凝血酶对食管癌EC109细胞增殖的影响

目的：探讨凝血酶对食管癌EC109细胞增殖的影响。方法：MTT比色法检测细胞增殖情况。凝血酶（终浓度1 U/mL）组和其作用于EC109细胞后收集的上清分别培养EC109细胞24、48、72 h。结果：凝血酶培养EC109细胞48 h后,细胞的490 nmOD值高于对照组（P〈0.05）。培养上清处理的各组与对照组比无统计学意义。结论：凝血酶有刺激EC109细胞增殖的作用。     

慢病毒载体的构建及低表达和过表达CEA EC9706细胞株的建立

目的:构建人癌胚抗原(CEA)慢病毒siRNA和表达载体并包装成感染性病毒颗粒,获得稳定低表达和过表达CEA的EC9706细胞.方法:构建CEA siRNA载体:依据siRNA靶序列设计原则,针对人CEA的cDNA序列,设计并合成编码siRNA的3对寡核苷酸序列,克隆入siRNA载体(pRNAT-U6.2/Lenti)中构建3个重组体pRNAT-U6.2-SCEA.构建CEA表达载体:以人CEA的测序质粒为模板,PCR扩增CEA全长,装入pLentiGFP转移质粒,经过测序证实其序列与标准序列完全一致.然后进行重组慢病毒分剐感染EC9706细胞,G418筛选得到稳定感染细胞株.分别用荧光定量Real-time RT-PCR和Western blot检测EC9706细胞中CEA基因表达抑制或增强的效果.结果:EC9706病毒感染48 h后荧光显微镜观察CEA慢病毒载体在细胞中高效表达.经过RTPCR和Western-blot验证:得到3株稳定CEA低表达的EC9706细胞株,其中一个CEA的表达几乎得到完全抑制,1株稳定CEA过表达的EC9706细胞株(EC9706-CEA).结论:建立了稳定低表达和过表达CEA的EC9706细胞株,成功调控食管癌EC9706细胞中CEA的表达,为进一步从分子水平探讨CEA的功能奠定了基础.     

Avian-specific TLRs and downstream effector responses to CpG-induction in chicken macrophages

Chickens possess toll-like receptor (TLR15), a pattern recognition receptor (PRR) absent in mammals. We characterized the regulation and mechanism of CpG responsiveness via TLRs in chicken macrophage HD11 cells. TLR15 was significantly upregulated after induction with B- and C-type CpG oligonucleotides (ODN), tripalmitoylated lipopeptide (PAM3CSK4), Escherichia coli- and Salmonella enteritidis-derived lipopolysaccharide (LPS). In response to CpG-ODN inhibitor, TLR15 and IL1B were downregulated, but TLR21 was upregulated. IL1B was upregulated with CpG-ODN and downregulated after inhibitor treatment. The results suggest that responsiveness to different types of CpG-ODN in chicken macrophages requires multiple receptors, each with unique variation in expression. We utilized RNA interference (RNAi) technology to examine myeloid differentiation primary response gene (MyD88) dependency of TLR15 and TLR21. HD11 macrophages transfected with multiple MyD88-target siRNAs exhibited 70% decrease in MyD88 mRNA expression. IL1B was upregulated with CpG induction in cells with no reduction of MyD88 mRNA levels, but not in cells with 70% MyD88 reduction. Therefore, induction through TLR15 in response to CpG-ODN operates via the MyD88-dependent pathway in chicken macrophages.     

Testing strategies for embryo-fetal toxicity of human pharmaceuticals. Animal models vs. in vitro approaches : A workshop report

Reproductive toxicity testing is characterized by high animal use. For registration of pharmaceutical compounds, developmental toxicity studies are usually conducted in both rat and rabbits. Efforts have been underway for a long time to design alternatives to animal use. Implementation has lagged, partly because of uncertainties about the applicability domain of the alternatives.     

A flavonoid component from Docynia delavayi (Franch.) Schneid represses transplanted H22 hepatoma growth and exhibits low toxic effect on tumor-bearing mice

The fruit of Docynia delavayi (Franch.) Schneid is a kind of popular food in southwestern areas of China. Additionally, its rhizome has been long used as a folk medicine in the treatment of liver cancer by local people. Chrysin is a kind of flavonoid which induces cancer cell death in vitro. However, its anti-tumor activity in vivo and toxicological effects on the tumor-bearing animals still remain poorly understood. In this study, we obtained four flavonoids from this herb. Among them, chrysin showed the strongest cytotoxic effect on an array of cultured tumor cells. Further investigations revealed that it significantly repressed transplanted H22 ascitic hepatic tumor cell growth in vivo. Moreover, this compound displayed little toxic effects. Additionally, we demonstrated that in transplanted tumor tissues, chrysin not only activated caspase-3 and induced apoptosis, but also inhibited the production of vascular endothelial growth factor (VEGF) and suppressed angiogenesis. These data showed that chrysin exhibited prominent anti-tumor activities and low toxic effects in vivo.     

RNA干扰Stat3基因对EC-1细胞侵袭转移和增殖能力的影响

目的：构建针对信号转导子和转录激活子3（Stat3）基因的siRNA表达载体,研究Stat3基因沉默对人食管鳞癌EC-1细胞侵袭转移及增殖的影响.方法：构建pSUPER-EG-FP-Stat3siRNA表达载体,转染EC-1细胞,RT-PCR,Western Blot检测Stat3的表达;Boyden-chamber体外侵袭实验检测siRNA对细胞侵袭转移能力的影响,MTT法检测siRNA对细胞增殖能力的作用.结果：酶切和测序证实pSUPER-EGFP-Stat3siRNA表达载体构建成功;RT-PCR,Western Blot结果表明,siRNA能有效地降解EC-1细胞中Stat3基因的mRNA,下调Stat3蛋白的表达;转染siRNA后,与对照组相比,EC-1细胞侵袭转移和增殖能力明显下降.结论：成功构建针对Stat3基因的siRNA表达载体,可有效沉默Stat3基因在食管鳞癌EC-1细胞中的表达,抑制EC-1细胞的侵袭转移及增殖能力,可为食管癌的基因治疗提供理论依据.     

做好伦理委员会主席之我见

伦理委员会主席在伦理委员会中扮演着一个相当重要的角色。他必须是一个相当热情和热心于伦理委员会工作的领导者,他必须是一个医学、法学、伦理学和管理学的专家。伦理委员会主席的主要职责就是要积极鼓励伦理委员会委员做好伦理评审工作,不要受任何外界因素的干扰。     

腹主动脉阻断术临床原理及其上中下段安全时限探讨——兼述缺血再灌注损伤机制与对策及“二叉树耐受法则”

目的深入探讨腹主动脉阻断术的临床原理及其高中低位阻断安全时限等问题,以期在临床上安全地使用该技术。方法1980年-1982年我们首次进行了腹主动脉阻断安全时限及其阻断与撤钳后病理生理变化的前瞻性实验研究。结果表明:犬腹主动脉阻断25min为安全时限;在阻断安全时限内,犬体内血液动力学、血气酸碱、血液生化和主要的脏器超微结构(脑肺肝肾胰肠脊髓)仅发生轻度可逆性改变;如超出安全时限,上述指标则会发生难以代偿的严重改变。基于广义生命时空场论学说,通过对腹主动脉阻断术解剖学基础,腹主动脉阻断后与撤钳后综合征群(呼吸宭迫综合征、急性高血压性脑水肿、多脏器微栓塞病细胞综合征、撤钳后中枢性呼吸衰竭)及腹主动脉阻断所致的缺血再灌注损伤病理生理与细胞分子生物学机制等讨论,并根据近期实验与临床研究结果和结合文献复习对相关问题做了探讨,对临床上预防和治疗腹主动脉阻断术所致的缺血再灌注损伤提出了一整套对策。此外还叙述了预防腹主动脉阻断术并发症的远端主动脉灌注、脑脊液引流以及硬膜外冷却、控制性再灌注与缺血预/后处理等几种新技术。结果如果超出腹主动脉阻断术安全时限,则在术中与术后将发生缺血再灌注损伤、全身炎症反应综合征和多脏器衰竭。临床上,人类一次性持续腹主动脉阻断术安全时限如下:高位(T12水平,腹腔干A上)、中位(L2水平,肾A上)与低位(L4水平,髂总A上)均不能轻易超越25-30min。如术中须延时宁可采用间断阻断法。结论如果"二叉树耐受法则"(即体内任何一处大血管阻断均不能超出其固有安全时限,否则受术者非死必伤)被严格遵守,则腹主动脉阻断术中与术后病人的安全将得到保证。