常染色体STR分型在同胞鉴定中的应用

目的研究常染色体STR基因座分型技术在同胞鉴定中的应用价值。方法采用24个STR基因座检测80对同胞个体和80对无关个体，ITO法计算同胞关系指数（PIFS）和同胞关系概率（wFS），分析两组的WFS值分布情况及每对个体等位基因全不同、全相同、半相同的基因座数，用Fisher＇s Exact Test方法比较后者的分布差异，并预测同胞与无关个体的界值。结果经X。检验，两组间24个STR位点基因型全相同与全不同的基因座数均存在显著性差异（P〈0．001），半相同的基因座数均无显著差异（P〉0．05）。结论选择24个STR基因座进行同胞鉴定具有一定的可行性，两个体24个STR基因座中全相同数≥8或全不同数≤3时，提示为同胞关系；全不同数≥9或全相同数≤1时，提示为无关个体。     

福建畲族人群15个短串联重复序列的遗传多态性及法医学应用

目的对福建畲族群体15个短串联重复序列等位基因遗传多态性进行分析,探讨其种族特异性及其在法医学个体识别和亲权鉴定的中的应用。方法荧光标记15个短串联重复序列(STR)位点和一个性别位点并进行PCR复合扩增,用ABI遗传分析仪以毛细管电泳技术对100例福建畲族无血缘关系健康个体PCR产物进行电泳分离基因,并以分析软件进行基因分型。结果共检出126个等位基因,基因频率范围在0.0050～0.5550。15个STR基因座的基因型分布均符合Hardy-Weinberg平衡。15个STR位点的HET在0.6300～0.8900之间,DP在0.7680～0.9590之间,EPP在0.3280～0.7750之间,PIC在0.5200～0.8600之间。累积DP和EPP分别达到0.99999999和0.99999288。结论15个STR位点均有较高的遗传多态性,并有丰富的信息含量,适合作为福建畲族地区的遗传标记,可用于个体识别和亲权鉴定。     

山西省高平地区汉族人群19个短串联重复系列基因座遗传多态性调查

目的:调查19个短串联重复系列(STR)基因座在山西省高平市汉族人群中的遗传多态性,并对其法医学应用进行评价。方法:采用Goldeneye20A STR荧光标记复合扩增试剂盒,对山西省高平市汉族210份无关个体进行扩增,利用3130XL遗传分析仪对扩增产物进行电泳分型,统计D19S433等19个STR基因座的等位基因频率和法医遗传学数据。结果:获得19个STR基因座的等位基因频率分布,分别检出10,9,15,13,13,6,8,7,7,7,10,8,9,5,17,6,11,13,17个等位基因,并分别获得19个STR基因座的杂合度观察值(Ho)、杂合度期望值(He)、个人设别能力(DP)、偶合率(PM)、非父排除率(PE)及多态信息总量(PIC)等法医遗传学参数,累积个人识别率和累积非父排除率分别为1~1.49×10-22和0.999 999 993。结论:Goldeneye20A STR荧光标记复合扩增体系的19个STR基因座在山西省高平市汉族人群中具有较高的个体识别能力和遗传多态性,对于法医学个体识别和亲子鉴定具有重要的应用价值。     

天津地区结核分枝杆菌可变串联重复序列多态性研究

目的:分析天津地区流行结核分枝杆菌(MTB)基因组中分布的可变串联重复序列(VNTR)拷贝数目多态性,探讨不同MTB菌株分辨能力强的VNTR基因分型方法.方法:根据VNTR位点PCR扩增产物片段长度分析临床分离的102株MTB菌基因组中22个VNTR位点中重复序列拷贝数目,使用Hunter-Gaston分辨指数(H)评估各个VNTB位点的多态性和VNTR基因分型方法对菌株的综合辨别能力.结果:13个VNTR位点对不同MTB菌株具有较强的分辨能力,其分辨指数达0.9963.其中QUB11b、11a、18、26,MIRU-26、31和Mtub21的多态性较好(H值为0.442～0.699);MIRU-10、27、39、40,QUB1895和ETR-A位点的多态性尚可(H值为0.203～0.316);而MIRU-2、4、16、20、23、24,QUB1451,ETR-B和C的多态性差(H值为0.000～0.150),其中MIRU-2、20、24和QUB1451在北京家族菌株中没有多态性(H=0).结论:选择多态性好的位点组合可以有效地区分不同的MTB北京家族菌株,建立起菌株分辨能力强的VNTR分型方法.     

应用六个STR基因座鉴定同胞关系

短串联重复序列（shor tandem repeat,STR）以核心序列重复单位数目的变化构成遗传多态性。在众多的STR基因座中,以三碱基重复或四碱基重复的STR基因座突变率低、稳定性好,且遵循孟德尔共显性遗传方式,是群体遗传学和法医遗传学高信息基因座的丰富来源[1]。本文参考有关文献[2-6],结合本实验室实验条件,筛选了6个STR座位进行扩增,并采用聚丙烯酰胺凝胶电泳及银染和（或）琼脂糖凝胶电泳EB染色进行带型分析,以多个同胞的STR片段长度等位片段,逆向推测其父母的相应四个位点,用于同胞间亲权鉴定。现报告如下。1 材料与方法     

应用六个STR基因座鉴定同胞关系

短串联重复序列(short tandem repeat,STR)以核心序列重复单位数目的变化构成遗传多态性.在众多的STR基因座中,以三碱基重复或四碱基重复的STR基因座突变率低、稳定性好,且遵循孟德尔共显性遗传方式,是群体遗传学和法医遗传学高信息基因座的丰富来源[1].     

荧光标记STR-PCR技术用于外周血干细胞移植监测

目的建立骨髓移植后监测的荧光标记STR-PCR检测方法,为白血病患者骨髓移植疗效评价提供可靠依据。方法建立荧光标记STR-PCR检测方法。于移植前、移植后7d至3个月不同时间采集供、受者血液提取DNA利用荧光标记STR-PCR方法进行分析。结果所检测的受者和供者均具有不同的DNA分型图。例1为HLA半相合母子,移植后监测结果仍为受者基因型,例2、3为HLA全相合的同胞,监测结果为完全供者基因型。结论荧光标记STR-PCR技术是一种准确、可靠的骨髓移植后监测方法。     

X染色体短串联重复序列基因座的遗传多态性

目的研究DXS7424基因座在山西汉族人群中的遗传多态性。方法随机抽取山西汉族人群250名无关个体静脉血样,乙二胺四乙酸抗凝后用酚-氯仿法提取DNA,经聚合酶链反应扩增,扩增产物用8%非变性聚丙烯酰胺凝胶电泳和银染法进行检测分型,对女性基因型频率分布进行Hardy-Weinberg平衡检验,并计算法医学常用参数。结果在山西汉族人群250名无关个体中共检出7个等位基因。等位基因频率分布在0.008～0.340。女性基因型频率分布符合Hardy-Weinberg平衡。个体识别率、多态性信息含量分别为0.7367、0.7039。结论DXS7424基因座在山西汉族人群中具有较高的遗传多态性,可用于法医学涉及女性的个人识别和亲权鉴定,并为X染色体短串联重复序列数据库的建立提供了山西汉族人群的遗传学数据。     

中国汉族人群遗传性聋基因座位STR位点的遗传多态性研究

目的 研究中国汉族人群10个遗传性聋基因座位的短串连重复序列(STR)的遗传多态性。方法 应用PCR方法对10个位点进行扩增，变性聚丙烯酰胺凝胶电泳分离，记录基因型。采用PPAP软件计算正常人各STR位点等位片段频率、基因型频率、预期基因型频率、多态信息量(PIC)和Hardy-Weinberg平衡吻合性检验。结果 中国汉族人群D6S287、D8S1132、D13S1275、D15S130、D1S2726、D4S3038、D2S2380、D22S282、D14S1042、D7S529各位点分别检出10个、9个、8个、7个、7个、7个、6个、6个、6个及5个等位片段，多态性分布均符合Hardy-Weinberg平衡定律。各位点PIC值分别为0．879、0．854、0．864、0．821、0．686、0．794、0．764、0．732、0．773、0．712；杂合度均高于0．7。结论 中国汉族人群10个位点STR均具有较高的杂合度和多态信息量，是较理想的遗传标记。     

Terminal telomere repeats are actually short in telomerase-negative immortal human cells

Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2-9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.     

抗菌肽cecropin-Xm在大肠埃希菌中的串联表达与抑瘤作用

在TNFα基因3’端连接抗菌肽cecropin—Xm基因多拷贝串联体，与温度诱导的表达载体pRCs组成表达载体pRCs—TNFα-（cecropin—Xm）。（n=1，2，3），并在大肠埃希菌BL21（DE3）中诱导表达融合蛋白TNFa-（cecropin—Xm）。（n=1，2，3）。SDS—PAGE结果显示，在同样表达条件下应用BL21（DE3）／pRCs—TNFα-（cecropin—Xm）2表达系统可获得比BL21（DE3）／pRCs—TNFα—cecropin—Xm系统多出-倍的目的物cecropin—Xm。融合蛋自经CNBr切割后用CM52纤维索柱分离纯化得到纯度大于95％的具有抑菌活性的抗菌肽cecropin—Xm，体外MTT抑瘤实验表明cecropin—Xm具有广谱杀灭肿瘤细胞的作用并且对正常细胞影响极小。     

The variable number of tandem repeats polymorphism of the dopamine transporter gene is not associated with significant change in dopamine transporter phenotype in humans.

A 40 base polymorphism of a variable number of tandem repeats (VNTR) has been described in the 3' untranslated region of the gene (SLC6A3) coding for the dopamine transporter (DAT). Despite being located in the untranslated region of the gene, this polymorphism has been associated with clinical phenotypes associated with dysregulation of dopamine transmission, such as attention deficit hyperactivity disorder and cocaine-induced paranoia. To examine the neurochemical phenotype associated with this polymorphism, we compared amphetamine-induced dopamine release (measured as displacement of the radiotracer [123I]IBZM) and DAT expression (measured with [123I]beta-CIT) in the striatum with Single Photon Computerized Emission Tomography (SPECT). Our sample included 59 subjects, 31 healthy controls and 29 patients with schizophrenia. No significant association was found between VNTR polymorphism and amphetamine-induced dopamine release or DAT density in the total sample, nor when each diagnostic group was considered separately. Thus, we did not replicate the findings of two previous studies, which had suggested that the 9 repeat allele was associated with either an increased or decreased DAT expression, albeit in different patient populations.     

Efficient identification of fungal antimicrobial principles by tandem MS and NMR database

The continuous re-isolation of the known and non-applicable compounds that is time-consuming and wasting resources is still a critical problem in the discovery of bioactive entities from natural resources. To efficiently address the problem, high performance liquid chromatography-diode array detector-microfractionation (HPLC-DAD-microfractionation) guided by disk agar diffusion assay was developed, and the active compounds were further identified using the tandem mass spectrometry (MS/MS)-based molecular networking. Of 150 fungal strains screened, the methanolic extracts of Phoma herbarum PPM7487, Cryptosporiopsis ericae PPM7405, and Albifimbria verrucaria PPM945 exhibited potent antimicrobial activity against Candida albicans SC5314 and Cryptococcus neoformans H99 in the preliminary agar diffusion assay. The concept of OSMAC (one strain many compounds) was employed in the fungal cultures in order to enrich the diversity of the 2^nd metabolites in this study. HPLC coupled with off-line bioactivity-directed profiling of the extracts enabled a precise localization of the compounds responsible for the conspicuous antimicrobial activity. The purified active compounds were identified based mainly on MS/MS database, and further supported by ¹³C nuclear magnetic resonance (NMR) spectral data compared to the literatures. In addition to nineteen known compounds, a new trichothecene derivative 1, namely trichoverrin D, was isolated and identified through this protocol. The antifungal activities of all the pure isolates were evaluated, and the structure activity relationships were also inferred. This report has demonstrated the combination of HPLC microfractination and MS/MS coupled by NMR spectral dereplication for speeding up the antimicrobial natural products discovery process.     

江苏汉族群体15个STR基因座遗传多态性分析

目的分析15个短串联重复序列(STR)基因座在江苏汉族群体中的基因多态性和基因频率。方法取2010年-2011年来自江苏省的1000例无血缘关系的汉族个体。用Sinofiler 16荧光标记复合扩增系统15个STR位点进行扩增,自动基因分析仪片段分析并进行基因分型。结果累积STR等位基因共发现211个,其中罕见等位基因36个。基因型频率超过0.1的基因座型22个,其中D3S1358基因座的15/16等位基因型频率最高达到0.2225。杂合度达到了0.802±0.051,随机个体相同表型偶合率值为0.068±0.031,多态信息总量值为0.778±0.060,个体识别能力和非父排除率值分别为0.932±0.032和0.607±0.091。FGA、D12S391基因座在发生24次突变的11个基因座中有较高的突变率。结论通过大量的样本实验和等位基因频率总结,得到了江苏人群更多法医学亲权鉴定和个人识别等位基因多样性的客观数据。     

First identification of dapagliflozin in human hair: Development of a new liquid chromatography with tandem mass spectrometry method

Sodium-glucose cotransporter 1 inhibitors are a new class of drugs used for the treatment of type II diabetes. Due to their diuretic capabilities and the glycosuria they induce, these molecules cause effective weight loss that could attract the interest of a wider public than diabetics with all the health consequences knowing the adverse effects of these substances. In order to reveal a past exposure to these substances, hair analysis can be very useful especially in the medicolegal context. There are no data in the literature about gliflozin testing in hair. In this study, a method was developed for the analysis of three molecules belonging to the gliflozin family (dapagliflozin, empagliflozin and canagliflozin) using a liquid chromatography system coupled to tandem mass spectrometry. After decontamination with dichloromethane, gliflozins were extracted from hair following incubation in methanol in the presence of dapagliflozin-d5. Validation showed acceptable linearity for all compounds between 10 and 10,000 pg/mg, with limit of detection and limit of quantification at 5 and 10 pg/mg, respectively. Repeatability and reproducibility were below 20% at three concentrations for all analytes. The method was subsequently applied to the hair of two diabetic subjects under dapagliflozin treatment. In one of the two cases, the result was negative, while in the second case, the concentration was 12 pg/mg. Due to the absence of data, it is difficult to explain the absence of dapagliflozin in the hair of the first case. Physico-chemical characteristics of dapagliflozin could explain its bad incorporation in hair, making detection difficult even after daily treatment.     

pU-VEGF-siRNA真核表达载体的构建及鉴定

目的构建针对血管内皮生长因子的发卡样siRNA真核表达载体pU-VEGF-siRNA,为进一步研究其对动脉粥样硬化的作用奠定基础。方法人工合成一对互补并编码相应短发夹状VEGF-siRNA的寡核苷酸链,将其插入到pSilencerTM2.1-U6 neo载体中,经测序鉴定所构建的重组载体是否正确。结果测序示pU-VEGF-siRNA序列正确。结论测序结果表明pU-VEGF-siRNA真核表达载体构建成功。