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1.
目的 观察应用RNA干扰技术沉默组蛋白去乙酰化酶1(HDAC1)对人宫颈癌SiHa细胞迁移和侵袭的影响,并探讨其可能机制。方法 体外合成针对HDAC1的小干扰RNA(siRNA),采用脂质体法转染人宫颈癌SiHa细胞。采用Western blot法检测细胞HDAC1蛋白和乙酰化组蛋白H4的表达,荧光定量PCR方法检测HDAC1、核因子-κB (NF-κB)和尿激酶型纤溶酶原激活剂(uPA)基因mRNA表达,划痕实验检测细胞迁移能力,Transwell侵袭实验检测细胞侵袭能力。结果 经转染HDAC1基因siRNA后,宫颈癌SiHa细胞HDAC1 mRNA和蛋白表达降低,NF-κB和uPA基因mRNA表达也降低,细胞内组蛋白H4乙酰化水平增加。SiHa细胞迁移速度降低,穿过Transwell滤膜的细胞数量减少。结论 HDAC1基因siRNA能够通过抑制宫颈癌SiHa细胞HDAC1基因表达,抑制人宫颈癌SiHa细胞迁移和侵袭能力。提高组蛋白乙酰化水平,下调NF-κB和uPA基因表达可能是其作用机制。  相似文献   

2.
目的探讨组蛋白去乙酰化酶1(HDAC1)对肝癌细胞增殖凋亡的影响。方法肝癌细胞SNU-449转染HDAC1小干扰RNA(HDAC1 siRNA)和小干扰RNA阴性对照(siRNA control),同时设置不转染的细胞为对照。采用RT-PCR检测细胞中HDAC1mRNA的表达水平,Western blot检测细胞中HDAC1、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、信号转导与转录因子3(STAT3)、磷酸化的STAT3(p-STAT3)蛋白表达水平,噻唑蓝(MTT)检测细胞增殖情况,流式细胞术检测细胞凋亡情况。结果肝癌细胞转染siRNA control后,细胞存活率、凋亡率及细胞中HDAC1、Cleaved Caspase-3、STAT3、p-STAT3表达水平与对照细胞相比无显著差异(P0.05)。肝癌细胞转染HDAC1 siRNA后,细胞中HDAC1mRNA和蛋白表达水平与对照细胞相比均受到抑制,并且细胞存活率只有(63.74±8.37)%,而细胞凋亡率升高为(32.84±6.92)%,细胞中Cleaved Caspase-3蛋白水平升高,p-STAT3蛋白水平降低。结论干扰HDAC1表达能够促进肝癌细胞凋亡,抑制肝癌细胞增殖,作用机制可能与STAT3信号通有关。  相似文献   

3.
目的探究沉默组蛋白去乙酰化酶4(HDAC4)基因表达对多发性骨髓瘤LP-1细胞增殖、凋亡的影响以及作用机制。方法脂质体Lipofectamine 2000转入LP-1细胞中,实验分为对照组(不做任何处理)、阴性组(转入siRNA NC)、siRNA HDAC4组(转入siRNA HDAC4)。CCK-8法检测细胞的增殖活性,Annexin V-FITC/PI双染色法检测细胞的凋亡率,蛋白质印迹法(Western Blot)检测细胞中HDAC4、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)、B细胞淋巴瘤/白血病-2(Bcl-2)的表达量。结果转染siRNA HDAC4后,LP-1细胞中HDAC4蛋白的表达量显著低于对照组(P 0. 05);与对照组相比,siRNA HDAC4组细胞的增殖活性显著降低(P 0. 05);而凋亡率显著增加(P 0. 05); siRNA HDAC4组细胞中Cleaved Caspase-3、Cleaved Caspase-9蛋白水平显著高于对照组(P 0. 05),但Bcl-2蛋白水平显著降低(P 0. 05)。结论沉默HDAC4基因可抑制多发性骨髓瘤LP-1细胞增殖,诱导其凋亡,可能通过降低Bcl-2水平,促进Cleaved Caspase-3、Cleaved Caspase-9表达发挥作用。  相似文献   

4.
目的探讨小干扰RNA(siRNA)沉默IκB激酶(IKK)/核因子κB(NF-κB)信号通路对子宫内膜异位症在位内膜腺上皮细胞中基质金属蛋白酶-9(MMP-9)的表达及细胞侵袭性的影响。方法根据基因数据库IKKα的c DNA序列,设计构建合成IKKαsiRNA转染10例EMs患者在位内膜原代腺上皮细胞,设立空白对照组(不加任何siRNA的等比例转染试剂)、阴性对照组(与IKKα同源性极低的无意义链siRNA)及实验组(IKKαsiRNA)。采用Realtime PCR、Western blot、Transwell等方法分别检测IKKαsiRNA转染前后NF-κB、MMP-9 mRNA和蛋白表达及腺上皮细胞侵袭性的变化。结果与空白对照组和阴性对照组比较,实验组EMs腺上皮细胞中NF-кB、MMP-9蛋白及mRNA表达降低,差异有统计学意义(P0.05);IKKαsiRNA转染后EMs腺上皮细胞的细胞侵袭性明显降低,差异有统计学意义(P0.05)。结论 RNA干扰可抑制IKK/NF-κB信号通路进而显著降低细胞的侵袭,这种抑制作用可能是通过下调MMP-9的表达进而改变其侵袭作用诱导EMs的发生。  相似文献   

5.
目的构建重组HDAC1基因的短发夹RNA(shRNA)慢病毒干扰质粒并研究其对卵巢癌COC1细胞HDAC1表达的影响。方法针对HDAC1基因设计特异性siRNA靶点,构建慢病毒干扰质粒载体,筛选获得的有效shRNA慢病毒干扰序列,将其转染卵巢癌COC1细胞。采用Real-time PCR方法检测靶基因在mRNA水平的沉默效果,Western blot方法检测靶基因在蛋白质水平的沉默效果。结果构建的慢病毒载体shRNA的PCR鉴定和测序正确。shRNA慢病毒干扰质粒转染COC1细胞后HDAC1基因的mRNA表达量和蛋白质水平较阴性对照质粒转染组均显著下调(P0.05)。结论成功构建HDAC1基因的shRNA慢病毒干扰质粒,该慢病毒干扰质粒能够在细胞水平有效沉默靶基因。  相似文献   

6.
目的:观察Bmi-1基因沉默对白血病耐药细胞株K562/ADR耐药性的影响并初步探讨其机制。方法:将2种序列的Bmi-1小干扰RNA SiRNA转染到耐药细胞K562/ADR,检测Bmi-1基因m RNA和蛋白的表达以确定转染效果。检测Bmi-1基因沉默后耐药蛋白P-gp及其编码基因MDR1的表达情况,并采用流式细胞术检测细胞内阿霉素蓄积情况。检测Bmi-1基因沉默后NF-κB蛋白的表达变化;应用NF-κB抑制剂PDTC处理K562/ADR细胞抑制NF-κB活性后,检测P-gp蛋白的表达及其功能的变化。检测Bmi-1基因沉默后PTEN、AKT和p-AKT蛋白表达的变化。应用PI3K/AKT通路抑制剂LY294002处理K562/ADR细胞抑制p-AKT表达后,检测NF-κB和P-gp蛋白的表达。应用PTEN抑制剂BPV(phen)处理Bmi-1基因沉默后的细胞,检测AKT、p-AKT、NF-κB和P-gp蛋白的表达。以上mRNA表达用RT-PCR法检测,蛋白表达用Western blot法检测。结果:在mRNA水平和蛋白水平2种序列的小干扰RNA均使K562/ADR细胞Bmi-1基因表达降低。MDR1/P-gp在Bmi-1 siRNA干扰细胞中的表达明显低于在K562/ADR细胞的表达(P0.05);干扰后细胞内阿霉素蓄积增多。Bmi-1基因沉默后,细胞NF-κB活性降低;NF-κB抑制剂抑制NF-κB活性后,K562/ADR细胞P-gp蛋白表达及药物泵出功能被抑制。Bmi-1基因沉默后PTEN蛋白表达增高,而p-AKT蛋白表达明显降低(P0.05)。PI3K/AKT通路抑制剂LY294002抑制p-AKT表达后,NF-κB活性和P-gp蛋白表达明显下降(P0.05)。PTEN抑制剂BPV(phen)处理Bmi-1基因沉默细胞后,NF-κB活性和P-gp蛋白表达得到重塑。结论:Bmi-1在MDR1/P-gp介导的K562/ADR细胞多药耐药中起关键作用,这种作用可能是通过激活PTEN/AKT途径调控NF-κB完成。  相似文献   

7.
目的研究组蛋白去乙酰化酶1(HDAC1)对喉癌细胞侵袭、迁移及血管内皮生长因子(VEGF)水平影响。方法喉癌细胞Hep2中转染HDAC1 siRNA和siRNA对照记为HDAC1 siRNA组和siRNA-NC组,同时以不做转染的细胞为对照组。qRT-PCR测定细胞中HDAC1 mRNA水平,Western blot检测细胞中HDAC1蛋白水平,Transwell小室检测细胞侵袭,细胞划痕实验检测细胞迁移,ELISA检测培养液上清中VEGF和碱性成纤维细胞生长因子(b FGF)水平,Western blot检测细胞中信号转导与转录因子3(STAT3)、磷酸化的STAT3(p-STAT3)、基质金属蛋白酶-2(MMP-2)水平。结果 HDAC1 siRNA组细胞中HDAC1 mRNA和蛋白水平均明显低于对照组(P0.05)。siRNA-NC组细胞中HDAC1 mRNA和蛋白水平与对照组相比无显著差异(P0.05)。siRNA-NC组细胞迁移率、侵袭数目及培养液上清中VEGF、b FGF含量均明显降低,细胞中MMP-2、p-STAT3/STAT3水平也下降,与对照组相比差异有显著性(P0.05)。siRNA-NC组细胞迁移率、侵袭数目、MMP-2水平、p-STAT3/STAT3水平及培养液上清中VEGF、b FGF含量与对照组相比无显著差异(P0.05)。结论下调HDAC1表达可降低喉癌细胞侵袭和迁移能力,降低细胞分泌VEGF、b FGF水平,作用机制可能与抑制STAT3信号通路有关。  相似文献   

8.
RNA干扰沉默急性白血病细胞SUV39H1基因表达的实验研究   总被引:1,自引:0,他引:1  
目的 应用小干扰RNA(siRNA)干扰人急性髓系白血病细胞株KG-1细胞SUV39H1(suppressor of variegation 3-9 homolog 1)基因的表达,研究SUV39H1基因沉默对KG-1细胞增殖、抑癌基因p15表达及组蛋白甲基化、乙酰化修饰的影响,探寻白血病基因治疗的新靶点.方法 用LipofectamineTM2000将体外合成针对SUV39H1基因的siRNA片段转染到KG-1细胞后,采用RT-PCR方法检测siRNA对SUV39H1 mRNA表达的抑制效果,用四唑氮化合物(MTS)法绘制细胞增殖抑制曲线,Western blot法检测目的 基因SUV39H1和抑癌基因p15的表达及对组蛋白甲基化、乙酰化修饰的影响.结果 SUV39H1 siRNA转染KG-1细胞可沉默该基因;沉默SUV39H1基因表达可抑制细胞增殖,30、60、120、240 nmol/L SUV39H1 siRNA转染48 h后,KG-1细胞的增殖抑制率分别为(23.57±1.98)%、(48.69±1.84)%、(62.69±1.61)%、(81.06±3.22)%,差异有统计学意义(P<0.05);沉默SUV39H1基因可上调p15基因的表达,下调组蛋白H3K9三甲基化,上调组蛋白H3K9、H3的乙酰化.30、60、120 nmol/L SUV39H1 siRNA处理KG-1细胞48 h后,组蛋白H3K9三甲基化水平较对照组分别减少25%、33%、49%;组蛋白H3K9乙酰化水平增强,分别为对照组的1.83、2.16、3.07倍;组蛋白H3乙酰化水平逐渐升高为对照组的1.35、1.87、2.37倍;p15表达水平呈递增趋势,分别为对照组的1.52、2.89、3.08倍;而组蛋白H4、H3K14、H3K27的乙酰化水平未见显著变化.结论 沉默SUV39H1基因表达可能通过改变抑癌基因启动子区域的组蛋白修饰,即上调组蛋白H3K9乙酰化及下调H3K9甲基化,使p15基因重新表达,从而抑制细胞增殖.  相似文献   

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目的观察沉默缺氧诱导因子HIF-1α对卵巢癌细胞化疗敏感性的相关影响并探讨其机理。方法构建HIF-1α基因短发夹RNA真核表达质粒,并转染卵巢癌细胞SKOV3(人卵巢浆液性囊腺癌细胞株)。实验可分为空白组,非特异对照组(转染pNonspecific-siRNA,即SKOV3NS),目的siRNA组(转染pHIF-siRNA,即SKOV3siRNA)3组,用RT-PCR及Western Blot法分别检测各组HIF-1α基因的mRNA和蛋白的表达水平。细胞经过20μmol/L顺铂作用后,用MTT法检测细胞生长抑制率,流式细胞术(FCS)检测细胞凋亡率。结果 SKOV3siRNA转染组HIF-1α基因在mRNA水平和蛋白水平表达均明显降低,SKOV3siRNA组肿瘤细胞的生长抑制率和细胞凋亡率均明显增高(P〈0.05)。结论 RNA干扰技术沉默HIF-1α能够有效地抑制卵巢癌SKOV3细胞HIF-1α基因表达,增强其对化疗药物顺铂的敏感性。  相似文献   

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This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.  相似文献   

12.
目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高<20%视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P<0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P<0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P<0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.  相似文献   

13.
The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.  相似文献   

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Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.  相似文献   

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Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.  相似文献   

18.
目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4%(55/72)]明显高于成功组[31.7%(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3%(42/72)]高于成功组[38.1% (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9% (28/72)]高于成功组[23.8%(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大<6 cm、胎头位于坐骨棘上及宫口开大8~10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1%(44/72)]高于成功组[38.9%(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2%(52/72)]明显高于成功组[34.1 %(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.  相似文献   

19.
ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly. Issue 4 for 2009 contains 4027 complete reviews, 1906 protocols for reviews in production, and 11447 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 600,000 randomized controlled trials, and 12,200 cited papers in the Cochrane methodology register. The health technology assessment database contains over 7500 citations. This edition of the Library contains 90 new reviews, of which 19 have potential relevance for practitioners in pain and palliative medicine.  相似文献   

20.
ZusammenfassungFragestellung Es wurde geprüft, wie sich der Differenziertheitsgrad zweier Schmerzmessmethoden auf Angaben zur Ausgedehntheit klinischer Schmerzen auswirkt. Zugleich wurde der Referenzzeitraum variiert, über den die Patienten berichten sollten.Methode Erfasst wurde der Einfluss zu Lasten der Befragungsdifferenziertheit durch den Vergleich zweier Körperschema-Bildvorlagen. Drei Referenzzeiträume (Schmerz aktuell, letzte Woche, letztes halbes Jahr) wurden vorgegeben.Ergebnisse Patienten mit ausgedehnten Schmerzen gaben bei differenzierter Befragung um so mehr Schmerzen an, je weiter die Schmerzen zurück lagen und je größer der Berichtszeitraum war. Patienten mit gelenknahen Schmerzen gaben bei hoch differenzierter Befragung weniger ausgedehnte Schmerzen in der Vergangenheit an als bei globaler Einschätzung. Patienten mit Rückenschmerzen berichteten bei differenzierter Befragung zum aktuellen Schmerz über weniger ausgedehnte Schmerzen als bei globaler Befragung.Schlussfolgerung Die Angaben zur Schmerzausdehnung variieren vor allem bei Patienten mit ausgedehnten Schmerzen in Abhängigkeit von der Differenziertheit der Befragung. In diesen Fällen ist die Wahrscheinlichkeit erhöht, dass sich die Beschwerdesymptomatik zumindest teilweise erst in der Reaktion auf die situativen Befragungsbedingungen konstituiert und daher nicht auf andere Befragungsbedingungen generalisiert werden kann.  相似文献   

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