产后抑郁症与雌激素受体β基因的多态性

目的 探讨汉族女性产后抑郁症与雌激素受体β (estrogen receptor β,ERβ)基因多态性的相关性.方法 收集产后抑郁症124例(观察组)和无抑郁症状的年龄匹配的健康女性144例(对照组),采用PCR-限制性片段长度多态性(PCRRFLP)法分析ERβ第5外显子Rsa-Ⅰ酶切多态性和第8外显子Alu-Ⅰ酶切多态性.结果 两组ERβ第5外显子Rsa-Ⅰ酶切多态性基因型rr、Rr、RR频率和等位基因r、R频率比较,差异无统计学意义(P＞0.05).两组ERβ第8外显子Alu-Ⅰ酶切多态性基因型aa、Aa、AA频率比较,差异无统计学意义(P＞0.05);产后观察组A等位基因频率高于对照组(P＜0.05).结论 ERβ第8外显子Alu-Ⅰ多态性可能与常州本地区汉族女性产后抑郁症的易感性有关,含A等位基因的基因型增加患产后抑郁症风险,而ERβ第5外显子Rsa-Ⅰ酶切多态性与本地区汉族女性产后抑郁症发病风险无相关性.     

中国人冠脉病变与载脂蛋白E基因多态性关系的研究

目的：研究中国人冠脉病变与载脂蛋白E（ApoE)基因多态性的关系。方法：选择72例经冠状动脉造影证实的冠心病患者及年龄，性别相匹配的90例正常对照组，聚合酶链反应－限制性片段长度多态性（PCR－RFLP）方法检测ApoE基因型。比较基因型及等位基因频率分布。结果：冠心病组ε4等位基因频率（12．50％）明显高于对照组（5．55％）（P＜0．05），1支，2支及3支冠脉病变间ApoE基因型及等位基因无显著差异。结论：ε4等位基因可能是冠心病的遗传易患因子之一。ApoE基因多态性与冠脉病变严重程度无关。     

PCR／RFLP用于黑龙江立克次体的鉴定

为探讨应用聚合酶链反应／限制性片段长度多态性图谱分析(PCR／RFLP)用于立克次体鉴定的高效性和特异性，将PCR／RFLP用于2株黑龙江立克次体的鉴定，并与以往其它鉴定黑龙江立克次体的分析结果作比较。结果表明，结论一致，均可鉴定出黑龙江立克次体为斑点热群立克次体的一新种。结论：PCR／RFLP以其简便、可信，采用菌体少等优点．适合在实验室进行立克次体研究时广泛应用。     

聚合酶链反应限制性片段长度多态性快速检测模拟尿标本中致病念珠菌的研究

目的研究常见的致病性念珠菌的分子生物学鉴定方法,为深部念珠菌的分子诊断奠定基础。方法用白色念珠菌、热带念珠菌、净平滑念珠菌和克柔念珠菌的标准菌株制备模拟尿标本,对其核糖体DNA内转录间隔区进行聚合酶链反应扩增,对扩增产物进行内切酶MspⅠ的酶切分析。结果4种念珠菌经聚合酶链反应后产生4种不同分子量的条带,扩增产物经酶切后产生4种特异性带型。结论聚合酶链反应—限制性片段长度多态分析方法是一种可靠、稳定、特异的鉴定常见致病念珠菌的方法。     

中国人冠脉病变与载脂蛋白E基因多态性关系的研究

目的 :研究中国人冠脉病变与载脂蛋白E(ApoE)基因多态性的关系。方法 :选择 72例经冠状动脉造影证实的冠心病患者及年龄、性别相匹配的 90例正常对照组 ,聚合酶链反应 -限制性片段长度多态性(PCR -RFLP)方法检测ApoE基因型。比较基因型及等位基因频率分布。结果 :冠心病组ε4等位基因频率(12 5 0 % )明显高于对照组 (5 5 5 % ) (P <0 0 5 )。 1支、2支及 3支冠脉病变间ApoE基因型及等位基因无显著差异。结论 :ε4等位基因可能是冠心病的遗传易患因子之一。ApoE基因多态性与冠脉病变严重程度无关。     

低密度脂蛋白受体基因多态性及其与慢性丙型肝炎的相关性

目的探讨我国汉族正常人群及慢性丙型肝炎(chronicviralhepatitisC,CHC)患者低密度脂蛋白受体(LDL-R)基因外显子12中HincⅡ多态性的分布及其与慢性丙型肝炎之间的关系。方法采用聚合酶链反应结合限制性片段长度多态性(PCR-RFLP)的方法,对84例慢性丙型肝炎病毒(HCV)感染者和72名健康献血员进行LDL-R基因HincⅡ多态性检测。结果在HCV感染组和健康对照组、慢性丙型肝炎组和HCV相关肝硬化组、丙氨酸转氨酶(ALT)正常组和ALT升高组,以及HCVRNA阴性组和HCVRNA阳性组之间,LDL-R基因外显子12HincⅡ多态性分布差异无统计学意义。结论人群LDL-R基因外显子12中存在着HincⅡ多态性位点,但该多态性位点可能与慢性HCV感染无相关性。     

结核病临床分离株及痰标本中embB基因型的快速测定

目的建立快速测定结核病耐乙胺丁醇(EMB)分离株和痰标本中结核分枝杆菌EMB耐药基因型的方法，以期为患者提供及时、有效地化验结果。方法应用16S rDNA聚合酶链反应(PCR)-单链构象多态性(SSCP)和PCR-限制性片段长度多态性(RFLP)对11株耐EMB分离株和46例结核病痰标本进行分子菌种鉴定和embB基因突变的部位与性质分析。结果以结核分枝杆菌H37Rv标准株为对照，11株耐EMB分离株和46例临床痰标本经16S rDNA PCR-SSCP分析电泳图谱均与结核分枝杆菌标准株相同；限制性内切酶NlaⅢ消化embB基因扩增产物显示，11株耐EMB分离株中4株(36．4％)不被NlaⅡ消化；46例结核病痰标本中10例(21．7％)不被NlaⅡ消化。结论部分结核分枝杆菌耐EMB是由于embB基因突变所致。采用PCR-SSCP和PCR-RFLP方法可直接快速测定结核病耐EMB分离株和痰标本中结核分枝杆菌耐EMB基因型。     

佛山市汉族人群HIFIA基因1790（G→A）多态性研究

目的研究低氧诱导因子-1α基因（HIF1A）第12外显子1790（G→A）单核苷酸多态性在广东佛山市汉族人群中的分布特征。方法随机选取佛山市汉族健康个体90人的血样,提取白细胞基因组DNA,利用限制性片段长度多态性-聚合酶链反应（restriction fragment length polymorphisrm-polymerase chain reaction, RFLP-PCR）技术检测HIF1A基因第12外显子1790（G＋A）的单核苷酸多态性基因型,并分析其基因多态性特征。结果HIF1A基因1790（G→A）单核苷酸多态性的GG、GA和AA基因型频率分别为75．56％、21．11％和3．33％,G、A等位基因频率分别为86．11％、13。89％。广东佛山汉族人群HIF1A基因1790（G→A）单核苷酸多态性等位基因分布频率与日本人群相比差异有显著意义（P〈0．05）。结论广东佛山汉族人群HIF1A基因1790（G→A）多态性以GG基因型分布频率高,具有一定的种族差异性。     

中国汉族人群中TNF-α-1031位点基因多态性分布的研究

目的 了解肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)启动子上游-1031位点基因多态性在中国汉族正常人群中的分布特点.方法 应用聚合酶链反应(PCR)和限制性片段长度多态性(RFLP)方法对正常个体基因组DNA中TNF-α-1031位点基因多态性进行了观察,探讨其基因型及等位基因分布特点.结果 正常汉族人群中TNF-α-1031位点基因型以TT型发生频率最高(64.4%),TC型较少(33.0%),CC型最少(2.6%),T等位基因的频率为80.9%,C等位基因的频率为19.1%,这种分布特点在正常男女间差异不显著(P＞0.05).与其它种族相比较,TNF-α-1031位点基因型在中国正常人群中的分布与日本人群中的分布相似(P＞0.05).结论 中国汉族正常人群中存在TNF-α-1031多态性,其分布特点与日本人群中的分布相似.     

佛山市汉族人群HIF1A基因1790(G→A)多态性研究

目的 研究低氧诱导因子-1α基因(HIFlA)第12外显子1790(G→A)单核苷酸多态性在广东佛山市汉族人群中的分布特征.方法 随机选取佛山市汉族健康个体90人的血样,提取白细胞基因组DNA,利用限制性片段长度多态性-聚合酶链反应(restriction fragment length polymor-phism-polymerase chain reaction,RFlP-PCR)技术检测HIFlA基因第12外显子1790(G→A)的单核苷酸多态性基因型,并分析其基因多态性特征.结果 HIF1A基因1790(G→A)单核苷酸多态性的GG、GA和AA基因型频率分别为75.56%、21.11%和3.33%,G、A等位基因频率分别为86.11%、13.89%.广东佛山汉族人群HIF1A基因1790(G→A)单核苷酸多态性等位基因分布频率与日本人群相比差异有显著意义(P<0.05).结论 广东佛山汉族人群HIF1A基因1790(G→A)多态性以GG基因型分布频率高,具有一定的种族差异性.     

Validation of a Ligand Binding Assay Using Dried Blood Spot Sampling

Dried blood spots (DBS) technology has been introduced as a microsampling alternative to traditional plasma or serum sampling for pharmacokinetics or toxicokinetics evaluation. The application of DBS has been established for many small molecule drugs at discovery, nonclinical, and clinical stages. However, the application of DBS for large molecule therapeutics development is not yet well-established. This article describes the method validation of a ligand binding assay (LBA) for DBS sampling of a therapeutic monoclonal antibody—AMG 162 (Denosumab). The original serum LBA was modified for the DBS method. A fit-for-purpose method validation was performed to evaluate accuracy and precision, selectivity, dilutional linearity, and stability. In addition, the parameters relevant to DBS, such as spot volume, extraction recovery, whole blood stability, and hematocrit effects, were evaluated. The validation results demonstrated assay robustness with inter-assay precision of ≤19%, inter-assay accuracy of ≤9%, and total error of ≤24%. Selectivity, extraction recovery, dilutional linearity, and stability were demonstrated. The validation results revealed some limitations of the possible effect of blood hematocrit on therapeutic concentration measurements and the caution required using whole blood for standards and quality controls preparation. This is the first article to describe a thorough method validation of an LBA using DBS for a therapeutic monoclonal antibody. The lessons learned can serve as a model process for future method validation of other LBAs for large molecule therapeutics or biomarkers using the DBS sampling method.     

Dried Blood Spot Technique Applied in Therapeutic Drug Monitoring of Anticancer Drugs: a Review on Conversion Methods to Correlate Plasma and Dried Blood Spot Concentrations

Pharmaceutical Research - Anticancer drugs are notoriously characterized by a low therapeutic index, the introduction of therapeutic drug monitoring (TDM) in oncologic clinical practice could...     

Activation of Estrogen Receptor by Bavachin from Psoralea corylifolia

In this study, we examined the estrogenic activity of bavachin, a component of Psoralea corylifolia that has been used as a traditional medicine in Asia. Bavachin was purified from ethanolic extract of Psoralea corylifolia and characterized its estrogenic activity by ligand binding, reporter gene activation, and endogenous estrogen receptor (ER) target gene regulation. Bavachin showed ER ligand binding activity in competitive displacement of [³H] E₂ from recombinant ER. The estrogenic activity of bavachin was characterized in a transient transfection system using ERα or ERβ and estrogen-responsive luciferase plasmids in CV-1 cells with an EC₅₀ of 320 nM and 680 nM, respectively. Bavachin increased the mRNA levels of estrogen-responsive genes such as pS2 and PR, and decreased the protein level of ERα by proteasomal pathway. However, bavachin failed to activate the androgen receptor in CV-1 cells transiently transfected with the corresponding receptor and hormone responsive reporter plasmid. These data indicate that bavachin acts as a weak phytoestrogen by binding and activating the ER.     

CAT基因突变热点区域的PCR扩增方法

目的探讨过氧化氢酶基因突变热点区域PCR扩增方法,提高PCR反应的特异性和灵敏度,有助于快速检测CAT基因相关疾病。方法从人静脉血液标本提取人血液基因组DNA,设计引物扩增特定的CAT基因片段,联合应用热启动PCR和降落PCR技术。结果建立了重复性好,分辨率高的PCR反应体系。结论建立了适用于CAT基因突变热点区域的PCR反应体系,有助于快速检测CAT基因相关疾病。     

广州地区绝经后妇女雌激素受体基因多态性与骨密度的关系

目的探讨雌激素受体（ER）基因XbaI多态性与广州地区部分汉族妇女骨密度相互关系。方法随机筛选年龄42～75岁广州汉族妇女157例,采用双能X线吸收法（DEXA）测定其全身、腰椎2～4（L2-4）、股骨颈（Neck）、Ward’S三角和大转子区等部位的骨密度（BMD）值,并采用聚合酶链反应限制性片段长度多态性（PCR-RFLP）方法检测外周血白细胞基因组ER基因型。结果157例受试对象中,ER基因型分别为XX型13例,Xx型67例,XX型77例,基因型分布符合Hardy—weinberg定律。携带XX基因型的妇女个体在各部位比Xx及XX型携带者较高的骨密度值,但无统计学意义。结论ER基因多态性与骨密度无相关性;ER基因多态性不是广州地区绝经后妇女骨质疏松的易感因子。     

Dried Blood Spot Analysis of Donepezil in Support of a GLP 3-month Dose-Range Finding Study in Rats

Donepezil hydrochloride is a reversible acetyl cholinesterase inhibitor approved for Alzheimer disease treatment. As an alternate therapy, a donepezil hydrochloride transdermal patch is in development. Recommended nonclinical safety studies include a 3-month Good Laboratory Practice (GLP) dose-range finding (DRF) study prior to conducting the 2-year dermal carcinogenicity study in rats. Demonstration of systemic exposure is necessary to interpret the in vivo data. Previous nonclinical reports supporting oral dosing have utilized liquid chromatography tandem mass spectrometry (LC/MS/MS) to quantify donepezil concentrations in plasma. Smaller species with limited blood volumes do not allow serial sampling to derive the full pharmacokinetic profile from a single animal. Therefore, the option of another analytical method requiring decreased sample volumes is desirable as it would decrease the required number of animals while obtaining the complete profile. The dried blood spot (DBS) technique allows drug level measurement from a few microliters; however, the method is still not widely utilized in GLP studies. Because donepezil plasma levels are known by the oral route, DBS was used to bridge the previous oral data and to support a 13-week GLP DRF study for repeated topical application in rats, comparing oral administration with 4 topical formulations. The DBS method was validated and demonstrated robustness and reproducibility for application to the DRF study. The assay results were comparable to a previously reported plasma LC/MS/MS assay-derived pharmacokinetic profile and provided justification for selection of the topical formulation and dose levels for the subsequent dermal carcinogenicity study.     

SLE患者PBMCs雌激素受体mRNA水平与IL-10 mRNA水平的研究

目的：探讨未经刺激外周血单个核细胞（PBMCs）雌激素受体（ER）mRNA水平和白细胞介素10（IL-10）mRNA水平在系统性红斑狼疮发病中的作用。方法：分离25例系统性红斑狼疮（SEE）患者（患者组）和15例正常人（对照组）新鲜外周血单个核细胞，提取总RNA，利用半定量RT-PCR方法逆转录并扩增雌激素受体和IL-10．通过凝胶图像扫描系统对PCR产物的电泳条带进行密度扫描。结果：患者组PBMCsERmRNA水平和IL-10mRNA水平均高于正常对照组（P〈0．01）。患者组和对照组PBMCsERmRNA水平与IL-10mRNA水平均呈正相关（r分别为0．442和0．557，P〈0．05）。结论：SLE患者外周血单个核细胞存在雌激素受体mRNA和IL-10mRNA表达异常．IL-10表达的上调可能与雌激素受体有关．过量的IL-10可能导致了SLE患者B细胞产生大量自身抗体。