膀胱癌高危乳头瘤病毒定位研究及临床意义

目的：进一步探讨高危ＨＰＶ１６、１５与膀胱癌的病因学关系。方法：运用改良的原位杂交技术对５２例福尔马林固定石蜡包理膀胱乳头状移行细胞癌组织进行高危ＨＰＶＤＮＡ定位检测。结果：ＨＰＶ ＤＮＡ的阳性信号存在于肿瘤细胞核内,呈点状,也可为点片状,位于乳头癌全层,癌旁不典型增生上皮、癌旁正常的上皮组织及Ｂｒｕｎｎ氏巢可同时有高危ＨＰＶ的点片状表达。５２例膀光乳头状移行细胞癌中高危型ＨＰＶ１６、１８ＤＮＡ阳     

利用TaqMan-MGB探针检测A(H3N2)亚型流行性感冒病毒E119V耐药突变

目的 建立快速检测A(H3N2)亚型流行性感冒病毒(简称流感病毒)神经氨酸酶(M)基冈E119V奥司他韦耐药突变位点的双重实时荧光定量逆转录PCR(rRT-PCR)方法.方法 由GenBank获取2000-2012年A(H3N2)亚型流感病毒NA基因序列26条,据此设计特异性靶向NA E119V突变位点的TaqMan-MGB探针进行双重rRT-PCR反应,并利用耐药参考株、临床分离株进行敏感性、特异性和重复性评价.结果 建立了快速检测NA基因E119V位点的双重rRT-PCR检测方法.本方法在A(H3N2)病毒(HA=8)稀释度至10-5仍可检测到荧光信号,病毒滴度的对数与Ct值之间呈线性相关,具有较高的灵敏度;与无耐药突变的A(H3N2)流感病毒及其他呼吸道病毒均无交叉反应,两条TaqMan-MGB探针之问不存在交叉反应,具有很高的特异性,并能够检测耐药株和敏感株混合病毒中的耐药突变,在较高浓度的混合病毒中对耐药突变的检测限是5％,在低浓度混合病毒中的检测限是50％.重复性试验得到H3N2-119E和H3N2-119V两组探针批内平均变异系数(CV)值分别为2.32％和0.57％,批间CV值分别为1.77％和0.97％,具有较好的重复性.对其中20株病毒的NA基因序列进行分析,证实这些毒株的NA基因119位点均为谷氨酸(E),表明本研究设计的方法与序列测定结果一致.结论 建立了基于TaqMan-MGB探针检测A(H3N2)亚型流感病毒E119V耐药突变的双重rRT-PCR方法,该方法灵敏、特异、重复性好,实验过程和结果判读简单易操作.     

荧光原位杂交技术分析人结肠菌群方法研究

建立荧光原位杂交技术分析人体内结肠菌群的方法。取受试者新鲜粪便 ,选用 5种特异性的 16SrRNA寡核苷酸探针 ,检测粪便样本收集后的保存时间、温度 ,离心条件及样本固定液存放时间对杂交计数结果的影响。结果建立最佳实验条件为 :粪便样本收集后应尽快在 4℃下保存 ,放置时间不要超过 12小时即作处理 ;样本的适宜离心条件为 70 0g 2分钟 ;样本用多聚甲醛固定后在 - 80℃下存放时间不要超过 5个月。该方法具有较好的稳定性 ,可以有效地检出个体之间结肠菌群的差异。     

反向杂交膜片检测结核菌耐利福平基因突变

目的利用一种新型的反向点杂交膜片,快速检测结核分枝杆菌耐利福平rpoB基因突变.方法根据结核分枝杆菌rpoB基因序列设计寡核苷酸探针并固定于尼龙膜上,用生物素标记的引物增扩结核分枝杆菌rpoB基因片断,与膜片上探针杂交,膜片检测结果与常规药敏试验结果和测序结果进行对照.结果对63株临床分离株进行检测,10株利福平敏感株未检测到突变,53株耐利福平株,其中48株检测到突变.灵敏度为90.6%,特异度为100%,阳性预测值100%,阴性预测值66.7%.用反向点杂交膜片检测27份肺结核患者的痰标本,17份非结核的痰液标本.12份耐利福平的痰标本中,11份检测到突变,膜片检测与药敏结果的符合率为92.3%;15份利福平敏感的痰标本中,膜片检测结果有6份出现突变信号;17份非结核病的其他患者的痰液标本中有1份检测到突变.检测痰标本的灵敏度为91.7%,特异度为78.1%,阳性预测值61.1%,阴性预测值96.1%.结论反向点杂交膜片可简便、快速、较准确地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变.     

Peptide-based diagnostic and therapeutic agents: Where we are and where we are heading?

Peptides are increasingly present in all branches of medicine as innovative drugs, imaging agents, theragnostic, and constituent moieties of other sophisticated drugs such as peptide-drug conjugates. Due to new developments in chemical synthesis strategies, computational biology, recombinant technology, and chemical biology, peptide drug development has made a great progress in the last decade. Numerous natural peptides and peptide mimics have been obtained and studied, covering multiple therapeutic areas. Even though peptides have been investigated across the wide therapeutic spectrum, oncology, metabolism, and endocrinology are the most frequent medical indications of them. This review summarizes the current use of and the emerging new opportunities of peptides for diagnosis and treatment of various diseases.     

乙型肝炎病毒核酸探针压电晶体传感器的初步研究

核酸探针压电晶体传感器,即由特异的单链DNA片断与压电晶体镀膜电板交联固定制成探针,并与标本中待测的变性DNA杂交成双链,杂交前后DNA量的变化,可通过压电晶体振荡频率的改变判定。从含pBR322-HBVDNA的大肠杆菌HB101中提取纯化重组质粒DNA,制成乙型肝炎病毒核酸探针压电晶体传感器,检测12例乙型肝炎感染者血清标本,结果显示5例阳性,与PCR检测结果一致,提示其具有良好的特异性与敏感性,并且更快速、简便。     

生物技术药物中残留DNA检测方法的比较

生物技术药物是所有以生物质为原料的生物活性物质及其人工合成类似物通过现代生物技术制得的药物,包括细胞因子、重组蛋白、抗体、疫苗和寡核苷酸等,临床上已开始广泛应用,为制药工业带来了革命性变化.但是生物技术药物中残留DNA可能存在安全性问题,因此应尽可能将产品中残留DNA的水平降到最低.新版美国药典将推荐实时定量PCR法作为生物技术药物中宿主残留DNA检定的唯一标准方法.该法的技术优势在于序列特异性高、灵敏度高、重现性好,还可以实现定量检测,使得结果更为精确,从而为生物技术药物企业在工艺研究和成品质量控制方面提供了可靠的检测手段.此综述对4类检测方法进行了比较,重点比较两种实时定量方法.     

荧光杂交探针技术检测单核苷酸多态性

目的 建立一种基于荧光杂交探针技术快速检测单核苷酸多态性方法。方法 提取线粒体DNA，在LightCycler仪器上进行实时PCR和熔解曲线分析，测序验证。结果 熔解曲线分析获得的150个基因型分析结果与测序结果100％一致。结论 实时PCR和杂交探针技术能快速、有效的检测单核苷酸多态性。     

人型结核杆菌克隆DNA探针的制备及其应用的研究

用限制性内切酶ＰｓｔⅠ消化人型结核杆菌Ｈ３７ＲｖＤＮＡ,与质粒ｐＵＣ１９ ＤＮＡ连接后转化大肠杆菌,经同位素３２Ｐ标记的人型结核杆菌Ｈ３７Ｒｖ全染色体ＤＮＡ探针筛选出６个阳性克隆（ＭＴ１￣６）,其中克隆ＤＮＡ片段ＭＴ２（４．２Ｋｂ）、ＭＴ４（３．５Ｋｂ）和ＭＴ５（３．０Ｋｂ）标记成探针,检测了２２种分支杆菌标准株和１５侏人型结核杆菌临床分离侏以及２种非分支杆菌,其杂交结果完全相同,与人型结核杆菌及     

Neuroprotective effects of brimonidine against transient ischemia-induced retinal ganglion cell death: a dose response in vivo study

The purpose of this study was to investigate the dose-response effects of topically administered brimonidine (BMD) on retinal ganglion cell (RGC) survival, short and long periods of time after transient retinal ischemia. In adult Sprague-Dawley rats, RGCs were retrogradely labeled with the fluorescent tracer fluorogold (FG) applied to both superior colliculi. Seven days later, the left ophthalmic vessels were ligated for 90 min. One hr prior to retinal ischemia, two 5 microl drops of saline alone or saline containing 0.0001, 0.001, 0.01 or 0.1% BMD were instilled on the left eye. Rats were processed 7, 14 or 21 days later and densities of surviving RGCs were estimated by counting FG-labeled RGCs in 12 standard regions of each retina. The following have been found. (1) Seven days after 90 min of transient ischemia there is loss of approximately 46% of the RGC population. (2) topical pre-treatment with BMD prevents ischemia-induced RGC death in a dose-dependent manner. Administration of 0.0001% BMD resulted in the loss of approximately 37% of the RGC population and had no significant neuroprotective effects. Administration of higher concentrations of BMD (0.001 or 0.01%) resulted in the survival of 76 or 90%, respectively, of the RGC population, and 0.1% BMD fully prevented RGC death in the first 7 days after ischemia. (3) Between 7 and 21 days after ischemia there was an additional slow cell loss of approximately 25% of the RGC population. Pre-treatment with 0.1% BMD also reduced significantly this slow cell death. These results indicate that the neuroprotective effects of BMD, when administered topically, are dose-dependent and that the 0.1% concentration achieves optimal neuroprotective effects against the early loss of RGCs. Furthermore, this concentration is also effective to diminish the protracted loss of RGCs that occurs with time after transient ischemia.