基因表达谱芯片杂交影响因素的初步研究

通过对K562细胞基因表达谱芯片杂交影响因素的研究表明,用限制性显示技术制备的cDNA探针长度较均一,适合基因芯片杂交;在42℃条件下含甲酰胺的杂交液中杂交16．20h,可保证样品的有效杂交。并表现出很好的杂交特异性;用RD-PCR或线性PCR对少量样品进行扩增,并用荧光(Cy3或Cy5)标记的通用引物对样品进行标记,可提高芯片检测的灵敏度;一次杂交反应总RNA的用量仅需0．5～10μg,在每cm^2约含1000～1500个点的阵列中杂交时,标记样品用量1～2μg为宜;用PCR产物纯化柱对荧光标记产物进行纯化,可大大减小背景荧光,提高信噪比;同一批芯片经同一样品杂交结果的重复性很好,相关系数高达97．8％     

芯片杂交中核酸靶样品的制备优化

样品来源、基因含量、检测方法和分析目的的不同,采用的核酸分离、扩增和标记方法各异。核酸样品制备条件的优化处理主要包括核改的单链化处理,片段化和标记方法。根据具体情况选用合适的处理方法,可显提高基因芯片检测的特异性和重现性。     

凋亡和周期基因芯片研究As2O3对NB4细胞凋亡相关基因表达谱的影响

目的:利用细胞凋亡和周期基因芯片研究As₂O₃作用前后NB4细胞基因表达谱的差异性,寻找As₂O₃诱导NB4细胞凋亡的相关基因并分析其可能机制。方法:流式细胞仪检测细胞凋亡率,抽提对照及诱导组细胞的mRNA,通过逆转录将As₂O₃处理前后的NB4细胞cDNA进行生物素标记,用含269个目的基因的细胞凋亡和周期基因芯片进行杂交,GEArray软件分析,筛选出As₂O₃诱导前后表达有差异的基因。芯片结果用荧光定量聚合酶链反应(realtimepolymerasechainreaction)进行验证。结果:筛选出As₂O₃作用前后表达有差异的基因共100条(占芯片基因总数的37.2%),其中97条(97/100,97%)基因表达上调,3条(3/100,3%)基因表达下调。表达上调的基因主要包括肿瘤坏死因子配体和受体家族、bcl2家族、半胱氨酸家族、DNA损伤检测和P53途径以及细胞分裂周期蛋白和激酶等基因。结论:As₂O₃主要通过上调促凋亡基因表达来诱导NB4细胞凋亡,其中TNFSF15、Apaf1、Caspase3和p16等基因可能参与As₂O₃诱导的NB4细胞凋亡,As₂O₃诱导NB4细胞凋亡的可能机制主要涉及TNF途径、线粒体途径、Caspase途径、细胞周期抑制途径和P53途径等。     

不同荧光定量PCR分析方法检测大鼠脑缺血再灌注损伤中Caspase-3基因的表达研究

目的:应用实时荧光PCR检测大鼠脑缺血/再灌注损伤组织中的Caspase-3基因表达变化,并比较了相对定量与绝对定量分析方法的技术优劣.方法:相对定量实验中,以β-actin基因作为内参,采用Delta-delta Ct法计算目的基因表达变化.而在绝对定量实验中,构建了含Caspase-3基因的重组质粒,以该重组质粒作...     

Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit（Qiagen）柱子法

目的比较RNeasy mini kit（Qiagen）柱子法和Trizol法提取RNA在雪貂肺和肠组织H3N2 SYBRGreen PCR定量中的去干扰作用,从而找到适合该定量体系的RNA提取方法。方法比较Trizol法,RNeasy minikit柱子法,改良RNeasy mini kit柱子法1和改良RNeasy mini kit柱子法2提取肺肠组织RNA的质量,RNA逆转录成cDNA后,利用SYBR Green PCR方法检测样品中H3N2的载量,比较产物的特异性,综合评价RNA提取方法。结果 4种方法提取的肺和肠组织的RNA质量不相同,紫外分光光度仪测得RNeasy mini kit柱子法提取的RNA的A260/280低于1.8,其余3种方法的A 260/280在1.8～2.1之间,A 260/230只有Trizol法能达到2.0左右,其余3种方法均远低于2.0。电泳可见Trizol法提取的RNA有3条带：28 s、18 s和5 s,而RNeasy mini kit柱子法的RNA除了有28 s、18 s外,还有基因组DNA,改良RNeasy mini kit柱子法1和2提取的RNA只有28 s和18 s带。RNA逆转录后进行荧光定量PCR,从溶解曲线看,不论是鼻甲刮取物还是肺肠组织的样品模板,4种方法获得的样品与标准品均为单一峰溶解曲线峰,并且波峰位置重叠。而鼻甲刮取物定量的产物跑琼脂糖凝胶电泳均为单一的特异性条带,而肺肠组织的产物电泳发现：Trizol法获得的模板定量产物与标准品一致,为单一的特异性条带,而其它3种方法获得的模板则均有非特异性条带。结论鼻甲刮取物的病毒定量选用RNeasy mini kit柱子法提取定量结果与Trizol法一样可靠,说明对于简单样品该体系及引物非常适用,结果可信。对于组织样品肺和肠,Trizol法获得的样品定量结果比其它3种方法可靠。     

标准型CD44基因蛋白在78例非小细胞肺癌中表达的定量研究

目的　探讨标准型CD44 (CD44s)基因表达与非小细胞肺癌 (NSCLC)的组织学类型、分化程度以及转移等之间的关系。方法　应用微波 -LSAB免疫组化染色及图像分析 ,测定了 78例NSCLC中CD44s阳性产物的光密度值(IOD)与组织学类型、分化程度和淋巴结转移间的关系。结果　 78例NSCLC的CD44s阳性细胞IOD值 (12 9.8± 12 .2 )与 10例癌旁组织IOD值 (91.3± 11.5 )之间差异非常显著 (P <0 .0 1) ;有淋巴结转移组 (10 9.8± 13.2 )与无淋巴结转移组 (136 .5± 14.8)间差异非常显著 (p <0 .0 1)。组织学类型、不同分化程度之间差异无显著性 (P >0 .0 5 )。结论　CD44s强阳性的病人转移率低、预后良好 ,CD44s可作为预测NSCLC病人预后的指标。     

实时荧光定量PCR检测RSV胁迫下抗病、感病水稻中与脱落酸相关基因的差异表达

采用实时荧光定量PCR方法测定了水稻条纹病毒(Rice stripe virus,RSV)胁迫下抗性不同品种水稻中与脱落酸相关基因的mRNA转录水平变化.结果表明:感病品种武育梗3号中WGPI、OsGASA2、Polcalcin、OsCBIA、Myb和OsCIPK15基因表达水平均上调,上调比率分别为4.96、5.17、2.01、5.17、12.04和7.84.而抗病品系KT 95-418中,OsGASA2和OsCIPK15基因表达水平下调,下调比率分别为1/5.40和1/2.08;Polcalcin和Myb基因表达水平上调,上调比率分别为4.20和3.86;WGPI和OsCBIA表达量变化不明显.这些结果表明,RSV胁迫能诱导脱落酸相关基因表达量的变化,并且在抗病、感病水稻品种中的表达特征不同,从而提示植物激素脱落酸可能调控了RSV胁迫条件下相关基因的表达.     

三氧化二砷对K562/A02 细胞的凋亡及细胞周期的影响

目的:研究三氧化二砷对多药耐药急性白血病细胞株K562/A02凋亡与细胞周期的影响及可能机制。方法:取阿霉素(Adr)的耐药白血病细胞株分为未加药的对照组及加入不同浓度的三氧化二砷(其终浓度为4.0μmol/L、5.0μmol/L)组,流式细胞仪检测细胞凋亡及细胞周期分布,Western blot方法检测不同浓度三氧化二砷对K562/A02细胞核NF-κBp65蛋白水平。结果:与对照组比较,三氧化二砷可显著增加Adr对K562/A02细胞凋亡率,阻滞细胞于G0/G1期,降低K562/A02细胞胞核中NF-kB p65的表达(P均<0.05)。结论:三氧化二砷可能是通过抑制NF-kB的胞内活化转位,从而促进K562/A02细胞凋亡及抑制细胞增殖。     

A Comparison of Two Methods for Quantifying Soil Organic Carbon of Alpine Grasslands on the Tibetan Plateau

As CO₂ concentrations continue to rise and drive global climate change, much effort has been put into estimating soil carbon (C) stocks and dynamics over time. However, the inconsistent methods employed by researchers hamper the comparability of such works, creating a pressing need to standardize the methods for soil organic C (SOC) quantification by the various methods. Here, we collected 712 soil samples from 36 sites of alpine grasslands on the Tibetan Plateau covering different soil depths and vegetation and soil types. We used an elemental analyzer for soil total C (STC) and an inorganic carbon analyzer for soil inorganic C (SIC), and then defined the difference between STC and SIC as SOC_CNS. In addition, we employed the modified Walkley-Black (MWB) method, hereafter SOC_MWB. Our results showed that there was a strong correlation between SOC_CNS and SOC_MWB across the data set, given the application of a correction factor of 1.103. Soil depth and soil type significantly influenced on the recovery, defined as the ratio of SOC_MWB to SOC_CNS, and the recovery was closely associated with soil carbonate content and pH value as well. The differences of recovery between alpine meadow and steppe were largely driven by soil pH. In addition, statistically, a relatively strong correlation between SOC_CNS and STC was also found, suggesting that it is feasible to estimate SOC_CNS stocks through the STC data across the Tibetan grasslands. Therefore, our results suggest that in order to accurately estimate the absolute SOC stocks and its change in the Tibetan alpine grasslands, adequate correction of the modified WB measurements is essential with correct consideration of the effects of soil types, vegetation, soil pH and soil depth.     

二烯丙基二硫诱导人白血病K562 细胞凋亡及其对Bax、Bag-1 表达的影响

目的:探讨二烯丙基二硫(Diallyl disulfide)诱导白血病K562细胞凋亡的作用及其机制。方法:采用吖啶橙/溴化乙啶(AO/EB)染色法观察细胞凋亡形态学变化;DNA琼脂糖凝胶电泳测定DNA梯度带;RT-PCR法检测BAG-1、BAX基因的mRNA表达变化。结果:DADS可诱导K562细胞凋亡。其对K562细胞的凋亡效用与药物浓度、有明显依赖关系;DNA琼脂糖凝胶电泳示:40mg/LDADS作用K562细胞48小时后能够产生明显的梯形电泳图谱(DNA ladder):DADS作用48h后,BAX mRNA表达水平较对照组上调;BAG-1 mRNA较对照组下调(差异具有统计学意义,P<0.05)。结论:DADS能够诱导K562细胞凋亡,其凋亡机制可能与上调BAX,下调BAG-1有关。     

Initial Value Dependence: A Comparison of Two Methods For Its Study

This article is concerned with the comparison of slope estimator precision in regression analysis and the structural relationships approach (e.g., Humak, 1983, ch. 3; Kendall and Stuart, 1977), as it is relevant for their applications when testing for initial value dependence in biomedical and behavioral contexts of repeated assessments (e.g., Blomqvist, 1977; Wall, 1977). As a basis for the comparison of the two methods, the mean square error is adopted. In the general case, it is argued for an informed (data-dependent) choice between regression analysis and the structural relationships approach. For the apparent majority of biomedical and behavioral studies of the phenomenon of initial value depenence, this comparison suggests that structural relationships is the preferable approach leading to more trustworthy substantive conclusions.     

The effect of intracellular ascorbate on the susceptibility of HL60 and Jurkat cells to chemotherapy agents

Chemotherapy agents initiate tumour cell apoptosis and this is thought to involve oxidative stress. In this study we have investigated the effect of the important antioxidant Vitamin C (ascorbate) on the response of HL60 and Jurkat cells to three chemotherapy drugs, namely etoposide, melphalan and arsenic trioxide (As₂O₃). Cells grown in routine culture media are deficient in ascorbate and to determine its effect on chemotherapy drug-induced apoptosis we supplemented the cells prior to drug exposure. We found that ascorbate had a varied effect on apoptosis and cell cycle progression. Etoposide-induced apoptosis in HL60 cells was significantly increased in ascorbate-loaded cells as measured by caspase-3 activation and DNA degradation, and this appeared to reflect a decrease in the number of necrotic cells rather than increased cytotoxicity. In contrast, ascorbate had no effect on etoposide-induced apoptosis in Jurkat cells. In both cell types melphalan-induced apoptosis was unaffected by intracellular ascorbate, whereas both apoptosis and growth arrest with low concentrations of As₂O₃ were diminished. These results indicate that intracellular ascorbate can affect cell responses to chemotherapy drugs in a complex and somewhat unpredictable manner and that it may play an important role in the responsiveness of tumour cells to chemotherapy regimes. This study was supported by the Health Research Council of New Zealand.     

Effect of phenols on natural killer (NK) cell-mediated death in the K562 human leukemic cell line

Cancer disease is a major cause of death in Western societies. Epidemiologically, antioxidant phenols have been associated with diminished incidence of cancer, while experimentally, they have cytotoxic effects on cancer cells. The aim of this study was to clarify whether natural antioxidant phenols render K562 human leukemic cells more susceptible to natural killer (NK) cell apoptosis and/or necrosis. K562 cells were pre-incubated with 7 different phenols (p-hydroxy benzoic acid, syringic acid, ferulic acid, p-coumaric acid, o-coumaric acid, gallic acid, and rutin) individually and afterwards targeted with NK cells at a ratio 1/5. Percentages of apoptotic and necrotic cells were assayed via flow cytometric analysis of annexin V and PI-stained cells. For the morphological assessment, cells were stained with acridine orange and ethidium bromide and were examined under a fluorescence microscope. Pre-treatment with gallic acid significantly rendered K562 cells more susceptible to NK cell-mediated necrosis, while pre-treatment with rutin significantly rendered K562 cells more susceptible to apoptosis. Gallic acid and rutin exert anticarcinogenic activity via the enhancement of K562 cell susceptibility to NK cell-mediated necrosis and apoptosis, respectively.     

A New Nonparametric Approach to the Comparison of K Independent Samples of Response Curves II: A K Sample Generalization of the FRIEDMAN Test

A K sample generalization of the FRIEDMAN test (1937) is introduced which can be used as a nonparametric procedure for testing the homogeneity of the profiles of K independent samples of response curves measured at T identical points of time. While a similar procedure in LEHMACHER & WALL (1978), section 3, is based on T combined tests, each of them at level a/T, here a finite and asymptotic test is presented which is based on a single test statistic. The application of the new multivariate test is illustrated by the same numerical example as in LEHMACHER & WALL (1978). The properties of this test are discussed and compared with the combined test mentioned above.     

当归多糖对K62细胞EpoR介导的信号转导通路研究

目的:研究当归多糖(APS)对K562细胞中EpoR介导的JAK2/STAT5信号转导通路的影响.方法:实验分为APS组(200mg/L APS)及阴性对照组(常规培养),两组细胞培养24h加入5×103IU/L Epo刺激,Western blot法检测Epo刺激不同时间后K562细胞核、浆蛋白中JAK2、STAT5的表达变化;免疫沉淀法检测其活性改变;采用免疫细胞化学技术和荧光显微镜观察其分布.结果:两组细胞培养24h加入Epo继续刺激对JAK2的表达影响不大,但APS组JAK2的活性较阴性对照组增强;Epo继续刺激K562细胞核中STAT5的表达及活性APS组都较阴性对照组增强.结论:APS在促进正常造血过程中,可能在EpoR介导的信号转导过程中增强JAK2、STAT5的酪氨酸磷酸化发挥重要作用.     

三氧化二砷联合氨氯地平对肝癌HepG2细胞体外作用的实验研究

目的:探讨三氧化二砷与氨氯地平单独用药及联合用药对肝癌Hep G2细胞增殖和凋亡的作用。方法:用不同浓度的三氧化二砷(4.0、2.0、1.0μmol/L)和氨氯地平(27×103、18×103、12×103 mg/m L)处理体外培养的人肝癌Hep G-2细胞,观察细胞形态的变化,采用CCK-8法检测两种药物单独及联合应用对Hep G-2细胞生长增殖的影响,并通过流式细胞术观察其对细胞凋亡及细胞周期的影响。结果:三氧化二砷和氨氯地平均可以浓度依赖性方式显著增加Hep G2细胞的生长抑制率及其凋亡率,以联合用药的作用较单独用药更显著(P0.05)。与三氧化二砷和氨氯地平单独用药相比,联合用药可显著阻滞Hep G2细胞于G2期及S期。结论:三氧化二砷和氨氯地平均可显著抑制人肝癌Hep G2细胞的生长和增殖,并促进其凋亡,且二者联合应用时具有协同作用。