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Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System
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作者 Tong Xie Hui Xu +5 位作者 congying zhao Yating Fang Yongsong Zhou Qiong Lan Chunmei Shen Bofeng Zhu 《Journal of Forensic Science and Medicine》 2023年第4期303-308,I0004-I0011,共14页
Background:A difficulty associated with forensic applications is the detection of degraded biological materials.Due to the large amplicon sizes of short tandem repeat alleles,valid genotyping results cannot be obtaine... Background:A difficulty associated with forensic applications is the detection of degraded biological materials.Due to the large amplicon sizes of short tandem repeat alleles,valid genotyping results cannot be obtained from degraded biological materials.Recently,insertion/deletion(InDel)polymorphisms have been used in forensic applications for their widespread distributions in the human genome,short amplicon sizes,and low mutation rates.Purpose:Human identification InDel panels have mostly been designed for European populations.Therefore,our laboratory independently developed a multiplex polymerase chain reaction(PCR)system with 35 polymorphic InDel loci to be used for human identification in China.Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system.Methods:The 35 InDel loci were screened in the database,and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology.Validation studies were performed on this novel panel,including accuracy,repeatability and reproducibility,species specificity,sensitivity,stability,forensic case sample detection,and mixture studies.In addition,forensic efficiency assessments were conducted in populations from different continents.Results:The data of validated studies indicated that the novel 35-InDel panel was accurate,stable,and efficient for forensic purposes.For human identification,the cumulative power of discrimination values for the these 35 InDel loci in East Asian,South Asian,European,American,and African populations were 0.999999999999995,0.999999999999995,0.999999999999971,0.9999999999999960,and 0.999999999998166,respectively.Conclusions:In this study,a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample,and this new assay was efficient and stable.The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations. 展开更多
关键词 35-InDel panel human identification insertion/deletion polymorphism validation study
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Genetic diversity analysis of forty-three insertion/deletion loci for forensic individual identification in Han Chinese from Beijing based on a novel panel 被引量:2
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作者 congying zhao Jinlong YANG +5 位作者 Hui XU Shuyan MEI Yating FANG Qiong LAN Yajun DENG Bofeng ZHU 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2022年第3期241-248,共8页
Due to the virtues of no stutter peaks,low rates of mutation,and short amplicon sizes,insertion/deletion(InDei)polymorphism is an indispensable tool for analyzing degraded DNA samples from crime scenes for human ident... Due to the virtues of no stutter peaks,low rates of mutation,and short amplicon sizes,insertion/deletion(InDei)polymorphism is an indispensable tool for analyzing degraded DNA samples from crime scenes for human identifications(Wang et al.,2021).Herein,a self-developed panel of 43 InDei loci constructed previously by our group was utilized to evaluate the genetic diversities and explore the genetic background of the Han Chinese from Beijing(HCB)including 301 random healthy individuals. 展开更多
关键词 INSERTION utilized SIZES
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负压微环境对人脐静脉血管内皮细胞新生的影响及其机制
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作者 董禹辰 黄容 +1 位作者 赵聪颖 李学拥 《中华烧伤与创面修复杂志》 CAS CSCD 北大核心 2022年第6期520-531,共12页
目的探究负压微环境对人脐静脉血管内皮细胞(HUVEC)新生的影响及其机制。方法采用实验研究方法。取第3~5代对数生长期HUVEC进行后续实验。取3批细胞,各批次细胞均分为常规培养24 h的正常对照组和单纯负压处理组以及加入17-丙烯胺基-17-... 目的探究负压微环境对人脐静脉血管内皮细胞(HUVEC)新生的影响及其机制。方法采用实验研究方法。取第3~5代对数生长期HUVEC进行后续实验。取3批细胞,各批次细胞均分为常规培养24 h的正常对照组和单纯负压处理组以及加入17-丙烯胺基-17-去甲氨基格尔德霉素(17-AAG)培养24 h的单纯17-AAG组与17-AAG+负压处理组,另采用自行设计研发的全自动三维细胞梯度负压加载装置对2个负压处理组细胞行持续8 h的间歇负压吸引(负压值为-5.33 kPa,吸引30 s、暂停10 s),第1个批次细胞处理完成后于培养0(即刻)、24、48、72 h,采用细胞计数试剂盒8法检测细胞增殖水平,样本数为6;第2个批次细胞处理完成后进行划痕试验,于划痕后12 h,在倒置相差显微镜下观察细胞迁移情况后计算细胞迁移率,样本数为3;第3个批次细胞处理完成后进行小管形成实验,于培养6 h,在倒置相差显微镜下观察成管情况后计算细胞成管总长度与分支节点数,样本数为3。取细胞,分为正常对照组、单纯负压处理组、17-AAG+负压处理组,同前相应组别进行处理,处理完成后,采用蛋白质印迹法检测细胞中热休克蛋白90(HSP90)、窖蛋白1、内皮型一氧化氮合酶(eNOS)、eNOS磷酸化位点1177蛋白表达并计算eNOS磷酸化位点1177/eNOS比值(样本数为3),采用免疫共沉淀(共沉淀HSP90与窖蛋白1、窖蛋白1与eNOS)与蛋白质印迹法检测各组细胞中窖蛋白1、eNOS的蛋白表达(样本数为3),采用免疫荧光双重染色法评估细胞中HSP90与窖蛋白1、窖蛋白1与eNOS的蛋白共定位情况。通过HADDOCK 2.4蛋白质-蛋白质对接程序对窖蛋白1和eNOS进行分子对接预测。数据分析采用析因设计方差分析、单因素方差分析、LSD法。结果与正常对照组相比,单纯17-AAG组细胞增殖水平于处理完成后培养24、48、72 h均明显降低(P<0.01),单纯负压处理组细胞增殖水平于处理完成后培养24、48、72 h均明显升高(P<0.01);与单纯17-AAG组相比,17-AAG+负压处理组细胞增殖水平于处理完成后培养48、72 h均明显升高(P<0.05或P<0.01);与单纯负压处理组相比,17-AAG+负压处理组细胞增殖水平于处理完成后培养24、48、72 h均明显降低(P<0.01)。划痕后12 h,与正常对照组的(39.9±2.7)%相比,单纯17-AAG组细胞迁移率明显降低[(10.7±2.7)%,P<0.01],单纯负压处理组细胞迁移率明显升高[(61.9±2.4)%,P<0.01];与单纯17-AAG组相比,17-AAG+负压处理组细胞迁移率明显升高[(37.7±3.7)%,P<0.01];与单纯负压处理组相比,17-AAG+负压处理组细胞迁移率明显降低(P<0.01)。处理完成后培养6 h,与正常对照组相比,单纯17-AAG组细胞成管总长度明显缩短(P<0.05)且分支节点数明显减少(P<0.05),单纯负压处理组细胞成管总长度明显延长(P<0.01)且分支节点数明显增加(P<0.01);与单纯17-AAG组相比,17-AAG+负压处理组细胞成管分支节点数明显增加(P<0.05);与单纯负压处理组相比,17-AAG+负压处理组细胞成管总长度明显缩短(P<0.01)且分支节点数明显减少(P<0.01)。蛋白质印迹法检测显示,处理完成后,3组细胞中eNOS、窖蛋白1蛋白表达总体比较,差异均无统计学意义(P>0.05);单纯负压处理组细胞中HSP90蛋白表达与eNOS磷酸化位点1177/eNOS比值均明显高于正常对照组(P<0.01),17-AAG+负压处理组细胞中HSP90蛋白表达与eNOS磷酸化位点1177/eNOS比值均明显低于单纯负压处理组(P<0.01)。处理完成后免疫共沉淀与蛋白质印迹法检测显示,与正常对照组相比,单纯负压处理组细胞中窖蛋白1蛋白表达明显升高(P<0.01),eNOS蛋白表达明显降低(P<0.05);与单纯负压处理组相比,17-AAG+负压处理组处理完成后细胞中窖蛋白1蛋白表达明显降低(P<0.01),eNOS蛋白表达明显升高(P<0.01)。处理完成后,与正常对照组相比,单纯负压处理组细胞中HSP90与窖蛋白1的蛋白共定位明显增多,窖蛋白1与eNOS的蛋白共定位明显减少;与单纯负压处理组比,17-AAG+负压处理组细胞中HSP90与窖蛋白1的蛋白共定位明显减少,窖蛋白1与eNOS的蛋白共定位明显增多。分子对接预测提示,窖蛋白1与eNOS相互作用较强,抑制eNOS 1177位点的磷酸化。结论负压微环境可能通过促进HUVEC中HSP90结合窖蛋白1进而抑制窖蛋白1结合eNOS,以解除窖蛋白1对eNOS 1177位点磷酸化的抑制,从而促进HUVEC增殖、迁移和成管,最终促进HUVEC新生。 展开更多
关键词 负压伤口疗法 细胞微环境 HSP90热休克蛋白质类 窖蛋白1 血管内皮细胞 内皮型一氧化氮合酶
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