METase在不同原核表达系统中的表达和活性比较

目的：构建L-蛋氨酸γ-裂解酶（Metase）基因的两种高效原核表达载体，建立最佳高效原核表达体系。方法：在Metase基因已经克隆的基础上，通过分子克隆技术构建两种重组表达质粒pBV220-Metase和pGEX-4T-1-Metase,重组质粒转化感受态大肠杆菌Dh5α,诱导表达，测定Metase活性，统计分析比较。结果：成功构建出两种高效重组表达质粒pBV220-Metase和pGEX-4T-1-Metase,经诱导后都能测出Metase活性。结果：重组表达质粒pBV220-Metase具有比pGEX-4T-1-Metase更强的活性，可作为Metase的高效表达体系。     

Age determines memory for face identity and expression

Background: The recognition of facial expressions is an important component of emotion processing which contributes to interactional behavior. One of the factors highly associated with potential decline of ability in behavioral tasks is age. Methods: We have investigated age‐related changes in facial identity and expression memory of healthy subjects in three age groups: young adults (20–40 years), elderly adults (60–80 years) and, for the first time in the literature, very old adults (over 80 years of age). Using a picture test, photographs of faces with happy or angry expressions were presented to study participants during the encoding task, and the memory for identity and emotional facial expression was investigated in a subsequent recognition task showing emotionally neutral faces. Half of the faces presented in the recognition task were initially shown in the encoding task. Results: Age interacted with the memory process: the ability to recognize both facial identity and emotional expression declined with advanced age. Happy facial expressions were better recognized in all age groups. Although there was a continuous overall decrease in recognition of both happy and angry expressions with advanced age, the effect favoring happy facial expressions was stable also in very old adults. Other factors such as gender or educational level did not affect the memory process for facial expressions. Conclusions: Our findings suggest that age is a significant determinant of memory for facial identity and emotional expression, and that, similar to younger adults, the recognition process of the elderly favors happy emotional facial expressions.     

凋亡素基因序列重组及其真核表达载体的构建

目的:提高凋亡素基因在肿瘤细胞中的表达效率,增强其诱导肿瘤细胞凋亡的生物学活性.方法:通过两次PCR,以pcDNA-VP3质粒为模板,扩增出带Kozak序列(真核核糖体结合位点)的凋亡素基因VP3.将其克隆到真核表达载体pRevTRE的多克隆位点,构建成凋亡素的真核表达载体pRevTRE-VP3,将该质粒分别以限制性内切酶BamHI和XhohI进行酶切鉴定,将初步鉴定显示可能克隆有目的基因的质粒进行测序鉴定.结果:测序结果表明,本研究合成的凋亡素基因与Noteborn报告的凋亡素基因序列一致,同源性为l 00%.在其起始密码子前添加了Ko zak序列的凋亡素基因已成功克隆到真核表达载体pRevTRE.结论:采用两次PCR法可提高表达效率的真核核糖体结合位点添加到凋亡素基因序列起始密码子前端,并构建成凋亡素真核表达载体.     

Gene expression profiling of human lymph node metastases and matched primary breast carcinomas: Clinical implications

The genetic program that drives tumor metastasis and the mode and timing of its initiation are of great practical significance to clinical management. Modern technical advances open new opportunities for gaining useful relevant information. Gene expression profiles of histologically‐verified viable tissue from lymph node metastases were compared with those of matched primary breast cancers from 10 different patients, among samples from over 400 cases, using high‐throughput oligonucleotide arrays comprising probes for 22,000 genes. It was observed that metastases have very similar expression signatures to their parent tumors. However, detailed computational analysis revealed that a small number of genes were consistently differentially expressed between 100% of tumors and metastases, suggesting that these are mechanistically important. Lists of such candidate genes, of potential clinical interest, are provided. We interpret these results in the framework of a meta‐analysis of previous investigations by others and ourselves and of existing clinical knowledge on the behavior of human tumors. The collective data show that metastases resemble their primary tumors but the signatures obtained in different studies are not sufficiently reproducible or informative to be prognostically useful, although they do give valuable insights into the pathogenesis and biology of human tumor metastasis. The findings indicate that the genetic program encoding metastasis is implemented progressively over time although, occasionally, this evolution can occur rapidly, early in the life of the neoplasm. The important clinical significance of this deduction is that, in most patients, early detection provides time for appropriate therapeutic intervention to be effective in obstructing metastasis.     

肝癌多药耐药产生与低糖环境的关系

目的探讨局部微环境低糖与肝细胞癌多药耐药性(MDR)产生的关系及影响机制。方法低糖培养HepG2细胞,应用流式细胞术Annexin V/PI法检测低糖培养的细胞在化疗药物5-氟脲嘧啶(5-Fu)作用后的凋亡情况,分别应用荧光定量聚合酶链反应(PCR)技术和Western blot技术检测低糖培养后HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP的mRNA和蛋白水平的表达。结果在低糖环境下生长时间越长的HepG2细胞对5-Fu的抵抗越强,而且随着低糖培养时间的增加,5-Fu诱导的HepG2细胞的凋亡高峰延迟。低糖培养的HepG2细胞内多药耐药相关基因mdr1、MRP1、LRP在mRNA和蛋白水平的表达随低糖培养时间的延长而升高,以LRP的改变最为显著。结论肝癌生长微环境葡萄糖耐量不足也是肝癌产生MDR的原因之一。低糖可通过上调一组多药耐药相关基因的表达而诱导肝癌的多药耐药性。     

巨噬细胞移动抑制因子与鼻咽癌微血管生成和淋巴结转移的关系

[目的]研究巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在鼻咽癌组织中的表达水平及其与肿瘤新生血管的关系,探讨细胞因子在鼻咽癌细胞侵袭转移过程中的作用.[方法]收集1999-2003年期间45例确诊未治的鼻咽原发癌活检组织标本,用LSAB免疫组化法检测鼻咽癌组织中MIF和VEGF表达,计数癌组织中新生血管热区的CD34阳性微血管密度(intratumoral microvessel density,IMD),并将各检测指标与患者的病理及临床资料相联系.[结果]鼻咽原发癌组织中,癌细胞MIF和VEGF的阳性表达率分别为78%(35/45)和71%(32/45),伴有淋巴结转移的癌组织中MIF和IMD水平均高于无淋巴结转移的癌组织,P=0.029,P=0.026,MIF阳性组的IMD计数为(35.1±13.3)/高倍视野,明显高于MIF阴性病例的(20.9±10.2)/高倍视野,P=0.003.以Schmincke型生长方式分布的癌细胞MIF表达水平(67.4%±35.2%)高于以Regaud型方式分布的癌细胞(32.9%±29.7%),P=0.007.癌细胞MIF与VEGF表达以及IMD计数均显示正相关关系,P＜0.05.但癌细胞VEGF的表达与患者性别、年龄、病理组织学分型以及临床分期无统计学显著意义.[结论]MIF在鼻咽癌细胞的淋巴结转移过程中可能具有重要的促进作用,并可能是影响肿瘤性微血管生成和癌细胞VEGF表达的重要因素.     

CAD基因原核表达载体的构建及表达产物的活性鉴定

目的构建pCool—GST—ICAD／CAD的表达载体，并存大肠杆菌BL21（DE3）中表达具有生物学活性的CAD核酸酶？方法用PCR扩增CAD基因，将扩增产物克隆入pCool—GST—ICAD载体中构建出pCool—GST—ICAD／CAD表达载体。经酶切和电泳鉴定正确后，在大肠杆菌BL21（DE3）中诱导表达，表达产物经亲和层析、离子交换层析及凝胶过滤等方法分离纯化。最后用SDS—PAGE和DNA降解实验进行鉴定。结果构建了pCool—GST—ICAD／CAD原核表达载体，重组载体转化后表达出毫克级水平的GST—ICAD／CAD蛋白复合体。经分离纯化后得到纯度很好的CAD—ICAD篮白复合体，在SDS—PAGE电泳上旱现清晰的两条蛋白带。经DNA降解实验证明纯化所得的CAD蛋白具有非特异性降解DNA的核酸酶作用。结论成功制备了具有生物学活性的CAD核酸酶，为进一步研究细胞凋亡的作用机制提供了有效的制剂。     

白介素-6、血管内皮生长因子、D-二聚体对CSDH的影响

目的探讨血肿局部炎症、假膜新血管生成、局部纤溶状况及其在CSDH发生、发展中的作用。进而探讨CSDH的发病机制，并为CSDH的治疗及预防复发提供理论依据。方法以78例CSDH患者作为病例组，20例健康人作为正常对照组。采用ELISA法测定患者血清及血肿液中VEGF及IL-6的含量。比较患者末梢静脉血及血肿液中四种因子的含量变化并与正常对照组比较。结果病例组血肿液FDP、d-dimer检测均为阳性，血液为阴性；正常对照组血液FDP、d-dimer检测均为阴性；病例组血清VEGF含量与正常对照组比较差异无统计学意义。血肿液中VEGF浓度高于血清中。病例组血清IL-6浓度与正常对照组差异无统计学意义，血肿液中IL-6浓度高于血清中。CSDH患者血肿液VEGF、IL-6水平没有相关性。结论CSDH患者血肿液局部纤溶亢进，局部VEGF分泌旺盛，新血管生成活跃，局部炎症活跃，可导致CSDH不断扩大而参与CSDH发病机制。抗炎治疗、抑制VEGF的生理作用、有选择的对病人施行促凝治疗，可成为部分CSDH病人保守治疗及预防复发的有效手段。     

GPC3在卵巢癌中的表达及其临床意义

目的 探讨GPC3在卵巢癌中的表达及其临床意义.方法 利用实时荧光定量RT-PCR技术检测2004年1月～2005年10月手术获得的35例卵巢上皮性癌组织及23例正常卵巢组织中GPC3 mRNA的表达量,并结合卵巢癌的临床病理特点进行分析.利用Western blot技术检测4例卵巢癌组织和3例正常卵巢组织中GPC3蛋白的表达水平.结果 GPC3 mRNA在卵巢癌组织中的表达量低于正常卵巢组织(P＜0.05),部分卵巢癌组织无GPC3 mRNA表达.GPC3 mRNA的表达量与卵巢癌临床分期有关,在Ⅲ、Ⅳ期卵巢癌组织中的表达量显著低于Ⅰ、Ⅱ期(P＜0.05).GPC3 mRNA与CA125在卵巢癌中的表达无相关性(P＞0.05).GPC3蛋白在正常卵巢组织中的表达水平是卵巢癌组织中的2～3倍.结论 GPC3在卵巢癌的发生发展中起着一定作用,其与CA125联合应用有可能提高卵巢癌的阳性诊断率.