Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells

While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25–200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.     

鸡胚胎腺垂体卵泡刺激素细胞的发育及其变化

应用免疫组织化学方法,对发育的第3.5～20.5天鸡胚腺垂体卵泡刺激素(FSH)细胞的发生及其在发育过程中的变化规律进行了研究。结果,鸡胚发育的中期(第10.5天),可观察到少量明显的FSH细胞分布于腺垂体后叶,随着胚胎的发育,FSH细胞数量显著增加(P<0.05),发育的第16.5天FSH细胞数量增加到了整个孵化期的最大值,分布于垂体后叶的腹侧,前叶仅有少量零散的FSH细胞;在发育的第18.5天至出生期FSH细胞数量显著减少。早期FSH细胞体积小、细胞浆少、细胞核大,单个或团状分布,随着胚龄的增加,细胞体积增大、细胞浆增多、细胞浆浓染。结果表明,鸡胚胎腺垂体FSH细胞发生于胚胎发育的中期,细胞的增殖和分化过程发生在胚胎发育的中后期;FSH细胞分布于垂体后叶腹侧。     

微量口腔脱落细胞检材的DNA检验

目的寻求提高微量口腔脱落细胞检材的DNA检验成功率的简便有效的提取方法。方法对不同载体上的100份微量口腔脱落细胞检材采用小体积Chelex-100法提取DNA,在ABI7500型荧光定量PCR仪上进行定量,同时用IdentifilerTM复合扩增系统扩增,在ABI3130遗传分析仪上进行STR分型。结果从25根饮料吸管上提取的DNA量在0.72～116.7.8ng,16个水杯杯缘提取的DNA量在2.15-142.5ng,31个饮料瓶(罐)口提取的DNA量在1～34.65ng,10根筷子上提取的DNA量在3.35～26.6ng,12个果核中提取的DNA量在0.294～21.4ng,6份吃剩的骨头中提取的DNA量在0.88～5.88ng。100份检材性别及9个以上STR位点分型成功率平均为59.38%。除了使用者的个人原因外,检材的提取送检方式、检材的质地、饮料的性质对提取的DNA量有显著影响,是否加蛋白酶K对提取的DNA量无显著影响。结论采用小体积Chelex-100法可对60%左右的微量口腔脱落细胞检材提取DNA进行STR分型。     

干细胞治疗神经性勃起功能障碍的研究现状

神经性勃起功能障碍(neurogenic erectile dysfunction,NED)的发生与勃起功能相关神经的损伤密切相关.最近,干细胞对阴茎勃起神经的修复和保护作用的临床前研究已经成为热点.本文综述了神经源性胚胎干细胞(NESCs)、肌源性干细胞(MDSCs)、脂肪源性干细胞(ASCs)和间充质干细胞(MSCs)等在NED方面的研究进展.早期研究显示干细胞或基因修饰的干细胞对ED治疗持久有效,并可能成功治愈ED.干细胞有望应用于NED的临床治疗.干细胞作为新的治疗方法应用于临床后,将会对法医临床学鉴定提出新的挑战.     

Three-Dimensional Analysis of Cartridge Case Double-Casts

Due to the shot-to-shot variability in tool mark reproduction on fired cartridge cases, a method of replication is needed for the creation of training and testing sets. Double-casting is one method that has been used for this application, but the accuracy and variability of this method needs to be characterized. Three firearms were used to fire 25 cartridges each to create the master cartridge cases. The double-casting method consists of creating a silicone mold of the master cartridge case. A plastic resin mix is then poured into the mold to create the double-cast reproduction. Fifteen double-casts of each of the 75 fired cartridge cases were created across different silicone molds to analyze within- and between-mold variability. The master cartridge cases and double-casts were scanned with a confocal microscope (Sensofar® S neox) to create three-dimensional representations of the surfaces. Two similarity metrics were used for the objective comparison of the double-casts to their master cartridge cases: the areal correlation coefficient (ACCF_MAX) and the number of congruent matching cells (CMC). The ACCF_MAX and CMC data, along with visual examinations, showed that the double-casting method produces accurate reproductions. Within-mold variability was found to be minimal, and between-mold variability was low. These results illustrate that double-casting can be applied for training and testing purposes.     

褪黑激素对犬脂肪干细胞向雪旺细胞分化过程中凋亡发生的抑制作用研究

为了探讨褪黑激素(Mel)对犬脂肪干细胞(ADSCs)向雪旺细胞(SCs)诱导分化过程中凋亡发生的影响。本实验将犬ADSCs诱导分化为SCs,并使用免疫荧光法进行鉴定。采用CCK8法选取Mel的最佳浓度。应用q RT-PCR法和Western-blot法检测无Mel组和Mel处理组中ADSCs向SCs诱导过程中细胞凋亡相关基因Caspase-3、Caspase-8、Bcl-2和Bax的m RNA和蛋白表达水平。结果显示,50 nmol/L Mel处理过的细胞活力最高;ADSCs向SCs诱导后S-100和GFAP呈阳性表达,并且观察到Mel的添加并不会影响到SCs的表型特征。ADSCs向SCs诱导后Bcl-2表达量明显低于对照组,Caspase-3、Caspase-8和Bax的表达量显著高于对照组,细胞凋亡较多;在加入Mel后Bcl-2表达量显著升高,Caspase-3、Caspase-8和Bax的表达量显著下降。结果说明Mel可以显著抑制犬ADSCs向SCs诱导分化过程中的凋亡发生。     

精子细胞定向捕获与分离技术初步研究

目的探索并研究精子细胞定向捕获与分离技术,初步建立精子细胞特异性分离与DNA提取的方法与试剂体系。方法通过特异性定向捕获复合体（精子特异性抗体一磁性纳米微球）的制备,在一定的试剂体系环境下,实现精子细胞的定向捕获与分离。结果能够实现精子细胞的定向富集与分离,通过后续的提取过程,获得了高质量的DNA,并获得了相应的完整STR分型结果。     

混合斑中精子细胞分离及其DNA制备方法

目的尝试建立一种检测混合斑中精子细胞的方法。方法使用显微操作法捕获精子细胞,全基因组扩增(多重置换扩增)精子细胞DNA。结果对10管精斑检材的全基因组扩增,获得了高产、保真的产物。使用50μL体系对20个精子细胞直接进行全基因组扩增,省去了对起始模板的纯化过程,DNA扩增倍数达30000倍以上,片段长度大多在15 kb以上,其STRs复合扩增分型结果有可参照性。结论显微操作法可以有效捕获精子细胞,排除干扰,多重置换扩增可以提供足够量的产物用于法医DNA分析,该方法具有可行性。     

损伤皮肤中组胺荧光显微定位定量与损伤时间推断的实验研究

本文应用显微荧光分光光度计法研究大白鼠皮肤创缘组织中组胺的分布和含量,并用甲苯胺蓝法观察创缘肥大细胞形态和数量的变化。结果发现,创缘真皮乳头层出现扩散的细胞外黄色荧光和肥大细胞脱颗粒现象为生前伤的重要特点;组胺荧光分布范围及增多的程度与损伤时间密切相关。从而为损伤时间推断提供了一种新的方法。     

The probability of achieving full allelic representation for LCN-STR profiling of haploid cells

Modern forensic techniques allow DNA to be extracted from ever decreasing amounts of cellular material. Low copy number (LCN) profiling enables the production of STR profiles from small numbers of cells. Moreover, methods such as laser micro-dissection enables forensic scientists to potentially isolate individual cells for PCR. The DNA derived from haploid cells (semen) is a common source of forensic evidence in sexual assault cases. Haploid cells contain only half the DNA complement of diploid cells (3 pg compared to 6 pg). The smaller the number of cells sampled, the smaller the probability that there is a full representation of the alleles comprising the donor profile. This paper investigates the relationship between the number of cells sampled and the probability of full representation of all alleles in the donor sample. It also considers the effect of typing several loci as opposed to just a single locus.