改进的亚硫酸氢钠测序法检测HepG2 RASSF1A基因CpG岛甲基化状态

目的 改进亚硫酸氢钠测序法，并检验改进后方法在甲基化检测中的可行性。方法 提取人肝癌细胞株HepG2基因组DNA，亚硫酸氢钠化学修饰，针对修饰后Ras相关家族1A基因（RASSF1A）启动子序列设计特异引物并结合沉降PCR扩增，T-A载体克隆、测序。结果 HepG2细胞RASSF1A基因启动子CpG岛的16个CpG位点均被甲基化。结论 改进后亚硫酸氢钠测序法能明显减少非特异性扩增，提高PCR效率，更适于基因甲基化状态的检测。     

中药治疗中风的分子靶点

“中风”是中医学对急性脑血管疾病的统称,具有极高的死亡率和致残率。中医药治疗中风有着悠久的历史,临床实验也证实了其疗效。然而由于中药常常需要多味药物联合使用,其有效成分很难明确,对其有效性评价具有挑战性,因此很难将中药制剂推向世界。综述中药治疗中风的分子靶点作用,列举了一些可能起效的天然化学物质,调查结果表明多成分多靶点的中药理论具有科学性。现代分子医学和传统中医理论的结合能促进医学界的广泛交流,值得深入探讨。     

职业女性经前期综合征心理干预效果评价

[目的]探讨对经前期综合征（PMS）患者进行心理干预的效果。[方法]对2004年11月至2005年10月在大同市第七人民医院体检的253名有PMS表现的职业女性进行调查。[结果]心理干预后症状自评量表（SCL-90）各因子均分中，恐惧因子无变化，其他各因子均分均有下降（P〈0．05或〈0．01）；心率变异频域分析，心理干预后与干预前比较，TP增加（P〈0．01）、LF／HF下降（P〈0．05）。[结论]对PMS患者进行心理干预可以调整其心理状态，使交感神经、迷走神经趋于平衡。     

Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1

Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release. Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1. This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs. Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1. These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs.     

三氟拉嗪诱发细胞凋亡及对DNA依赖蛋白激酶催化亚单位表达和P38磷酸化的抑制作用

目的　探讨三氟拉嗪(TFP)对肿瘤细胞生长的抑制作用和分子机制,寻找抑制肿瘤生长的作用靶分子。方法　用不同剂量TFP作用于人宫颈癌上皮细胞(HeLa) ,体外细胞计数绘制生长曲线;荧光染料染色及DNA琼脂糖凝胶电泳检测细胞凋亡;γ射线及TFP处理细胞观察TFP对细胞辐射敏感性的影响;Western印迹分析蛋白表达或磷酸化。结果TFP作用后HeLa细胞的生长受到抑制,对γ射线的敏感性增加,且随着剂量的增加细胞凋亡的比率也增加。TFP还可有效诱导DNA修复蛋白DNA依赖蛋白激酶催化亚单位(DNA-PKcs)被切割,且可抑制辐射诱发的P3 8的磷酸化。结论　TFP可抑制肿瘤细胞的生长,对DNA-PKcs的表达和P3 8的磷酸化都有抑制作用     

A new membrane-attack complex/perforin (MACPF) domain lethal toxin from the nematocyst venom of the Okinawan sea anemone Actineria villosa.

The Okinawan sea anemone Actineria villosa causes severe cases of stinging. We isolated the 60 kDa A. villosa toxin (AvTX-60A) as the major toxin from the isolated nematocysts of this species. AvTX-60A showed fatal toxicity to mice with intraperitoneal injection at a minimum lethal dose of less than 250 microg/kg. The N-terminal amino acid sequence was determined and the corresponding cDNA encoding AvTX-60A was sequenced. The deduced amino acid sequence of AvTX-60A showed high similarity with PsTX-60A, which had been isolated as one of the major toxins from the venomous sea anemone Phyllodiscus semoni. These sea anemone toxins are new members of the family of proteins containing membrane-attack complex/perforin (MACPF) domains, best known in pore forming proteins such as perforin. These are the first examples of MACPF domain proteins as toxins for prey acquisition or repelling predators in nature.     

III期结直肠癌中NLRC3与预后和肿瘤免疫的关系

目的：探讨核苷酸寡聚化结构域样受体家族半胱天冬酶募集结构域蛋白3(nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 3,NLRC3)与III期结直肠癌的预后及肿瘤免疫相关指标 的关系。方法：回顾性收集中南大学湘雅医院2012年至2013年122例经手术根治切除的III期结直肠癌患者的相关资 料。利用免疫组织化学法分析NLRC3与CD8+ T细胞的表达情况,利用患者的术前临床资料计算中性粒细胞/淋巴细 胞比值(neutrophil to lymphocyte ratio,NLR),并检测其微卫星稳定性。采用χ2检验分析NLRC3与临床病理因素之间的 关系,采用COX回归模型分析III期结直肠癌的独立预后因素。结果：在III期结直肠癌中,肿瘤组织CD8+ T细胞浸润 分期(χ2=27.79,P<0.01)、NLR值(χ2=6.35,P<0.05)、淋巴结转移分期(χ2=10.12,P<0.01)以及微卫星稳定性(χ2=6.05, P<0.05)与NLRC3的表达有关。NLRC3(OR=0.066,95% CI：0.020~0.218)、血管癌栓(OR=3.119,95% CI：1.547~6.286) 及NLR(OR=5.103,95% CI：2.465~10.563)对III期结直肠癌的5年总生存期(overall survival,OS)有影响(均P<0.05);另 外,NLRC3(OR=0.144,95% CI：0.055-0.377)、血管癌栓(OR=3.589,95% CI：1.859~6.932)及NLR(OR=2.939,95% CI： 1.509~5.723)对III期结直肠癌的无病生存期(disease free survival,DFS)同样有影响(均P<0.05)。结论：NLRC3,血管癌栓 和NLR是III期结直肠癌的独立预后因素。NLRC3通过抑制系统性炎症、促进局部抗肿瘤免疫而使III期结直肠癌患者 具有良好的预后。     

Type I secretion systems – a story of appendices

Secretion is an essential task for prokaryotic organisms to interact with their surrounding environment. In particular, the production of extracellular proteins and peptides is important for many aspects of an organism's survival and adaptation to its ecological niche. In Gram-negative bacteria, six different protein secretion systems have been identified so far, named Type I to Type VI; differing greatly in their composition and mechanism of action (Economou et al., 2006). The two membranes present in Gram-negative bacteria are negotiated either by one-step transport mechanisms (Type I and Type III), where the unfolded substrate is translocated directly into the extracellular space, without any periplasmic intermediates, or by two-step mechanisms (Type II and Type V), where the substrate is first transported into the periplasm to allow folding before a second transport step across the outer membrane occurs. Here we focus on Type I secretion systems and summarise our current knowledge of these one-step transport machineries with emphasis on the N-terminal extensions found in many Type I-specific ABC transporters. ABC transporters containing an N-terminal C39 peptidase domain cut off a leader peptide present in the substrate prior to secretion. The function of the second type of appendix, the C39 peptidase-like domain (CLD), is not yet completely understood. Recent results have shown that it is nonetheless essential for secretion and interacts specifically with the substrate of the transporter. The third group present does not contain any appendix. In light of this difference we compare the function of the appendix and the differences that might exist among the three families of T1SS.     

谱域光学相干断层扫描对孔源性视网膜脱离术后黄斑微结构的观察

目的 运用谱域光学相干断层扫描仪对孔源性视网膜脱离(RRD)术后患者的视网膜结构进行观察.方法 在我院眼科行RRD复位术后1年以上的患者17例(17眼),包括行玻璃体切除术8例,巩膜外环扎外加压术8例,玻璃体切除联合巩膜外环扎1例.17例中,巩膜外冷凝12例,眼内光凝5例.OCT检查观察视网膜结构变化.并用SPSS 19.0软件进行统计学分析及独立样本t检验.结果 发现10只眼有黄斑区的组织结构异常(58.82％),这些异常包括视网膜变薄2例(11.76％),视网膜前膜6例(35.29％),视网膜水肿2例(11.76％),板层视网膜裂孔1例(5.88％)、脉络膜视网膜萎缩1例(5.88％)、感光细胞内节和外节(IS/OS)连接线的异常4例(包括IS/OS连接线断裂及萎缩)(23.53％)、视网膜外界膜断裂2例(11.76％),外核层高反射点2例(11.76％).在视网膜脱离累及黄斑的患者中,视网膜前膜和感光细胞IS/OS连接线异常所占的比例均为50％,比例较高.结论 OCT因其能够清晰观察黄斑微结构改变,为RRD术后视力预后评估及随访治疗提供了极大方便.     

Heart rate variability during wakefulness as a marker of obstructive sleep apnea severity

Study ObjectivesPatients with obstructive sleep apnea (OSA) exhibit heterogeneous heart rate variability (HRV) during wakefulness and sleep. We investigated the influence of OSA severity on HRV parameters during wakefulness in a large international clinical sample.Methods1247 subjects (426 without OSA and 821 patients with OSA) were enrolled from the Sleep Apnea Global Interdisciplinary Consortium. HRV parameters were calculated during a 5-minute wakefulness period with spontaneous breathing prior to the sleep study, using time-domain, frequency-domain and nonlinear methods. Differences in HRV were evaluated among groups using analysis of covariance, controlling for relevant covariates.ResultsPatients with OSA showed significantly lower time-domain variations and less complexity of heartbeats compared to individuals without OSA. Those with severe OSA had remarkably reduced HRV compared to all other groups. Compared to non-OSA patients, those with severe OSA had lower HRV based on SDNN (adjusted mean: 37.4 vs. 46.2 ms; p < 0.0001), RMSSD (21.5 vs. 27.9 ms; p < 0.0001), ShanEn (1.83 vs. 2.01; p < 0.0001), and Forbword (36.7 vs. 33.0; p = 0.0001). While no differences were found in frequency-domain measures overall, among obese patients there was a shift to sympathetic dominance in severe OSA, with a higher LF/HF ratio compared to obese non-OSA patients (4.2 vs. 2.7; p = 0.009).ConclusionsTime-domain and nonlinear HRV measures during wakefulness are associated with OSA severity, with severe patients having remarkably reduced and less complex HRV. Frequency-domain measures show a shift to sympathetic dominance only in obese OSA patients. Thus, HRV during wakefulness could provide additional information about cardiovascular physiology in OSA patients.Clinical Trial Information: A Prospective Observational Cohort to Study the Genetics of Obstructive Sleep Apnea and Associated Co-Morbidities (German Clinical Trials Register - DKRS, DRKS00003966) https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00003966