FISH联用激光捕获显微分离技术对混合斑进行分离检验

目的建立荧光原位杂交(FISH)联用激光捕获显微分离技术(LCM)精确分离男女混合斑中精子的检验方法。方法收集健康志愿者精子和女性阴道上皮细胞制备模拟混合斑,经过预处理后用Vysis CEPX SpectrumOrangeTMY SpectrumGreenTM试剂盒进行荧光原位杂交,并用PALM激光捕获显微分离系统分离男、女性细胞,使用Identifiler试剂盒结合低体积扩增技术分别对男女成分进行STR扩增。结果荧光原位杂交后,可清晰分辨混合斑中的男女细胞。捕获20个精子细胞可以得到完整的STR分型,检出率为80%。随着精子数目增多,检出率提高而等位基因丢失率降低。30个精子检出率最高,为95%。结论激光捕获显微分离联用FISH技术可用于混合斑中男女细胞DNA的分离检验。     

免疫磁珠法分离混合样本中血液成分的探究

目的 基于免疫磁珠法分离脱落上皮细胞中的白细胞，消除或减弱混合检材中血液来源STR分型对结果分析的干扰。方法 分别取两名不同个体的血液和口腔脱落上皮细胞，按不同比例制备成混合样本作为实验组，并同时制备细胞量相等的对照组。应用免疫磁珠法分离实验组样本中白细胞，并与对照组以相同条件进行DNA提取、扩增、分型，对比两组STR分型结果。结果 当血液量较少时，对照组为混合STR分型，经免疫磁珠法分离混合检材中白细胞后，实验组为单一来源STR分型；当血液量较多时，此时对照组为混合STR分型，但口腔脱落上皮细胞来源的STR分型谱带峰高较低甚至丢失，经免疫磁珠法后，实验组仍为STR混合分型，但口腔脱落上皮细胞来源的STR分型谱带成为主峰；当混合样本中口腔脱落上皮细胞微量，血液占比极大时，此时对照组为血液来源的单一STR分型结果，经免疫磁珠法后，实验组为STR混合分型，包含口腔脱落上皮细胞来源的所有等位基因分型。结论 该方法可用于分离脱落上皮细胞中血液成分，降低血液来源的DNA比例，能够改善此类混合检材中目的细胞的STR分型结果，为刑事案件中含有血迹浸染的混合生物检材的检验提供了一种新思路。     

龙胆紫染色法在显微捕获单细胞分离技术中的应用

目的建立用于显微捕获单细胞技术的龙胆紫染色法并评价其应用效果。方法制作20枚口腔拭子脱落细包悬液,配制0．05g／mL龙胆紫染液。取100μL口腔细胞悬液加入0．15、0．25、0．5、1．0、2．0μL染色液,考察最佳染色浓度;争别染色5、10、30、60min,考察最佳染色时间;用最佳条件染色后抓捕3个细胞,采用联合LV-PCR技术扩增并进行DNA分驯佥测,进行重复试验20次,并对同一检材未经染色的抓捕细胞进行检测,用于比对STR分型成功率。将龙胆紫染色法用于案例检材的口腔上皮细胞分离检验。结果1001μL细胞悬液加入0．5止0．05g／mL龙胆紫染液,染色5min,胞核呈紫工色,与胞浆对比明显;染色时间延长不影响染色效果及细胞分离检验结果。优化后的龙胆紫染色法对低体积扩增无归显抑制（P〉0．05）。应用此染色法于案例检材,胞核标识清晰,STR分型结果达同一认定标准。结论龙胆紫染色法刚于细胞核的发现,有助于提高显微捕获单细胞技术的检测效能。     

激光捕获显微分离技术在DNA检验中的应用研究

目的建立一套显微细胞捕获技术用于法医学生物检材DNA检验方法。方法使用VeritasTM LCM激光捕获仪,采用紫外加红外的捕获方式,对框架覆膜玻片上经苏木素染色口腔上皮细胞进行捕获,采用改良硅珠法提取细胞DNA,使用Identifiler TM试剂盒在5μL体系中进行PCR扩增。结果成功获得20个口腔上皮细胞的13个以上完整STR基因座分型谱带。结论本研究建立的方法适合法医学生物检材制成的染色涂片上细胞的DNA检验。     

激光显微捕获口腔上皮细胞的DNA分型

目的探索激光显微技术(lasercapturemicrodissectionsystem,LCM)捕获口腔上皮细胞,并进行STR-DNA分型检测的方法。方法用VERITAS显微切割仪红外低能激光显微捕获一定数量口腔上皮细胞,进行ProfilerPlus试剂盒STR复合扩增,检测DNA基因型。结果7～8个口腔上皮细胞能成功获得STR-DNA分型。3～4个口腔上皮细胞不能成功获得STR-DNA分型。结论激光显微捕获作为一种分离单个细胞的新技术,对于微量口腔上皮细胞的STR-DNA分型是可行的。     

单细胞分离荧光原位杂交法用于男女混合血DNA分型

目的采用单细胞分离荧光原位杂交法精确分离混合血样中男性和女性细胞并进行分型检验。方法收集男、女性血,按照男∶女为1∶5、1∶10、1∶20制备混合血样,加入0.075mol/L KCl 600μL,轻混、放置30min后加入150μL固定液离心留沉淀涂片,利用Vysis 30-161050试剂盒进行荧光原位杂交,并用PALM激光显微捕获系统分离出男、女性细胞,使用Identifiler试剂盒复合扩增并进行检测。结果捕获8个血细胞即可得到完整的DNA分型,且随着细胞数目的增多,检出率逐渐提高而等位基因丢失率逐渐降低。10个血细胞的检出率最高,为93.75%。5μL男性血液与本实验各比例女性血混合用本文方法检验均可获得男性分型。案例混合血斑经检验获得单一男性和女性分型。结论单细胞分离荧光原位杂交法可用于男女混合血样本中DNA分型检验。     

激光捕获显微分离技术分离多人混合精斑

目的利用激光捕获显微分离技术(LCM)分离多供体混合精斑。方法将3名自愿者供给的精液混合后,制备成混合精斑,并涂于覆膜玻片上。用PALM激光捕获显微分离系统切割并捕获样本中的单个精子,利用低体积扩增技术(LV-PCR)扩增并进行检测,并对分型结果进行统计。结果联用LCM和低体积扩增技术捕获样本中单个精子细胞进行扩增,可获得每位自愿者的Y-STR分型,分型成功率为73.15%。结论激光捕获显微分离联用低体积扩增技术可完成多供体混合精斑的分离检验。     

MiniFiler试剂盒进行微量细胞STR分型可行性探讨

目的探讨采用MiniFiler试剂盒进行微量细胞STR分型的可行性。方法利用显微操作法捕获微量口腔上皮细胞,采用MiniFiler试剂盒进行STR复合扩增,ABI 3130遗传分析仪检测STR分型。结果10个口腔上皮细胞能够获得稳定的STR分型;1、3、5个口腔上皮细胞能够获得完整的STR分型,但存在随机性。结论微量细胞进行STR分型具有不稳定性,目前还难以在实际检案中应用,但可以通过增加模板量来提高分型的成功率。     

口腔拭子DNA检验的实时定量研究

目的寻求提高口腔拭子的DNA检验效率的方法。方法对来源于不同个体或同一个体不同擦拭次数的口腔拭子用Chelex-100或磁珠法提取的DNA用实时定量PCR技术进行定量,同时用Identifiler复合扩增系统在ABI3100遗传分析仪上对这些DNA样品进行STR分型。结果在建立的Identifiler系统8μl扩增体系中,3个月内的口腔拭子用Chelex-100法提取的DNA模板1μl量较3μl量扩增效果好,磁珠法提取的DNA模板用量大小对复合扩增检测影响较小。结论用棉签擦拭颊粘膜5次制备口腔拭子,取其头部的约1/4用200μlChelex-100法提取DNA,然后用1μl模板进行复合扩增,是提高口腔拭子的DNA检验效率的简便可行的方法,但陈旧口腔拭子用磁珠法提取更能保证复合扩增分型成功。     

尼龙膜套管分离技术对混合斑中精子细胞DNA的提取

目的建立新型的混合斑中精子细胞分离的方法,并评价其应用价值。方法收集性侵案件中的40份混合斑检材,分别采用常规差异裂解法和尼龙膜套管分离技术进行精子细胞分离,使用硅膜试剂盒(Forensic DNA Extraction Kit for Soft Tissues)提取精子细胞DNA,Amp FlSTR~ Identifiler~ Plus PCR扩增试剂盒进行PCR扩增,3500x L基因分析仪进行电泳检测,并对两种分离方法的结果进行对比。结果 40份混合斑检材中,采用尼龙膜套管分离技术的检材仅3份有女性成分微弱残留,余均获得了完整的单一男性分型。而采用常规差异裂解法分离的检材中,有25份完全未检出男性精子细胞STR分型,15份检出男性精子细胞STR分型(7份不完整男性精子细胞STR分型,6份有女性成分残留,2份单一的男性精子细胞STR分型)。结论本研究建立的尼龙膜套管分离技术适用于混合斑中精子细胞的分离,特别是对含有大量女性细胞而精子细胞较少的检材提取效果明显,整个提取实验成本低廉、快速简便。     

Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis

This report focuses on the development of a method for chemically induced enhancement of cell elution and recovery from cotton swabs. The method exploits the exclusive use of detergents for intact cell removal, and can be utilized in conjunction with, or to circumvent, conventional differential extraction (DE). Samples treated with Sarkosyl (54.4 +/- 1.8%) and sodium dodecyl sulfate (SDS) (78.5 +/- 0.7%) yielded higher sperm cell recoveries than a conventional DE buffer (39.4 +/- 2.1%). The results indicated that the choice of detergent affected sperm cell yield, with anionic detergents having the greatest effect. Storage time of samples affected the concentration of detergent required for optimal sperm cell recovery, longer times requiring increased detergent concentrations. In addition, the extent of sperm cell lysis by proteinase K digestion was evaluated. The results indicate that the exclusive use of SDS enhances the release of sperm and epithelial cells from a cotton swab as compared with DE buffer, providing for a more effective DNA analysis.     

兔阴茎海绵体平滑肌细胞培养及其RyRs的表达

目的探讨兰尼碱受体(RyRs)在兔阴茎海绵体平滑肌细胞(CCSM)的正常表达。方法取雄性性成熟新西兰兔CCSM细胞,用组织块法进行细胞培养,差速贴壁法进行细胞纯化,利用倒置显微镜观察、免疫荧光染色法鉴定细胞纯度;取三代以内培养细胞,通过Genebank查出RyRscDNA序列,针对RyRs3种不同亚型的非同源部分用PrimerPremier5.0软件设计3对引物,以逆转录PCR方法分别检测RyRs3种亚型mRNA的表达,凝胶成像系统分析结果。结果培养7d开始有细胞从组织块长出,15～20d后长满瓶底,呈典型的“峰-谷”现象,经倒置显微镜观察见培养的细胞呈现典型的梭形,免疫荧光染色可见细胞内染为绿色的肌丝,证实培养的细胞为CCSM细胞,经差速贴壁法纯化后,细胞纯度可接近100%。经RT-PCR检测,CCSM只表达RyRs1,未见RyRs其余亚型的表达,提示CCSM表达RyRs1蛋白。结论CCSM表达RyRs中的1亚型,提示其可能参与CCSM细胞的钙调节,进而能调节阴茎勃起功能。     

氯胺酮对PC12细胞增殖及凋亡的影响

目的 探讨氯胺酮对肾上腺嗜铬细胞瘤(pheochromocytoma,PC12)细胞的增殖抑制和诱导凋亡的作用及其机制. 方法 以大鼠PC12细胞为多巴胺能神经元的细胞模型,分别以0.9、1.2、1.5、1.8、2.1 mmol/L浓度的氯胺酮加入培养的PC12细胞中,分别培养12、24、48、72h后,用MTT实验检...     

Enhanced elution of sperm from cotton swabs via enzymatic digestion for rape kit analysis

This report describes development of a method for enhanced cell elution from cotton swabs. The method exploits an enzyme mixture for digestion of the cotton to remove intact cells, and can be utilized in conjunction with or to circumvent conventional differential extraction (DE). Samples digested with Aspergillus niger cellulase yielded sperm cell recoveries (18+/-3.5%) similar to conventional DE buffer (23+/-7.8%) while providing intact epithelial cells. Storage time affected the concentration of enzyme required for optimal sperm cell recovery, with longer times requiring increased cellulase concentrations. Cellulase from A. niger yielded a twofold enhancement in sperm cell elution over buffer alone, and preliminary testing of higher activity cellulases from Trichoderma reesei and Trichoderma viride showed even greater enhancement. These results indicate that cellulose-digesting enzymes enhance the release of sperm and epithelial cells from a cotton swab over buffer alone, providing for efficient DNA analysis.     

Separation of sperm and epithelial cells based on fluorescence-activated cell sorting

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.     

激光捕获显微切割技术用于分离混合斑中精子细胞

目的 评估激光捕获显微切割(laser capture microdissection.LCM)技术在分离混合斑中少量精子细胞的应用价值.方法 配制不同比例的精液-阴道上皮细胞混合液.分别用差异裂解法和LCM法分离精子细胞,用磁珠法提取精子细胞DNA,并用IdentifilerTM试剂盒进行STR基因型检测.结果 LC...     

毒鼠强诱导细胞DNA损伤的彗星电泳检测

目的　研究毒鼠强对小鼠淋巴细胞和脑细胞DNA的损伤作用。方法　分离健康小鼠的淋巴细胞和脑细胞 ,以彗星电泳的方法测定不同浓度毒鼠强处理后的细胞DNA损伤。结果　 1/2 0～ 1/2LD50 剂量组的毒鼠强均可引起淋巴细胞和脑细胞不同程度的DNA损伤 ,与对照组呈极显著性差异 (P <0 0 0 1)。结论　毒鼠强引起细胞DNA断裂损伤 ,并呈现明显的剂量 -效应关系。     

Differentiation of epithelial cell types by cell diameter

Genital swabs play an important role in cases of alleged sexual assault. The aim of our study was to see if epithelial cells from the vagina, glans penis, or mouth could be distinguished on the basis of size. Vaginal swabs were taken from 12 women in different phases of their menstrual cycles; penile swabs were taken from 5 men, and mouth swabs were taken from 6 men and 6 women. For each swab, a sample was smeared across a microscope slide and allowed to dry. The dried epithelial samples were then viewed without any further processing with a "SteReoLumar.V12" stereo microscope. The microscope slide surfaces were divided into grids and all single epithelial cells whose contours could be clearly distinguished were photographed. The maximum diameter for each photographed cell was digitally determined using the Axiovision software. In total, 995 vaginal epithelial cells, 211 penile epithelial cells, 329 male oral epithelial cells, and 525 female oral epithelial cells were measured. Menstrual cycle phase did not affect vaginal epithelial cell diameter. The mean vaginal epithelial cell diameter was 63.95 microm (min. = 28.08 microm, max. = 108.06 microm, s = 11.50 microm). The mean penile epithelial cell diameter was 39.24 microm (min. = 28.38 microm, max. = 51.02 microm, s = 4.84 microm). The diameter of oral epithelial cells hardly differed for both sexes, although the female cells were, on the whole, slightly larger. On the basis of these results, it is not possible to conclude that epithelial cells of less than a certain diameter found in the assessment of a vaginal swab must be of penile origin. It is also not possible to usefully distinguish vaginal epithelial cells from male or female oral epithelial cells on the basis of the diameter. However, finding epithelial cells with a diameter distinctly greater than 50 microm in a penile swab sample suggests the presence of vaginal or oral epithelial cells. Epithelial cells examined with the presented method can be used without restrictions for further examinations, such as single-cell DNA analysis after single-cell picking with the micromanipulator developed by Aura Optik (Jena).