardium, and Western blot was used to analyze alpha actin (alpha -actin), GRP78. CRT, CHOP, and the expression of apoptosis related proteins.
The results showed that the abdominal aorta stenosis induced the hypertrophy of the rat myocardium. Compared with the sham operation group, the blood pressure of the 7d model group was increased, the cardiac function was increased and the HW/BW and LVW/BW increased significantly. The expression of the myosin light chain expression of the 28 d cardiac hypertrophy was up, and the expression of the a-actin precursor and the alpha myosin heavy chain expression was down, W Estern blot confirmed that the expression of a-actin in the myocardial tissue of the model group was significantly higher than that of the sham operation group. Secondly, it was found that the expression of CRT mRNA in the model group was significantly up-regulated after the operation and increased by 136% (P0.01) than that of the sham operation group. The expression of GRP78 in the model group increased significantly after the operation, and the high expression of the GRP78 was increased to the end of the experiment. Long term ER stress triggered CHOP apoptotic pathway and model. The expression of CHOP and apoptotic protein Bax in myocardial tissue of group rats increased significantly after operation, but the expression of anti apoptotic protein Bcl-2 decreased. The results suggested that ER stress could be triggered at the early stage of abdominal aorta stenosis and the expression of ER molecular chaperone increased, and ER stress reaction may be involved in hypertension in rats caused by abdominal aorta stenosis and.CH of cardiac hypertrophy. OP mediated ER stress related apoptosis pathway may be involved in the regulation of myocardial hypertrophy and decompensation, determining the process of hypertrophic cardiomyocyte decompensation,.2 ER stress inducer TG and TM induced primary cultured cardiomyocytes hypertrophy and significant ER stress
These experiments showed that ER stress was involved in the occurrence of myocardial hypertrophy in rats with abdominal aorta stenosis. The development process was that ER stress was independent to induce myocardial hypertrophy independently. In this part of this experiment, the primary cultured rat cardiomyocyte model was treated with different concentrations of TG (1,2.5,5,10,50,70100 nmol / L) to treat the primary cultured cardiomyocytes 48 h or 50 nmol / LTG. 12,24,36,48,60,72 h of cardiac myocytes was treated with TM (1,10100 ng / M1) at the same time, 48,72,96 h in cardiac myocytes was treated respectively. 10-7mmol / L angiotensin (angiotensin, Ang) II was used to treat cardiac myocytes as positive control. The activity of culture based lactate dehydrogenase and the rate of apoptosis were used. The detection of myocardial cell damage changes, RT-PCR technique was used to observe the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (brain natriuretic peptide, BNP) mRNA, and 3H- leucine ([3H]-Leucine) technique was used to detect the protein synthesis rate of cardiac myocytes. RT-PCR technique was used to observe the expression of ER stress molecules CRT, GRP78, PERK, ATF4 and CHOP mRNA. Western blot technique was used to observe CRT, GRP78, protein and protein levels. Detection of CRT fluorescence changes.
The results showed that ER stress inducer TG and TM induced cardiomyocyte injury in time and dose dependent manner, the activity of LDH and the rate of apoptosis increased significantly, and the cardiac myocytes were significantly hypertrophic, which showed that TG and TM induced ANP and BNP mRNA form with time and dose dependent manner, and the protein synthesis rate and cell surface area increased; F increased. -actin staining showed that the fluorescence intensity of the cytoskeleton induced by TG and TM was significantly enhanced and the stress fibers were increased. Moreover, 50 nmol / L TG action was found in 48 h and 10 ng / ml TM, and the 72 h myocardial cells were significantly hypertrophy, and the cell damage was lighter. Furthermore, TG induced the culture heart in a dose and time dependent manner. The expression of CRT and GRP78 in the muscle cells was significantly increased by ER stress, and the expression of ER stress molecules CRT and GRP78 was significantly increased. It was worth noting that the expression of PERK and ATF4 mRNA was significantly increased in the action of TG, but decreased significantly at the time of 60-72 H. The ER staining showed that the ER morphology was significantly expanded, the distribution of fluorescent particles was uneven, and the vacuoles appeared. The fluorescence intensity of CRT was enhanced after the immunofluorescence of TG and the concentration of.10 ng / ml TM to 72 h cardiomyocytes was also confirmed. The above-mentioned results suggested that ER stress inducer TG and induce the culture of myocardium Significant cell hypertrophy and ER stress, and the activation of CHOP apoptosis pathway involved in ER induced cardiomyocyte hypertrophy.3 CaN-MEF2c signaling pathway involved in ER induced hypertrophy of cardiomyocytes.
ER is an important Ca2+ processor in cells. The increase of Ca2+ in cells is the basic signal to trigger cardiac myocyte hypertrophy, and CaN can be directly regulated by intracellular Ca2+. This part of the experiment aims to explore the role of CaN-MEF2c signal pathway in the process of cardiac hypertrophy induced by ER stress. The different concentration TG (1,2.5,5,10,50,70100 nrnol / L) deals with the original generation. Cardiac myocytes were treated with 48 h or 50 nmol / L TG respectively, and 12,24,36,48,60,72 h was treated respectively with 10NG / ml TM treatment of cardiac myocytes 72 h. followed by the observation of cardiomyocyte hypertrophy and ER stress changes after the inhibition of CaN activity. 5 mol/L) the pretreated cardiomyocytes were followed by 10 min and cultured in the culture base to add 50nmol/LTG to 48 h.. Fluo-3AM staining was used to detect the level of free Ca2+ in cardiac myocytes. Ultra micro Ca2+-ATP enzyme activity test box was used to detect the activity of sarcoplasmic reticulum / endoplasmic reticulum calcium -ATPase (sarco / ER Ca2+-ATPase, SERCA) activity, and the substrate chromophore was used to detect cardiac myocytes Activity, cell apoptosis rate and culture medium LDH activity were used to detect cell damage after inhibition of CaN activity, ANP and BNP mRNA were used, protein synthesis rate and cell surface area were detected to evaluate the hypertrophy of cardiomyocytes after CaN activity inhibition,.Western blot technique was used to detect cardiac myocyte SERCA, phosphoprotein (phospholamban, PLB), MEF2c, MEF2c and CaN activity inhibited ER stress molecules CRT, GRP78, CHOP, Bax and Bcl-2 protein expression, and immunofluorescence technique was used to detect MEF2c fluorescence changes.
The results showed that ER stress inducer TG induced the increase of intracellular free Ca2+ level in dose and time dependent manner, while SERCA activity and expression decreased, and PLB expression increased. In addition, the CaN activity and the expression of MEF2c/p-MEF2c protein in cardiac myocytes significantly increased.TM action myocardial cells also confirmed the increase of free Ca2+ level in cardiac myocytes induced by ER stress. The activity of addition and SERCA decreased and the activity of CaN increased. The immunofluorescence showed that MEF2c in normal cardiac myocytes was mainly distributed in the cytoplasm. After the action of TG, the transposition of MEF2c to the nucleus.CsA significantly inhibited the increase of CaN activity induced by TG, while blocking TG induced cardiomyocyte hypertrophy. The expression of CsA significantly blocked TG induced cardiomyocyte ANP. Expression of protein synthesis rate and cell surface area increased. Immunofluorescence showed that the expression of MEF-2c and nuclear transposition of TG induced cardiomyocytes were significantly inhibited by CsA. The cell damage was significantly increased after the inhibition of myocardial hypertrophy, and the apoptosis rate and the activity of LDH were increased. Furthermore, the inhibition of CaN activity did not block CRT, GRP78 and CH induced by TG. The above results suggest that the decrease of SERCA activity and the increase of PLB expression may participate in the increase of intracellular free Ca2+ in cardiac myocytes. Ca2+ may participate in ER stress induced cardiomyocyte hypertrophy by activating CaN-MEF2c signal pathway. Cardiomyocyte hypertrophy may be a compensatory response to ER stress injury, and CaN-MEF2c pathway blocking is significantly inhibited. ER stress induced cardiomyocyte hypertrophy, resulting in increased cellular damage, at least partly through CHOP mediated ER stress pathway.
Based on the above analysis, the following conclusions are drawn: ER stress is not only involved in the occurrence and development of cardiac hypertrophy in hypertensive rats induced by abdominal aortic stenosis.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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