构建Loa22基因去信号肽片段原核重组表达载体

目的：构建赖型钩端螺旋体OmpA膜蛋白Loa22基因去信号肽片段的原核表达载体，并对其进行克隆表达。方法：实验于2004—12／2005—12在四川大学华西医学中心感染免疫研究室完成。以赖型钩端螺旋体017株基因组DNA为模板，PCR扩增Loa22基因去信号肽片段，亚克隆至原核表达载体pGEX-4T-1，经双酶切、PCR鉴定，筛选出阳性重组质粒克隆。经DNA测序正确后，转化大肠杆菌，利用IPTG进行诱导表达，通过SDS—PAGE鉴定表达产物。结果：PCR获得长516bp的片段。Loa22基因去信号肽片段与pGEX-4T-1的重组质粒构建成功。重组质粒经IPTG诱导后能在大肠杆菌中表达Mr45000的融合蛋白。结论：制备了Loa22基因去信号肽片段原核重组表达载体，为钩体新型疫苗的研究奠定基础。     

聚合酶链反应检测SEN病毒D亚型方法的建立

目的：研究建立SEN病毒D亚型（SENV－D）聚合酶链反应（PCR）检测方法。方法：选择SENV－D开放读码框1高度保守序列，设计合成2对特异性引物，建立检测SENV－D感染的套式PCR方法。对327例无偿献血和职业献血者进行SENV感染的检测，并对部分PCR阳性产物进行克隆测序，结果：该方法特异性和灵敏度均较高，检测结果显示无偿献血人群SENV－D感染率为5．5％，职业献血人群为6．7％，两者无统计学差异，结论：我国广东部分地区无偿献血和职业献血人群中存在SENV－D亚型感染；建立的该方法适用于SENV感染的检测。     

Borrelia burgdorferi basic membrane proteins A and B participate in the genesis of Lyme arthritis

Lyme arthritis results from colonization of joints by Borrelia burgdorferi and the ensuing host response. Using gene array-based differential analysis of B. burgdorferi gene expression and quantitative reverse trancription-polymerase chain reaction, we identified two paralogous spirochete genes, bmpA and bmpB, that are preferentially up-regulated in mouse joints compared with other organs. Transfer of affinity-purified antibodies against either BmpA or BmpB into B. burgdorferi-infected mice selectively reduced spirochete numbers and inflammation in the joints. B. burgdorferi lacking bmpA/B were therefore generated to further explore the role of these proteins in the pathogenesis of Lyme disease. B. burgdorferi lacking bmpA/B were infectious in mice, but unable to persist in the joints, and they failed to induce severe arthritis. Complementation of the mutant spirochetes with a wild-type copy of the bmpA and bmpB genes partially restored the original phenotype. These data delineate a role for differentially produced B. burgdorferi antigens in spirochete colonization of mouse joints, and suggest new strategies for the treatment of Lyme arthritis.     

自建NK细胞表面受体KIR2DS1基因mRNA表达的方法及验证

摘要:目的：建立检测NK细胞表面免疫球蛋白样受体基因 KIR2DS1 mRNA表达水平的荧光定量PCR方法并评价其性能。 方法：以行异基因造血干细胞移植的供患者共57对为研究对象，针对 KIR2DS1 基因设计特异性引物及探针，构建TaqMan-MGB荧光定量PCR反应体系，并评价该实验方法的性能，包括：总符合率、重复性、灵敏度及仪器适用范围、技术人员再现性。 结果：以KIR-SSO基因定性分型法为金标准，通过检测其中35例样品，荧光定量PCR方法对 KIR2DS1 基因阳性及阴性检测率均为100%。在重复性试验中，选取Ct值分别为高、中、低值3个样品的批内、批间CV分别为0.09%～0.46%、0.71%～1.13%。该方法的灵敏度达10² copies/μL，且对拷贝数为10² copies/μL的3个样品进行5次重复试验的CV分别为5.37%、2.71%、5.51%。仪器比对结果显示，参比仪器ABI-7500与实验仪器LC-480之间的拟合直线回归方程为Y=0.973 6X+0.118 3（R²=0.961 9，R²＞0.95）。2名技术人员对10个 KIR2DS1 基因阳性样品的再现性比对试验结果显示其偏倚（%）均＜±5%。 结论：建立了TaqMan-MGB荧光定量PCR检测 KIR2DS1 mRNA表达水平的检测方法，其性能良好。     

持续腰穿外引流防治脑血管痉挛的观察与护理

目的　探讨持续腰穿外引流对防治脑血管痉挛的疗效与护理。方法　 6 4例动脉瘤性蛛网膜下腔出血患者 ,在超早期行电解可脱卸弹簧圈 (guglielmidetachablecoil,GDC)栓塞术后 ,34例行持续腰穿外引流 ,另 30例行间断腰穿作为对照组 ,2组同样给予常规的尼莫通及 3H治疗 ,通过临床症状及经颅多谱勒超声 (TCD)判断血管痉挛的发生率及严重程度。结果　引流组发生症状性血管痉挛 4例 ,对照组 8例 ,其中 1例遗留神经功能障碍。引流组的脑血管痉挛发生率明显低于对照组。无引流相关并发症发生。结论　腰穿持续外引流可以有助于清除蛛网膜下腔积血 ,严密观察及合理护理对于减少并发症是必要的。     

中国汉族人群凝血因子Ⅸ基因限制性片段长度多态性的研究

为探讨中国人群中凝血因子Ⅸ（FⅨ）基因SalⅠ，NruⅠ和MseⅠ酶切位点的限制性片段长度多态性（RFLP），采用聚合酶链反应（PCR）方法，以三代内相互间无亲缘关系的正常汉族人为研究对象，对其FⅨ基因内含子1第192位以及5′端－793位核苷酸A和G的频率和5′端－698位核苷酸T和C的频率进行了分析，其中对FⅨ－192共分析了214条X染色体，对FⅨ－793共分析了210条X染色体，对FⅨ－698共分析了206条X染色体。结果表明，FⅨ基因内含子1第192位A和G的频率分别为0．878和0．122，杂合体频率为0．213；－793位核苷酸A和G的频率分别为0．552和0．448，杂合体频率为0．494，FⅨ－698位核苷酸T和C的频率分别为0．311和0．689，杂合体频率为0．429。结论提示：FⅨ基因SalⅠ,NruⅠ和MseⅠ酶切位点的RFLP有利于我国血友病B的携带检测和产前诊断。     

Abnormalities in Monocyte Recruitment and Cytokine Expression in Monocyte Chemoattractant Protein 1–deficient Mice

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1–deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1^−/− mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1^−/− mice, as was expression of IL-4, IL-5, and interferon γ in splenocytes. In contrast, MCP-1^−/− mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.     

不同体位干预对第三脑室肿瘤手术病人的影响

第三脑室的肿瘤手术一般需要采用枕部小脑幕入路，由于手术时间长．操作空间狭小，对手术体位的摆放要求甚高。既要充分暴露视野便于医生的手术操作．又要保护病人的神经、呼吸、循环等系统的重要生理功能，还要有利于麻醉师观察病人，避免病人神经、血管及各部位肌肉的损伤。     

Molecular diagnostics of genetic eye diseases

Eye diseases can be simple or complex, and mostly of heterogeneous molecular genetics. Some eye diseases are caused by mutations in a single gene, but some diseases, such as primary open angle glaucoma, can be due to sequence variations in multiple genes. In some diseases, both genetic and epigenetic mechanisms are involved, as was recently revealed in the mechanism of retinoblastoma. Disease causative mutations and phenotypes may vary by ethnicity and geography. To date, more than a hundred candidate genes for eye diseases are known, although less than 20 have definite disease-causing mutations. The three common genetic eye diseases, primary open angle glaucoma, age-related macular degeneration, and retinitis pigmentosa, all have known gene mutations, but these account for only a portion of the patients. While the search for eye disease genes and mutations still goes on, known mutations have been utilized for diagnosis. Genetic markers for pre-symptomatic and pre-natal diagnosis are available for specific diseases such as primary open angle glaucoma and retinoblastoma. This paper reviews the molecular basis of common genetic eye diseases and the available genetic markers for clinical diagnosis. Difficulties and challenges in molecular investigation of some eye diseases are discussed. Establishment of ethnic-specific disease databases that contain both clinical and genetic information for identification of genetic markers with diagnostic, prognostic, or pharmacological value is strongly advocated.     

呼吸运动周期中成人上呼吸道形态的动态观察

目的 观察正常人和阻塞性睡眠呼吸暂停综合征(Obstructive sleep apnea syncirome，OSAS)患者在呼吸运动周期中上呼吸道的形态学变化，比较正常人和OSAS患者间的差别。方法 经PSG确诊的OSAS患者共34人，年龄性别相近的正常成年人30人，以纤维内窥镜观察清醒平静呼吸时上呼吸道的形态变化，对受试者上呼吸道吸气和呼气时的形态分别拍照，利用计算机图像测量分析技术计算吸气和呼气之间上呼吸道形态学改变的幅度，比较正常人和OSAS患者间的差别。结果 正常人和OSAS患者，均表现为吸气时上呼吸道横截面积明显缩小，吸气中期达到最小，呼气开始则横截面积增大，呼气中期达到最大。OSAS患者在呼气和吸气间的改变幅度明显大于正常对照组，左右径的改变幅度大于前后径。软腭后区的改变幅度大于舌后区。结论 呼吸运动周期中上呼吸道的形态存在周期性的变化，OSAS患者的变化幅度明显大于正常对照者，这种较大的变化幅度是OSAS患者睡眠中发生上呼吸道阻塞的基础。