C57BL/6J小鼠超数排卵的研究

目的　确定C57BL 6J小鼠超排的最佳激素剂量和最合适的注射间隔时间 ,提高超排率。方法　 40只C57BL 6J雌鼠随机分为四组 ,分别用 5IU或 10IU的PMSG和HCG ,间隔 48h或 72h注射 ,比较排出卵母细胞的数量。结果　 5IU +5IU剂量的PMSG和HCG、间隔 48h注射组超排效果最好 ;8～ 10周龄雌鼠较 6～ 8周龄雌鼠超排效果好。结论　C57BL 6J小鼠超排的最佳激素剂量为 5IUPMSG +5IUHCG ,最合适的注射间隔时间为 48h ,处于繁殖期的雌鼠超排效果好。     

电磁脉冲对C57BL/6J 胸腺的作用

目的:观察电磁脉冲Electromagnetic Pulse(EMP)对C57BL/6J小鼠胸腺的影响。方法:50只C57BL/6J小鼠按体重区组随机化分为对照组和辐照组,每组25只。EMP每天照射400次,连续照射7天,照后1d(天)、3d、7d、14d、28d共5个时间点杀取胸腺。2只辐照组和2只对照组杀取的胸腺做HE染色,观察其病理改变;3只辐照与3只对照组的小鼠,杀后称取小鼠的体重和胸腺的重量,计算胸腺指数;然后提取T淋巴细胞进行计数;同时取小鼠外周血检测其中的IL-4的水平。结果:照后1d胸腺的切片没有明显改变。7天后,胸腺开始有出血,结构不清;胸腺指数呈现递减的趋势,但辐照组与对照组没有的差异没有统计学意义;T淋巴细胞数的变化也呈现先减后增的趋势,在第1d、14d、28d辐照组与对照组的差异没有统计学意义,在第3d和7d辐照组的细胞数小于对照组的细胞数(P<0.05);辐照组与对照组的外周血IL-4水平的差异也没有统计学意义。结论:电磁脉冲对雄性Balb/c小鼠胸腺结构造成一定的损伤,但胸腺指数改变不显著,T淋巴细胞数量增加。表明EMP对胸腺有一定的作用,但是胸腺不是EMP作用的敏感器官。     

Determination of RBC Survival in C57BL/6 and C57BL/6-Tg(UBC–GFP) Mice

Although several methods for determining erythrocyte lifespan are used in research studies that involve mice, all involve the alteration of RBC to allow for its tracking over time, which may affect overall RBC survival. The aims of this study were to determine 1) whether sex affects RBC survival; 2) whether RBC survival differs between the biotin method and an alternative method that uses GFP; and 3) whether repeat exposure of mice to biotin results in an antibiotin antibody response or decreased RBC survival. The results suggest no difference in the RBC half-life between male and female C57BL/6 mice (22.9 ± 1.2 and 22.4 ± 0.9 d, respectively). In addition, RBC half-life did not differ between the biotin- and GFP-based methods (20.5 ± 2.1 d and 22.7 ± 2.1 d, respectively). Finally, retransfusion of mice 90 d after an initial transfusion with biotin-labeled RBC did not induce detectable antibiotin antibodies nor alter the half-life of transfused biotin-labeled RBC (initial transfusion, 22.0 ± 1.2 d; subsequent transfusion, 23.4 ± 1.4 d, respectively).Abbreviations: T_1/2, half-lifeRBC lifespan and senescence are important parameters used both clinically and in research studies of hereditary disorders of erythrocyte metabolism, transfusion medicine, and sepsis.^{8,21,27,32,35} Labeling RBC with a biotinylating reagent is a common method used to determine their circulating lifespan. Other methods involve using radioactive isotopes, such as ⁵¹Cr and ⁵⁹Fe.^7,20 Biotinylating reagents are preferred for various research applications with humans,^8,23,24 and are used in a variety of animal models.^{1,25,33,34,37} Once biotin attaches to RBC surface proteins, streptavidin (a protein derived from Streptomyces avidinii) that is labeled with a fluorescent dye is used to form a strong and rapid complex with biotin, thereby allowing for its detection through flow cytometry. Blood samples analyzed sequentially over a period of weeks will show a linear decline in biotin–streptavidin signal as labeled cells age and are cleared from the circulation through the reticuloendothelial system.The characteristics of an ideal label for performing RBC survival studies include: 1) stable presence on or within the cell throughout its normal lifespan; 2) specificity for RBC; 3) inertness, such that the cell does not become prone to accelerated destruction; 4) nonrecycling (that is, the label does not reenter the circulation and bind to new cells after destruction of the labeled RBC); and 5) easy and accurate measurement by using available assays. Radioactive isotopes and other labels fulfill several of these criteria, but their limitations include elution from RBC as well as safety concerns.^7,22 In contrast, biotin poses little to no risk of accumulation or toxicity. The sulfo- N-hydroxysuccinimide–biotin ester used for RBC tracking studies in humans and animals can be administered directly or through the transfusion of biotinylated RBC. Although it is generally accepted that biotinylation of RBC does not affect their function, antibodies to biotin have been demonstrated in some human studies, posing the question of whether repeated administration of biotin ester or biotinylated RBC could interfere with subsequent results within the same subject.^4,20 Repeat transfusions of biotinylated RBC to mice have not been described in the literature. One aim of this study was to determine whether exposure to biotinylated RBC induces an antibiotin antibody response in mice. Furthermore, we tested whether the survival of biotinylated RBC changed after repeat exposure.Recently GFP-expressing RBC have been used to track the posttransfusion survival and recovery of stored RBC administered to nonGFP-expressing recipient mice.^9,12,36 The C57BL/6-Tg(UBC–GFP)30Scha/J mouse strain is characterized by GFP expression under the control of a human ubiquitin C promoter. All tissues of these mice express GFP, including blood.²⁶ GFP expression appears to be consistent throughout life and does not otherwise alter the normal structure, physiology, or function of RBC. In addition, GFP is unaffected by ambient light contamination or degradation, drawbacks that are associated with fluorescent dyes.¹⁵ In addition, GFP allows for the separation of cell populations through flow cytometry.^9,11 Many qualities of GFP suggest that it may serve as a useful surrogate marker in place of other labeling techniques in mice. Therefore, we sought to evaluate the utility of UBC–GFP transgenic mice as an alternative to labeling RBC with biotin esters. Our aim for this work was to determine the survival of RBC in wild-type C57BL/6 mice and in the UBC–GFP strain and to compare methods for determining RBC half-life (T_1/2).     

C57BL/6 J小鼠黑色素瘤肺转移模型的构建

目的探讨建立C57BL/6 J小鼠黑色素瘤肺转移模型的影响因素,包括肿瘤的接种方式、细胞接种数量和成瘤周期。方法体外培养小鼠黑色素瘤细胞B16F10。1)取6~8周龄,雄性小鼠18只,随机分三组,每组6只,分别采取尾静脉注射、腹腔注射和皮下注射方式,每只小鼠注射100μL(3×10~6个细胞)B16F10细胞悬液,2周后,解剖小鼠并观察黑色素瘤的生长和转移情况;2)分3组,同上,经尾静脉分别注射3×10~6个细胞、1×10~6个细胞、3×10~5个细胞,2周后,解剖小鼠并观察黑色素瘤的生长和转移情况;3)分3组,同上,尾静脉注射1×10~6个细胞,分别于1周、2周、3周解剖小鼠,观察黑色素瘤的生长和转移情况。结果 1)尾静脉注射小鼠黑色素瘤细胞,小鼠发生肺转移的成功率为100%,而腹腔注射和皮下注射未发生肺转移。2)接种小鼠黑色素瘤细胞数量为1×10~6时,发生肺部转移的黑色素瘤细胞数量适中;接种细胞数量为3×10~6时,发生肺部转移的黑色素瘤细胞数量过多;接种细胞数量为3×10~5时,发生肺部转移的黑色素瘤细胞数量较少。3)尾静脉注射1×10~6个小鼠黑色素瘤细胞,饲养2周后,可以观察到黑色素瘤细胞明显的肺部转移,且不会导致小鼠死亡;饲养3周,黑色素瘤细胞肺部转移数量过多,且小鼠死亡过半;饲养1周,黑色素瘤细胞肺部转移数量较少。结论经尾静脉注射1×10~6个小鼠黑色素瘤细胞,生长2周时间,为构建C57BL/6 J小鼠黑色素瘤肺转移模型的推荐方法。     

不同方法建立C57BL/6J小鼠抑郁症模型的探讨

目的用不同方法建立C57BL/6J小鼠抑郁症模型,为探讨GalR蛋白对小鼠抑郁症的治疗作用打下基础。方法 C57BL/6J小鼠经体重、敞箱实验及反抗抓获实验初筛后,随机分为4组:Ⅰ.CUMS组,Ⅱ.CUMS+CORT组,Ⅲ.CORT组,Ⅳ.正常对照组。每天记录小鼠体重及摄食量。28d后进行液体消耗及强迫游泳实验测试。结果小鼠抑郁模型在第28d建立成功。Ⅱ组小鼠短期内体重迅速下降并死亡。与Ⅰ组小鼠相比,Ⅲ组小鼠液体消耗和强迫游泳实验指标改变更明显。结论成功建立C57BL/6J小鼠抑郁症模型。CUMS和CORT模型结合,小鼠不能耐受,短期内死亡。单独CORT模型造模效果要优于CUMS模型。在后续试验中,将用CORT法建立C57BL/6J小鼠抑郁症模型。     

Dual Opposing Roles of Metallothionein Overexpression in C57BL/6J Mouse Pancreatic β-Cells

Background

Growing evidence indicates that oxidative stress (OS), a persistent state of excess amounts of reactive oxygen species (ROS) along with reactive nitrogen species (RNS), plays an important role in insulin resistance, diabetic complications, and dysfunction of pancreatic β-cells. Pancreatic β-cells contain exceptionally low levels of antioxidant enzymes, rendering them susceptible to ROS-induced damage. Induction of antioxidants has been proposed to be a way for protecting β-cells against oxidative stress. Compared to other antioxidants that act against particular β-cell damages, metallothionein (MT) is the most effective in protecting β-cells from several oxidative stressors including nitric oxide, peroxynitrite, hydrogen peroxide, superoxide and streptozotocin (STZ). We hypothesized that MT overexpression in pancreatic β-cells would preserve β-cell function in C57BL/6J mice, an animal model susceptible to high fat diet-induced obesity and type 2 diabetes.

Research Design and Methods

The pancreatic β-cell specific MT overexpression was transferred to C57BL/6J background by backcrossing. We studied transgenic MT (MT-tg) mice and wild-type (WT) littermates at 8 weeks and 18 weeks of age. Several tests were performed to evaluate the function of islets, including STZ in vivo treatment, intraperitoneal glucose tolerance tests (IPGTT) and plasma insulin levels during IPGTT, pancreatic and islet insulin content measurement, insulin secretion, and islet morphology assessment. Gene expression in islets was performed by quantitative real-time PCR and PCR array analysis. Protein levels in pancreatic sections were evaluated by using immunohistochemistry.

Results

The transgenic MT protein was highly expressed in pancreatic islets. MT-tg overexpression significantly protected mice from acute STZ-induced ROS at 8 weeks of age; unexpectedly, however, MT-tg impaired glucose stimulated insulin secretion (GSIS) and promoted the development of diabetes. Pancreatic β-cell function was significantly impaired, and islet morphology was also abnormal in MT-tg mice, and more severe damage was detected in males. The unique gene expression pattern and abnormal protein levels were observed in MT-tg islets.

Conclusions

MT overexpression protected β-cells from acute STZ-induced ROS damages at young age, whereas it impaired GSIS and promoted the development of diabetes in adult C57BL/6J mice, and more severe damage was found in males.     

Quantitative trait loci for growth traits in C57BL/6J × DBA/2J mice

Quantitative trait loci (QTLs) for body weight and tail length are mapped in an F₂ population of 927 C57BL/6J × DBA/2J mice. We test the concordance between the locations of the mapped QTLs with those detected by changes of marker frequency under artificial selection in a previous experiment with the same base population. The directions of effects of the QTLs are generally in agreement, and in many cases significant QTLs are found in similar map positions, but there are also discrepancies between the two experiments. There are indications of age-specific QTL effects on growth. For body weight traits, the genetic variation in the F₂ appears to result from many loci with relatively small effects. For tail length at 10 weeks, however, a single QTL on Chromosome (Chr) 1 with a peak LOD score of ∼33 contributes most of the genetic variation detected, changes the trait value by about 6%, and explains about 20% of the phenotypic variance of the trait. Received: 4 August 1998 / Accepted: 17 November 1998     

Genetics of ethanol-induced locomotor activation: detection of QTLs in a C57BL/6J × DBA/2J F2 intercross

Moderate doses of ethanol (1–2 g/kg) markedly increase locomotor activity in some inbred mouse strains, for example, the DBA/2J (D2), but have relatively little effect in other strains, for example, the C57BL/6J (B6). In the present study, we conducted a genome-wide search in a B6D2 F₂ intercross (N = 925) for quantitative trait loci (QTLs) associated with the locomotor response. A QTL with a LOD score of 8.4 was detected on Chromosome (Chr) 2; this QTL accounted for 11.4% of the phenotypic variance and approximately 30% of the genetic variance. The QTL on Chr 2 is in the same general region as QTLs previously described for ethanol preference/consumption (Rodriguez et al. Alcohol Clin Exp Res 19, 367, 1995; Melo et al. Nat Genet 13, 147, 1996; Phillips et al. Mamm Genome, in press), acute ethanol withdrawal (Buck et al. J. Neurosci 17, 3946, 1997) and nitrous oxide withdrawal severity (Belknap et al. Behav Genet 23, 213, 1993). A logical candidate gene in the region of interest is the enzyme which synthesizes GABA, glutamic acid decarboxylase 1 (GadI). Received: 15 September 1998 / Accepted: 8 October 1998     

Why does a sucrose choice reduce the consumption of alcohol in C57BL/6J mice?

To determine why animals reject alcohol when offered palatable solutions of sucrose, male C57BL/6J mice were challenged first with 5% sucrose then with 10% sucrose, while given continuous free-access to alcohol and water. The 5% sucrose dramatically reduced the intake of alcohol and increased the intake of total fluid by an average of 7.3 ml/day. The suppression of alcohol intake could not be attributed to a volumetric ceiling since access to 10% sucrose produced a further large increase in total intake (8.8 ml/day). The results support the interpretation that animals consume alcohol for characteristics it shares with sucrose.     

高脂饮食诱导C57BL/6J肥胖小鼠骨关节炎模型的建立

目的通过高脂饮食诱导C57BL/6J小鼠肥胖来建立骨关节炎模型并观察其病理特征,为肥胖相关骨关节炎研究中动物模型的建立提供实验依据。方法 C57BL/6J雄鼠20只随机分为对照组和高脂组,分别喂饲基础饲料和高脂饲料。每周测体重及摄食量,16周后收集血清,ELISA法检测血清Ⅱ型胶原C端肽水平;取小鼠左下肢膝关节,固定、脱钙、石蜡包埋,切片后行HE及番红O染色并进行修改后的Mankin评分;TUNEL法检测软骨细胞凋亡。结果与对照组相比,高脂组小鼠体重、血清Ⅱ型胶原C端肽水平显著升高,而摄食量方面则无明显差异。HE及番红O染色结果显示,高脂组小鼠的膝关节软骨细胞减少,排列混乱,基质染色不均匀,可出现潮线部分缺失,颜色变浅等;且高脂组的修改后的Mankin评分也显著高于对照组。TUNEL检测结果显示,高脂组小鼠关节软骨凋亡细胞数增多,但与对照组相比差异不显著。结论利用高脂饮食诱导C57BL/6J小鼠肥胖的方法可以建立骨关节炎模型,该模型可用于肥胖相关骨关节炎的研究。     

C57BL/6J慢性应激小鼠血中胆固醇和甘油三酯的变化

目的观察慢性应激对C57BL/6J小鼠行为和血液中血脂的影响。方法将20~25 g雄性C57BL/6J小鼠分成应激组及相应的对照组。应激组小鼠独笼饲养,将6个日相和6个夜相刺激及1个全天刺激随机安排到1周内,每周刺激顺序随机重新组合,连续8周。对照组动物则每5~6只小鼠合笼饲养,自由给水,整个实验过程中不接受任何刺激。2组动物均于刺激后的1、2、4和8周经内眦取血,用于血脂的检测。结果行为学实验结果显示:除第8周外,刺激后各时间点模型组小鼠体重均明显低于对照组(P<0.05)。空腹胆固醇及甘油三酯检查结果显示:与对照组相比较,模型组小鼠自刺激后的第2、4、8周显著增加(P<0.01),相反甘油三酯则在个时间点均显著降低(P<0.05)。结论复合式慢性应激是一种建立应激动物模型的有效方式;应激形成过程中动物体内胆固醇的增加提示长期的应激很可能对心血管系统造成一定危害。     

从C57BL/6J小鼠胚胎中分离ES细胞系(英文)

ＥＳ细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍＣｅｌｓ）是来源于小鼠早期胚胎的细胞系,它可以在体外大量培养而不失去其发育的多潜能性。ＥＳ细胞不仅可以用来制作转基因动物,而且能够作为载体进行基因打靶等多种研究。目前,国际上常用的胚胎干细胞系都是来源于１２９小鼠的胚胎。因而,有必要探讨用其它品系小鼠建立ＥＳ系。１９９１年,Ｌｅｄｅｒｍａｎｎ等人首次报道从Ｃ５７ＢＬ／６Ｊ小鼠胚胎中建成了ＥＳ细胞系,但是没有对建立的细胞系进行特性分析。国内柴桂萱等人虽然做了特性分析,但是他们所建细胞系的细胞直径较大,生长速度较慢,不同于常见的ＥＳ细胞系。我们从Ｃ５７ＢＬ／６Ｊ品系小鼠胚胎中共分离了四个ＥＳ细胞系,分别命名为ＣＥ１、ＣＥ２、ＣＥ３、ＣＥ４（Ｆｉｇ．１ａ＆ｂ）。这四个细胞系核型正常率均达到７０％以上。我们检查了ＣＥ２细胞的分化能力,当将ＣＥ２细胞注入同基因型小鼠的皮下后,获得的畸胎瘤（Ｆｉｇ．３）组织切片检查的结果表明：该细胞能够分化成多种组织（Ｆｉｇ．２）。我们也研究了ＥＳ细胞的嵌合能力,用ＩＣＲ小鼠胚胎作为受体胚胎,采用囊胚显微注射法构建嵌合鼠。在幸存的幼鼠中我们获得了来源于ＣＥ２细胞的嵌合鼠（Ｔａｂｌｅ１,Ｆｉｇ．４）。综     

多次注射乌拉坦诱导的BALB/C及C57BL/6J小鼠肺癌模型的比较

目的探讨一种稳定的多次乌拉坦注射诱导小鼠肺癌模型的构建方法,比较BALB/C及C57BL/6J小鼠对该肺癌模型的敏感性。方法 10只BALB/C小鼠及10只C57BL/6J小鼠适应性饲养3周,随后每只小鼠分别被给予每周1次腹腔注射1 g/kg体重乌拉坦,连续注射10周,继续喂养15周后处死小鼠取肺组织。由3名不同的检测者在解剖显微镜下观察计数肺组织表面肿瘤数目并以直径记录肿瘤大小;HE染色检测肺组织病理变化。结果多次乌拉坦注射诱导的BALB/C及C57BL/6J小鼠肺癌发生率均为10/10(100%);BALB/C小鼠荷瘤数明显多于C57BL/6J小鼠(P0.01),同时,直径也大于C57BL/6J小鼠(P0.05);HE染色显示多次乌拉坦注射诱导的肺癌有非典型性腺瘤增生及腺瘤两种病变类型。结论 BALB/C和C57BL/6J小鼠均可以作为多次注射乌拉坦诱导性肺癌模型的动物,BALB/C小鼠对该肺癌模型的敏感性高于C57BL/6J小鼠。     

成对框基因2蛋白在C57BL/6J小鼠脑发育进程中的表达

目的 研究正常C57BL/6J小鼠脑发育进程中的组织形态学变化以及成对框基因2(Pax2)蛋白在胚胎期和出生后脑组织中的表达规律。方法 取C57BL/6J小鼠胚胎期11.5 d、13.5 d、15.5 d的胎鼠头部和胚胎期17.5 d、出生后0 d、7 d、14 d和30 d小鼠的全脑组织进行石蜡包埋,连续矢状位切片后进行HE染色和免疫组织化学染色。结果 胚胎期11.5d可见小鼠完整的5个脑泡结构,15.5 d大脑皮质出现5层结构,小脑皮质外颗粒层明显可见。出生后各脑区按照时空顺序继续发育,到P30大脑皮质和小脑皮质均发育成熟出现典型的结构特征。Pax2蛋白在脑组织中的表达主要定位于细胞核和神经纤维上,在胚胎期的表达主要集中在脑桥延髓部位且呈弥漫性表达,E13.5达到高峰,出生后在脑桥延髓的表达逐渐减弱,但在小脑中的表达较胚胎期增强;E13.5时在小脑内开始少量表达,P7时达到高峰,P14后表达量逐渐降低;Pax2蛋白在外颗粒层中于E17.5时开始表达,出生后主要在内颗粒层和髓质中强烈表达,到P30时表达减弱,仅分布在内颗粒层中。结论 小鼠脑发育在一定的时空顺序下,经历了一个复杂的过程...     

GM-CSF体外诱导培养C57BL/6J小鼠BMDCs的动态观察及鉴定

本研究旨在通过培养并观察C57BL/6J小鼠骨髓来源树突状细胞(BMDCs)集落、形态、表型的动态变化,为BMDCs的形态、表型等方面提供参考数据。取C57BL/6J小鼠骨髓,用粒细胞-巨噬细胞集落刺激因子(GM-CSF)诱导和培养BMDCs,在第3、6、9、12天拍照、HE染色观察BMDCs的集落、形态;FACS分析第3、6、9、12天以及LPS刺激后BMDCs的CD11c、CD80和CD86的表达;扫面电镜拍照LPS刺激后BMDCs的树突与形态。发现GM-CSF诱导和培养的C57BL/6J小鼠BMDCs在第3天形成明显的集落、第6天集落释放较多的BMDCs;显微镜下HE染色显示,第3天可见BMDCs有突起、第6天明显,第12天有明显突起的细胞数量增多;FACS分析显示,随着培养时间的增加,BMDCs的CD11c、CD80、CD86的表达逐渐上调,第12天表达较明显,LPS刺激能提高CD80、CD86的表达;扫描电镜显示,LPS刺激的BMDCs有明显的树突状突起。因此GM-CSF诱导和培养的C57BL/6J小鼠BMDC具有典型的树突状突起、表达CD11c、CD80、CD86等树突状细胞(DCs)分子标记,LPS刺激能促进CD11c、CD80、CD86的表达。     

三个近交系C57BL/6J小鼠群体微卫星遗传变异分析(英文）

应用微卫星遗传标记对近交系C57BL/6J(B6)小鼠遗传稳定性进行分析。用FAM标记的引物PCR扩增了来自北京和上海三个实验动物生产单位提供的三个B6小鼠群体共15个微卫星位点并进行分型。结果显示,所有位点均处于纯合状态,其中7个位点为多态位点。研究表明各B6群体虽然为高度近交群体,但不同生产单位维持的B6群体之间存在遗传分化。     

高效建立129/ter、C57BL/6J小鼠胚胎干细胞系的方法学探讨

小鼠胚胎干细胞 (ＥＳ细胞 )是从小鼠囊胚内细胞团(ＩＣＭ)分离出来的、在体外培养过程中可维持未分化状态、正常二倍体核型及无限增殖能力 ,具有多能性或全能性的细胞系[1～ 3 ] 。ＥＳ细胞广泛应用于克隆动物制作、转基因动物生产、动物医学模型建立、真核细胞基因表达与调控的研究、细胞分化机制的探索、人及哺乳动物基因功能的研究以及细胞、组织、和器官的修复与移植研究。胚胎干细胞的研究和应用已成为生命科学研究的热点和前沿领域之一[4～ 8] 。自第一株 12 9小鼠ＥＳ细胞系建立以来 ,人们在生物学和医学等多个领域进行了广泛深入的…     

C57BL/6J-HBV转基因小鼠的繁育与检测

目的　摸清C57BL 6J -HBV转基因小鼠的繁育规律 ,建立可靠的HBsAg表达检测方法。方法　对C57BL 6J-HBV转基因小鼠两种不同交配方式的繁殖性能和HBsAg的表达情况进行比较研究 ;应用免疫组化的方法证实血清学诊断HBsAg的准确性并用激光扫描共聚焦显微镜确定HBsAg在肝细胞中的表达部位。结果与结论　从繁殖性能来看 ,两种交配方式窝产仔数差异不显著 (P >0 .0 5 ) ,而离乳数差异具有显著性 ( 0 .0 1

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