天津市儿童急性上呼吸道感染病毒病原学分析

目的了解天津地区儿童急性上呼吸道感染中病毒分布情况,探讨不同病毒的流行趋势,为临床诊断与治疗提供病原学依据,同时为今后制定正确的预防策略奠定基础。方法采用多重RT—PCR法,对249份咽拭子标本同时检测腺病毒,人偏肺病毒,人冠状病毒229E／NL63,1型、2型、3型副流感病毒,甲型、乙型流感病毒,甲型、乙型呼吸道合胞病毒,A／B型人鼻病毒,人冠状病毒0C43／HKUl共12种常见呼吸道病毒。结果249份标本中共检出阳性标本144份,阳性率为57．83％,2种以上病毒混合感染为7份,占2．8l％;在阳性检测结果中流感病毒占53．64％,腺病毒占21．19％,呼吸道合胞病毒占8．61％;流感病毒中H3N2亚型占49．38％,甲型H1N1亚型占34．57％,B型占16．05％。结论流感病毒、腺病毒和呼吸道合胞病毒为天津地区儿童急性上呼吸道病毒感染的主要病原体,不同的病毒在时间上有一定的流行规律。     

Quantitative detection of metastasis-associated 1 mRNA and protein expression in breast cancer

Objective Understanding the mechanism of breast cancer metastasis will benefit those patients in need of aggressive treatment and avoid side effects caused by chemotherapy over treatment.Recently,a pot...     

OGGl基因表达的半定量RT-PCR检测方法的建立

目的培养的犬肾细胞（MDCK）内8-羟基鸟嘌呤DNA糖苷酶（OGGl）基因表达的半定量RT-PCR检测体系的建立。方法培养MDCK细胞后提取总RNA,经优化进行RT-PCR反应,检测细胞内OGGl基因的表达。扩增产物经测序同GenBank中OGGl基因序列比对验证产物的准确性,条带经ImageJ软件分析并与内参基因甘油醛-3-磷酸脱氢酶（GAPDH）相比,对体系的重复性进行分析。结果扩增产物证实与GenBank中该细胞的OGGl基因序列一致;重复性测定发现,MDCK细胞OGGl与GAPDH灰度值比值的均值为（0．84±0．025）,CV值为2．99％。结论本方法的准确性、重复性均较好,可用于半定量检测培养细胞尤其是MDCK细胞内OGGlmRNA的表达量。     

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

Background and Purpose

Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca²⁺-activated K⁺ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function.

Experimental Approach

We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function.

Key Results

We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions.

Conclusions and Implications

Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity.     

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-l-methionine or its demethylated product S-adenosyl-l-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-l-homocysteine partly explained catechol-enhanced erythroid differentiation.     

桑黄肌动蛋白基因片段的克隆及序列分析

目的 对桑黄肌动蛋白（Actin）基因进行克隆及序列分析。方法 搜索网上已知数据库中Actin基因,与本实验室测得的桑黄转录组数据进行生物信息学比对,找到转录组数据中与Actin相似性最高的片段,并根据其设计引物,提取桑黄菌丝体总RNA为模板,采用RT-PCR的方法扩增Actin基因片段并对PCR产物进行测序。结果 得到一段839 bp的序列,序列分析表明,该片段编码279个氨基酸,与其他物种Actin基因核苷酸序列同源性在87%以上。结论 首次从桑黄中克隆出了Actin基因,为有效利用该基因奠定了基础。     

CD44在胃癌细胞株MKN45悬浮球体细胞中的表达及意义

目的：检测干细胞相关因子CD44在胃癌细胞株MKN45悬浮球体细胞中的表达。方法：选择人胃癌细胞株MKN45。在含有纤维生长因子-2及表皮生长因子无血清培养液中培养,观察球体形成情况;采用逆转录聚合酶链反应和免疫印迹分析法,检测干细胞相关基因CD44在球体细胞及原代贴壁细胞两组细胞中的表达差异。结果：胃癌细胞株MKN45在无血清培养条件下能形成悬浮球体,球体细胞中CD44的相对表达量高于原代贴壁细胞（P〈0．05）。结论：悬浮球培养法在胃癌细胞株MKN45中培养形成的球体细胞富集类肿瘤干细胞。该方法是分选和鉴定肿瘤干细胞的行之有效的方法。     

人类NY-ESO-1基因编码在肝癌中的表达

目的 研究NY-ESO-1在肝细胞肝癌中的表达情况并探索其表达与肝癌预后的相关性.方法 我们收集了124例肝癌患者的肝癌组织和相应的癌旁组织,37例肝硬化患者的肝组织,以及43例非肝硬化的肝组织.应用实时荧光定量聚合酶链技术(RT-PCR)检测了上述组织中NY-ESO-1基因的表达水平,并利用DNA免疫杂交技术和测序技术检测RT-PCR产物.收集健康志愿者外周血基因组DNA作为正常对照.结果 在124例肝癌组织中,56例(45.2％)检测到有NY-ESO-1基因的表达,而在癌旁组织中未检测到表达.NY-ESO-1基因的RT-PCR产物长度为329 bp,利用测序方法检测NY-ESO-1基因突变.在56例表达NY-ESO-1的肝癌组织中,有4例检测到位点2的突变.该突变导致一个氨基酸的变异,这种氨基酸的变异对蛋白结构和功能具有决定性的影响.NY-ESO-1基因的表达与临床特征如肿瘤标志物(甲胎蛋白,半乳糖酸苷酶)、转移、复发和肿瘤的大小无显著关联性.结论 NY-ESO-1在肝癌组织中呈高表达,但是与预后无相关性.NY-ESO-1蛋白能否作为肝癌治疗的候选疫苗有待进一步研究.     

Involvement of the essential metal transporter Zip14 in hepatic Cd accumulation during inflammation

Upregulation of Zip14 contributes to hepatic zinc (Zn) and non-transferrin-bound iron (Fe) uptake during infection and inflammation. We investigated whether this essential metal transporter is also involved in hepatic cadmium (Cd) uptake under these conditions. An injection of lipopolysaccharide (LPS), turpentine oil (Tur) and n-hexane (Hex) resulted in an decrease in plasma Zn and Fe concentrations to 25–50% and an increase in hepatic concentrations of both metals to 150–200% of control mice. LPS significantly increased plasma interleukin (IL)-6 levels more rapidly than Tur or Hex. Tur or Hex significantly increased hepatic Zip14 mRNA expression and decreased ferroportin 1 mRNA expression following continuous increase of IL-6 level. Hepatic Cd and Zn concentrations increased significantly after repeated injections of Cd in Tur- or Hex-treated mice fed a control diet. Treatment with Tur or Hex additionally increased hepatic Cd accumulation in Zn-deficient mice, unlike in Fe-deficient mice. These results suggest that Zn transporters, such as Zip14, may be involved in hepatic Cd uptake during inflammation.     

Comparison of DNA and RNA extraction methods for mummified tissues

Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.